The American Stroke Association/American Heart Association recommended the criteria for diagnosis of vascular cognitive impairment and memory impairment (MI) is a feature in the classification of vascular mild cognitive impairment (VaMCI). VaMCI patients with MI may differ in terms of infarct location or demographic features, so we evaluated the clinical characteristics associated with MI in patients with VaMCI.A prospective multicenter study enrolled 353 acute ischemic stroke patients who underwent evaluation using the Korean Vascular Cognitive Impairment Harmonization Standard Neuropsychological Protocol at three months after onset. The association between MI and demographic features, stroke risk factors, and infarct location was assessed.P = 0.03, 66.7 vs. 47%, P = 0.02). Multiple logistic analyses revealed that male sex , left-side infarcts , and basal ganglia/internal capsule infarcts  were associated with MI after adjusting other demographic variables, vascular risk factors, and subtypes of stroke.VaMCI was diagnosed in 141 patients, and 58 (41.1%) exhibited MI. Proportions of men and of left side infarcts were higher in VaMCI with MI than those without (75.9 vs. 57.8%, MI is associated with sex and infarct location in VaMCI patients. Vascular mild cognitive impairment (VaMCI) is a subtype of vascular cognitive impairment (VCI) and commonly follows stroke ,2. WhileCognitive impairment after stroke is characterized by disturbance of frontal or executive function, however, several previous studies also indicated that clinical stroke causes subsequent poorer performance in multiple cognitive domains including memory -8. MemorThe prevalence of MI is about 23–55% from a review paper  and is 2In addition, neuropsychological protocols previously used to assess VCI have varied with respect to examined cognitive domains ,4,15. VCSix hundred twenty acute ischemic stroke patients from 12 hospitals were enrolled in the study within 7 days of symptom onset from October 2007 to August 2008. Exclusion criteria and the exact number of patients for each stage of the study are shown in Figure The 60-min K-VCIHS-NP includes 7 tests that assess 4 cognitive domains: the Hopkins Verbal Learning Test for memory; Semantic  and phonemic fluency tests, the Digit Symbol Coding, and the Trail Making Test for executive function/activation; the Rey Complex Figure Test-Copy for visuospatial skills; and the Boston Naming Test for language .We also administered the Korean version of the Informant Questionnaire on Cognitive Decline in the Elderly (K-IQCODE) to examine pre-morbid history of cognitive impairment; the Instrumental Activities of Daily Living scale (IADL) to assess instrumental complex daily functioning; and the Korean version of the Mini-Mental Status Examination (K-MMSE) and Clinical Dementia Rating (CDR) as supplementary tests of cognitive function at 3 months post-stroke. All tests and scales were previously validated for the Korean language and standardized in Korean subjects by age and educational levels ,21.th percentile on more than 2 tests were identified as having impaired functions. Abnormal functional status was indicated by a score greater than 0.43 on the Korean IADL [The diagnostic procedure included a clinical interview and the administration of the K-VCIHS-NP, K-IQCODE, and IADL. Impaired cognitive function was defined as impairment in at least one cognitive domain on the K-VCIHS-NP, as indicated by a score below the 10th percentile using normative corrections for an individual domain. For executive function/activation domains, which consisted of 4 tests, patients who scored below the 10ean IADL . Pre-strean IADL . A vascuWe compared the demographic features and characteristics of index infarcts of VaMCI patients based on the presence of MI, using a Mann-Whiteny test for continuous variables and chi-square tests for categorical variables. Univariate and multivariate logistic analyses examined the influence of baseline demographic features, infarct locations, and classification of stroke subtypes on memory impairment in VaMCI at 3-month after stroke. We entered age, sex, duration of education, all locations and subtypes of ischemic stroke in the logistic model for detailed description. Analyses were performed with SPSS (Windows version 18.0).http://hallym.hallym.or.kr/irb/). All subjects provided informed consents. This study followed Good Clinical Practice guidelines and was consistent with the International Conference on Harmonization of ethical principles for medical research involving human subjects.This study was approved by the Institutional Review Board/Ethics Committee of each participating hospital , while no sex differences were observed in the other domains.At 3 months post stroke, 506 patients were follow-up and 353 patients completed K-VCI-HP. Cognitive impairments were present in 221 patients, and VaMCI was diagnosed in 141 patients . Correlations were observed between language and memory impairments  and language and visuospatial impairments .Sixty-nine patients (48.9%) exhibited impairments in two or more domains: There were 17 amnestic single domain VaMCI patients, 41 amnestic multiple domain VaMCI patients, 55 non-amnestic single domain VaMCI patients, and 28 non-amnestic multiple domain VaMCI patients. Combined memory and executive/activation impairments were present in 35 patients, but these were not correlated . VaMCI with MI tended to have a higher chance of having basal ganglia/internal capsule  or cerebellum  as the location of index stroke than those without. The two groups did not significantly differ in education levels; the vascular risk factors; the other location or the number of infarcts; TOAST classification  and showed a tendency of lower score in K-MMSE  three months after stroke. There are no difference in K-IQCODE (3.0 ± 0.53 vs. 3.0 ± 0.49) and instrumental ADL (0.19 ± 0.37 vs. 0.19 ± 0.32) between two groups.VaMCI with MI had a higher score in sum of box of CDR , left-side infarct location , and basal ganglia or internal capsule infarct location  were associated with MI  and the proportion of cognitive assessing was somewhat low compared to the previous studies ,22. ThisSex differences in VaMCI have been indicated by several studies ,14,23. FInfarct location influenced the domain of cognitive impairment. Basal ganglia and thalamic infarcts have been associated with MI in previous studies ,28-31. TThe strength of this study is that it investigated the clinical details of VaMCI with MI using ASA/AHA guidelines for VaMCI diagnosis and VCIHS protocols ,16. The This study has several limitations. First, we did not measure white matter or infarct volume; or Alzheimer’s biomarkers, such as hippocampal volume and apolipoprotein E subtypes ,14,34. TSex and stroke location differ between VaMCI with MI and those without MI. VaMCI with MI was more frequently men and associated with infarcts on the left side or within the basal ganglia or internal capsule. These differences might be considered in the interpretation of cognitive testing or rehabilitation of VaMCI patients.


Oryza sativa L.) are direct seeded rices grown on hill slopes of different gradients. These landraces have evolved under rainfed and harsh environmental conditions and may possess genes governing adaptation traits such as tolerance to cold and moisture stress. In this study, 64 hill rice landraces were collected from the state of Arunachal Pradesh of North-Eastern region of India, and assessed by agro-morphological variability and microsatellite markers polymorphism. Our aim was to use phenotypic and genetic diversity data to understand the basis of farmers’ classification of hill rice landraces into two groups: umte and tening. Another goal was to understand the genetic differentiation of hill rices into Indica or japonica subspecies.Hill rices  and tening . We did not find significant difference in days to 50 % flowering between the groups. Principal component analysis revealed that two groups can be distinguished on the basis of kernel length-to-width ration (KLW), kernel length (KL), grain length (GrL), grain length-to-width ration (GrLW) and plant height (Ht). Stepwise canonical discriminant analysis identified KL and Ht as the main discriminatory characters between the cultivar groups. Genetic diversity analysis with 35 SSR markers revealed considerable genetic diversity in the hill rice germplasm . Pair-wise allelic difference between umte and tening groups was not statistically significant. The model-based population structure analysis showed that the hill rices were clustered into two broad groups corresponding to Indica and Japonica. The geographic distribution and cultivars grouping of hill rices were not congruent in genetic clusters. Both distance- and model-based approaches indicated that the hill rices were predominantly japonica or admixture among the groups within the subspecies. These findings were further supported by combined analysis hill rices with 150 reference rice accessions representing major genetic groups of rice.According to farmers’ classification, hill rices were categorized into two groups: japonica or admixture among the subpopulations of Indica or Japonica. The findings are useful for utilization and conservation of hill rice germplasm.This study collected a valuable set of hill rice germplasm for rice breeding and for evolutionary studies. It also generated a new set of information on genetic and phenotypic diversity of hill rice landraces in North-Eastern region of India. The collected hill rices were mostly The online version of this article (doi:10.1186/s12863-016-0414-1) contains supplementary material, which is available to authorized users. Oryza sativa L.) is one of the important crops in the world. Being a model organism with fully sequenced genome, it also affords unique opportunities to use genomic approaches to study its domestication, adaptive selection, and the history of crop improvement [Indica and Japonica, three to seven genetically distinct groups have been identified within global rice (Oryza sativa) germplasm sets in different studies [O. sativa, resulting in >120,000 distinct rice varieties [Rice is the primary food source for more than half of the world’s population . Over 90rovement , 3. The rovement –6). In a studies –14. Nume studies –22. Such studies , and for studies . The cularieties . An assejhum) and sedentary agriculture. Shifting or ‘slash-and-burn’ cultivation is the earliest form of agriculture and is still practiced in vast areas of the state. About 76 % of the total cropped area in the state is under jhum cultivation.The North-Eastern (NE) region of India, due to its unique eco-geographical features and high ethnic diversity, possesses at least 10,000 indigenous rice landraces which cultivated under upland, lowland and deep water conditions . The impjhum fields), plateaus, terraces and river beds. A wide range of rice landraces were found throughout the state. Sometimes it depends on the tribal communities who inhabit the area. It was estimated that more than 70 % of the total rice varieties grown in the state are traditional cultivars or landraces having low to very low productivity [jhum cultivation system possess many adaptive traits such as cold tolerance important for crop improvement programmes. Cold-tolerance in hill rice landraces was also observed in high altitude (1300–2200 m) rice areas in Nepal [jhum) rice cultivars of Arunachal Pradesh.Rice is the principal crop of Arunachal Pradesh and it is grown in 44.7 % of the total cropped land and accounts for 70.3 % of the total food grain production . Rice pructivity . Despitein Nepal . Similarviz, East Kameng, Papum Pare and Kurung Kumey of the state of Arunachal Pradesh was conducted for the collection of hill rice landraces. These rices are direct seeded, grown on upland fields, usually on sloping hill sides (jhum lands), and are grown as rainfed during June to September. During the collection trip, we interviewed local farmers to gather information on significance of naming a landrace, its cultural value and popularity, and production practices of local farmers. The indigenous farmers of the surveyed region rely on gathering forest products, hunting and cultivation of rice and maize for their subsistence. In the present study, we phenotyped these rice landraces for 16 agro-morphological and grain characteristics, and genotyped them with 35 SSR markers. The specific objectives of this study were to: (i) collect information on the meanings of the variety names, special uses or properties, and important agronomic characteristics; (ii) study the agro-morphological variability; and (iii) understand the genetic relatedness and structure of these landraces.In 2012, an exploration trip to three districts A total of 67 hill rice accessions were collected from 32 villages in three districts  of Arunachal Pradesh, India during a collection trip from November 2–15, 2012 , two aus , three Basmati , three short-grain traditional aromatic  and a single accession each of tropical japonica (‘Azucena’) and japonica admixture (‘Dular’). Pure seeds of these accessions were obtained from ICAR-National Rice Research Institute, Cuttack, India. These varieties were used to understand genetic grouping of the hill rice accessions, and also to act as a molecular weight reference in DNA gel electrophoresis. We did not include these varieties in phenotypic evaluation as the plant growth was very poor for most of the accessions.A total of 15 control varieties were selected for SSR genotyping along with the hill rices . We sampled 150 accessions from the originally reported 234 accessions used in Garris et al. [To provide a reference for the rice subpopulation classification in the current study, we also used the SSR fingerprinting data of 150 rice accessions (here we denoted these as reference accessions) previously reported in the genetic diversity study by Garris et al. . The bans et al.  based ons et al. .Kharif season (June-November) under rainfed upland conditions. The soil of the field is an acid alfisol with a pH of 5.3, organic C 1.13 % and available N 211 kg/ha. Farmyard manure (10 tonnes/ha) was incorporated into the soil during land preparation. No chemical fertilizers and pesticides were used during crop growth period. A manual weeding was conducted to keep the crop weed free. The accessions were line-sown in 1 m x 1.5 m plots (4 lines/accession) using an augmented block design during first week of June. A spacing of ~0.25 m between the plants and 0.45 m between the rows was maintained. Phenotypic data, listed in Table Sixty four hill rice accessions and three check varieties  in 2015 http://www.gramene.org/markers/microsat/). Information about these markers is given in Additional file Thirty five SSR markers were designed from the ‘Gramene’ marker database  was performed on the correlation matrix of the data to understand the most important variables contributing to the total phenotypic variation among the accessions. PCA represents the simplest and most commonly used multivariate method to visualize the grouping of accessions based on component loadings. Ward’s hierarchical clustering was used to understand the relationships among rice accessions based on phenotypic data. All these analyses were performed using IBM-SPSS Statistics version 20.0 [Summary statistics such as mean, standard deviation (SD), minimum and maximum values, and coefficient of variation (CV) were determined. The mean data were standardized by deriving ion 20.0 .K) evaluated here ranged from 1 to 8. The analysis was performed using five replicate runs per K value, a burn-in period length of 5000, a run length of 50,000, and a model allowing for admixture and correlated allele frequency. ‘Structure harvester’ programme (http://taylor0.biology.ucla.edu) was used to determine the final K value(s) based on both the LnP(D) and Evanno’s ΔK [K = 2–4 were then performed with a burn-in period of 10,000 and a run length of 100,000. The membership coefficient from the run with the lowest likelihood value was used to assign each accession to the K = 1 to 4 subpopulations based on the estimated membership coefficients.Population structure of 64 hill and 15 control rice accessions was examined using the Bayesian model-based approach implemented in STRUCTURE V2.3.4 . The numnno’s ΔK . SubsequHe), heterozygosity (Ho) and polymorphism information content (PIC) were calculated using PowerMarker V3.25 [Rs) and Wright’s fixation index (Fst) values were calculated using FSTAT V2.9.3.2 [The average number of alleles per locus (AN), major allele frequency (MAF), gene diversity . The reference set also included some ‘admixture’ accessions. For clustering, a pair-wise genetic distance matrix was calculated in PowerMarker following C. S. Chord distance method [Fst and allelic differences (FPTest) between the groups were calculated as stated earlier.Cluster analysis was conducted on hill and reference accessions to understand the grouping of hill rices in respect to different genetically defined groups , large grained-late maturing rice, and (ii) tening, small grained-early maturing types  umte and tening groups of hill rice are given in Table tening group showed higher average value of DtF (120.8 d) than umte (115.8 d). However, the difference was prominent for grain and kernel characteristics. The umte accessions had longer grains with an average value of 6.9 mm than that of tening (6.2 mm). Similarly, the value of KL in umte (5.4 mm) was higher than that recorded in tening (4.6 mm). Both the highest values for GrLW and KLW were recorded in umte. On average, the plants of umte accessions (81.1 cm) were taller than the tenings (69.9 cm). The tenning accessions produced larger leaves (59.7 cm).Descriptive statistics for 16 agro-morphological characteristics in umte and tening groups clustered separately based on the phenotypic traits  using 16 traits produced five principal components  which cumulatively accounted for 76.4 % of the total phenotypic variance  of 15 tenning accessions which had highest values for KW, GrLW, DtF, LL and leaf width (LW).Ward’s hierarchical cluster analysis revealed a similar grouping of accessions as found in PCA Fig. . OverallHe) ranged from 0.41 (RM55) to 0.94 (RM259) and the average observed heterozygosity (Ho) was 0.051. Allelic richness (Rs) varied from 2.0 (RM338) to 14.6 (RM259) with an average value of 6.04 across the study. The polymorphism information content (PIC) values ranged from 0.37 (RM338 and RM507) up to 0.93 (RM259) with an average of 0.65.The summary statistics of 35 SSR markers are given in Additional file K values by increasing K from 1 to 8, we found that LnP(D) values increased with K, with the highest log likelihood score at K = 2, while ∆K value was also highest at K = 2  including ‘Azucena’, ‘TKM9’ and ‘Pakistani Basmati’ were identified in this group. Group 2 consisted of a total of 47 accessions including ‘Dular’, a japonica admixture variety, as admixture. At K = 3, ten control varieties  formed group 1 along with 11 hill accessions. Group 2 has 15 hill rices along with ‘Dular’ as admixture. A majority of hill rices (37 accessions) were included in group 3 along with the control varieties such as ‘Azucena’, ‘TKM9’, ‘Pakistani Basmati’ and ‘Mohanbhog’. The subpopulation structure in these rice accessions was more clear at K = 4. The four subpopulations (StrGr1 to 4) had a population-specific Fst value of 0.33, 0.33, 0.06 and 0.20, respectively, with an average of 0.23, indicating a moderate population structure. At this level, indica control varieties were separated from aus and aromatic varieties. The aromatic tropical japonica variety ‘Azucena’ showed separate grouping along with 50 % of hill accessions. A group of 15 hill accessions formed a distinct group (StrGr1). At K = 4, a total of 25 accessions were identified as an admixture.The inferred population structures are given in Fig. indica and aus controls, along with four hill accessions collected from Kurung Kumey and a single accession from East Kameng. Cluster 2 grouped all aromatic control varieties and four hill accessions collected from East Kameng. The rest hill rices grouped either with tropical japonica variety ‘Azucena’ (53 accessions) or with ‘Dular’ (3 accessions). The cluster membership of each accession is given in Additional file K = 4). However, the PCoA results were consistent with STRUCTURE  analysis of pair-wise genetic distances detected four major clusters  among and within subpopulation groups corresponding to districts, STRUCTURE groups and overall hill and control accessions  0.8 % of the total variation among umte and tening groups was recorded. Considering the districts, the highest genetic diversity was observed in East Kameng accessions (0.65) with a mean of 6.31 alleles per locus. The lowest genetic diversity was recorded in the accessions from Papum Pare (0.50) with a mean of 3.54 alleles per locus  was identical. The umte group had the highest PIC value in spite of having lower number of accessions. Among four STRUCTURE subpopulations, the highest genetic diversity was observed in StrGr3 (0.69) with an AN of 6.26, followed by StrGr4 (0.59) with an AN of 5.69. Similar trends were observed for PIC and allelic richness (Rs) values. The overall genetic diversity in hill and control accessions was at par, but the hill rices had the highest Rs (5.47) and AN (7.4). Note that, despite having only 15 accessions, the control group recorded the highest genetic diversity and PIC. This may be due to inclusion of diverse rice control varieties from different genetic groups.The rice accessions displayed different pattern of SSR variation with respect to district, cultivar group and STRUCTURE grouping Table . The AMOus Table . Betweenumte and tening, or between controls and either of the two groups. Among the four STRUCTURE subpopulations, allelic count of StrGr3 was significantly different with that of StrGr1 and StrGr2  is an important measure of diversity. Pair-wise tests of allelic differences between different groups of accessions are given in Fig. Gr2 Fig. . Pair-wiFst values among and between subpopulations are given in Additional file umte and tening was very low (0.008). Overall, the rice accessions from three districts had a low level of differentiation with Fst values ranging from 0.043 to 0.126. Among the STRUCTURE groups, the greatest differentiation was observed between StrGr1 and 2 (0.290), and the lowest was between StrGr3 and 4 (0.086).Pair-wise estimates of indica, aus, aromatic, tropical japonica and temperate japonica , followed by the indica (0.231) .We sampled 150 diverse rice cultivars representing all five major groups identified in previous studies , 28 to aica Fig. . The paiumte and tenning based on grain morphology and crop duration. These two groups of hill rice germplasm varied significantly for the traits such as plant height, leaf length, grain/kernel length and grain/kernel length-to-width ratio, but non-significant genetic differentiation between the two groups were observed. Population genetic analysis showed that these hill rices were mostly japonica or admixture among the subpopulations of Indica or Japonica. These results are useful for understanding the agro-morphological variability, genetic diversity and structure of hill rice landraces, and significant for utilization of hill rices in crop improvement programmes. Our study also provides baseline information for planning in-situ conservation of the hill rice landraces, which are adapted to local environments in harmony with cultural preferences of the farmers.Our characterization of hill rice germplasm of North-Eastern region of India has revealed that a diverse array of rice landraces was grown by the farmers which traditionally classified as pitha and sweets. Therefore, knowledge about the local names of rice landraces is useful for understanding their origin as well as grain quality and other characteristics. The traditional rice landraces possess wide genetic variability valuable for rice breeding [The traditional names of rice cultivars are generally found to be a valuable concept in describing the landraces worldwide. In the present study, the names of rice cultivars varied among the districts. Few rice accessions ‘Taba’, ‘Kilung’ and ‘Sarpung’ were cultivated in all three districts. The farmers in Arunachal Pradesh most often name the rice landraces after their place of origin. For instance, ‘Nepali dhan’ is originated in Nepal. Likewise, ‘Manipuri dhan’ and ‘Naga dhan’ are from the state of Manipur and Nagaland, respectively. Rice landraces often named after a community/tribe. For example, ‘Taba dugu’ and ‘Adi’ named after ‘Taba’ and ‘Adi’ communities of Arunachal Pradesh, respectively. During collection trip the farmers expressed that hill rice cultivars generally taste better than the low land cultivars, and thus used for making local delicacies like breeding , 43. Thebreeding . In Nepabreeding . It was breeding .umte and tening). Our analysis apparently showed that two groups of hill rice are mainly differentiated by the traits such as grain/kernel length and length-to-width ratio. We did not find significant variation in days to flowering between umte and tening. However, this is a key trait considered by the farmers in categorizing the hill rices. The non-significant variation in days to flowering in the present study may be due to change in crop growing conditions including soil and other environmental factors. We also performed a step-wise canonical discriminant analysis (CDA) taking umte and tening as priori groups to identify the most important discriminating traits contributing to the grouping of hill rices. Kernel length and plant height were found to be the best predictors for classifying the cultivar groups (results not shown). In both PCA and cluster analysis, we found that the accessions with similar names formed close groups, indicating the consistency in naming the cultivars by traditional farmers. However, in a different study with 130 indigenous rice accessions from a neighbouring state  in NE India, we found inconsistency in naming the cultivars [The tribal farmers of Arunachal Pradesh usually classify hill rice cultivars on the basis of grain morphology and/or quality characteristics, in addition to other agronomic traits such as crop duration. In the present study, we have assessed the variability in agronomic and grain/ kernel morphological traits to understand the morphological factors differentiating the two groups  and naming of rice cultivars. We cannot explain why geographical distribution did not match with genetic relationship among accessions. Seed exchange between the villages might be the reason. We noted that the farmers in Arunachal Pradesh most often gift seeds of good rice varieties during wedding ceremonies. It has been observed that the landraces with the same name collected from different villages/district are not always genetically identical. Similar observations were also reported in other studies , 48. TheHe = 0.66; PIC = 0.62) similar to the 107 aromatic rice cultivars of NE India  [umte and tening groups of hill rice was at par, and pair-wise allelic difference was non-significant. This is perhaps due to similar genetic makeup of the accessions of two morphological groups. The genetic diversity estimates among four STRUCTURE subpopulations were higher for group 3 and 4 , which can be categorized as japonica groups. However, the gene diversity in nine accessions of groups 2 was slightly lower (He = 0.53). This group was characterized by indica accessions, and higher genetic diversity of these accessions supports the fact that indica accessions are more diverse than japonica accessions [The current hill rice accessions revealed overall genetic diversity ( = 0.62) . Slightl = 0.62) . The gen = 0.62) . Althoug = 0.62) . The divcessions , 56.japonica or admixture among the subpopulations of Indica or Japonica. Both FPTest and Fst results indicated that the hill accessions are genetically diverse from temperate japonica and indica. The rice germplasm prevailing in NE India has great importance in studying the evolution and population genetics of cultivated rice. This region is considered to be the domestication origin of Indica rices [Japonica rice [Japonica rices has also been recorded in NE Indian rice germplasm [Indica-Japonica evolution.The present research, although with a limited number of SSR markers, indicated that hill rices were mostly ca rices . While, ica rice , 57. Theica rice . Prevaleermplasm , 59. TheThe findings presented here also have some implications for the utilization and conservation of hill rice germplasm. First, considerable diversity, both in agro-morphological and genetic terms, has been maintained by the traditional farmers. This is most likely the result of maintaining different sets of landraces in different regions in response to local heterogeneities in climate and soil type, as well as indigenous culture, as demonstrated by Fu et al.  in Thai AMOVA, analysis of molecular variance; AN, average number of alleles per locus; ANOVA, analysis of variance; CDA, canonical discriminant analysis; CGIAR, Consultative Group on International Agricultural Research; CV, coefficient of variation; ICAR, Indian Council of Agricultural research; MAF, major allele frequency; NJ, neighbour joining; PCA, principal component analysis; PCoA, principal coordinate analysis; PCR, polymerase chain reaction; PIC, polymorphism information content; SD, standard deviation; SSR, Simple sequence repeats


Equisetum giganteum L. (E. giganteum), Equisetaceae, commonly called “giant horsetail,” is an endemic plant of Central and South America and is used in traditional medicine as diuretic and hemostatic in urinary disorders and in inflammatory conditions among other applications. The chemical composition of the extract EtOH 70% of E. giganteum has shown a clear presence of phenolic compounds derived from caffeic and ferulic acids and flavonoid heterosides derived from quercitin and kaempferol, in addition to styrylpyrones. E. giganteum, mainly at the highest concentrations, showed antimicrobial activity against the relevant microorganisms tested: Escherichia coli, Staphylococcus aureus, and Candida albicans. It also demonstrated antiadherent activity on C. albicans biofilms in an experimental model that is similar to dentures. Moreover, all concentrations tested showed anti-inflammatory activity. The extract did not show cytotoxicity in contact with human cells. These properties might qualify E. giganteum extract to be a promising alternative for the topic treatment and prevention of oral candidiasis and denture stomatitis. Candida fungus, the use of proper hygiene aids by the denture wearer is fundamental, by physical means, such as brushing, and, if necessary, chemical, and immersion of dentures in disinfectant solutions. Local or systemic antifungal therapy may still be an option in some cases of DS − with collision energy to 10–25%. After being dissolved in MeOH-H2O (85 : 15), the extract was infused in the ESI source by flow injection analysis (FIA) using a syringe pump at 5 μg/mL.The chemical profile of the 70% EtOH extract of Staphylococcus aureus (ATCC 6536), Escherichia coli (O:124), and C. albicans (SC 5314). C. albicans were grown in YEPD broth  and tested in Sabouraud broth . Bacteria were grown and tested in brain-heart infusion broth (BHI) .For this study, we usedIn vitro antimicrobial action was analyzed by the broth microdilution method with the objective of obtaining the minimum inhibitory concentration (MIC) of the E. giganteum extract. Bacterial and fungal suspensions (1 × 105 cells/mL) were, respectively, inoculated into BHI and Sabouraud broth using 96-well plates. Wells containing each inoculum with the 1% NaOCl solution (CTRL/NaOCl) and culture medium (Sabouraud or BHI broth) (CTRL/Medium) served as the positive and negative control, respectively.Ninety specimens of heat-polymerized acrylic resin (2.5 × 2.0 × 0.5 cm) were fabricated according to the manufacturers' directions . Each surface of specimens was abraded manually with high grade silicon carbide abrasive paper  , to prom E. giganteum extract; CTRL/PBS, sterile phosphate buffered saline (pH 7.2); or CTRL/NaOCl-1% NaOCl solution. The treatments were performed in 6-well plates, at room temperature for 10 min. Sequentially, the specimens were incubated for 90 min at 37°C at 75 rpm  with fungal suspension (1 × 107 cells/mL), washed with PBS, and incubated in RPMI-1640  at 37°C for 12 h at 75 rpm. Three specimens for group were stained by LIVE/DEAD BacLight Bacterial Viability Kit solution  [According to the different treatments, the resin specimens were divided into five groups: E50, E25, and E16  ofOR, USA)  and incuμm3. The more the biofilm mass on the specimens decreased, the higher the antiadherent activity was.Subsequently, the specimens were conditioned in the confocal laser scanning microscopy , from which digital images were obtained. The viable cells were visualized in green, while dead cells were visualized in red. With the use of BioImage Software program, the biofilm mass was quantified including both viable and nonviable cells and expressed as 2. Following, nonadherent cells were removed by aspiration. Monocytes were cultivated in RPMI-1640 , supplemented with 10% FBS (GIBCO) and 1% penicillin-streptomycin (GIBCO). The medium was replaced every other day until the cells grew to about 70% confluence. The experimental protocol used in this study was approved by the Committee on Ethics in Human Experimentation, of Bauru School of Dentistry, University of São Paulo , and written informed consent was obtained from all patients. This study was performed in accordance with the Declaration of Helsinki.HPEC were cultivated as previously described by Klingbeil et al.  and huma5 cells/well) suspended in 200 uL of medium were plated into 96-well tissue culture plates for 24 h. Thereafter, 200 uL of fresh medium containing various concentrations of E. giganteum extracts  was added to each well and incubated for time intervals of 1, 12, and 24 hours and compared with control cells (untreated and water). After these periods, the plates were washed twice in PBS and 200 uL of fresh medium mixed with 10 uL of 5 mg/mL MTT dye in PBS was added to the wells for 4 h in a humidified 5% CO2 atmosphere at 37°C. After the removal of MTT solution, 150 uL of dimethyl sulphoxide  was added and mixed to dissolve the dye crystals. The absorbance was measured at 500 nm by means of a multiplate reader . The same assay was performed with human palatal epithelial cells (HPEC) (104 cells/well) for 12 and 24 hours.The different extracts were evaluated regarding cytotoxicity by using MTT  colorimetric assay . Briefly, monocytes (2 × 105 cells/well) were in vitro incubated with E. giganteum extract  for 1 hour at 37°C in 5% CO2 atmosphere. After, they were stimulated with LPS at 5 μg/mL or C. albicans SC5314 (1 × 107 cells/mL) for three hours and incubated with 5 μM of Cell Rox at 37°C in 5% CO2 atmosphere for 30 min. The fluorescence intensities (FIs) of the cells were measured using the multiplate reader at 640/665 nm, at 37°C.Endogenous amounts of ROS in human monocytes were determined using the fluorescent probe 2′,7′-dichlorofluorescin diacetate . Monocytes . We considered Rt in RP-UHPLC, UV-vis, and MS/MSn spectra analysis and comparison with data from the literature [The constituents present in the hydroethanolic extract were identified by terature . In totan analysis of the hydroethanolic extract highlighted UV spectrum characteristic of styrylpyrones with bands at 253–258 and 355–373 nm [ m/z 379, 301, and 285 confirm the presence of styrylpyrones and flavonoid glucosides derivatives of quercetin and kaempferol, respectively. The signals of mass fragments at m/z 178 and 192 were related to presence of caffeic and ferulic acid derivatives. The neutral losses of 162 mass units allowed the identification of hexoses (sophoroside or glucoside).The UHPLC-PAD-ESI-MS5–373 nm  and flav5–373 nm . Diagnos E. giganteum 70% EtOH extract is presented.In  E. giganteum extract  showed antimicrobial activity against all strains studied ( E. giganteum extract was S. aureus. At the highest concentration of the extract (50 mg/mL), the results were similar to 1% NaOCl for all strains tested. Even at its lowest concentrations, the extract still showed a microbicidal action.The studied . The str C. albicans on the acrylic resin specimen surface, thereby revealing biofilms at the stage of proliferation and filamentation  showed an expressive presence of biofilm mass ofentation . As expeentation . In contentation , leavingentation . E. giganteum extract  revealed that increasing the concentration of the extract improved its antiadherent activity, because biofilm formation decreased with an increase in amount of herbal extract used , showing marked antiadherent activity of the E. giganteum extracts against C. albicans biofilms. At 25 and 16 mg/mL, the values of biofilm mass were similar to those obtained for PBS; however, they differed statistically from both control (NaOCl) and E50. No statistical differences were observed between E50 and CTRL/NaOCl with respect to the total number of remaining nonviable cells.The surface of resin specimens pretreated with the different concentrations of Figures . As show Figures . All gro C. albicans, returning to baseline levels. Values for cells treated with E. giganteum extract, which were not stimulated with LPS or C. albicans, remained at baseline levels  were able to decrease the levels of ROS production by cells stimulated with LPS ore levels . E. giganteum extract; but, at the lowest concentration (E4) for 12 hours on monocytes, there were significant differences between the cells treated with extract and untreated cells. In addition, at highest concentrations of E. giganteum, the percentage of cell viability was higher than it was for untreated cells, suggesting an increased cell metabolism . This was shown not only against C. albicans, but also against the S. aureus and E. coli bacteria used as parameters for comparison in our study. At a concentration of 50 mg/mL, by means of antimicrobial assay based on the broth microdilution method, the herbal extract showed antimicrobial action similar to 1% sodium hypochlorite, an agent widely applied for immersion of dentures during treatment of DS.Although Escherichia coli (E. coli). In our study, another aspect that highlights the significant antimicrobial activity of E. giganteum is that this phytotherapeutic agent was effective against a clinical strain of E. coli (O1124), which usually presents more resistance and higher pathogenicity than standard strains when they are isolated from patients [ E. coli ranges from 65 to 54% of death.We emphasize the values of antimicrobial action obtained in our study, which were higher than those reported in a previous study  that asspatients . However E. giganteum has shown a clear presence of phenolic compounds derived from caffeic and ferulic acids and flavonoid heterosides derived from quercitin and kaempferol, in addition to styrylpyrones  from the hydroxyl groups present in their composition, stabilizing these free radicals [ E. giganteum extract. This group of substances is characterized by their antioxidant properties, since they were able to reduce the levels of reactive oxygen species due to trapping free radicals directly or scavenging them through a series of combined reactions with antioxidant enzymes [ E. giganteum extract could negatively modulate monocyte-mediated inflammatory responses.In addition to the beneficial results with respect to the antimicrobial and antiadherent activities, all concentrations ofavonoids . In factradicals , 53 or sradicals , 54. The enzymes , 53, 55. C. albicans during the establishment of DS.Another interesting finding was that the extract did not alter the viability of monocytes and HPEC for up to 24 hours. This has important clinical repercussions, since these cells are closely related to the inner surface of the denture, in denture wearers. The oral epithelial cells are the first cells to interact with E. giganteum extract has the following properties: antimicrobial against C. albicans, S. aureus, and E. coli; antiadherence of C. albicans to heat-cured acrylic resin specimens; and anti-inflammatory effects on human monocytes activated by C. albicans. In addition, the phytotherapeutic extract did not compromise the viability of human monocytes or human palatal epithelial cells. These properties might qualify E. giganteum extract to be a promising alternative for the topic treatment and prevention of oral candidiasis and denture stomatitis.In summary, our results suggest that the phytotherapeutic


These results suggested that SQP could ameliorate NASH in rats, and that quercetin may be the critical active compound that exerts the therapeutic effect.Shenqi pill (SQP), a famous traditional Chinese medicine (TCM) herbal formula derived from Jinguiyaolue (Synopsis of Prescriptions of the Golden Chamber), has long been used to treat kidney yang deficiency syndrome. According to the TCM treatment principle that the liver and kidney are homologies, the clinical use of SQP in the treatment of nonalcoholic steatohepatitis (NASH) has achieved a good effect. However, the active targeted genes and underlying mechanism remain unclear. In this study, we aimed to explore the treatment mechanism of SQP in NASH rats, which may further contribute to the in-depth exploration of SQP in clinical applications. Network pharmacology analysis was used to screen the target genes of SQP for NASH treatment based on public databases. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, and protein–protein interaction (PPI) analysis were used to search for crucial target genes and mechanisms. UPLC–MS/MS was used to verify the active compounds of the SQP screened. The hepatic pathology and biochemical indicators of rats were used to judge the modeling results and the curative effect of SQP. Western blotting and qRT–PCR were used to verify the expression of crucial target genes at the protein and RNA levels, respectively. Network pharmacology analysis and bioinformatics analysis showed that PTGS2, JUN, MYC, and CDKN1A might be crucial target genes in the primary mechanism of SQP in treating NASH and improving the inflammatory response. The UPLC–MS/MS results confirmed that the hub active compound, quercetin, screened out through the TCMSP database, is indeed present in SQP. Hepatic injury and lipid metabolism indicators of NASH rats were significantly improved after SQP treatment. The results of WB and qRT–PCR showed that the expression of PTGS2, JUN, MYC, and CDKN1A was higher in NASH rats than in normal rats and decreased after SQP treatment. The expression of inflammatory cytokines (IL-1 Nonalcoholic fatty liver disease (NAFLD), a liver injury disease caused by metabolic disorders , includeNASH patients could be improved by control measures such as exercise and diet adjustment. However, it is difficult to adhere to these measures. Additionally, compared with NAFL, NASH accompanied by inflammation is more challenging to reverse . It is pIn the cognition of traditional Chinese medicine (TCM), according to its clinical manifestations, NASH belongs to the categories of “Xie-tong” (hypochondriac pain) and “Ji-ju” (accumulation). The statistical results showed that the median age of the NASH population is 55 years old, which means that NASH patients are mostly middle-aged and elderly . ChineseGuizhi), Aconiti Lateralis Radix Praeparata (Fuzi), Rehmanniae Radix Praeparata (Dihuang), Cornus officinalis Sieb. et Zucc. (Shanzhuyu), Cortex Moutan (Mudanpi), Rhizoma Dioscoreae (Shanyao), Poria cocos (Fuling), and Alisma orientale (Zexie). The herbal name has been checked by The Plant List (http://www.theplantlist.org) and Chinese Pharmacopoeia (http://db.ouryao.com/yd2015/).SQP is comprised of Cinnamomi ramulus  in combination with oral bioavailability (OB) and drug-like (DL) [The active compounds of SQP were queried through the Traditional Chinese Medicine Database and Analysis Platform (TCMSP) (ike (DL) . The scrike (DL) . DL refeike (DL) . The calA represents the measured compounds. B is a fixed value representing the mean DL index of all known components of the Drugbank database (https://drugbank.ca).A combination of these indicators can effectively select the bioactive compounds of SQP.(ii) GSE89632, a NASH-related sequencing dataset, was downloaded from the Gene Expression Omnibus (GEO) database to screen the differentially expressed genes (DEGs) between NASH patients and healthy adults; and (iii) “Nonalcoholic steatohepatitis” was used as a keyword to screen NASH-related disease genes in the DisGeNET (https://www.disgenet.org/home), GeneCards (https://www.genecards.org), and MalaCards  databases. The intersection of the above results was regarded as the target genes of SQP in treating NASH(i) Screening the predicted target genes of active compounds through the TCMSP database is as follows; R package cluster Profiler [The Profiler  was usedCombined with the gene functions and pathways of SQP target genes, we can explain the primary mechanism of the above genes and then clarify the mechanism of SQP in treating NASH, which is also helpful for the subsequent screening of hub genes.To clarify the relationship between the active compounds and targets of SQP, the screened compounds and therapeutic targets of SQP were connected into a “compounds-target” network.We could determine which active compounds in SQP play a significant treatment role and clarify which hub target genes are affected by multiple active compounds simultaneously through the network.https://string-db.org) database was used to clarify the protein interaction relationship of SQP target genes (the screening threshold was interaction score confidence ≥ 0.9). CytoNCA, the plug-in of Cytoscape 3.8.0, was used for network topology analysis according to degree centrality (DC), betweenness centrality (BC), and closeness centrality (CC) to screen the hub target genes at the core of the “protein–protein interaction (PPI)” network.The STRING  was used to verify the previously screened crucial compounds of SQP existing in various Chinese herbs or acting on multiple target genes. This verification process could clarify the exact compounds of SQP and further judge the reliability of the results selected based on the public databases.μm membrane, and separated on a CORTECS UPLC T3 column  at 4°C. The mobile phases consisted of solvents A (0.1% formic acid) and B (acetonitrile). Gradient elution is as follows: 0–2.00 (min), A: 5%; B: 95%; 2–32.00 (min), A: 100%; 32–33.00 (min), A: 100%; 33–33.50 (min), A: 5%; B: 95%; 33.5–35.00 (min), A: 5%; and B: 95%. The total running time was 35 min. The solvent flow rate was 0.3 ml/min. The column temperature was 30°C, and the injection volume was 2 μl.UPLC–MS/MS was performed at the Academy of Chinese Medicine, Zhejiang Chinese Medical University. The SQP extracts were dissolved in 80% methanol to a concentration of 10 mg/ml, filtered through a 0.22 The MS monitoring was run in both positive ion mode and negative ion mode. For positive ion mode, the heater temperature was 325°C, the sheath gas flow rate was 45 arb, the aux gas flow rate was 15 arb, the sweep gas flow rate was 1 arb, the spray voltage was 3.5 kV, the capillary temperature was 330°C, and the S-Lens RF level was 55%. For negative ion mode, the heater temperature was 325°C, the sheath gas flow rate was 45 arb, the aux gas flow rate was 15 arb, the sweep gas flow rate was 1 arb, the spray voltage was 3.5 kV, the capillary temperature was 330°C, and the S-Lens RF level was 55%. The MS scan mode was Full Scan . The MS/MS scan mode was set as dd-MS2 (TopN = 10). MS spectra were acquired at a resolution of 120,000, and MS/MS scans were acquired at 60,000.https://pubchem.ncbi.nlm.nih.gov/).Finally, we compared the mass charge ratio (m/z) and structure of the active compound to determine whether the above components, which were selected via the TCMSP database, were actually present in SQP. A deviation range between 5 m Dalton is accepted. The molecular weight and structural formula of the compounds were referenced in the PubChem database, which records chemical information from authoritative sources (https://pubchem.ncbi.nlm.nih.gov), a database of small organic molecules' biological activity. We also downloaded the protein structure of the hub target genes (receptors) of SQP in the treatment of NASH through the Protein Data Bank (PDB) (http://www.rcsb.org), a protein structure database, and set up the active pocket of the receptor for subsequent molecular docking. A binding energy < 0 indicates that the ligand could bind with the receptor. The smaller the value of the binding energy is, the stronger the binding ability of the ligand to the receptor.AutoDock Vina 1.2.0 was used for molecular docking. The 3D structures of the crucial active compounds (ligands) of SQP were downloaded through PubChem . Subsequently, we referred to the Lipinski (rule of five) principle   24 g, Rhizoma Dioscoreae (Shanyao) 12 g, Cornus officinalis Sieb. et Zucc. (Shanzhuyu) 12 g, Alisma orientale (Zexie) 9 g, Poria cocos (Fuling) 9 g, Cortex Moutan (Mudanpi) 9 g, Cinnamomi ramulus (Guizhi) 3 g, and Aconiti Lateralis Radix Praeparata (Fuzi) 3 g, as recorded in Jinguiyaolue (Synopsis of Prescriptions the Golden Chamber).The Traditional Chinese Medicine Preparation Room of The First Affiliated Hospital of Zhejiang Chinese Medical University made a liquid extract with an herbal ratio of 8 : 4 : 4 : 3 : 3 : 3:1 : 1. The specific dosages of SQP are Rehmanniae Radix Praeparata  rats  were purchased from Shanghai Sippe-Bk Lab Animal Co., Ltd. and were housed in the Experimental Animal Research Center of Zhejiang Chinese Medical University. The animal license was SCXK (Hu) 2008-0016. This study was approved by the Laboratory Animal Management and Ethics Committee of Zhejiang Chinese Medicine University (Resolution number: ZSLL-2010-26) and followed the guidelines of the National Guidelines for Experimental Animal Welfare.−1·d−1 SQP liquid extract for 12 weeks. The reason for the selected SQP dosage is to achieve a total of 81 g per SQP serving. Divide the total per serving of SQP by the weight of a typical adult (70 kg) to obtain an adult human dose of 1.15 g·kg−1. The rats were treated with intragastric administration at 12 times the normal human adult dose. Finally, 14 g·kg−1·d−1was obtained as the dose for rats in the study. The remaining two groups of rats were given an equal volume of normal saline for 12 weeks. After modeling, all rats were sacrificed under anesthesia. Serum and livers were collected and stored at -80°C until further use.After one week of adaptive feeding, 15 SD rats were randomly divided into three groups: the normal diet (ND) group, the high-fat diet (HFD) group, and the group treated with SQP (HFD-S), with five rats per group. ND group rats were fed a normal diet. The rats in the other groups were provided a high-fat diet of 82.5% normal diet, 10% lard, 2% cholesterol , 0.5% sodium cholate , and 5% egg yolk powder. Meanwhile, rats in the HDF-S group were given 14 g·kgSerum alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG), and total cholesterol (CHOL) were detected by an automatic biochemical analyzer. TG and CHOL levels in the rat liver were also detected. Hepatic pathology of the rat livers (steatosis/inflammation) was detected by Oil Red O and hematoxylin-eosin (HE) staining. The pathological diagnosis of NAFLD was based on the “Guidelines for the Diagnosis and Treatment of Nonalcoholic Fatty Liver Disease (2010 Revision),” and the NAFLD Activity Score (NAS) was calculated .μg/μl. Proteins (10 μl) were loaded on 10% SDS–PAGE gels and transferred to PVDF membranes by the semidry transfer method. Then, the PVDF membranes were incubated overnight with the corresponding primary antibodies directed against CDKN1A (1 : 800), MYC (1 : 1000), JUN (1 : 600), and PTGS2 (1 : 2000) at 4°C, followed by incubation with a secondary antibody. An ECL Kit was used to reveal the protein binding. An Odyssey infrared imaging system was used to collect images and analyze the values.Rat hepatic proteins were extracted according to the instructions of the total protein extraction kit. The protein concentration was determined by a BCA kit and diluted to 5 The total protein extraction kit  was purchased from APPLYGEN. The BCA protein quantification kit  was purchased from MULTI SCIENCES. 2× SDS protein loading buffer  was purchased from Solarbio. The Western ECL substrate kit  was purchased from Bio-Rad. The prestaining Western Blot Marker  was purchased from Haigene. The anti-c-JUN antibody  was purchased from Cell Signaling Technology. The anti-MYC , anti-PTGS2 , and anti-GAPDH antibodies  were purchased from Proteintech. CDKN1A  was purchased from HUABIO. HRP-conjugated goat anti-rabbit IgG (H + L)  and HRP-conjugated goat anti-mouse IgG (H + L)  were purchased from Multi Science.μl lysis buffer, three grinding beads, and 50 mg hepatic tissue were mixed in a grinding tube and subsequently broken with a homogenizer . The tube was then centrifuged , and 300 μl of supernatant was placed in a new 1.5 ml RNase-free tube. Next, RNA was extracted using a Universal RNA Extraction Kit. The reverse transcriptase reaction was performed for 15 min at 37°C and 5 seconds at 85°C. Finally, a 10 μl PCR mix was applied, including 2 μl cDNA, 5 μl 2× TB green, 0.4 μl forward primer (10 μm), 0.4 μl reverse primer (10 μm), and 2.2 μl ddH2O.Then, 400 The Universal RNA Extraction kit , reverse transcription (RT) kit , and RNA amplification kit  were purchased from Takara.https://www.ncbi.nlm.nih.gov) and PrimerBank (https://pga.mgh.harvard.edu/primerbank/), synthesized by Sangon Biotech, as shown in We designed primer sequences according to the mRNA sequences obtained from the NCBI database . One-way analysis of variance was used to analyze the statistical significance between the ND, HFD, and HFD-S groups. A From the TCMSP database, we screened 91 unique SQP active compounds of 8 Chinese herbs, meeting the screening conditions with OB ≥ 30% and DL ≥ 0.18  We used “non nonalcoholic steatohepatitis” as the keyword to screen NASH-related genes in the DisGeNET, GeneCards, and MalaCards databases. A total of 816 intersecting genes were called NASH-related disease genes.(i) We searched the TCMSP database and found 182 predicted target genes of the SQP active compounds. (ii) Downloading the GSE89632 dataset from the GEO database, we selected NASH and HC  samples and used Then, we took the intersection of 187 predicted target genes of SQP compounds, 344 DEGs, and 816 NASH-related disease genes, which were seen as the target genes of SQP for the treatment of NASH . A totalR package clusterProfiler was used for enrichment analysis and visualization. The screening thresholds were p.adj < 0.05 and q value <0.05. The results showed that a total of 870 BP, 17 MF, 0 CC, and 70 pathways were identified. The top 5 enrichment results of each category are shown in p value, indicating the highest credibility, and they may be associated with the secretion of inflammatory cytokines. The results of the KEGG enrichment analysis showed that the IL-17 signaling pathway and TNF signaling pathway with low p values were inflammation-related pathways  is the most crucial compound with the most targets. Quercetin could act on all eight selected target genes of SQP simultaneously. PTGS2 is the top gene affected by the most active compounds, suggesting that PTGS2 may be the hub gene of SQP in treating NASH.The above 8 target genes were input into the STRING database, and CytoNCA was used to calculate the gene connections and screen the core therapeutic genes according to DC, BC, and CC. The results showed that JUN was the hub gene with the highest DC, BC, and CC among the eight therapeutic target genes .In addition, JUN has an exact protein interaction relationship with CDKN1A and MYC (determined by experiments –25), sugTo further clarify whether the selected hub genes PTGS2 and JUN are related to the active mechanism of SQP in treating NASH by improving inflammation, we constructed an enrichment analysis network, showing that PTGS2 and JUN are both genes related to inflammation . CDKN1A Finally, we selected two hub target genes (PTGS2 and JUN) and two genes (MYC and CDKN1A) that may synergize with JUN to aggravate hepatic inflammation via the eight SQP target genes.α) by regulating the expression of PTGS2 and JUN and affecting the interactions of JUN, CDKN1A, and MYC.In summary, we speculated that PTGS2  and JUN  are the hub genes for the treatment of NASH. SQP may improve inflammation in the liver and reduce the secretion of inflammatory factors . Therefore, we selected quercetin and four crucial SQP target genes for molecular docking verification. As shown in Furthermore, we analyzed the docking results of quercetin and the hub target genes. The hydrogen acceptor/donor can be calculated according to the molecular docking visualization . The resp < 0.05). SQP treatment significantly alleviated the liver injury in rats and restored their abnormally elevated ALT and AST levels (p < 0.05)  in the HFD-induced NASH rats were significantly increased ( < 0.05) .p < 0.05), while SQP could significantly reduce CHOL (p < 0.05) (p < 0.05). After SQP treatment, the TG content in the liver of rats was reduced. Therefore, we deem that compared with rats and mice, golden hamsters and guinea pigs with lipid profiles closer to Homo sapiens are more suitable for observing the metabolic changes in serum TG [In addition, HFD also caused lipid metabolism disorder in rats. There was a significant increase in CHOL in the rat serum and liver after long-term HFD ( < 0.05) . The chaserum TG .p < 0.05). In addition, the NAS of rats in the HFD group was greater than 5, which suggested that these rats could be diagnosed with NASH. Treatment with SQP significantly reduced the NAS score of rats (p < 0.05) and improved NASH.As the gold standard for the clinical diagnosis of NASH, the pathological results combined with the previous biochemical indicators are sufficient to demonstrate the success of the animal model in this study, and that SQP can alleviate the hepatic fat accumulation and inflammation and treat NASH.p < 0.05). The expression of PTGS2, c-JUN, and CDKN1A was reduced significantly in the HFD-S group treated with SQP (p < 0.05). The expression of MYC decreased after SQP treatment, but the difference was not significant (p > 0.05).The protein expression results of the hub target genes of SQP in treating NASH rats  are shown in In summary, the protein expression of the SQP hub target genes was elevated after long-term HFD feeding and downregulated by SQP treatment. SQP could regulate the protein expression of the hub genes, which further supports the reliability of the screening.p < 0.05). The RNA level of PTGS2 in the HFD group was significantly upregulated compared with that in the normal group (p < 0.05). After SQP intervention, the expression level showed a downwards trend but without significance (p > 0.05). The RNA expression levels of the SQP hub target genes were both elevated after long-term HFD feeding and downregulated with treatment, consistent with the trend of the protein levels.To increase the reliability of the previous analysis, we detected RNA changes in the SQP hub target genes and inflammatory cytokines by qRT–PCR. β, IL-6, TNF-α) to estimate hepatic inflammation. β, IL-6, and TNF-α were upregulated in the HFD group and downregulated in the HFD-S group, but the difference was not significant (p > 0.05). These results showed that long-term HFD feeding promoted the secretion of inflammatory cytokines by the hepatocytes of rats, which led to hepatic inflammation. SQP could improve inflammation and treat NASH by reducing the secretion of inflammatory cytokines.We also measured the RNA levels of inflammatory cytokines  via network pharmacology and bioinformatics analysis.PTGS2, also known as cyclooxygenase 2 (COX-2), is an essential gene in cell death signaling. Its overexpression significantly promotes hepatocyte death, causing hepatic inflammation . PTGS2 iWhile the tissue is under stress, its cells will produce inflammatory memory: fixing inflammation-related proteins on its deoxyribonucleic acid (DNA), remembering the workplace, and thus constructing memory regions, contributing to a rapid response of subsequent inflammation . These mThe system can be activated unilaterally to respond to almost any stimulus but may not always benefit the affected tissues, such as the liver. Continuous guarding of the inflammatory memory regions by JUN will make the tissue try to clear even the slightest abnormality, which can easily cause continuous chronic inflammation. Persistent chronic hepatic inflammation is crucial to the occurrence of NASH, and it is the primary mechanism leading to the progression of NASH to fibrosis and cirrhosis .CDKN1A and MYC were selected as hub target genes due to their direct interactions with JUN. CDKN1A is a crucial gene in ageing, also known as a biomarker of cellular ageing, and it can promote the production of senescence-associated secretory phenotype (SASP) by reducing the phosphorylation of Rb . With thKalra and Kumar found that MYC is an essential gene in regulating the apoptosis of cells . The apoIn this study, we clarified the crucial mechanism of SQP in treating NASH, identified four hub target genes, and completed the verification. These results may provide theoretical support for promoting the clinical usage of SQP in NASH treatment. We have discovered the potential feasibility of treating HCC with SQP by inhibiting the expression of MYC, but this has not been verified. SQP may treat both NASH and HCC by targeting a common mechanism.This result is consistent with the advantages of TCM formulas that treat diseases via multiple targets and channels. Because they contain many Chinese herbs and various active compounds, it is not easy to fully clarify the therapeutic principles of TCM formulas. However, this is precisely the advantage of TCM formulas: TCM formulas are not limited to treating single or similar diseases. TCM has a history of thousands of years, and formulas, the essence of TCM, are undoubtedly a treasure trove of undiscovered drugs. We should strengthen their research and applications.(1) Our study verified that it is feasible and reliable to screen the therapeutic targets of TCM through network pharmacology analysis. (2) Combining the screening of network pharmacology and the validation of animal experiments, we confirmed that SQP could improve excess lipid accumulation and inflammation in the rat liver and treat NASH rats. (3) Quercetin may be an essential active compound for SQP to exert its curative effect. (4) PTGS2, JUN, MYC, and CDKN1A are the hub target genes of SQP in the treatment of NASH rats.


Among peatland categories, median C concentrations were highest in bogs, poor fens and tropical swamps (446–532 g kg-1) and lowest in intermediate and extremely rich fens (375–414 g kg-1). The C:OM ratio in peat was similar across most peatland categories, except in deeper samples from ombrotrophic tropical peat swamps that were higher than other peatlands categories. Peat N concentrations and C:N ratios varied approximately two-fold among peatland categories and N concentrations tended to be higher (and C:N lower) in intermediate fens compared with other peatland types. This study reports on a unique data set and demonstrates that differences in peat C and OM concentrations among broadly classified peatland categories are predictable, which can aid future studies that use land cover assessments to refine global peatland C and N stocks.Peatlands account for 15 to 30% of the world’s soil carbon (C) stock and are important controls over global nitrogen (N) cycles. However, C and N concentrations are known to vary among peatlands contributing to the uncertainty of global C inventories, but there are few global studies that relate peatland classification to peat chemistry. We analyzed 436 peat cores sampled in 24 countries across six continents and measured C, N, and organic matter (OM) content at three depths down to 70 cm. Sites were distinguished between northern (387) and tropical (49) peatlands and assigned to one of six distinct broadly recognized peatland categories that vary primarily along a pH gradient. Peat C and N concentrations, OM content, and C:N ratios differed significantly among peatland categories, but few differences in chemistry with depth were found within each category. Across all peatlands C and N concentrations in the 10–20 cm layer, were 440 ± 85.1 g kg Peatlands store a disproportionate amount of the global terrestrial carbon (C), covering approximately 3% of the earth’s surface yet accounting for between 15 and 30% of the world’s soil C stock . Around et al. [et al. [et al. [Peatland C stocks have been most widely estimated for northern peatlands, with Gorham’s  estimateet al.  who cons [et al.  reported [et al.  estimate [et al.  summariz-1 [et al. [-1, with notable regional variation in measured C concentrations. Relatively small differences in assumed “average” C concentrations can have a major impact on estimates of C stocks at the global scale. As an example, two recent meta-analysis studies have examined the potential impact of using mean global C values on two other major terrestrial C stocks (trees and dead wood) and suggested that assuming a mean C proportion of 50% may overestimate C stocks in both forests and dead wood [et al. [Carbon stock estimates in peatlands are a function of the assumed C concentration in peat with the most widely cited mean global estimates of peat C concentration falling within a narrow range of 500–517 g kg-1 , despite-1 , 17–19.  [et al.  analyzedead wood , 22. ForAn additional factor to consider is that many studies measure organic matter (OM) rather than C concentration in peat and use an assumed C:OM ratio to convert organic matter stocks into C . The C:OSphagnum and non-Sphagnum peat, Loisel et al. [et al. [Peatlands also store large quantities of nitrogen (N) and can have a large impact on the global N cycle , 25. Stul et al.  reported [et al.  also fou [et al. .More recent efforts to characterize soil C and N stocks in northern regions combine empirical measurements with land-cover classification that differ in physical  properties –32. WhilWe quantified global variation in peatland C and N concentrations and organic matter content in 1226 individual peat samples at different depths, from 436 cores, collected across 24 countries and six continents . The samIn the lab, sub-samples were immediately oven dried at 60°C and ground to a fine powder in a ball mill. Organic matter was determined by combusting 0.1–0.5 g of dry ground peat in a 10 ml crucible at 550°C in a muffle furnace for 6 hours. Samples were then re-weighed to determine the proportion of mass loss (OM %). Peat pH was measured in a slurry at a 2:1 ratio (volume to volume) of distilled water to peat. A small number (< 20) of samples were freeze-dried by collaborators, prior to shipping to our lab; freeze dried samples were rewetted to 90% water content using distilled water and left to sit for several hours prior to measuring pH. For C and N analysis dried ground peat was weighed to within 0.0001 g on a digital balance. Approximately 75 mg of peat was placed into a formed tin foil capsule along with 150 mg of tungsten (VI) oxide powder. The tin foil was folded over sealing the sample and was compressed in a pill-shaping device. Packed samples were analyzed using an Elementar VarioMacro CNS Analyzer and precision of results was confirmed using blanks and sulfadiazine for CNS recalibration and QA standard (NIST-1515-SRM apple leaves). Laboratory processing and analyses for all samples was done consistently by the same people in Erik Lilleskov’s group at the Northern Research Station  for milling and pH determination; in Nathan Basiliko’s group at Laurentian University  for OM measurements and preparation for CNS analysis, and in Shaun Watmough’s group at Trent University  for CNS analysis.To classify habitats, sites were binned into categories based on pH ranges put forth by Rydin and Jeglum ; bog pH depth in the peat profile, peatland type, and a peatland type x depth interaction as fixed effects. Each model also included individual peat core, 1 km diameter and 100 km diameter spatial clusters as random intercept effects, to account for samples from the same core, wetland or region being less independent from each other than samples from much more distant locations. Spatial clusters were generated with the leaderCluster package [lmerTest package [F-tests utilizing the Kenward-Roger approximation. Partial R2 values for each fixed effect were obtained with the package r2glmm [emmeans [Linear mixed models were used to examine patterns of C, N, C:N, OM, and C:OM among peatland habitats, utilizing R 3.5.2 . Models  package , using l package , and fixe r2glmm . Post-ho[emmeans . For eac-1, depending on peat depth . Peat C:N ratios were generally lowest in minerotrophic peat swamps and both intermediate and extremely rich fens except at the 30–40 cm depth  in their synthesis of northern peatlands and found that mean C concentrations for each category varied between 460–509 g kg-1 but did not clearly sample or distinguish minerotrophic fens that have the lowest C concentration in our data set. The difference in the types of peatlands used in our study compared with Loisel et al. [In our global data set, peat C concentration exhibited little variation with depth (to 70 cm) but varied considerably among the six peatland categories that we used in this study, with tropical peat swamps, bogs and poor fens having higher C concentrations than intermediate and extremely rich fens. Linking differences in peat chemistry to peat or land cover classification is essential in upscaling studies designed to characterize regional or global C stocks . Loisel et al.  identifil et al.  is likelet al. [-1 in Sphagnum dominated peat with a pH of 3.9 to 387 g kg-1 in peatlands dominated by ash (Fraxinus) with a pH of 5.8. We acknowledge that some peatland types may not fall along the broad acidity gradient that we identify here, and we could have attempted to divide our four categories along the bog-fen gradient into treed and non-treed sites. Nevertheless, the pattern we observed in our classification suggests that categorizing peatlands simply on their degree of ombrotrophy (bog–rich fen gradient) is very useful for quantifying C and N in peatlands, especially when combined with improved land cover classification [In our study, all peatlands were placed into one of six broad categories  that encompassed a wide variety of peatland types. Differences in peatland classification are also widely evident in the literature  and canet al.  similarlfication , 32.Sphagnum-derived peat that is more typical in acidic northern peatlands has a lower C:OM ratio than peat derived primarily from herbaceous vegetation and woody plants [The predominant reason for the difference in C concentration among peatland types is due to varying amounts of ash  in peat as patterns in OM values are very similar to patterns observed for C concentration. Intermediate and extremely rich fens contain a much greater mineral fraction than bogs or peat swamps, which is reflected in the much lower OM values, especially in the deeper samples. As a result, the median C:OM ratio was quite similar among peatland categories at the upper depths, with a median value in the global data set of 0.48 to 0.51 (depending on depth), which is within the range reported in other studies , but dify plants , 39. Indy plants  used FTIy plants . In thisy plants .et al. [Patterns in median N concentrations and C:N ratios in peat also varied significantly among peatland type. While our median C:N ratios in peat across all sites were between 27.2–31.6 (depending on depth), median peatland categorical values ranged from lows of around 20–25 in base rich peatlands to 30–40 in more acidic bogs, fens, and tropical peat swamps. Wang et al.  similarlet al. . A high Sphagnum that has much lower N concentrations and higher C:N ratios than most other peatland vegetation [Sphagnum is the dominant vegetation in bogs and poor fens compared with richer fens and peat swamps it plays a major role in the low N concentration and high C:N ratio in peat in these systems [Variations in peat C and N concentration (and C:N ratios) among these peatland types are due to several factors including differences in vegetation composition (litter inputs), litter decomposition rates , aciditygetation , 45. Bec systems . In addi systems , 46. Dur systems , 47. In  systems , 46, 48.Most previous estimates of global C and N stocks are estimated using data from northern peatlands as they account for 80% of global peatlands and have been historically more widely studied . The preet al. [-1 in eastern Russia and Asia to a high of 540 g kg-1 in the eastern European Islands. Similarly, mean N concentrations in peat are between 7.0 g kg-1 in continental Europe and 16.0 g kg-1 in several regions including Hudson Bay and James Bay, western European islands and Western Russia [Even within northern peatlands, considerable regional variation in mean C and N concentrations in peat exists. Loisel et al.  for examn Russia .While wen Russia , 53 to bWe established six broad peatland categories, consisting of ombrotrophic tropical peat swamps, minerotrophic tropical peat swamps, bogs, poor fens, intermediate fens, and extremely rich fens, that fell along an increasing pH gradient and were sampled in 24 countries across six continents. Peat C, N and OM values were relatively consistent within each category but varied considerably among the six peatland categories. Peat C concentrations were highest in bogs, poor fens and tropical peat swamps, while N concentrations were highest in minerotrophic peat swamps and intermediate fens. The C:OM ratio exhibited less variation among peatland category suggesting that differences in mineral content were largely responsible for differences in C concentration among peatlands. Peat C and C:N ratios in our data set followed a predictable pattern among broadly classified peatland categories, and data sets such as these may be used to better constrain global assessments of peat C and N in surface peat, especially when coupled with improved land cover classification.


The patients were divided into control and baseline DM complication groups. The primary endpoint was the evaluation of CIN incidence between the groups. Propensity score-matching was performed to confirm the robustness of the primary analysis results. CIN occurred in 6.8% of control and 27.0% of DM patients, respectively, with a significant difference in all-patient populations (P = 0.001). In addition, variation of serum creatinine and creatinine clearance significantly worsened in DM patients. Similar results were obtained in a propensity-matched population. Multivariate logistic regression analysis found that DM complication is a singular risk factor for CIN development . In conclusion, our study revealed that baseline DM complications significantly worsen CIN.Cisplatin (CDDP)-induced nephrotoxicity (CIN) is dose-limiting. We revealed that co-administration of non-steroid anti-inflammatory drugs and baseline comorbidity of diabetes mellitus (DM) are associated with CIN development in the short hydration method; however, the results were accessorily obtained without appropriate power calculation. This study aimed to demonstrate the influence of DM complications on CIN incidence in a real-world setting. Lung cancer patients receiving CDDP (≥ 75 mg/m In contrast, it induces nausea, vomiting, hearing impairment, neuropathy, and nephrotoxicity3. CDDP-induced nephrotoxicity (CIN) is dose-relatedly reversible and is known to be its dose-limiting toxicity3. CIN used to occur in 30–40% of the patients3. However, the progress of CIN management, including magnesium supplementation, quality antiemetic therapy, and appropriate diuretics administration, significantly reduced its occurrence to 0–10%11.Cisplatin (CDDP) is a key chemotherapeutic agent in treating lung cancer in combination with radiotherapy and surgery22. We have revealed that co-administration of non-steroid anti-inflammatory drugs (NSAIDs) and baseline comorbidity of diabetes mellitus (DM) are significantly associated with CIN incidence in the short hydration method, the most advanced CDDP administration method4. However, the results were obtained without sufficient power calculation. The prevalence rate of DM is increasing worldwide, and people with prediabetes are also growing23. Therefore, the CDDP administration to DM patients is increasing. It is important to manage CIN in higher-risk patients although there is a lack of evidence in the population. This study aimed to determine the real-world impact of DM complications on CIN incidence.Many reports evaluate CIN risk factors, although most were conducted using old CDDP administration methods without sufficient CIN prophylaxis described above, suggesting some do not reflect real-world settings19) were more included in the DM group than control patients. In contrast, no background differences were confirmed between the groups in the propensity score-matched population. Patients with co-administered NSAIDs, an independent CIN risk factor, accounted for 13.7% of the control group and 13.5% of the DM group in all populations, and 6.1% and 12.1% in propensity-matched populations, respectively, without significant difference. Total administration cycles were not different between the groups . All DM patients were type 2 with well-controlled HbA1c . During the evaluation periods, none of the patients had type 1 DM. Detailed DM medication is shown in Supplemental Table In total, 227 patients were enrolled in this study based on its eligibility criteria Fig. . The basCIN occurred in 6.8% of control and 27.0% of DM patients, respectively, with a significant difference in the all-patient population, meeting the study's primary endpoint  according to current guidelinesP = 0.003, Table Multivariate logistic regression analysis in the all-patient population revealed that DM complication is a singular risk factor associated with CIN development , which transports CDDP to the proximal tubule, has a critical role in CDDP accumulation33, and its expression level reportedly decreases with aging34. These suggest that there is compensation between renal function decline and CDDP accumulation reduction due to aging. Further studies evaluating these mechanisms are necessary for better CIN management in DM patients.DM has been reported to accelerate kidney aging31, the results may vary for type 1 DM. Third, in all-patient population analysis, patients in the DM group were significantly older than the control patients. Aging is associated with glomerulosclerosis and arteriosclerosis of intrarenal vessels, causing nephron losss19. However, age was not a risk factor, and CIN was more common in DM patients, even in propensity-matched groups in this study. Evaluation of a balanced population in all-patient population analysis was desirable. Finally, it has been suggested that OCT2 has single-nucleotide polymorphisms (SNPs)35 and that the 808G > T SNP in OCT2 ameliorates CIN without alteration of disposition36. We did not assess patients' genetic backgrounds in this study so they might have affected the results.This study has some limitations. First, this study was retrospectively conducted with a relatively small population from a single institution. Second, as all DM patients in this study were type 2, and renal pathology differs between type 1 and 2 DMIn conclusion, our study revealed that DM complications significantly worsen CIN. Therefore, we should monitor DM patients cautiously and evaluate its mechanisms and countermeasures for appropriate CIN management.2) from May 2014 to December 2021. We evaluated CDDP-including regimens such as CDDP  + pemetrexed  ± bevacizumab , CDDP  + etoposide  ± radiation, and CDDP  + vinorelbine  ± radiation.This retrospective observational study enrolled lung cancer patients who received CDDP (≥ 75 mg/mAll patients met the following baseline criteria: (1) age ≥ 20 years; (2) detailed patient information available from medical records; (3) 0 to 2 Eastern Cooperative Oncology Group performance status (ECOG-PS); (4) sufficient renal or liver function for CDDP-containing treatment induction. Patients who were previously administered CDDP, transferred hospital during the chemotherapy, could not complete the first cycle, and those who received dose reduction from treatment initiation were excluded. Patients administered immune checkpoint inhibitors were also excluded as we evaluated direct DM influence on CIN. The patients were divided into two groups: the DM group, which includes patients who required pharmacotherapy for DM treatment at baseline between May 2014 and December 2021, and the controls, who did not exhibit DM during the treatment between May 2014 and March 2021. If the patient had uncontrolled DM, they were treated by diabetologists before treatment induction.4. To achieve 80% power with an alpha error of 5%, the required sample size was 190 subjects in the control group and 38 subjects in the DM group. Finally, 190 patients in the control group and 37 in the DM group were analyzed for eligibility.We hypothesized that the CIN incidence would be 10% in the control group and 30% in the DM group, with a patient ratio of 5:1, according to our previous researchThe present study was approved by the Ethical Review Board for Life Science and Medical Research of Hokkaido University Hospital  and was carried out following the Declaration of Helsinki and the STROBE statement. However, given the retrospective nature of the study, informed consent from the subjects was waived by the Ethical Review Board for Life Science and Medical Research of Hokkaido University Hospital.4. In addition, all regimens included the same antiemetic therapy according to the current guidelines24; intravenous palonosetron 0.75 mg on day 1, oral aprepitant 125 mg on day 1 and 80 mg on days 2, 3, and dexamethasone 9.9 mg infusion on day 1 and 8 mg orally on days 2–4.All patients received the CDDP short hydration method, including 8 mEq of magnesium sulfate administration as described previously4. The primary endpoint of this study was the evaluation of CIN incidence between the groups. Secondary endpoints were assessment of SCr and CCr variation and CDDP-related adverse effects. Furthermore, propensity score-matching was performed to adjust the baseline factors between the two groups, and matched data were additionally analyzed to confirm the robustness of the primary analysis results.Toxicities in all subsequent treatment cycles were evaluated under the Common Terminology Criteria for Adverse Events, version 5.0, by physicians and pharmacists. Pharmacists confirmed appropriate CDDP administration, including oral hydration in each treatment cycle. Renal function was assessed based on the SCr variation measured by an enzymatic method. CCr was calculated using the Cockcroft-Gault formula. CIN was defined as grade 2 or higher SCr elevation in this study as well as our previous reportsU test for continuous parameters. Incidence of CIN was compared using Fisher's exact probability test, and differences in variation of SCr and CCr between the two groups were assessed using the Mann–Whitney U test. Assessment of adverse effects other than CIN was conducted using Fisher's exact probability test for the incidence and the Mann–Whitney U test for severity. The univariate and multivariate logistic regression analyses were used to identify the independent risk factor(s) for CIN incidence. We included the following baseline covariates: sex, age, ECOG PS, staging, adjuvant chemotherapy setting, radiation combination setting, prior chemotherapy existence, body surface area (BSA), hemoglobin, serum albumin levels, co-administration of proton pump inhibitors (PPI) and NSAIDs, complication of DM and cardiovascular diseases according to previous reports22. Variables that demonstrated potential associations with CIN incidence in univariate logistic regression analysis (P < 0.10) were considered when building the multivariable model. Propensity score-matching was performed using the following baseline variables: sex, age, ECOG PS, staging, adjuvant chemotherapy setting, prior treatment existence, BSA, baseline hemoglobin, albumin, SCr levels, NSAIDs and PPI co-administration, and cardiovascular disease complication. To reduce bias with these potential confounding factors, 1:1 matching (without replacement) in the two groups was achieved using the nearest neighbor method with a 0.20- width caliper of the standard deviation of the logit of propensity scores.The differences in baseline clinical characteristics between control and DM groups were assessed using Fisher’s exact probability test for categorical outcome variables and the Mann–Whitney P-values were less than 0.05.All analyses were performed using JMP version 16.2 statistical software . Differences were considered statistically significant when All procedures performed in this study were carried out in accordance with the ethical standards of the institutional and/or national research committee and the 1964 Helsinki declaration and its later amendments or comparable ethical standards. The study was approved by the Ethical Review Board for Life Science and Medical Research of Hokkaido University Hospital (Approved Number: 022-0107). Given the retrospective nature of the study, informed consent from the subjects was waived by the Ethical Review Board for Life Science and Medical Research of Hokkaido University Hospital.Supplementary Information.


Lactobacillus, but the mechanisms involved are not well understood. The purpose of this study is to investigate differentially expressed genes (DEGs) of Limosilactobacillus reuteri (L. reuteri) LR08 responding to soybean protein and peptides using transcriptome. The results showed that both digested protein (dpro) and digested peptides (dpep) could enhance a purine biosynthesis pathway which could provide more nucleic acid and ATP for bacteria growth. Moreover, dpep could be used instead of dpro to promote the ABC transporters, especially the genes involved in the transportation of various amino acids. Interestingly, dpro and dpep played opposite roles in modulating DEGs from the acc and fab gene families which participate in fatty acid biosynthesis. These not only provide a new direction for developing nitrogen-sourced prebiotics in the food industry but could also help us to understand the fundamental mechanism of the effects of dpro and dpep on their growth and metabolisms and provides relevant evidence for further investigation.Soybean protein and peptides have the potential to promote the growth of  Limosilactobacillus reuteri (L. reuteri), as one of the most important probiotics, has been gradually used in the functional food and dairy industry [Lactobacillus have various beneficial effects including amelioration of symptoms of lactose intolerance [industry ; this isindustry . It has olerance  and the olerance , and thaolerance  or improolerance ,7.Lactobacillus. Researchers found that whey protein hydrolysate can promote the proliferation of Lactobacillus acidophilus [L. reuteri growth and metabolism and were highlighted as potential prebiotic candidate [Lactobacillus in order to understand the preliminary mechanisms at RNA level [Food proteins and their hydrolysates also have many bioactivities, such as antihypertension, reducing cholesterol, antioxidant, etc. ,9,10. Redophilus . Others dophilus . These cdophilus ,14. Howeandidate ,16. WhilNA level .L. reuteri LR08. The results can provide a theoretical basis for discovering a nitrogen source with potential probiotic function for prebiotic families as well as exploring the mechanism of protein and peptides on probiotics.The purpose of this article is to investigate the preliminary mechanism of soybean protein and soybean peptides affecting the growth and metabolism of w/w). The mixture was stirred at 37 °C for 1 h. The pH was then adjusted to 5.3 with a saturated NaHCO3 solution and further to pH 7.5 with 5 N NaOH. Pancreatin was added to the mixture (enzyme to substrate ratio of 1:25 w/w), followed by incubation at 37 °C for 2 h. After proteolysis, the reaction was terminated by heating to 95 °C for 10 min, and the supernatant was lyophilized for 36 h and stored at −20 °C for future analysis. The nitrogen content of digested soybean protein (dpro) and digested soybean peptide (dpep) was 10.93 g/100 g and 10.14 g/100 g, respectively. The molecular weight of dpro was lower than 35 kDa, and dpep was mostly lower than 500 Da (71.65%) [The sources of original soybean protein and peptides were consistent with those described by Zhao . Soybean(71.65%) .L. reuteri was obtained from China General Microbiological Culture Collection Center (CGMCC). The MRS, dpro, and dpep treatments were prepared according to Zhang’s method [L. reuteri bacteria stored in the refrigerator at −80 °C were activated in 3 mL MRS medium, and then cultured at 37 °C for 12 h. Next, 200 microliters of culture solution were inoculated in 50 mL MRS medium for 12 h at 37 °C for follow-up experiments under different treatments. After being incubated for 10 h with different treatments, samples of L. reuteri (the logarithmic growth phase of L. reuteri LR08) were selected as samples for transcriptome analysis. MRS was used as the positive control group, and digested soybean protein and digested soybean peptides were added into the culture medium, replacing half of the nitrogen content . The compositions of related culture medium are shown in s method . The L. L. reuteri LR08 was extracted using a Bacterial Total RNA Extraction Kit . The RNA-seq transcriptome library was constructed using Illumina TruSeqTM RNA Sample Prep Kit. The mRNA was separated by oligo-dT magnetic beads. First, fragmentation was performed with fragment buffer, and then double-stranded cDNA was synthesized using SuperScript double-stranded cDNA synthesis kit . The target cDNA fragment of 200 bp–300 bp was selected after completing the repaired end of the synthesized cDNA. A total of 15 cycles of PCR amplification were conducted using Phusion DNA polymerase (NEB). The two-terminal RNA-seq sequencing library for computer sequencing was performed using Illumina Novaseq 6000 (2 × 150 bp) after TBS380 quantification.Total RNA of L. reuteri LR08 extraction, the reverse transcription was conducted for preparing cDNA with the help of a Reverse Transcription Kit . The specific method was as follows: the 5×FastKing-RT SuperMix and RNase-Free ddH2O was thawed at room temperature (15–25 °C) and was then quickly put on ice. Each solution was blended evenly using vortex oscillation before being prepared for use and centrifuged briefly to collect the liquid remaining on the pipe wall. The reverse transcription reaction system that consisted of 4 μL 5× FastKing-RT SuperMix, 1 μg total RNA, and 15 μL RNase-Free ddH2O was configured on ice, and then the reaction was carried out. The genome and reverse transcription reaction were removed by reacting for 15 min at 42 °C, and then the enzyme activity was inactivated for 3 min at 95 °C. The resulting cDNA solution was stored at −80 °C for subsequent use.After total RNA of Lpla16rsRNA was referred to [−ΔΔt method was used to evaluate the fold change with relation to the control group of selected genes.The reaction of qPCR was performed following the program of “95 °C for 10 s for denaturation, 63 °C for 20 s for annealing, and 72 °C for 32 s for extension” using a SuperReal PreMix Plus Kit . Primers sequences were designed using Beacon Designer 8.0 software  for 16 crucial differentially expressed genes, and the sequence of the internal reference gene p < 0.05 and |log2FC| ≥ 2 as the screening conditions, and genes with different expressions between the experimental group and the blank group were screened. Excel 2016 was used to analyze the variance of experimental data which were expressed as mean ± standard deviation and to draw a graph. * p < 0.05, ** p < 0.01 or *** p < 0.001 was statistically significant. The analysis of variance (ANOVA) was performed a as one-way method for the analysing the data. Tukey–Kramer and Games–Howell tests were performed to compare the groups’ mean values.SeqPrep and Sickle software were used to perform the clipping and quality control of primary end-numbers with default parameters. TopHat software was used to compare with the reference genome in the orientation mode Clean reads. The Mapped Reads were spliced using StringTie software, based on the selected reference genome sequence, and then compared with the original genome annotation information. Statistical analysis was carried out for Raw counts using DESeq2 software, based on L. reuteri LR08 when reacted with dpep and dpro. The three groups of sample clusters were analyzed by principal component analysis (PCA) and correlation analysis methods [The transcriptomic analysis was performed to deepen the understanding of overall gene changes of  methods . In FiguL. reuteri growth than both the MRS and dpro groups.Compared with MRS, dpep elevated transcription of genes mainly enriched in. the biological process (BP) of IMP biosynthesis and purine biosynthesis, which is critical for cell division during bacteria growth A. OrganoL. reuteri, and that dpep can also regulate nutrients absorption and protein production of bacteria through ABC transporters and ribosome. Compared with dpro, dpep can also regulate the energy metabolism of L. reuteri through genes’ enrichment in oxidative phosphorylation, pyruvate metabolism, and citrate cycle.As can be seen in accA (N134_RS05255), accC (N134_RS05265), accD (N134_RS05260), fabI (N134_RS05250), and fabZ (N134_RS05270). They were significantly upregulated in the dpep/MRS group and dpep/dpro group (accD) [accC), and acetyl-CoA carboxylase carboxyl transferase subunit α (accA) [acc gene family could enhance the carboxylation of acetyl-CoA, providing enough malonyl-CoA serves as a two-carbon extender unit for the synthesis of fatty acids. β-hydroxacyl-ACP-dehydratase  and enoyl-ACP-reductase  are closely related in the subsequent fatty acid prolongation reaction [FabZ regulates the activity of 3-hydroxyacyl-ACP dehydratase, which is the dehydration step in the elongation of fatty acids promoting the conversion of 3-hydroxyacyl-ACP to enoyl-ACP [fabI and fabZ would enhance unsaturated fatty acid synthesis, which might alter the composition of fatty acids in plasma membrane. It has been observed that the increased level of unsaturated fatty acid synthesis in Lactobacillus helveticus prevented major damage to viability in cells exposed to the various stress combinations, thereby providing survival advantages [L. reuteri in our previous investigation [L. reuteri to produce more organic acids in metabolism. Conversely, the downregulated expression of fabZ and fabI affected by soybean protein might not be conducive to the growth of L. reuteri, relatively.DEGs were significantly enriched in a fatty acid biosynthesis pathway both in the dpro/MRS group and dpep/MRS group, and there were five enriched genes, namely ro group , but dowp (accD) , ATP-graα (accA)  belong tα (accA) . The uprN134_RS055, accC . Interestingly, bovine lactoferrin even inhibited purine synthesis of LGG, which may be related to the diversity of composition of various protein and strain of Lactobacillus.DEGs in both dpep and dpro treatment were, compared with MRS group, significantly enriched in purine metabolism pathway, within which a series of DEGs involved in purine biosynthesis were upregulated . The expand purL , which wynthesis . Additio and DNA , but alstoferrin  and wheytoferrin  were alsTcyA (N134_RS01535), tcyB (N134_RS01525), and tcyC (N134_RS01530) encoding transporter substrate-binding domain-containing protein [N134_RS08600) [N134_RS00920) also increased by relatively high folds, while the glnH (N134_RS00495) and glnQ (N134_RS00500) responsible for glutamine transport [artQ (N134_RS04200) and artR (N134_RS04205) responsible for arginine lysine or histidine transport [L. reuteri LR08. However, the expression of these genes has no significant difference in dpro/MRS comparison, probably since that it is easier and more direct for L. reuteri to use smaller molecule weights of nitrogen sources. The expression of opuC, opuBD, and opuA as transport system proteins of osmoprotectants also increased [L. reuteri still showed strong growth under the increased osmotic pressure, despite higher accumulation of metabolites in dpep. A similar result was also elucidated in LGG under bovine lactoferrin treatment [ABC transporter protein is one of the largest superfamilies found in bacteria, which is responsible for cells to absorb essential nutrients and transport molecules from outside cells to organelles . CompareRS08600)  and glycransport  and the ransport  were signcreased . Glycinencreased  and can ncreased . This mareatment . Moreove N134_RS000 and armetC (N134_RS01520), tycB (N134_RS01525), tycC (N134_RS01530), and opuC (N134_RS00920) in the 3 groups were all in accordance with those of in RNA-seq. These results were deemed to be relatively reliable.16 genes in L. reuteri LR08 regulated by soybean protein and peptides, as well as the relationship between physiological functions and gene expressions affected by dpep and dpro. The results of this study provide a new direction for the selection of nitrogen-sourced prebiotics in the food industry, as well as a new idea for the development of soybean protein products. Further research is needed to clarify how soybean protein and peptides affect specific gene expression within various pathways.In conclusion, soybean peptides could promote DEGs from the acc and fab gene families as a critical role in fatty acid biosynthesis, while soybean protein modulated them oppositely, which was consistent with the results of organic acids secretion reported before. Both soybean protein and peptides improved gene expression enriched in purine biosynthesis and IMP biosynthesis pathways to provide more abundant nucleic acid and ATP for bacteria growth. Additionally, dpep instead of dpro induced genes enriched in ABC transporters pathway, promoted and prevented the accumulation of some amino acids in cells, and maintained the balance of osmotic gradient to improve the nutrition absorption. These results contribute to profound understanding of the mechanism of DEGs of