Background: The increase in the medical use of cannabis has revealed a number of beneficial effects, a variety of adverse side effects and great inter-individual variability. Association studies connecting consumption, addiction and side effects related to recreational cannabis use have led to the identification of several polymorphic genes that may play a role in the pharmacodynamics and pharmacokinetics of cannabis. Method: In total, 600 patients treated with cannabis were genotyped for several candidate polymorphic genes , encoding receptors CNR1 and TRPV1; for the ABCB1 transporter; for biotransformation, bioactivation and biosynthesis; and CYP3A4, COMT and UGT2B7 conjugation. Results: Three polymorphic genes  were identified as being significantly associated with decline in pain after treatment with cannabis. Patients simultaneously carrying the most favourable allele combinations showed a greater reduction (polygenic effect) in pain compared to those with a less favourable combination. Considering genotype combinations, we could group patients into good responders, intermediate responders and poor or non-responders. Results suggest that genetic makeup is, at the moment, a significant predictive factor of the variability in response to cannabis. Conclusions: This study proves, for the first time, that certain polymorphic candidate genes may be associated with cannabis effects, both in terms of pain management and side effects, including therapy dropout. Significance: Our attention to pharmacogenetics began in 2008, with the publication of a first study on the association between genetic polymorphisms and morphine action in pain relief. The study we are presenting is the first observational study conducted on a large number of patients involving several polymorphic candidate genes. The data obtained suggest that genetic makeup can be a predictive factor in the response to cannabis therapy and that more extensive and planned studies are needed for the opening of new scenarios for the personalization of cannabis therapy. The therapeutic use of cannabis dates back to 2800 B.C. in China, and its diffusion in Europe to treat various diseases occurred mainly in the 18th and 19th centuries ,4 and grAn “open-label”, multi-centric, non-randomized observational study to assess the feasibility of possible associations between the outcomes of treatment with cannabis in patients suffering from chronic pain, for different diseases and certain polymorphic candidate genes.In total, 600 Italian patients of Caucasian origin, undergoing cannabis therapy for chronic pain, were recruited by Azienda USL Toscana Sud-Est, Zona Arezzo, San Donato Hospital , Department of Pain Medicine and Palliative Care, starting in 2018. The study was approved by the Tuscan Regional Ethical Committee (n. 1287) in 15 May 2018. None of the patients refused to take part in the project, reaching a total participation rate of 100%.Patients were informed of the project and gave written consent to participating in the study and permitting their genotyping. They were interviewed and assessed for pain severity, in addition to collecting information on their age, sex and pain treatment. Exclusion criteria: age below 18 years, organic or psychiatric disorders interfering with judgement and comprehension abilities, persistence of pain for less than three months, no treatment with painkillers from traditional pharmacopoeia, pregnancy or breast-feeding, arrhythmia or ischemic cardiopathy, patients with severe psychiatric disorders, patients with a history of abuse or addiction to cannabis or other psychoactive substances.Inclusion criteria: chronic pain, use of painkillers from traditional pharmacopoeia for at least three months without complete efficacy, full ability to understand.Procedures were compliant with the ethical rules of the committee responsible for human experimentation and with the 1975 Helsinki Declaration, as reviewed in 2008.Patients were grouped into five categories based on the type of disease originating the pain: (1) central nervous system diseases; (2) rheumatoid, arthritic, inflammatory and autoimmune diseases; (3) headache and migraine; (4) neuropathic pain; (5) cancer pain.(1)Pain intensity measured with the visual analogue scale (0–10);(2)Presence/absence of benefits ;(3)Presence/absence of side effects ;(4)Anxiety and depression symptoms, measured with the hospital anxiety and depression scale (HADS) .Interviews and data collection were carried out during the first examination (T0), after 4 weeks (T1) and after 12 months. During the study, a few patients decided to drop out of therapy, either because of unresponsiveness to cannabis therapy or because of relevant side effects. Measured variables were, mainly:® , Cannabis FlosBedrolite® , Cannabis FlosBediol® , Cannabis FM2®  and Cannabis FM1® . The different preparations of cannabis inflorescence were extracted in accordance with the SIFAP  method [Administered cannabis preparations were: Cannabis FlosBedrocan) method , CannabiMDR1/ABCB1 rs1045642; TRPV1 rs8065080; 5-UGTB7 rs7438135; CYP3A4 rs2242480; CNR17B rs1049353; COMT rs4680.Genetic variants  in candidate genes were selected from previous publications with at least one positive association with a proven biological activity in individuals using cannabis for recreational purposes, given the lack of suitable indications in patients using cannabis for medical purposes. The 6 selected SNPs were: The DNA was obtained with an oral swab and extracted with DNA Extract All Lysis Reagent , and SNPs were determined with TaqMan Assay (Applied Biosystem) with an RT-PCR One-Step-Plus system (Applied Biosystem by Thermo Fisher).The main dependent variable was pain reduction (∆VAS), calculated as the difference between basal pain and the pain assessed in each subsequent time. Each patient, therefore, also represented their own control. A multi-variate linear regression model (MLR) was used to assess the significance of the effect of variables on ∆VAS: year and centre of recruitment, age, gender, genotype, disease, painkillers and medication taken, type of cannabis and THC and CBD doses. The χ2 test and/or multi-nomial logistic regression were used for associations between categorical variables and the Kruskal–Wallis t (K–W) test for non-normally distributed variables. Associations with genotypes were initially tested within a co-dominant inheritance model (three separate genotypes). Simplified models were then applied: a dominant model  and a recessive model  when effects were not significantly different. Being an explorative study, Bonferroni correction was not applied. All statistical analysis have been performed by Stat Graphics Centurion XVI, by Statgraphics Technologies, Inc. The Plains, VA, USA.p < 0.05). The allelic proportions of the genes considered did not vary significantly in different disease groups or according to the different types of cannabis used. Patient demographics are reported in Out of 600 patients recruited in the study, 564 were eligible for the analysis, as there was a complete database including correct genotyping. For the genotype quality control, a Hardy–Weinberg (H–W) equilibrium test was performed; all genotypes tested statistically in the H–W equilibrium  and of CBD , despite doses having doubled overall.Pain reduction was approximately 20% in the first month, subsequently reducing by half in the third month and then further decreasing to 43% after one year. During the whole study, 443 patients (75.7%) dropped out of cannabis therapy, 179 of them putting forward various reasons, such as poor or no pain reduction, side effects or both, whereas the reasons for the remaining 264 were unknown. Polymorphism frequencies did not change significantly as a result of the drop-out factor. A decrease in ∆VAS during the first month was not significantly correlated with doses (squared) of THC , only polymorphisms for the ABCB1 , as displayed by p < 0.05).The MLR (multi-variate linear regression) analysis of ∆VAS showed a highly significant effect in new groupings . Consequently, we concluded that the poor responders to cannabis were patients carrying three non-favourable alleles.Patients were further grouped based on the amount of favourable allele combinations for each genotype: group one with two or three favourable alleles, i.e., CC + CT for p < 0.05) different pain decreases. Group three, therefore, showed a 0.56 ∆VAS, classifying Group three as non-responders. Group two showed a 1.29 ∆VAS—that is, 2.3 times higher than group three—and could be considered to constitute responders. Group one showed a 1.73 ∆VAS—that is, 3.1 times higher than group three—and could be considered to have the highest number of responders.Patients grouped on the basis of their genotypes being more or less favourable to a pain decrease could be classified in groups with statistically significantly , with a larger proportion of women .At the beginning of the study, 98 patients presented evidence of depression, and this condition was significantly associated with p = 0).In total, 78 patients dropped out of treatment with cannabis during the first month for various reasons, mainly for the lack of pain reduction, side effects or both. The average pain reduction in drop-out patients was null or negative (∆VAS = −0.17) compared to the others (∆VAS = 1.7), and the difference was highly significant  between the drop-out patient group and those suffering from multiple adverse events, such as tachycardia, drowsiness, dry mouth and hyperactivity. Furthermore, there was a significant association (χ2 = 5.6 p < 0.018) between the drop-out phenomenon and the polymorphism (recessive model) of the gene CNR1 (CB1) rs1049353. The odds ratio for patients with the GG + GA genotypes vs. AA homozygotes was 2.4 (CL 1.1–5.2), indicating that the former were 2.4 times more likely to continue treatment with cannabis than AA homozygotes. Logistic regression confirmed the role of CNR1 polymorphism in the risk of treatment drop-out for AA homozygotes, excluding significant associations with all the other possible factors we tested.The χ2 test showed a strongly significant association , as group two was more strongly associated with anxiety and the use of medication (p = 0.0002). Those who took medication were at higher risk, probably because they were affected by more serious diseases.In respect to anxiety, the logistic analysis showed significant associations between the presence of anxiety and disease  not only showed no significant association, but remained very far from significance. It is very likely that the associations observed were in part disguised by the constant adjustment of cannabis dosages, being approximately doubled on average, but reduced in certain cases, and by the fact that approximately 30% of patients changed their type of cannabis during the first month of treatment to the “titrate” type.In this study, we showed that, despite the complexity of cannabis pharmacology and the difficulty in precisely quantifying the results of treatment with cannabis, it is possible to assess the role of certain genetic polymorphisms in patients and to explain part of the great inter-individual variability observed in pain reduction. On average, the pain reduction in remaining patients (non-drop-out) at the end of the first month amounted to approximately 20%, but with great variability. Part of this was significantly associated with three polymorphic candidate genes: C3435T, of the MDR1/ABCB1 gene, which encodes a transporter protein (MRP1), produces a “silent” missense variant, which, however, determines the reduction in the expression of the transporter protein [MDR1/ABCB1 gene in pain control, the pharmacological treatment aside.The rs1045642 polymorphism, also known as  protein . Patient protein . In this protein , confirm protein . An incr protein . It is i protein , suggestTRPV1 polymorphism (rs8065080), also known as c.1191A > G, is a common variant in the TRPV1 gene , which encodes, among others, vanilloid receptor subtype-1 and the capsaicin receptor. The polymorphism determined by rs8065080 was associated with salt sensitivity [The sitivity , the tolsitivity  and withsitivity . In our sitivity , as by msitivity . Cannabisitivity ,26.UGT2B7 gene encodes UDP-glucuronosyltransferase-2B7, involved in phase II metabolism, conjugating a variety of compounds such as analgesics (morphine), carboxylic non-steroidal anti-inflammatory drugs (ketoprofen) and anti-cancer drugs  [The ic acid) . The rs7ic acid)  and in tic acid) . In the ic acid) .CNR1 gene, rs1049353, was a therapy drop-out risk factor. This gene encodes the most relevant cannabinoid receptor, the polymorphism of which was extensively studied in depression therapy [The fact of having found three polymorphic genes which individually significantly influence pain decrease after treatment with cannabis poses the problem of the impact of their possible combinations in patients. As expected, patients simultaneously carrying the most favourable allelic combinations showed a greater pain reduction (∆VAS = 1.73) compared with those with a less favourable combination (∆VAS = 0.56). Considering genotype combinations, we could, therefore, group patients into good responders, intermediate responders and poor or non-responders. We are aware that this study included many variables, such as cannabis types, contents of active principles, different groups of diseases and patients’ overall clinical situations, the interactions of which were difficult to assess as they were not planned a priori, but could have important effects on the assessment of pain reduction. However, the results suggested that a patient’s genetic makeup is, at the moment, a significant descriptive factor of variability in the response to cannabis. Changes in cannabis dosages, doubled on average, which occurred during the first month and were empirically guided, were not associated with any factor, including genetic factors, thus, suggesting that the initial dosage, apparently low, was due to the caution of physicians who administered a drug which was not well known to patients who were treated for the first time. Furthermore, the association study showed that the A allele of the  therapy ,32.This study proved, for the first time, that certain polymorphic candidate genes may be associated with cannabis effects, both in terms of pain control and side effects, including therapy drop-out. This approach is, therefore, feasible and, if adequately developed, could possibly contribute to shedding light on the complex pharmacokinetics and pharmacodynamics of cannabis, making physicians more confident in its therapeutic use. We are aware that only by extensively expanding the polymorphic genes panel and the number of their polymorphisms could it be possible to hypothesize a more personalized therapy, aided by the knowledge of patients’ genetic characteristics. It is our opinion that an association study on the whole genome  could lead to important progress in this field.


Chronic kidney disease (CKD) is a common chronic condition with high incidence and insidious onset. Glomerular injury (GI) and tubular injury (TI) represent early manifestations of CKD and could indicate the risk of its development. In this study, we aimed to classify GI and TI using three machine learning algorithms to promote their early diagnosis and slow the progression of CKD.Demographic information, physical examination, blood, and morning urine samples were first collected from 13,550 subjects in 10 counties in Shanxi province for classification of GI and TI. Besides, LASSO regression was employed for feature selection of explanatory variables, and the SMOTE (synthetic minority over-sampling technique) algorithm was used to balance target datasets, i.e., GI and TI. Afterward, Random Forest (RF), Naive Bayes (NB), and logistic regression (LR) were constructed to achieve classification of GI and TI, respectively.A total of 12,330 participants enrolled in this study, with 20 explanatory variables. The number of patients with GI, and TI were 1,587 (12.8%) and 1,456 (11.8%), respectively. After feature selection by LASSO, 14 and 15 explanatory variables remained in these two datasets. Besides, after SMOTE, the number of patients and normal ones were 6,165, 6,165 for GI, and 6,165, 6,164 for TI, respectively. RF outperformed NB and LR in terms of accuracy , sensitivity , specificity , and AUC  for both GI and TI; the four variables contributing most to the classification of GI and TI represented SBP, DBP, sex, age and age, SBP, FPG, and GHb, respectively.RF boasts good performance in classifying GI and TI, which allows for early auxiliary diagnosis of GI and TI, thus facilitating to help alleviate the progression of CKD, and enjoying great prospects in clinical practice. Chronic kidney disease (CKD) is a common chronic condition worldwide, with a prevalence of 13.4% , emerginIn 2012, Luxia Zhang employed logistic regression to predict CKD, but it comes with drawbacks . The firFeature selection is of great necessity in constructing classifiers, since the presence of irrelevant features may be responsible for the poor model performance . L1 reguIn this study, we aimed to (1) employ LASSO algorithm to conduct feature selection for GI and TI; (2) use the classical and widely accepted SMOTE (Synthetic Minority Over-sampling Technique) algorithm to handle the imbalanced classes of GI and TI; (3) employ the mature machine learning algorithm, Random Forest (RF) to make a classification of GI and TI, respectively, and compare its performance with logistic regression (LR) and Naive Bayes (NB), thus achieving the auxiliary diagnosis of GI and TI and providing a new idea for clinical practice in delaying the progression of CKD.Shanxi Provincial People’s Hospital conducted CKD screening for permanent residents aged ≥ 40 years in the northern region of Shanxi Province (Ningwu County), the central regions , and the southern regions  from April 2019 to November 2019. A total of 13,550 residents volunteered for this screening, and 12,285 were eventually enrolled in the study, including 5,206 men and 7,079 women aged 41–91 years.Inclusion criteria: (1) residents aged 40 years or older; (2) conscious participants without communication impairment; (3) participants understanding the significance of the study and willing to sign a written informed consent; (4) participants with no cognitive impairment or mental illness; (5) more than 1 year of local residents as of the survey date. Exclusion criteria: (1) Severely incomplete information recorded; (2) poor compliance; (3) pregnant women or those with a history of substance abuse.Questionnaires, physical examination, and laboratory analysis were used to collect data. (1) The questionnaire comprised demographic information , lifestyle . The questionnaire was administered online and completed by the subjects themselves or their family members. (2) Physical examination included height, weight and blood pressure , which were measured twice and then the mean value was calculated. All data were measured by a medical professional. Body mass index (BMI) was calculated by weight in kilograms divided by the square of height in meters.(3) Fasting venous blood was collected from subjects for fasting blood glucose (FPG), glycated hemoglobin (GHb), homocysteine (Hcy), total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein (HDL-C). (4) Morning urine specimens were collected from subjects. After centrifugation at 3,000 r/min for 10 min, the supernatant was extracted (low-speed centrifuge Anhui Zhongke Zhongjia SC3616), and α1-microglobulin (α1MG), urinary creatinine (UCr), and microalbuminuria (MAU) were determined by latex turbidimetry, sarcosine oxidase, and immunoturbidimetry, respectively.2), normal weight (18.5–24.0 kg/m2), overweight (24.0–28.0 kg/m2), obesity (≥28 kg/m2). ACR was defined as urinary microalbumin divided by urinary creatinine multiplied 8.84; MCR was defined as urinary microglobulin divided by urinary creatinine multiplied 8.84.Information on the annual income, educational levels, health history, and lifestyle of the study participants was obtained from the questionnaire. Annual income was defined as < 5K yuan, 5K–10K yuan, 10K–20K yuan, > 20K yuan; education levels were defined as ≤ primary school, ≤ middle school, ≤ high school, ≥ bachelor’s degree; smoking was classified as yes or no; alcohol consumption was classified as always (>100 g/time and 3 times/week), sometimes (<3 times/week or < 100 g/time) and rarely; exercise was classified as “none or a little” or “regular” . BMI was defined as underweight (<18.5 kg/m(1) Questionnaire: demographic information ; lifestyle . (2) Morning blood: HDL, LDL, TG, TC, Hcy, FPG, GHb. (3) Physical examination: SBP, DBP, BMI. 20 variables in total.ACR ≥ 30 mg/g was defined as GI; MCR > 23 mg/g was defined as TI. The presence of GI, TI was assigned 1; otherwise, they were defined as 0. In this study, we employed RF, LR and NB to make a classification of GI and TI, respectively.Absolute Shrinkage and Selection Operator (LASSO) is one of the common methods for feature selection. It is characterized by the inclusion of an L1 regularization penalty term in fitting generalized linear regression, which makes the sum of the absolute values of the regression coefficients of the model lower than a particular value. It aims to minimize the sum of squared residuals, forcing the regression coefficients of variables that contribute less to the model to be compressed to zero and achieving a feature sparse process , 15. LASThe Synthetic Minority Over-Sampling Technique (SMOTE) is an oversampling technique that is an effective algorithm for dealing with imbalances between data classes . It’s emRF, a data-driven integrated learning algorithm, could obtain multiple new training data by an autonomous sampling of the training set, constructing multiple classification trees based on the parallelization of these new data, and achieving de-correlation between the trees by introducing the selection of independent variables. By doing so, the diversity of the classification trees originates from both sample and independent variable perturbations to achieve the effect of reducing the model variance, and finally to vote on the classification results of multiple trees to obtain the final classification results , 24. TheSD) or median ± interquartile [)], as appropriate.Qualitative data are expressed as percentages (%), and quantitative data are expressed as mean ± standard deviation (M±The datasets were divided into training set (80%) and testing set (20%). The former ones were used for models training, i.e., RF, NB, and LR, while the latter ones were employed for evaluation of model performance. All analyses were implemented in R software (version 4.0.3).The evaluation parameters comprised Accuracy (1), Specificity (2), Sensitivity (3) and area under the receiver operating curve (AUC). The predicted result was defined as True Positive (TP) when patients with renal conditions were classified as patients and True Negative (TN) when healthy ones were classified as healthy. Besides, the predicted result was False Positive (FP) if healthy subjects are considered patients; similarly, False Negative (FN) if patients are considered healthy subjects. Accuracy is to evaluate how accurate the machine learning algorithms are to detect what it is supposed to measure. Specificity is the ability to correctly exclude those without renal conditions and Sensitivity is to correctly identify those with renal conditions.A total of 12,330 people were included in this study, 5,230 men and 7,100 women, the number of GI was 1,587 (12.8%) and the number of TI was 1,456 (11.8%). Besides, the number of participants with both GI and TI was 2,439 (19.7%). Other parameters are detailed in As shown in As shown in When constructing model for GI, the number of GI and non-GI in the training set were both 4,932, and 1,233 in the testing set, respectively. When constructing model for GI, the number of TI and non-TI in the training set were 4,973 and 4,891, respectively, and 1,273 and 1,192 in the testing set. The accuracy, sensitivity, specificity and AUC of RF for classification of GI and TI performed better than NB and LR in both the training set and testing set, which shows that RF does have a high diagnostic value for classification .We indicated the contribution of the explanatory variables to the model by %IncMSE, and the larger the %IncMSE, the more important the variables were for the RF model. The four variables that contributed most to the classification of GI in the RF model represented SBP, DBP, sex, and age. The four variables that were most important for the classification of TI constituted age, SBP, FPG, and GHb .In this study, ACR and MCR levels were used as screening indicators for GI and TI, and ACR ≥ 30 mg/g was considered GI; MCR > 23 mg/g was considered TI. Besides, the machine learning model RF was used to classify them, and we compared its classification performance with NB, and LR.This study suggested that the accuracy, sensitivity, specificity and AUC of the RF algorithm outperformed other classifiers in both the training set and testing set. Yet its performance in the testing set was comparatively lower than that in the training set, because the classification performance based on the training set was prone to overfitting , while tOf note, the RF model is sensitive to response variables with unbalanced data. Imbalanced classes in the data would leave the output of the model tending to larger classes, causing some classification errors, and leading to a less accurate classification performance . As suchSince RF model is data-driven, a visible functional equation is unavailable to determine the extent to which the explanatory variables contribute to the model based on the regression coefficients. However, a more intuitive alternative for the model is that by outputting feature importances, the model could explain the importance of the variables on the explanatory variables. This study demonstrated that the four explanatory variables with the greatest output weight of RF classifier for GI represented SBP, DBP, sex, and age; and the four explanatory variables for TI constituted age, SBP, FPG, and GHb.In a hypertensive state, abnormal glomerular hemodynamics, spasmodic constriction of renal arteries would reduce renal blood flow, leading to renal ischemia and long-term hyperperfusion and hyperfiltration of glomerular capillaries, resulting in damage to glomerular vascular endothelial cells and podocytes , 27. Mea2 for the definition of CKD may lead to overdiagnosis of CKD in women (It has been documented that kidney disease in China is more prevalent in male patients, suggesting that sex is also one contributor to degenerative changes in kidney structure and function. The prevalence of CKD has been reported to be higher in women than in men. A representative study pooling 33 population-based studies worldwide evaluated the global prevalence of stage 1–5 CKD at 10.4% among men and 11.8% among women aged 20 years and older . The reain women . A folloin women . The effAfter the age of 40, the glomerular filtration rate decreases at a rate of 1 ml/min/1.73 per year, resulting in stiffening of the renal vessel wall, glomerular atrophy, sclerosis, tubular atrophy, and interstitial fibrosis, which eventually lead to renal hypofunction . In diabThere are also some limitations in this paper. Firstly, the study constructed the models with data from Shanxi Province, and no other external datasets are available to validate the model performance. Our ongoing work is to collect samples from other areas, to validate the generalization capabilities of the model. Secondly, this study was initially considered for cost-effectiveness and other indicators reflecting CKD were not collected, such as blood creatinine, which will also be the focus of our next step. Additionally, as CKD was more prevalent in people aged ≥ 40 years, this study centered on those over 40 years. In the future, we consider surveys on those aged 18–40 years to improve the prediction model in younger groups. Finally, GI and TI were defined only by surrogate parameters, ACR and MCR, which may not well accurately reflect the renal conditions. In our future work, we would conduct a follow-up for those with positive urine protein.In short, as early manifestations of CKD, GI and TI have emerged as a global public health issue; their early diagnosis and corresponding treatment are of great importance. Our results demonstrate the potential value of machine learning algorithms in GI and TI-assisted diagnosis, which facilitates reducing the workload of doctors, while achieving automated diagnosis and treatment decisions, and thus could be promoted in clinical practice.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethics Committee of Shanxi Provincial People’s Hospital. The patients/participants provided their written informed consent to participate in this study.WS was responsible for the data analysis and the writing of the manuscript. XZ, QD, QW, and YahL helped polish the manuscript. AL, WZ, LS, LQ, and RL gave precious advice on the statistical methods. YafL was responsible for the conception and design of the research. All authors read and approved the final draft.


Although respiratory diseases (RD) are rapidly becoming a global health issue due to their high mortality and prevalence, there are limitations to the currently available treatments. Acupuncture has been recognized to mitigate many diseases by reducing inflammation and modulating cytokines. However, no systematic analysis has been performed to examine the effects of acupuncture on RD. We aimed to evaluate the effects of acupuncture on rodent animal models of RD.PubMed, EMBASE, MEDLINE, and the Research Information Service System were searched to retrieve studies that met our inclusion/exclusion criteria. The quality of each included study was evaluated using a 10-item checklist modified from the Collaborative Approach to Meta-Analysis and Review of Animal Data from Experimental Studies. With adequate data extracted, meta-analysis was performed using RevMan software.A total of 18 studies were included, and the mean quality assessment was 5.7. The meta-analysis revealed that acupuncture had a significant effect on changing the cytokine levels, including pro-/anti-inflammatory, Th1-, Th2- and Th17- specific cytokines.Although there were limitations in the number of included studies, the results suggest that acupuncture can be a possible treatment for RD through its modulation of various cytokines, leading to reduced inflammation. Respiratory diseases (RD) are a major global health issue that have been a serious financial burden owing to the difficulty in treatment. RD encompasses various disorders such as sinusitis, asthma, chronic obstructive pulmonary disease (COPD), and even acute lung injury caused by other diseases. Although the lesions and symptoms of the aforementioned diseases may differ, most of them are associated with acute/chronic inflammation . InflammAcupuncture, which originates from traditional Chinese medicine, has long been acknowledged to mitigate many diseases. Acupuncture at the specific acupoint of ST36 has been reported to promote anti-inflammatory, anti-oxidative, and immune-enhancing effects –7. MoreoIn this review, we performed a meta-analysis on inflammatory cytokines in various animal models of RD to determine the effects of acupuncture on RD.Reports that examined the effect of acupuncture on inflammatory cytokines in rodent models of RD that were written in English were included in the present study. PubMed, EMBASE, MEDLINE, and Research Information Service System were searched from inception until December 2021 using the following search terms: “mouse (mice)” or “rat (rats)”, and “acupuncture (electroacupuncture)” and “respiratory disease.”Studies were included based on the following criteria: subjects (rodent models of RD), interventions , and outcomes . Lung function test data were included as the subsequent outcomes. Along with those that did not provide access to the full text, studies that were not written in English or focused on unrelated topics were excluded. After screening the full text, studies that did not meet our criteria in the methods and results were also excluded.Two authors (Kim and Lee) independently extracted the data on the publication year, name of the first author, type of rodent RD model, disease/condition, sample size, type of acupuncture, and type of specimen. Along with the mean value of inflammatory cytokines within each group , standard deviation or standard error of the mean were extracted to determine the effect measures and effect size of acupuncture on RD. Each value from the studies was extracted using the Engauge Digitizer software version 12. We estimated the number of animals in groups by calculating the mean number of animals per study. Lung function data, including the type of lung function test and the result, were collected as a secondary outcome.The methodological quality of each included study was assessed by two authors (Kim and Lee) using a 10-item checklist modified from the Collaborative Approach to Meta-Analysis and Review of Animal Data from Experimental Studies (CAMARADES) checklist , 15: puba p-value < 0.05 was considered statistically significant. For the assessment of study heterogeneity, chi-squared and I2 statistics were used.In the present study, the ratios of cytokine levels in the disease and intervention groups to the control group were considered as continuous data. To compare studies that used different units for cytokine levels, the difference between the disease/control ratio and acupuncture/control ratio was calculated. Since all 18 studies set RD as an independent variable and analyzed the correlation with the outcomes, the standardized mean difference (SMD) was estimated based on a fixed-effect model. The meta-analysis was performed on each cytokine subgroup using RevMan version 5.4 . The confidence interval (CI) was established at 95%, and Among the 100 studies selected, 57 studies were excluded because 50 were not written in English, five did not cover relevant topics, and two did not allow access to the full text. The full-text screening was performed on the remaining 43 studies, of which 25 were excluded due to deficiencies in the methodology and results. Eight studies used intervention other than manual acupuncture or electroacupuncture, 16 did not evaluate inflammatory cytokines, and 1 study did not include proper control group. Therefore, a total of 18 studies were included in this review. A flow diagram of the study selection process is shown in The quality assessments of the included studies are summarized in The characteristics of the included studies are summarized in p < 0.00001; heterogeneity X2 = 89.74, I2 = 72%, p < 0.00001; heterogeneity X2 = 20.14, I2 = 90%, X2 = 28.75, I2 = 73%, p < 0.00001; heterogeneity X2 = 15.77, I2 = 30%, p < 0.00001; heterogeneity X2 = 1.92, I2 = 0%, Cytokines, the principal mediators of inflammation, are composed of various subfamilies that play different roles. First, they are known to have function as pro- or anti-inflammation. Second, cytokines also can be categorized with the immune cells where they are released from, including CD4+ Th1, Th2 or Th17 cells. 0.1), forced expiratory volume in 0.3s (FEV0.3), the ratio between the aforementioned two and forced vital capacity , inspiratory capacity, peak expiratory flow, and minute volume. Among the 18 studies, seven measured RL and Cdyn, three conducted PFTS, and eight did not mention any lung function tests. Acupuncture was found to increase Cdyn and alleviate RL. It was also shown that acupuncture improved lung function in general by increasing FEVs, FVC, inspiratory capacity, peak expiratory flow, and minute volume.Recently, a number of clinical studies have reported the effect of acupuncture on RD. Acupuncture enhanced the strength of diaphragm while relieving the respiratory muscle fatigue of COPD patients , mitigatThe following inflammatory cytokines were examined in lung tissue, fluids, or serum/plasma samples from animal models. Inflammatory cytokines can be specifically classified according to their functions: those that promote inflammation are known to be pro-inflammatory, while those that engage in restoring damages are considered anti-inflammatory cytokines. In pulmonary diseases, pro-inflammatory cytokines are known to trigger mucus secretion and airway fibrosis , while avia Th17 cell control. As Th2 and Th17 cells play a crucial role in RD pathology  (NRF-2020R1C1C1004107) and from the Ministry of Health & Welfare through the Korea Health Industry Development Institute (KHIDI) (grant no. HF21C0018).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Nutrition and health knowledge play a crucial role in promoting healthy dietary behaviors, and have been found to be related to sociodemographic characteristics. However, the existing evidence is limited and inconsistent. We aimed to evaluate the awareness level of nutrition and health knowledge and its influencing factors among Wuhan residents, and to provide scientific basis for carrying out targeted nutrition education programmes.By stratified random sampling, residents aged 18–64 in Wuhan were selected for self-administered questionnaire survey. We adopted the structured questionnaire to investigate respondents' sociodemographic characteristics, nutrition and health knowledge, and the way to acquire knowledge. Among them, nutrition and health knowledge includes the following four parts: dietary guidelines recommendations, food and nutrients, nutrition and disease prevention, and nutrition skills. Chi-square tests were used to analyze the associations between total awareness rate and sociodemographic characteristics. Multiple linear regression models were used to analyze the influencing factors of nutrition and health awareness.p < 0.001). The multiple linear regression models showed that age, gender, education level, occupation, residential address, and the history of chronic disease were the potential factors affecting individual nutrition awareness.A total of 33,436 valid questionnaires were obtained, with a response rate of 97.8%. The total awareness rate was 20.4%, with the highest in nutrition and disease prevention (72.7%) and the lowest in nutrition skills (46.3%). Responders aged 35–44 (23.3%), females (22.8%), educational workers (24.8%), obtaining a master's degree or above (34.1%), living in downtown area (23.1%), and without a history of chronic disease (24.6%) were more likely to have higher awareness rates (all The total awareness rate of nutrition and health knowledge among Wuhan residents was not optimistic. Besides, our findings suggested that sociodemographic characteristics are closely related to nutrition awareness, which may provide important clues for carried out nutrition education campaigns. With the rapid economic development and the improvement of living standards, significant changes have taken place in the lifestyle and dietary patterns of Chinese people. Meanwhile, the prevalence of nutrition-related chronic diseases such as obesity, hypertension and diabetes has increased year by year, causing huge burden on individuals and society , 2. CurrIt is well accepted that a higher level of nutrition knowledge contributes to promoting healthier dietary behaviors, while a lower level of nutrition knowledge is closely related to poor eating habits, unbalanced dietary patterns , 6, and Individual sociodemographic characteristics, including age, education level, economic status, and residential area, are key factors affecting their nutrition and health knowledge , 20. HowTo date, however, there are few large-scale studies on nutrition and health knowledge of the Chinese population and its possible socio-demographic factors. Thus, we aimed to acquire the level of nutrition and health knowledge of Wuhan residents and to explore its influencing factors. Our findings will help to provide scientific basis for carrying out targeted nutrition education programmes.The China Nutrition and Health Knowledge Survey is an ongoing cross-sectional study launched in April 2021 in 31 provinces, cities and autonomous regions in China. The survey aims to explore the awareness level and its influencing factors of nutrition and health knowledge among Chinese adult residents and providing a scientific basis for the policy decision on nutrition intervention. Wuhan, a representative city in central China, conducted part of the survey from April 2021 to October 2021, and the permanent residents aged 18–64 from Wuhan were included in the analysis. Based on the results from the sixth national population census in China , we adopThe questionnaire was designed by the National Institute for Nutrition and Health, Chinese Center for Disease Control and Prevention, and formed after expert review, pre-survey, reliability and validity test. We adopted the structured questionnaire to investigate respondents' sociodemographic characteristics , nutrition and health knowledge, and the way to acquire knowledge.The nutrition and health knowledge consisted of 32 items, including 22 single choice questions and 10 multiple choice questions, involving the following four parts: (a) dietary guidelines recommendations (items 1–18); (b) food and nutrients (items 19–22); (c) nutrition and disease prevention (items 23–26); and (d) nutrition skills (items 27–32). For single choice questions, a score of 1.5  or 2.0  was assigned to the correct answer, and 0 was assigned to the wrong or “don't know” answers. For multiple choice questions with 5 options , a score of 0 was assigned to the “don't know” answer, and 1.5 was assigned to each correct answer. Besides, if the wrong answer was not selected, 1.5 points will be further added, with a total score of 6.0 (except for item 18). The specific scoring criteria was shown in For multiple choice questions, we defined both choosing the correct answer and not choosing the wrong answer as knowing this option. To assess the responders' awareness of each multiple choice questions, we also calculated the proportion of knowing 0, 1, 2, 3 and 4 options.Definition of analysis indicators as follows:Awareness rate of each item (single choice question):Awareness rate of each item (multiple choice questions):Awareness rate in each part:The total awareness rate:Age was divided into five groups . Gender was divided into two categories . Education level was allocated into six categories . The occupation was allocated into five categories . The residential address included the downtown area and the remote area. A history of chronic diseases  was divided into three categories (Yes/No/Don't know).The questionnaire was revised after review by an expert group, preliminary investigation and reliability and validity test. Prior to the online survey, the investigators at each monitoring site must receive professional training and assessment. During the survey, all investigators carried out the investigation strictly in accordance with the unified survey manuals, and the participants should be informed of the purpose and filling method prior to answering the questionnaire. After completing a questionnaire, an on-site inspection is conducted immediately. Additionally, after the investigation of all monitoring sites is completed, the quality control personnel of the Centers for Disease Control and Prevention will conduct a telephone return visit to the respondents according to the specially designed review questionnaire. If there are 3 or more unqualified questionnaires at one monitoring site, the on-site investigation was considered unqualified and should be re-investigated.p < 0.05 was considered indicative of statistical significance.The sociodemographic characteristics of respondents were all set as categorical variables and expressed as frequencies (percentages). Chi-square tests were used to analyze the associations between the total awareness rate of nutrition and health knowledge and sociodemographic characteristics. The awareness level of nutrition and health knowledge was a continuous variable with skewed distribution, presented as median (lower and upper quartile) and the differences between the two groups were compared using the Mann-Whitney U test. Multiple linear regression models were used to analyze the influencing factors of nutrition and health awareness. All statistical analyses were performed using SPSS version 25.0 (IBM Corporation). A two-tailed value of The average age of 33,436 respondents was (37.76 ± 12.29) years. 9430 (28.2%) were aged 35–44 years, and 7393 (22.1%) were aged 25–34 years. Males accounted for 47.0% and females for 53.0%. In terms of education level, 6.5% of respondents had a master's degree and 28.1% had a bachelor's degree or above. For the remainder, 23.7, 23.5, 15.6, and 2.7% of respondents had junior college, high school, junior school, and primary school diplomas or below respectively. In terms of occupation, respondents working in medical institutions, catering industry, other health-related industries, educational institutions and other work units accounted for 11.2, 4.0, 1.9, 8.5, and 74.4%, respectively. More than half of the responders (51.5%) were resident in the downtown city while the rest (48.5%) were resident in the far city. Additionally, respondents with chronic history accounted for 20.5% .Among the four parts of nutrition and health knowledge, the part of nutrition and disease prevention knowledge had the highest awareness rate of 72.7%, followed by the knowledge of core recommendations of dietary guidelines (59.6%), food and nutrients (49.4%), and nutrition skills (46.3%). The top three items for the awareness rate in single choice questions were: Q12 (84.3%), Q23 (82.6%), and Q27 (80.6%). The bottom three single choice questions were Q29 (21.5%), Q10 (21.6%) and Q11 (28.7%). The top three items for the awareness rate in multiple choice questions were: Q18 (80.9%), Q17 (78.3%), and Q14 (71.0%). The bottom three items for the awareness rate in multiple choice questions were: Q21 (21.0%), Q22 (35.2%), and Q15 (56.8%) . Besides2 = 90.519, p < 0.001). A higher proportion of the female respondents (22.8%) were aware of nutrition and health knowledge compared with the male respondents (17.7%) . The results also showed that there was a significant correlation between the awareness rate of nutrition and health knowledge and education level , occupation , residential address , and a history of chronic disease  . Across the age groups, the awareness rate of responders aged 35–44 years was the highest (23.3%), while responders aged 18–24 years had the lowest awareness rate (17.6%). A significant association was found between age and the nutrition awareness rate (χ< 0.001) .p < 0.001). Females were more aware of nutrition and health knowledge than males . Responders with higher education levels were more likely to have higher awareness levels . Compared with medical workers, catering service workers  and other health-related workers  had lower awareness levels, while educational workers  and other workers  had higher awareness levels. The awareness level of residents in a remote area was lower than that of downtown residents . Besides, compared with those without a history of chronic disease, those with chronic disease and those who did not know whether they had a chronic disease had a lower awareness level , especially in nutrition skills and food and nutrients aspects. Besides, we found individual sociodemographic characteristics, including age, gender, education level, occupation, residential address, and the history of chronic disease, were significantly associated with their awareness level of nutrition and health knowledge, suggesting that divergent demographic characteristics should be considered in carrying out various forms of nutrition education and promotional activities.The awareness rate of nutrition and health knowledge in this study was similar to the results of the survey among adults in 9 provinces in China (21.1%), but much lower than that of adult residents in 3 municipalities  , Suzhou In terms of food and nutrition, the awareness rate of each item ranged from 21.0 to 73.5%. The lower awareness rate of items 21 and 22 highlights the fact that the nutritional value of food is not present in many responders' minds and is not factored into their daily food choices. Therefore, more efforts are needed to increase people's awareness of food nutrition in order to facilitate people to choose food more rationally.Given that awareness of dietary guidelines is a key factor in whether people are likely to adopt the recommended behavior, adherence to dietary guidelines may be particularly valuable in promoting rational dietary intake and reducing the risk of nutrition-related chronic disease , 35, 36,A considerable amount of literature has been published on the relationship between sociodemographic characteristics and nutrition knowledge , 41. SimOur study has several strengths. Firstly, the large sample size and representative study population came from 15 monitoring sites in Wuhan, which provided strong evidence for examining the nutrition awareness level of Wuhan residents. Secondly, we used validated questionnaires to evaluate participants' awareness of nutrition and health knowledge from various aspects. The results of our study can provide a reference for future nutrition questionnaire design. Finally, we have adopted strict quality control before, during and after the investigation to minimize potential deviations.Our study also has some potential limitations. Firstly, since this survey uses a self-administered questionnaire, there might be some cases where responders filled in the questionnaire carelessly, however, three-level quality control measures were taken to reduce the information bias as much as possible. Secondly, although the participants in this study were representative of the Wuhan population in terms of age, gender, and residential area, we could not rule out the potential sampling bias. Thirdly, various definitions of nutrition awareness may contribute to different results, therefore, the results of interpolation and extrapolation need to be treated with caution. Last but not least, different scoring criteria and questionnaire design, such as the representative questions selected in each part may contribute to different results. However, in order to acquire the shortcoming of residents' nutrition and health knowledge in a large scale, the questions designed in each part should not only be closely related to nutrition and health, but also easy to understandable and representative. Additionally, the questionnaire used in this study were determined by expert review, pre-survey, reliability and validity test, which can ensure the design quality of the questionnaire.In conclusion, in this large-scale cross-sectional study, the total awareness rate of nutrition and health knowledge among Wuhan residents was far from reaching the national requirements, especially in terms of nutrition skills. Besides, we found evidence that sociodemographic characteristics were associated with nutrition and health knowledge. Further nutrition education campaign is needed to target certain subgroups of the population or specific nutrition concerns.The original contributions presented in the study are included in the article/YW, SW, MS, LH, and XW designed the research. LH and XW supervised the study conduct. YW, SW, MS, XW, HL, SG, and LT conducted the research. SW analyzed the data and wrote the manuscript. YW and MS coedited, revised, and finally reviewed the manuscript critically for important intellectual content. All the authors read and approved the final version of the manuscript.This work was supported by the DSM-Chinese Nutrition Society Science and Technology Award (CNS-DSM2018A34).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The concern with human health has increased the interest in producing foods enriched with polyunsaturated fatty acids (PUFA), directly or naturally, by inclusion in the animals’ diet. The positive effects such as antithrombotic, anti-inflammatory, and hypolipidemic have been observed in pigs and rats, used as human models for study. The present study evaluated the effect of cow’s milk with different lipid profiles on performance, serum fatty acid profile, biochemical analysis, and a complete blood count of gilts used as a human model. At 34 days, thirty gilts were equally distributed in three treatments. Experimental treatments were milk from cows without the oil supplementation (C), milk from cows fed an enriched diet with linseed oil (n-3), and milk from cows fed an enriched diet with soybean oil (n-6). Milk supplementation was performed until 190 days old, provided once in the morning. The n-3 and n-6 milk reduced the concentration of myristic acid in the blood and increased the leukocytes. Milk enriched with n-3 compared to n-6 reduced the stearic acid. In conclusion, milk with a better PUFA profile can reduce saturated fatty acids in the blood and alter the concentration of cells in the defense system. The consumption of cow’s milk and its derivatives in human nutrition has been reduced or even replaced by other beverages of vegetable origin due to the lipid profile  and lactIt is known that the composition of fatty acids in cow’s milk can be changed and is highly influenced by the nature of the forage consumed, supplementation with vegetable or oilseed oils, and even vitamin E, and this change can be verified in a few hours ,4. ReseaAccording to , the incMilk enriched with PUFA showed a reduction in the concentration of LDL in the blood of rats , and in All activities performed in this study were reviewed and approved by the Animal Care Committee of the School of Animal Science and Food Engineering, University of Sao Paulo (#4939070317).C, n = 10), basal diet + milk from cows without the oil supplementation, omega-3 , basal diet + milk from cows fed an enriched diet with linseed oil, and omega-6 , basal diet + milk from cows fed an enriched diet with soybean oil (Thirty hybrid gilts (Landrace x Large White) were evaluated from 34 to 190 days old. They were randomly allocated into three groups: control . The gilts were fed ad libitum twice a day (at 07:00 am and 1:00 pm) during the grower phase (130 days). After this period, the feeding program was designed to ensure that the gilts did not become overly fat. The feed amount provided to gilts was adjusted based on the nutritional requirements of the females according to [ad libitum water.At 34 days of age and initially weighing 9.59 ± 1.28 kg, the gilts were housed individually in pens , 300 mL (from 77 to 128 days), 400 mL (from 129 to 174 days), and 500 mL (175 to 190 days) of cow’s milk. The enriched milk provided to the gilts in this current study was obtained from the study carried out by  using HoData of gilts were collected at 34, 76, 112, 129, 155, and 190 days.®, model MGR-3000, São Bernardo do Campo, SP), consisting of initial (from 34 to 76), grower (from 77 to 112), and finisher (from 113 to 190) phases. Additionally, average daily feed intake (ADFI), and from these measurements, the average daily gain (ADG) and the feed conversion ratio (FCR) were calculated.Body weight (BW) was assessed using a digital scale . After collection, all blood samples were centrifuged at 2000 x g at ~ 25°C for 10 min and immediately submitted to the laboratory .Whole blood, serum, and plasma were collected by jugular venipuncture using a 1.2 × 40 mm collection needle . Whole blood and serum were collected into 2 mL tubes with a clot activator, and plasma into 2 mL EDTA tubes . The serum FA profile was quantified by gas chromatography-mass spectrometry as follows [®, Minas Gerais, Brazil) using a spectrophotometer. Triacylglycerol and LDL were analyzed by enzymatic kits  using a spectrophotometer. The very-low-density lipoprotein (VLDL) concentration was determined indirectly by the following equation [The complete blood count was evaluated by Sysmex follows . The plaequation : VLDLchoAll statistical analyses were performed using SAS 9.4 . The data were analyzed in a completely randomized design, and the animal was considered an experimental unit. The animals were randomly distributed in 1 of 3 treatments. The normality of the residues was verified by the Shapiro-Wilk test (UNIVARIATE procedure of SAS), and information with student residues greater than +3 or less than -3 was excluded from the analysis. The Levene test compared the homogeneity of the variances. Variables with a continuous distribution such as body weight, average daily gain, average daily feed intake, feed conversion rate, complete blood counts, biochemical parameters, and fatty acids profile of blood were analyzed using the MIXED procedure of SAS. When the time factor was present, repeated measures models were fitted by multiple mixed linear models using the MIXED procedure of SAS. The model included: the fixed effects of treatment, time and their interaction, and the random effect of the animal. The experimental unit was the gilt. The SLICE option using the LSMEANS/PDIFF command was used to explore the interactions between treatment and the day of data collection. The Kenward-Roger method was used to correct the degree of freedom of the denominator for the F test. The covariance structure was determined based on the lowest Akaike information criteria value. The treatments were analyzed using two orthogonal contrasts: contrast 1 (C1) control group vs. n-3 + n-6 groups, and contrast 2 (C2) n-3 group vs. n-6 group. Significance level was set at P < 0.05 and tendency towards significance at 0.05 < P < 0.10.All gilts consumed their supplementation successfully so that the milk remains were not significant.There was no difference in the treatment x time interaction on performance. Females supplemented with n-3 and n-6 had similar growth performance between themselves, and in comparison, to those of the control groups see . Only tiAt 77 days old, the control group had greater myristic acid concentration than those that received enriched cow’s milk , platelets, and lymphocytes compared to those that received enriched cow’s milk , but in female serum, the ratio was similar  after 22 weeks of supplementation. Growth performance results were similar to those reported by Liu and Kim . On the The importance of lipids consumed in the diet on the effects on human health has been widely discussed, as they influence the lipid composition of blood, colostrum, milk, and tissues, and it is even possible to observe the manipulation of gene expression and immunity –29. Myride novo lipogenesis, but it represents a small fraction [Cow’s milk from the study by  was suppfraction . Thus, wde novo lipogenesis due to the higher elongation activity of palmitic acid by fatty acid elongase. Furthermore, PUFA is known to repress the stearoyl-CoA desaturase-1 (SCD-1) gene expression [A higher concentration of stearic acid was observed in milk from cows that received soybean oil and linseed compared to control , and no differences between the PUFA-supplemented groups were reported by . The higpression  that it pression –36. The pression . The linpression . Therefopression .In the present study, control gilts tended to have a reduced RBC, platelets, and lymphocytes and enhanced MCH compared to those that received enriched cow’s milk. These females also had lower leukocytes and higher monocytes than the gilts of the PUFA group.4, C4, D4, and E4, and LTB4 is a potent chemotactic agent for leukocytes [Inflammation is a positive state for the body, as it denotes an immediate response to injury or infection. Thus, several cells are moved towards the affected site, including leukocytes, which can be modulated by fatty acids. This is because leukocytes have an essential role in inflammation and infection through the synthesis of important cytokines, some with more inflammatory functions, arising from PUFA n-6, and others with more anti-inflammatory functions, mainly from PUFA n-3 ,40. In tukocytes .Thus, it is possible that the PUFA-enriched milk, treatments n-3 and n-6 in this study, have stimulated this higher concentration of leukocytes due to the performance they can perform in the body in terms of cytokine production, cell proliferation, adhesion molecules, and cell death.The consumption of PUFA can cause different effects on the cells that make up the blood plasma. According to Xia et al. , dietaryThe increased intake of EPA and DHA replaces the arachidonic acid in the cell membranes of erythrocytes, platelets, neutrophils, monocytes, and liver cells  and accoLymphocytes become active and initiate cytokine secretion stimulating the immune response, although their proliferation may be impaired due to γ-linolenic acid or EPA ,47. CaugIngestion of PUFA leads to their distribution to virtually every cell in the body, resulting in effects on membrane composition and function, cellular signaling, and regulation of gene expression through various transcription factors. Although the results obtained for gilt in growing were not expressive in the current study, more studies are necessary to clarify how the consumption of cow’s milk enriched naturally with PUFA can influence the development of animals e humans.In conclusion, gilt that received the cow’s milk enriched naturally with PUFA n-3 or n-6 had similar growth performance, but the blood fatty acid profile and hematological parameters were altered without impairing the health of gilts. This study provided insight on how food enriched can modulate the serum lipid profile and cells of the defense system, promoting health benefits.S1 Fig(TIFF)Click here for additional data file.S2 Fig(TIFF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


Cardiopulmonary exercise testing (CPET) is used widely in the diagnosis, exercise therapy, and prognosis evaluation of patients with coronary heart disease (CHD). The current guideline for CPET does not provide any specific recommendations for cardiovascular (CV) safety on exercise stimulation mode, including bicycle ergometer, treadmill, and total body workout equipment.The aim of this study was to explore the effects of different exercise stimulation modes on the occurrence of safety events during CPET in patients with CHD.A total of 10,538 CPETs, including 5,674 performed using treadmill exercise and 4,864 performed using bicycle ergometer exercise at Peking University Third Hospital, were analyzed retrospectively. The incidences of CV events and serious adverse events during CPET were compared between the two exercise groups.P < 0.001], premature ventricular contractions (PVCs) , angina pectoris , and ventricular tachycardia (VT)  were significantly higher in the treadmill group compared with the bicycle ergometer group. No significant difference was observed in the incidence of bradyarrhythmia and atrial arrhythmia between the two groups. Logistic regression analysis showed that the occurrence of overall CV events (P < 0.001), PVCs (P = 0.007), angina pectoris (P < 0.001), and VT (P = 0.008) was independently associated with the stimulation method of treadmill exercise. In male subjects, the occurrence of overall CV events, PVCs, angina pectoris, and VT were independently associated with treadmill exercise, while only the overall CV events and angina pectoris were independently associated with treadmill exercise in female subjects.Cardiovascular events in enrolled patients occurred during 355 CPETs (3.4%), including 2 cases of adverse events (0.019%), both in the treadmill group. The incidences of overall events [235 (4.1%) vs. 120 (2.5%), In comparison with treadmill exercise, bicycle ergometer exercise appears to be a safer exercise stimulation mode for CPET in patients with CHD. Cardiopulmonary exercise testing (CPET) is a comprehensive and practical examination of individuals conducted to investigate the interaction among the heart, lung, muscular, and circulatory system and their reserve capacity during incremental-load exercise. CPET is used widely in the diagnosis, exercise therapy, and prognosis evaluation of patients with coronary heart disease (CHD). CPET is also commonly used in other heart-related conditions, such as heart failure, cardiomyopathy, and pulmonary hypertension . In certPatients diagnosed with CHD who underwent CPET at Peking University Third Hospital between July 2006 and September 2019 were enrolled in the present retrospective study. The inclusion criteria were as follows: age > 18 years, diagnosis of CHD, and having undergone CPET. Patients with incomplete information were excluded from this study. The study protocol was approved by the Ethics Committee of Peking University Third Hospital [2019 No. 266–01]. Informed consent was obtained from all included subjects.The collected data included demographic characteristics , history of hypertension, hyperlipidemia, diabetes, smoking, and exercise habit, and the current medication for cardiovascular (CV) disease.2peak), oxygen uptake at the anaerobic threshold (VO2@AT), peak oxygen pulse (VO2/HR), and peak RER (RERpeak). The anaerobic threshold was determined using the V-slope method.The CPET system  was adopted for testing. The treadmill exercise was conducted following the Bruce protocol . The bicAccording to the objective of the present study, investigators reviewed the CPET data of all included patients, which contained the records of all the events that occurred during CPET. A CV event was defined as the event with any of the following conditions : (1) PVCt-test for normally distributed data and the Mann–Whitney U test for non-normal data. Categorical variables were compared using the chi-square test. Logistic regression analysis was performed to explore the correlation between different types of exercises and the occurrence of CV events. P < 0.05 was considered the threshold of statistical significance.SPSS 20.0 was employed for statistical analysis. Quantitative data with normal distribution were presented as mean ± standard deviation (mean ± SD), while the non-normal data were presented as median (interquartile range) [median (IQR)]. The data of the treadmill group and bicycle ergometer group were compared using an independent-samples P < 0.05) compared with the bicycle ergometer exercise group. The mean age and the proportions of subjects with hyperlipidemia were significantly lower in the treadmill group compared with the bicycle ergometer group .A total of 10,538 CPETs conducted with 8,975 patients were included in the present study. The CPETs included 5,674 performed using treadmill exercise and 4,864 performed using bicycle ergometer exercise. The mean age in the treadmill exercise group was 59.5 ± 10.6 years, and there were 4,650 men (82.0%). The mean age in the bicycle ergometer exercise group was 61.1 ± 10.5 years old, and there were 3,808 men (78.3%). As shown in P < 0.001). The VO2peak, VO2@AT, HRpeak, and peak SBP*HR of the treadmill group were significantly higher than the corresponding values in the bicycle ergometer group, while the RERpeak of the treadmill group was significantly lower than that of the bicycle ergometer group .The median RERpeak of all subjects was at 1.11 , suggesting a good level of exertion during CPET. As shown in P < 0.001], PVCs , angina pectoris , and VT  were significantly higher in the treadmill group compared with the bicycle ergometer group. No significant differences were observed in the incidences of bradyarrhythmia and atrial arrhythmia between the two groups.Cardiovascular events occurred during a total of 355 CPETs (3.4%) in the enrolled patients, including 2 male patients with serious adverse events (0.019%). Both patients with serious adverse events were hospitalized after sustained ventricular tachycardia during CPET performed using treadmill exercise. As depicted in 2peak. The results suggested that the occurrence of overall CV events was independently associated with the exercise stimulation mode in the treadmill exercise group . The subsequent logistic regression analysis performed with the occurrence of various types of CV events as the dependent variable and adjusting for the above covariates revealed that the occurrences of PVCs , and VT  were independently associated with treadmill exercise, while bradyarrhythmia and atrial arrhythmia were not associated with the exercise stimulation modes . The mean age and the proportion of subjects with hyperlipidemia were significantly lower in the treadmill group  compared with the bicycle ergometer group.A total of 8,458 CPETs were conducted in male subjects, including 4,650 in the treadmill group and 3,808 in the bicycle ergometer group. As presented in 2peak, VO2@AT, HRpeak, and peak SBP*HR were significantly higher in the treadmill group compared with the bicycle ergometer group, while SBPpeak and RERpeak were significantly lower in the treadmill group .Furthermore, as presented in P < 0.001], PVCs  and angina pectoris  were significantly higher in the treadmill group compared with the bicycle ergometer group. No significant differences were observed in the incidences of VT, bradyarrhythmia, and atrial arrhythmia between the two groups  vs. 99 (2.6%), o groups .2peak, it was revealed that the occurrences of overall CV events, PVCs, angina pectoris, and VT were independently associated with the exercise stimulation mode in the treadmill exercise group . The proportions of subjects consuming β-blockers, statins, ACEI/ARB, calcium antagonists, antiplatelet drugs, and nitrates were also significantly higher in the treadmill group , while the mean age was significantly lower in the treadmill group (P < 0.05). Furthermore, as presented in 2peak, VO2@AT, HRpeak, SBPpeak, and peak SBP*HR were significantly higher in the treadmill group compared with the bicycle ergometer group, while RERpeak was significantly lower in the treadmill group .A total of 2,080 CPETs were conducted with female subjects, including 1,024 in the treadmill group and 1,056 in the bicycle ergometer group. As presented in P < 0.001] were significantly higher in the treadmill group compared with the bicycle ergometer group. No significant difference was observed in the incidences of PVCs, angina pectoris, VT, bradyarrhythmia, and atrial arrhythmia between the two groups  vs. 21 (2.0%), o groups .2peak revealed that the occurrences of overall CV events and angina pectoris were independently associated with the exercise stimulation mode in the treadmill exercise group  for the publication of any potentially identifiable images or data included in this article.CR, JZ, and TS participated in the study design, analyzed the data, and drafted the manuscript. YS collected the data for this study. LT assisted in the statistical analysis. SX assisted in drafting the manuscript. WZ and WG contributed to the conception of the study and also participated in its design and coordination. All authors read and approved the final version of the manuscript and also agreed with the order of presentation of the authors.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Plant biostimulants (PBs) are a potential strategy to improve crop growth and grain quality. In the present study, 100 mg/L trehalose, chitosan, humic acid and gamma-aminobutyric acid treatments were applied to analyze the effects of maize production and reproductive characteristics. The contents of nitrogen, phosphorus and potassium and grain quality were significantly affected by the PBs, but not yield. The seed germination rate of all PB treatments was significantly reduced, but the drought resistance of progeny seedlings was significantly improved, with humic acid having the strongest effect. Liquid chromatography mass spectrometry analysis indicated that the disruption of the tricarboxylic acid cycle, probably due to the blockage of intermediate anabolism, reduced the supply of energy and nutrients in the early stages of germination, thus inhibiting seed germination, while the increased resistance of the offspring seedlings may be due to the up-regulation of the synthesis of unsaturated fatty acids and alkaloids by humic acid treatment. This study revealed the similarity and heterogeneity of the effects of different PBs on nutrient accumulation, yield characteristics and grain quality of maize, providing guidance for the application of PBs in intensive and sustainable agricultural production. In 2018Anthurium flowers treated with gamma-aminobutyric acid exhibited improved resistance to chilling injury by modulating antioxidant enzyme activity , yield traits and grain quality at maturity were analyzed. Next, we conducted a germination test on the collected cornkernels and treated the germinated seedlings with 15%PEG-6000 to explore the resistance of progeny seedlings todrought stress. These results were used to evaluate the potential value of PB application to the grain produced. In addition, we used metabolomics to analyze the metabolite changes in corn grain treated with humic acid to reveal its regulatory mechanisms. This study reveals the effects of different PBs on maize growth and reproduction, and provides guidance for the application of PBs in sustainable agricultural development.22.12. The average monthly temperature and rainfall during maize growth are shown in Field experiment: The experimental site is located in Pingyuan New District, Xinxiang City, Henan Province, China. The corn variety used in this study, ‘Jundan 20,’ is cultivated by the Academy of Agricultural Sciences of Junxian County, Henan Province and was sown on June 8, 2021. Untreated water was used as the control, and 100 mg/L trehalose , chitosan , humic acid  and gamma-aminobutyric acid  mixed with 0.1% Tween 20 were sprayed during the three-leaf stage once every 5 days for a total of three times. The supplementary material provides more information on PBs, which are thought to have minimal effects as carbon sources or nutrients due to their low concentration . AdditioGermination test: Three hundred seeds treated with different PBs were randomly selected, disinfected with 10% hydrogen peroxide for 10 minutes, washed three times with deionized water, soaked in distilled water for 12 hours and allowed to germinate under protection from light at 25°C.Indoor seedling growth test: Germinated seeds from different PB treatments were transferred to nursery pots and placed in a light incubator . The growth medium was 2:1 vermiculite and nutrient soil, and all treatments were supplemented with water until the three-leaf stage of maize. Half of the samples were under control conditions with a standard water supply, and the other half were treated with 15% PEG-6000 to simulate drought stress . Leaves 2.22.2.1Four representative plants from each growth period  that were treated with PBs and water were collected, washed several times with deionized water, dried, ground and used for analysis . Nitroge2.2.2Seeds with radicals longer than 1 mm were considered to have germinated . Over th2.2.3Malondialdehyde, proline, soluble sugar and total antioxidant capacity (T-AOC) in leaves were measured according to the method of Wang et al. . The con2.2.4V:V:V), containing an isotope-labeled internal standard mixture). This sample was flash frozen in liquid nitrogen and quickly mixed for 30 s after thawing, and this process was repeated two to three times. The samples were sonicated in an ice-water bath for 10 min, allowed to stand at -40°C for 1 h, frozen and centrifuged, after which the supernatants were collected.Non-targeted metabolomic analysis of corn kernel samples from the humic acid and control treatments was performed using liquid chromatography-mass spectrometry (LC-MS). Mixtures of four biological replicates were prepared per treatment as quality control samples (QC). To 100 mg of ground corn powder was added 400 μL of extraction solution , the data were preprocessed using R (version 3.3.2), and substance annotation was performed using a secondary mass spectrometry database. The cutoff value for algorithm scoring was set to 0.3. KEGG and HMDB databases were used for functional annotation and functional enrichment analysis of metabolites.2.3P < 0.05).One-way ANOVA was performed using IBM SPSS Statistics 23 software , and the error was shown as the mean ± standard error. Physiological and biochemical analyses were performed with at least three replicates, while metabolomic analyses were performed with four biological replicates. For metabolites, principal component analysis, partial least squares discriminant analysis, Pearson correlation analysis and cluster heat map analysis were conducted with the R statistical computing environment. The criteria for differential metabolites included variable importance of projection (VIP) of partial least squares discriminant analysis (VIP > 1), fold difference between humic acid treatment and control treatment (FC > 2 or FC < 0.5) and statistical significance   were subjected to a 7-day germination test. On days 1 and 2, there were no significant differences among all treatments. By the third day, the seed germination rate was significantly inhibited by PBs compared with the control treatment, while there was no difference among different PB treatments. By day 5, the seed germination rates of the control and PB-treated groups were about 96% and 73%, respectively. From the 6th day onward, the remaining ungerminated seeds did not continue to germinate. In addition, owing to the long-term immersion of seeds, mildew began to appear, so the data were not counted thereafter.3.5The application of PBs mainly changed the stress resistance of progeny seedlings under drought stress, rather than affecting the growth of seedlings under a normal water supply. Compared with the control under drought, all PB-treated progeny seedlings showed significantly reduced contents of malondialdehyde (MDA) . Of these metabolites, 3491 were involved in metabolic activities, accounting for 66.2% of the total number of metabolites, and they can be categorized as follows: 1432 global and overview maps, 450 amino acid metabolism and metabolism of other amino acids, 371 chemical structure transformation maps, 308 biosynthesis of other secondary metabolism metabolites, 189 carbohydrate metabolism, 174 lipid metabolism and relatively fewer nucleotide and energy metabolism -reticuline, protopine, quinidine, tubocurarine, tetrahydroharmine were significantly increased by more than 2.1 times), zeatin biosynthesis (map00908) , glycine, serine and threonine metabolism (map00260) (d-glyceric acid was increased 1.73 times) and drug metabolism - cytochrome P450 (map00982)   , biosynthesis of alkaloids derived from the shikimate pathway (map01063) , vitamin B6 metabolism (map00750), phenylpropanoid biosynthesis (map00940), histidine metabolism (hercynine and ergothioneine were decreased by 58.4–68.4%) and ABC transporters (map02010)  , alanine, aspartate and glutamate (map00250) , 2-oxocarboxylic acid metabolism (map01210) (3.8S)-reticuline and morphine in downstream metabolites. The differential regulation of these metabolites may explain the stability of dopamine content. Compared with the control, the content of oxoglutaric acid was significantly decreased in the humic acid treatment, and its synthesis was bidirectionally regulated by phosphoenolpyruvate l-glutamine and 4-imidazolone-5-propanoate upstream and l-glutamate downstream. Pyruvate was not detected in the metabolome, possibly owing to its high transformation efficiency or low expression abundance. Its regulation resulted in a significant decrease in the abundance of arbutin, citraconic acid and 2-isopropyl-3-oxosuccinate downstream. In addition, we also noticed that arachidonate is the intermediate hub of arachidonic acid metabolism and biosynthesis of unsaturated fatty acids pathways, and its changes are regulated by dihomo-gamma-linolenate in upstream metabolites, 5,6-DHET in downstream metabolites and feedback effects of prostaglandin G2 and leukotriene B4.Based on the abundance changes of metabolites, we mapped their regulatory networks in different metabolic pathways Figure 4Collectively, our results indicate strong interactions among differential metabolites in different metabolic pathways.3.9P-values of the differential metabolites (L-histidine trimethylbetaine (meta_pos_745), trans-aconitate (meta_pos_386), thiamine aldehyde (meta_pos_1125) and arbutin (meta_neg_558), among others. We performed a Pearson correlation analysis  Table 1.4.2Corn is one of the most economically important crops globally, with more than 35,000 uses . Maize qCrude protein and crude lipid were significantly increased by trehalose and chitosan treatment compared to the control treatment. All PB treatments significantly reduced the total starch content of corn kernels Figure 54.3Plant breeding and molecular techniques have driven the improvement of maize varieties and accelerated the commercialization of superior germplasm resources, resulting in greater benefits for producers and consumers . After dMalondialdehyde (MDA) is a characteristic substance of membrane lipid peroxidative damage . Soluble4.4Metabolites are key participants in plant stress responses, and their content changes are considered to be the ultimate manifestation of plant responses to environmental stress . Regulatl-histidine trimethylbetaine and arbutin, indicating convergent changes in amino acid metabolism, glycolysis/glycogenesis and 2-oxocarboxylic acid metabolism linked by the TCA cycle. In addition, the vitamin B6 metabolic pathway was significantly downregulated  is a central pathway linking almost all individual metabolic activities and is generally considered to be responsible for the oxidation of respiratory substrates and the synthesis of ATP in energy metabolism . The enregulated Figure 3ndosperm . This acTaken together, our findings suggest that humic acid causes starvation of key metabolites in the TCA cycle. Therefore, reducing the intensity of nutrient cycling and energy conversion processes, inhibiting early seed germination but not affecting the growth of seedlings after germination.4.5Germination is the beginning of the life of a new individual plant and probably its most vulnerable stage, during which any small changes in the environment can be fatal to the development of the embryo . A large2-involved buckwheat resistance to AI stress  were further enhanced by humic acid treatment, which may expand the application prospects of maize seeds. In conclusion, this study revealed differential regulatory activities of different types of PBs on crop growth and yield characteristics, and most importantly, they altered seed fertility. This has important implications for the application of PBs in sustainable agricultural production.The original contributions presented in the study are included in the article/DS, TG, XX and CA provided help on sample processing, and WZ supported this research. BL completed the experiment, analyzed the data and wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors gratefully acknowledge funding from the Science and Technology Innovation Project of Chinese Academy of Agricultural Sciences.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.