Spermidine is a naturally occurring polyamine compound that has many biological functions, such as inducing autophagy and anti-inflammatory and anti-aging effects. Spermidine can affect follicular development and thus protect ovarian function. In this study, ICR mice were fed exogenous spermidine drinking water for three months to explore the regulation of ovarian function by spermidine. The results showed that the number of atretic follicles in the ovaries of spermidine-treated mice was significantly lower than that in the control group. Antioxidant enzyme activities  significantly increased, and MDA levels significantly decreased. The expression of autophagy protein (Beclin 1 and microtubule-associated protein 1 light chain 3 LC3 II/I) significantly increased, and the expression of the polyubiquitin-binding protein p62/SQSTM 1 significantly decreased. Moreover, we found 424 differentially expressed proteins (DEPs) were upregulated, and 257 were downregulated using proteomic sequencing. Gene Ontology and KEGG analyses showed that these DEPs were mainly involved in lipid metabolism, oxidative metabolism and hormone production pathways. In conclusion, spermidine protects ovarian function by reducing the number of atresia follicles and regulating the level of autophagy protein, antioxidant enzyme activity, and polyamine metabolism in mice. Mammalian ovaries consist of follicles as basic functional units. The reproductive life span of the organism mainly depends on follicular development that maintains the primordial follicle pool in the cohort of follicles within the ovary . The tot2O2 [HSP12, GAD1, GPX2, etc.) [In addition, studies in recent years have shown that spermidine can reduce ROS levels and delay cell aging in humans and other models, such as yeast, Drosophila and nematode models . Adding 2O2 ,19. Usin2, etc.) , suggest2, etc.) ,22. In a2, etc.) . Spermid2, etc.) ,25. To dUnder normal physiological conditions, autophagy maintains the physiological activities of cells by specifically degrading damaged or redundant organelles, so autophagy is an important cytoprotective mechanism. However, excessive autophagy is detrimental. Studies have shown that nonapoptotic forms of programmed cell death (PCD), such as autophagy, may also be involved in the process of follicular atresia ,27,28. MIn recent years, proteomics technology has provided technical support for ovarian-related research. For example, comparing the differences in serum protein expression levels between patients with polycystic ovary syndrome (PCOS) and non-PCOS patients is helpful for finding potential serum markers . Additio−1 spermidine for 3 months, it was found that the weight of the mice increased steadily but did not differ significantly between the two groups , 3 β-hydroxysteroid dehydrogenase (3β-HSD), and 17 β-hydroxysteroid dehydrogenase (17β-HSD) and other enzymes are expressed in growing follicles, and the corpus luteum, and their main function is to convert estrogen into E2, thereby affecting follicular development. The results showed that the protein expression of CYP17A1 and HSD3B2 in mouse ovaries treated with spermidine was significantly increased C,DAPAO) gene was significantly upregulated in the treatment group. The relative expression of the spermidine synthase (SPDS), spermine synthase (SPMS) and spermine oxidase (SMO) genes was significantly lower than in the control group. The relative expression of the ornithine decarboxylase (ODC) and N’-spermidine/spermine acetyltransferase (SSAT) genes was not significantly different , nuclear receptor subfamily 5 group A member 1 (Nr5a1), BCL2-related ovarian killer (Bok), and BH3 domain apoptosis-inducing protein (Bid). There were 257 downregulated proteins, including xanthine dehydrogenase (XDH) and thioredoxin-interacting protein (TXNIP)  . These pMost of the DEPs have transporter, transducer, binding and catalytic activities and participate in cellular biological processes A. We claIn terms of biological processes, DEPs are involved in steroid biosynthesis, cholesterol metabolism, lipid homeostasis and transport, and cellular hormone metabolism A. In terIn order to study the physiological functions of proteins, we enriched protein domains. Protein domains were mainly enriched in the MCM domain, MCM OB domain, MCM N-terminal domain; EGF-like domain, extracellular; START domain; alkaline phosphatase-like, core domain; AMP-dependent synthase/ligase, etc. . A total of six DEPs were screened, namely, XDH, TXNIP, p53, Nr5a1, Bok and Bid. XDH is an enzyme that catalyzes purine metabolism, which may be related to oxidative stress. TXNIP is also related to oxidative stress, and there seems to be some connection to excessive autophagy. p53 is a cell cycle factor that participates in the cell block. Nr5a1 is a transcription factor for steroidogenesis. Bid and Bok are both proapoptotic proteins and are involved in inducing apoptosis .−1 spermidine for 3 months, the expression of the ODC gene in the ovary of mice was statistically insignificant, and the level of putrescine in the ovary did not change, indicating that 3 mmol·L−1 spermidine did not affect the synthesis of putrescine in the ovary of mice. Furthermore, the expression of the SPDS, SPMS and SMO genes in the ovaries of mice in the spermidine group was significantly decreased. APAO had the opposite effect . It is speculated that a portion of the ingested spermidine is converted into spermine for storage, and part of the ingested spermidine forms N 1-acetyl-spermidine under the action of SSAT to maintain spermidine in the ovary. In addition, the decreased expression of SMO is also one of the reasons for the increased spermine level in the ovary. Natural polyamines themselves can bind to negatively charged biological macromolecules such as nucleic acids, proteins, and biofilms, thereby exerting the functions of antioxidative stress and scavenging ROS. However, excess polyamines are catabolized by various amine oxidase enzymes, which produce many reactive aldehydes (such as acrolein) and H2O2, which are toxic and detrimental to proteins, DNA in mammalian cells, and other cellular components that are susceptible to oxidative damage [2O2, oxidation by spermine oxidase is more likely to cause damage because spermine oxidase is present in the nucleus and cytoplasm, while acetylpolyamine oxidase is a peroxidant. The bioenzyme itself has a protective effect on cells [SMO in the ovary decreased significantly after spermidine feeding. Our experimental results are consistent with previous results [Putrescine, spermidine and spermine are interconverted through the polyamine metabolic system to maintain the stability of the body’s polyamine pool, and this process requires the participation of a variety of polyamine metabolizing enzymes . In our e damage ,36. Theron cells . In this results . We spec2O2 and indirectly inhibits lipid peroxidation and membrane damage. The activity of CAT can indirectly reflect antioxidant capacity [−1 spermidine significantly increased the T-AOC/SOD and CAT enzyme activities of mouse ovarian tissue without affecting the daily weight gain and ovarian organ index of mice. It significantly reduced the MDA content and decreased the level of ovarian lipid peroxidation. It also significantly reduced the number of atretic follicles in mice. Moreover, the protein expression of CYP17A1 and HSD3B2 in ovarian tissue was increased, thereby promoting follicle development. In conclusion, adding spermidine to drinking water can significantly reduce the atresia rate of follicles in mice and significantly improve the antioxidant capacity of mouse ovaries by increasing the activities of various antioxidant enzymes and reducing lipid peroxidation.Studies have shown that many pathological phenomena in the body are closely related to lipid peroxidation caused by free radicals ,39. In rcapacity . GSH-Px capacity . MDA is capacity . As a secapacity ,45. Oxidcapacity ,47. Jantcapacity  indicatecapacity . During capacity ,51 that 2a) cells. This contributes to mitochondrial health and function, which in turn has anti-aging effects [−1 spermidine feeding can induce autophagy. At the same time, the immunohistochemistry results showed that the positive products of the autophagy marker proteins LC3 and p62 were mainly distributed in the cytoplasm and concentrated in the granulosa cell layer of antral follicles. Phagocytosis marker proteins were specifically distributed in the ovary, mainly in granulosa cells. Properly increasing the level of autophagy can help to improve oxidative stress and alleviate neural deafness due to aging or drugs [It was found that autophagy levels were significantly increased in various tissues  from 4 h to 24 h by acute intraperitoneal injection of spermidine in mice supplemented with spermidine in drinking water. Changes in mitochondrial structure and function are an initial factor in cellular senescence ,53, and  effects ,54. Rece effects . In the or drugs . The aboThe specific mechanism by which spermidine induces autophagy in mouse ovaries to protect against oxidative stress is unclear. Therefore, our research group used proteomic sequencing to screen differentially expressed proteins and provide theoretical support for subsequent experiments. In this study, proteomic methods revealed 681 DEPs in the ovarian tissue of spermidine drinking water-fed mice, including 424 upregulated proteins and 257 downregulated proteins, including p53, Nr5a1, Bok, Bid, XDH and TXNIP, etc. These proteins are mainly involved in functions such as “cell cycle, steroid synthesis, apoptosis, autophagy and oxidative stress”. The expression of several ribosomal proteins and histones was increased, and the upregulated proteins are known to be involved in apoptosis and other processes, indicating that spermidine can promote protein synthesis and apoptosis in ovarian tissue. At the same time, the expression levels of several ribosomal proteins and molecules related to oxidative metabolism were downregulated, and the downregulated proteins are known to be involved in energy metabolism, signal transduction, RNA processing and modification, transcription, lipid transport and metabolism, and other processes. The treatment has an inhibitory effect on some functions of ovarian tissue. GO annotation and functional enrichment analysis showed that the functions of most of the DEPs were related to cell binding, enzyme catalytic ability and molecular function regulation, indicating that spermidine acts on ovarian granulosa cells and through the regulation of related molecular functions, it can change the interactions among ovarian granulosa cells. In our research, protein domains were mainly enriched in the MCM domain, MCM OB domain, MCM N-terminal domain; EGF-like domain, extracellular; START domain; alkaline phosphatase-like, core domain; AMP-dependent synthase/ligase, etc. The MCM protein family is an important part of the DNA prereplication complex and plays an important role in the DNA replication initiation process and DNA damage repair. EGF may regulate reproductive function by controlling the secretion of transferrin; aging can affect the transcription of EGF and EGFR. START and AMP-dependent enzymes are closely related to ovarian steroidogenesis. The KEGG pathway enrichment results showed that the DEPs were involved in multiple cell signaling pathways, including the p-ERK, JNK, p53, TNF, TXNIP and DR signaling pathways. Some research has shown that the p53 [TXNIP and insulin-like growth factors (IGF-1) in ovarian tissue decreased after TXNIP was knocked down, and the level of FSH increased. This result indicated that inhibiting the expression of TXNIP could reduce the expression of ovarian function-related hormones. The results of Illumina sequencing and qRT-PCR showed that the CYP19A1 and TXNIP genes might take part in promoting the development of bovine follicles and ultimately cause follicle ovulation [TXNIP is highly expressed in bovine cumulus cells [An appropriate level of autophagy in the body is a prerequisite for the body to maintain normal physiological functions. Insufficient or excessive autophagy can lead to cell death. TXNIP is a widely expressed protein that is induced by various cellular stresses, including oxidative stress and apoptosis . TXNIP ivulation . TXNIP ius cells , porcineus cells  and porcus cells . This geus cells . These rus cells . These dus cells , which ius cells . Spermidus cells . This reOur current findings suggest that spermidine regulates the activity of antioxidant enzymes and the expression level of autophagy proteins, and autophagy activated by spermidine plays an antioxidant role in preventing follicular atresia. Using proteomics, we found that it is mainly involved in steroid hormone production, oxidative stress, lipid metabolism, autophagy and apoptosis, and the main proteins are Nr5a1, XDH, P53, LDLR, TXNIP, Bok and Bid. In summary, spermidine protects ovarian function by reducing the number of atresia follicles and regulating the level of autophagy protein, antioxidant enzyme activity, and polyamine metabolism in mice.−1 spermidine solution instead of drinking water), with 20 mice in each group. During the feeding process, the water intake, cleaning of the drinking water bottle, and replacement of the drinking water were recorded every three days, and the animals were weighed and recorded every 15 days. After 3 months, the reproductive cycle was identified by the vaginal smear method, and mice in oestrus were selected. The mice were killed by cervical dislocation, and the bilateral ovaries were quickly collected, washed with 0.9% normal saline, dried with filter paper, and placed on an electronic balance. Some of the ovarian tissue samples were fixed with 4% paraformaldehyde for HE staining, some were sequenced by proteomics, and the rest were stored in a −80 °C freezer for later use. Seven-week-old healthy female ICR mice  were selected and kept in separate cages, and the mice were fed ad libitum. The mice were randomly divided into a control group  and a spermidine group .Putrescine standard, spermidine standard, spermine standard and 1,6-hexanediamine were purchased from Sigma ; Protein Maker, ECL chromogenic solution and PVDF membrane were purchased from BIO-RAD Biological Company; PBS, PMSF, RIPA, primary antibody diluent and secondary antibody diluent were purchased from Beyotime Co., Ltd. ; chromatographic methanol was purchased from Bio-Rad Yu ; SYBR Green Supermix was purchased from TaKaRa ; PMSG was purchased from Ningbo Sunshine Biological ; perchloric acid, sodium hydroxide, concentrated hydrochloric acid, benzoyl chloride, anhydrous ethanol and all other reagents were analytically pure and purchased from Ruijinte .The proteins obtained by liquid chromatography-mass spectrometry were analyzed by means of bioinformatics. The tools and websites used for the analysis are shown in The ovarian tissue was fixed in 4% paraformaldehyde for 24 h, after which the tissue was removed, dehydrated in a series of alcohols with different concentrations, embedded in paraffin, and sectioned. Next, hematoxylin-eosin staining was performed. The slides were sealed with neutral gum and dried in an oven at 37 °C. The changes in the tissue morphology of the ovary, the size of the follicles, and the granulosa layer of the follicles were observed under an optical microscope, and the follicles of different grades were counted.First, the tissue was embedded and sectioned, the sections were dewaxed and then stained, and the sections were infiltrated with 3% methanol and hydrogen peroxide for 9 min, washed three times with PBS, and immersed in 0.01 mol/L citrate buffer (PH 6.0). After heating for 5 min, the slices were allowed to cool completely, and then the slices were washed 2–3 times with PBS. At room temperature, goat serum blocking solution was used to block the slices for 20 min. The primary antibody was diluted according to the antibody instructions, added dropwise to the slice, and incubated at 4 °C overnight. The secondary antibody was incubated at 37 °C for 30 min. The slices were washed three times with PBS for 4 min each time. After DAB color development at room temperature, the slices were placed under a microscope, and the reaction was observed and rinsed with distilled water after approximately 2–3 min. Counterstain was performed, followed by dehydration and xylene clearing. Finally, the sections were mounted with neutral gum. The sections were observed microscopically, and images were acquired for subsequent analysis.An appropriate amount of ovarian tissue sample was added to prepare the solution. Then, a hand-held electric homogenizer was used to homogenize the samples on ice, and the supernatant was collected as the sample to be tested. The protein concentration was determined with a BCA protein concentration kit for quality control for subsequent detection. The working solution was prepared according to the instructions of the kit, the sample was diluted, the 96-well plate was gently shaken to fully mix the solution, the plate was incubated in an incubator at 37 °C, and the absorbance of the sample was detected at 450 nm. The enzyme activity was calculated according to the instructions of the kit {CAT , SOD , GSH-Px , total antioxidant capacity  and MDA }.v/v), the detection wavelength of the UV detector was 229 nm, and the column temperature was 25 °C. The peak time and peak area of three polyamines and internal standard in each sample were obtained by liquid chromatography, and the concentration of each polyamine was calculated according to the standard curve.Approximately 0.1 g of mice ovary tissue was added to an internal standard and 1 mL 5% HClO4 grinding sample and placed on ice for subsequent detection. Vortex vibration and ultrasonic crushing were performed for 10 min, followed by centrifugation and supernatant extraction, which was repeated 1–2 times. Aliquots of 2 mL 2.5 mol/L NaOH and 7 μL benzoyl chloride were added to the above homogenate solution, vortexed and mixed, then derivation was performed in a water bath without light, with the derivatization solution adjusted to pH 7.0 with 6 mol/L HCl. The samples were activated with chromatographic methanol and ultrapure water in advance, and the polyamines were completely filtered and separated, washed with 15 mL double distilled water and 15 mL 15% chromatographic methanol, and dried. Then, 0.5 mL chromatographic methanol was used to elute the components to be tested. A 0.22 μm filter was used to filter the prepared sample solution, and the filtered sample solution was detected by HPLC. The detection conditions were as follows: the mobile phase was methanol: water , according to the instructions of the reverse transcription kit , and the total RNA sample was reverse transcribed into a cDNA template. Primer information is as follows  and protein lysis buffer (PIPA) were added and centrifuged at 4 °C at 12,000 r/min for 10 min, and the protein of the sample was detected according to the BCA protein concentration detection kit  after concentration and denaturation. After preliminary separation by SDS-PAGE, the proteins were transferred to PVDF membranes for sealing, followed by 4 °C, overnight incubation of primary antibody {1:1000, rabbit polyclonal antibody CYP17A1 , HSD3B2 , p62/SQSTM1 , LC3B , rabbit monoclonal antibody Beclin 1 , mouse monoclonal antibody GAPDH  and rabbit monoclonal antibody β- Tubulin }. The membrane was incubated with secondary antibody {1:1000, goat anti-rabbit secondary antibody , goat anti-mouse secondary antibody } for 60 min at 37 °C. An ECL reagent kit was used for color development, exposed with an AI 600 imager, and analyzed with image analysis software .−1 spermidine drinking water for three months, ovarian tissue was collected. The samples were ground in liquid nitrogen, lysis buffer (inhibitor) was added, each group of samples was lysed by ultrasonication, and the supernatant was collected after centrifugation. SDS-PAGE was used for quality control. The Pearson correlation coefficient (Pearson) was used to measure the degree of linear correlation between the two groups of data; the relative standard deviation (RSD) was used to calculate the ratio of the standard deviation to the arithmetic mean of the measurement results, which reflected the degree of dispersion of the data; the principal component analysis (PCA) method extracts the key components in the sample data to effectively distinguish the samples, intuitively reflects the relationship between the samples, and assesses the consistency and repeatability of samples.After the mice were fed with 3 mmol·Lv/v) formic acid in water), the Nano Elute ultrahigh-performance liquid system was used for separation. The peptides were separated by an ultrahigh-performance liquid phase system, injected into the capillary ion source for ionization, and then analyzed by a tims-TOF Pro mass spectrometer.After dissolution with liquid chromatography mobile phase A . The nonstandard quantitative calculation method was used to calculate the nonlabeled quantitative intensity of protein in each sample, and the relative quantitative value of each sample was obtained. The average value was obtained, and the final differential expression amount was calculated. Differential proteins were screened based on the p-value was calculated by t-test. When p ≤ 0.05, a fold change (FC) change >1.2 was used as the threshold for significant upregulation. FC < 0.67 was taken as the threshold for significant downregulation. Fisher’s exact paired-end test was used to detect the enrichment of DEPs for all identified proteins, and p ≤ 0.05 was considered statistically significant. The DEPs were considered significantly enriched at this time.The relative quantitative value of each sample was taken as log 2 , and the p < 0.05 indicates a significant difference, and p < 0.01 indicates an extremely significant difference. p > 0.05 indicates an insignificant difference. GraphPad Prism 8.0.1 was used to create graphics.The data are expressed as the mean ± S.E.M. All statistical analysis was performed with SAS 9.4 statistical software. One-way analysis of variance (ANOVA) was applied to analyze differences in data for the biochemical parameters among the different groups, followed by Dunnett’s significant post hoc test for pair-wise multiple comparisons.


Urban green areas are critically important for maintaining biodiversity in urban ecosystems. The pill bug is an ecological bio-indicator of soil health that is widely distributed around the world. In this study, we studied the relationship between the characteristics of the pill bugs population and soil properties of urban green spaces. The characteristics of the population vary significantly among selected habitats. The pH and soil organic matter may be the main factors for explaining the variation in body size of pill bugs among the habitats in spring. Significant positive regressions were detected between body length and body weight.Armadillidium vulgare population characteristics in Yancheng, China. Results indicate that soil water content, pH, soil organic matter, and soil total carbon varied significantly among habitats, as well as the body length and body weight of pill bugs. The higher proportion of larger pill bugs was found in the wasteland and the lower proportion in the grassland and the bamboo grove. The body length of pill bugs was positively related to pH. Soil total carbon, soil organic matter, and the number of plant species were correlated with the body weight of pill bugs.Rapid urban development poses a threat to global biodiversity. At the same time, urban green spaces offer opportunities for holding biodiversity in cities. Among biological communities, the soil fauna plays a crucial role in ecological processes but is often ignored. Understanding the effects of environmental factors on soil fauna is critical for ecological conservation in urban areas. In this study, five typical green space habitats were selected including bamboo grove, forest, garden, grassland, and wasteland in spring, for detecting the relationship between habitats and  Urban environmental degradation threatens the sustainable development of the human society worldwide, and the degradation occurs not only in developed regions with a high level of urbanization, but also in developing regions with an accelerated urbanization process ,2,3. NowSoil fauna is an important ecological indicator for assessing urban soil quality and is involved in many soil functions ,7,8. MacArmadillidium vulgare (Isopoda: Armadillidae), also known as the pill bug, is distributed worldwide, with particularly dense populations in temperate climates, and is one of the most important soil macrofauna in urban green spaces [n spaces ,17. The n spaces ,19,20. Pn spaces ,18,21,22n spaces ,17,22,23Different plant coverage may affect soil environments, such as soil moisture, soil nutrient status, pH, and so on ,25. TherWe conducted this study on Xinchang campus of Yancheng Teachers University , Jiangsu Province, China . The cliPhyllostachys nigra) with lush growth and partial leaf cover on the ground surface. The forest was mainly dominated by cherry (Prunus subg. Cerasus sp.), privet (Ligustrum lucidum), and golden raintree (Koelreuteria paniculata). Chinese violet cress (Orychophragmus violaceus) was the main vegetation in the garden. The grassland was mainly covered by mascarene grass (Zoysia tenuifolia), bermudagrass (Cynodon dactylon), and cogongrass (Imperata cylindrica). The wasteland has been unmanaged for about 8 years and has more plant species, including Chinese tallow tree (Sapium sebiferum), paper mulberry (Broussonetia papyrifera), sweet woodruff , etc. Five replicates of separate patches were used as sampling sites in each habitat, with a minimum distance of 2 m among the patches in the same habitat. All sampling sites were distributed in an area with a radius of 250 m. The compact distribution of the sampling sites was designed to reduce spatial variability. Details of the habitats are shown in On the campus, we selected five different habitats of green spaces with different land covers, including bamboo grove, forest, garden, grassland, and wasteland. The bamboo grove was dominated by pale bamboo  were taken by using a soil loop cutter. The soil cores were mixed and immediately packed in polyethylene plastic bags. The vegetation coverage and the number of plant species at each sampling site were recorded. The height of the highest plant was measured as the habitat description in each habitat. A total of 25 soil samples were collected from five habitats.Representative green spaces were selected and randomly sampled, and 5 replicate sample plots (50 cm × 50 cm) were set in each habitat. In each plot, debris such as surface plant and animal residues were removed, and three soil cores (100 cmPart of the fresh soil samples was used for the determination of soil water content (WC) after oven-drying the samples at 105 °C for 24 h. Part of the fresh soil samples was sieved through an 8-mm sieve and air-dried at room temperature (about 20 °C) for three weeks. The air-dried soils were then sieved through a 2-mm sieve to remove coarse debris and stones. Afterward, the soil samples were used for the determination of electrical conductivity (EC), pH, available phosphorous (AP), and available nitrogen (AN). Then each of the prepared soil samples was ground and passed a 0.149-mm nylon sieve for determination of soil organic matter (SOM), total carbon (TC), and total nitrogen (TN) using the method introduced in the literature ,29,30,31l) on 1 mm × 1 mm grid paper, and the wet weight (m) by an electronic balance.Pill bugs were captured by searching under fallen leaves, rocks, and topsoil in the selected habitats. They were then placed in bottles in the field and labeled, and were killed and kept by freezing at −20 ℃ in a refrigerator. Then, every individual was thawed and flattened for measurements of body length  was conducted to analyze the soil properties and population characteristics of pill bugs in various habitats. Multiple comparisons were performed using Tukey’s test or Dunnett’s T3 test depending on whether they passed Levene’s test for homogeneity . Principp < 0.05. A map of sampling sites was created using ArcGIS 10.5 and statistical analyses were performed using Microsoft Office Excel 2021, SPSS18.0, R version 4.2.1, and PAST 4.03 software [In this study, no variables were transformed. Significant statistical differences were used as software ,39,40.4,20 = 5.908, p < 0.01). The forest had the highest pH while the bamboo grove and the garden had the lowest . There were similar results for SOM , and TC , which had their highest values in the bamboo grove and the lowest values in the wasteland. There were significant differences in the number of plant species among habitats, with higher values in the forest and wasteland . The coverage in the bamboo grove, garden, and grassland was higher, while the coverage in the forest was the lowest . There were no significant differences in soil TN , AN , EC  and AP .Soil properties varied considerably among habitats . The WC 2 = 62.736, df = 15, p < 0.001). Results of PCA showed that soil samples could be classified by habitat characteristics, and pc1 and pc2 could respond to 53.97% and 21.73% of habitat differences  demonstrated the adequacy of sampling in this study. The sphericity of Bartlett’s test for PCA showed that these approximately normal data for multiple variables were acceptable  and body weight  of pill bugs among habitats. The mean body length and weight of all individuals in the five habitats were (8.21 ± 2.07) mm and (48.06 ± 32.68) mg, respectively. The mean body length and weight in the bamboo grove and grassland were smaller (7.52 ± 1.89) mm (36.34 ± 24.15) mg, (7.58 ± 1.86) mm (37.96 ± 25.33) mg, while the average body length and body weight of wasteland were larger (10.30 ± 1.44) mm (87.27 ± 31.17) mg. The body length and body weight of pill bugs in the forest and the garden were on the medium level  and weight  were significantly different among different habitats. There was a moderate correlation between habitat type and body length , and between habitat type and body weight .As the specific data obtained from the statistics indicate that most pill bugs were of medium body length. As shown in l >10 and m >100) were found in the wasteland with the highest residuals (>12). Furthermore, the lower proportion of bigger individuals (l >10 and m >100) were found in the grassland and bamboo grove with the lower residuals (<−4). The variation of the proportion and the residuals are shown in The post-hoc testing of the grouping composition indicated a higher proportion of bigger individuals (m) and body length (l) of the pill bugs  and the linear regression equation is obtained as m = 14.78 l − 73.22 . We found that the power function equation is better able to present the relationship between body weight and body length. There was a significant positive regression relationship between body length and body weight in the selected urban spaces , with the larger and heavier individuals in the wasteland   . Meanwhill > 12) . BesidesChanges in soil properties along the vegetation types have a significant effect on the population characteristics of pill bugs. Land use management influenced soil physicochemical properties in urban spaces, and population characteristics changed along with the variation of soil properties. There were significant differences in WC, pH, TC, and SOM of soils among selected habitats, and no significant differences in other soil properties. In addition, the regression equation between body length and body weight was set and well-fitted with a significant positive correlation. The larger pill bugs were found in the wasteland, which may relate to the lower SOM and TC and less disturbance; the smaller pill bugs were found in the grassland and the bamboo grove, which may relate to the lower pH. Furthermore, the food resource and human disturbance may also contribute to the variation of body size and weight among habitats.


Cardiovascular diseases are currently one of the most important causes of morbidity and mortality in liver transplant patients over the long term. Therefore, evaluating prognostic factors for cardiovascular events (CVEs) in this population is essential for taking preventive measures. The aim of this study was to identify the impact of diabetes and other metabolic disorders on CVEs in liver transplant patients. Three hundred fifty-six liver transplant recipients who survived at least 6 months after surgery were enrolled. Patients were followed for a median time of 118 months (12–250 months). All cardiovascular events were carefully recorded and detailed in the patients' charts. Demographic data, diabetes, hypertension, dyslipidemia, weight changes, and a diagnosis of metabolic syndrome both before and after transplantation were noted to assess their possible relationship with CVE. The presence of a diagnosis of metabolic-associated fatty liver disease (MAFLD) was also evaluated. Immunosuppressive therapy was included in the analysis. Diabetes mellitus (DM), especially when present before transplantation, was strongly associated with CVEs . Metabolic syndrome was found to be associated with CVEs in univariate analysis , while pretransplantation and de novo MAFLD were not. Immunosuppressive therapy had no influence on predisposing transplanted patients to CVEs during follow-up. Further prospective studies may be useful in investigating the risk factors for CVEs after liver transplantation and improving the long-term survival of transplant patients. Liver transplantation (LT) has become a well-established treatment for end-stage liver disease due to advancements in surgical techniques and immunosuppressive therapies. As a result, the survival rate for liver transplant patients has significantly improved over the years. In Europe, survival rates are 84% and 72% at 1 and 5 years after transplantation, respectively [While the short-term survival rates of liver transplant patients continue to improve, various long-term complications have drawn the attention of clinicians. These complications might be related to liver disease recidivism, posttransplant metabolic changes, and chronic immunosuppressive therapy , 3.Furthermore, the progressive increase in age of the surviving population is associated with increased cardiovascular morbidity and mortality, with the cardiovascular risk being even higher in transplant recipients than in the age- and sex-matched control population .This increased risk has been attributed to various factors, including those intrinsic to the patient and those related to transplantation, such as the requirement for long-term immunosuppressive therapy to prevent organ rejection , 6. MetaDiabetes is a significant risk factor for cardiovascular events (CVEs) in liver transplant patients –14, and Recently, a new definition called MAFLD (metabolism-associated fatty liver disease) has been coined . Indeed,The aim of this study was to evaluate the association of metabolic alterations, including MAFLD, and immunosuppressive treatment with the incidence of CVEs in liver transplant patients over time.This retrospective study aimed to analyze the demographic and clinical data of patients who underwent elective liver transplantation at the University Hospital Policlinico Umberto 1 between January 2000 and December 2020. The Ethics Committee of the Sapienza University of Rome approved this study . Before transplantation, all patients from our center undergo a rigorous cardiovascular assessment, which includes electrocardiography, echocardiography, and intracardiac pressure measurements if pulmonary hypertension is suspected. In some cases, cardio-CT or coronarography is used to exclude cardiac ischemia.To be eligible for this study, patients had to have a follow-up of at least 12 months at the center. After excluding 33 patients with less than 12 months of follow-up and 27 patients who continued their follow-up at other regional tertiary centers, a total of 356 patients were enrolled in the study.A composite variable “cardiovascular event” was created for the purpose of the study, and it included transient ischemic attack (TIA), ischemic and hemorrhagic stroke, cardiac decompensation, cardiac arrest, acute myocardial infarction (MI), symptomatic non-MI ischemic heart disease, and ischemia-based peripheral vasculopathy.The data analyzed were extracted anonymously through a thorough review of patients' charts. The data collected before transplantation included age, sex, reason for transplantation, body mass index (BMI), and presence of metabolic comorbidities such as diabetes (transient posttransplant diabetes lasting less than 6 months during steroid treatment was not considered) , arteriaClinical and demographic characteristics were reported using the mean and standard deviation or median and interquartile range (IQR) based on the distribution's normality. Categorical variables were expressed as absolute counts and percentages.We did not perform prior sample size calculations because of the retrospective nature of the study. However, all eligible data available were considered to maximize the power and generalizability of the results.Univariable and multivariable models were analyzed using complete case analysis. The only potentially relevant predictors with missing values were metabolic syndrome  and MAFLD . Multiple imputation for missing data was performed for the metabolic syndrome by replacing missing values with predicted group membership according to a multivariable logistic model. For MAFLD, no imputation was conducted due to the large number of missing values (28%) compared to the entire sample.p values <0.10 in the simple model were included in the multivariable model using a change-in-effect criterion. Absolute risks were calculated as the number of events that occurred in a group divided by the number of people in that group.Variables with de novo DM patients, traditional proportional-hazard Cox regression and Fine-gray competing risk models were utilized, with CVEs as the primary outcome and CVE-unrelated mortality as a competing event. In the multivariable model, we adjusted for demographic characteristics that may be associated with CVEs or death, such as age at LT, sex, etiology of liver disease, hypertension, hyperlipidemia, MAFLD, metabolic syndrome, and treatment.To compare the risk of CVEs among nondiabetic, pretransplant DM patients and F) or Mann–Whitney tests with Bonferroni corrections.The significance of differences in continuous variables between groups was tested using Fisher  men and 79 (22.2%) women.The median age at the time of transplantation was 56 years in the whole population, with no differences between males and females.n = 107, 30.1%) and HCV and/or HBV infection , followed by autoimmune and/or cholestatic liver disease  and nonalcoholic hepatitis , with other diseases accounting for 56 cases (15.7%). Hepatocellular carcinoma (HCC) was concurrently diagnosed in 173 patients (45.8%)  .Regarding the immunosuppressive maintenance therapy, 192 (53.9%) patients were treated with monotherapy of calcineurin inhibitors, while 47 (13.2%) were prescribed everolimus or sirolimus monotherapy. The remaining 117 patients (32.9%) received various combined therapies. Of these 60 patients (16.8% of the entire cohort) treated with tacrolimus and mycophenolate mofetil, 28 patients (7.8% of the entire cohort) were treated with cyclosporine and mycophenolate mofetil, 1 patient (0.3% of the entire cohort) was treated with sirolimus and mycophenolate mofetil, 4 patients (1.2% of the entire cohort) were treated with azathioprine and tacrolimus, and 24 patients (6.7% of the entire cohort) were treated with tacrolimus and everolimus.During the follow-up period, a total of 78 (21.9%) patients died. Among them, 21 (26.9%) patients died due to the recurrence of liver disease , 18 (23.1%) died due to the diagnosis of malignancies , 11 (14.1%) died due to infections, and 5 (6.4%) died due to cardiovascular disease.2 (IQR 22.6–28.1), whereas the median value after transplantation was 25.9 kg/m2 (IQR 23.2–28.7). In particular, 15.8% of patients were obese (31.9 kg/m2—IQR 30.8–34 kg/m2), and 34.5% were overweight before transplantation (26.8 kg/m2—IQR 25.7–28.1 kg/m2). After transplantation, the percentage of obese patients increased to 18.0% (32.3 kg/m2—IQR 31.2–34.2 kg/m2), while the proportion of overweight patients increased to 40.9% (27.3 kg/m2—IQR 26.0–28.4 kg/m2). Overall, after transplantation, the majority of patients (58.9%) had a BMI >25 kg/m2. BMI at the time of transplantation was significantly related with the origin of liver disease  = 9.584, p < 0.001) since patients with alcohol-related cirrhosis  and with nonalcoholic steatohepatitis (NASH)  had a higher BMI than patients with viral  or autoimmune hepatitis . This significant difference persisted even during the posttransplant follow-up period. The modifications of BMI after LT were not significantly different depending on the origin of liver disease  = 0.46; p=0.807).The median BMI value at the time of transplantation was 25 kg/mPrior to transplantation, 93 (26.1%) out of 356 patients had been diagnosed with diabetes. Among them, 8 patients exhibited regression of diabetes following the surgery. Seventy-four (20.8%) patients developed de novo diabetes after transplantation, resulting in a total of 159 (44.7%) patients with diabetes after transplantation. We investigated whether patients who developed de novo diabetes had different demographic and clinical characteristics compared with patients with pretransplant diabetes and nondiabetic patients. However, no significant differences were found .Metabolic syndrome was present in 17 patients (5.3%) prior to liver transplantation. However, its prevalence increased from 5.3% to 10.6%, as 34 patients developed this syndrome following liver transplantation.Moreover, the percentage of patients developing de novo metabolic alterations after transplantation is noteworthy: 20.8% of patients became diabetic, 40.2% of subjects developed arterial hypertension, and 43% of patients showed dyslipidemia. A smaller percentage of patients also developed either MAFLD or complete metabolic syndrome  cases of cardiovascular events (CVEs) were documented. The characteristics of the two cohorts of patients (with CVE and without CVE) are reported in During the follow-up, as reported in The occurrence of CVEs took place on average 71 months after transplantation, with a range of 12 to 228 months. Of the entire cohort, 5 (7.9%) patients died due to CVE-related complications.Simple and multiple Cox regression analyses and the fine-gray competing risk regression analysis are reported in A diagnosis of MAFLD, neither before nor after liver transplantation, was not associated with CVE, similar to viral hepatitis and the type of immunosuppressive therapy. On the other hand, a diagnosis of metabolic syndrome before liver transplantation was significantly associated with CVEs .Patients with pretransplant DM had the highest incidence of CVE (31.1%), while those with de novo DM had a lower incidence at 16.1%, and nondiabetic patients had the lowest incidence at 10%. At 5 years postliver transplantation, 20% of subjects with pretransplant DM had experienced a CVE, whereas only 5% of those with de novo DM had experienced one. After 10 years, the respective percentages were 30% in patients with pretransplant diabetes mellitus (DM) and 10% in patients with de novo DM .Over time, success and survival rates following liver transplantation have risen significantly, resulting in an increasing prevalence of long-term complications. Among these, metabolic complications are common as a consequence of multiple etiologic factors. Cardiovascular events are one of the leading causes of morbidity and mortality in the Western world and are closely linked to metabolic comorbidities.Our retrospective study aims to explore how metabolic features can be modified in a population of transplant patients and how they may impact on CVEs after transplantation. For this purpose, we analyzed multiple parameters before and after liver transplantation.In the case of our cohort, viruses and alcohol were the most frequent causes of liver cirrhosis. A moderate percentage of these patients had already been diagnosed with arterial hypertension, diabetes, and dyslipidemia during the pretransplant period . The recent increase in metabolic cirrhosis as a condition leading to liver transplantation may increase these numbers further in more recent cohorts .During a follow-up period of an average of 10 years after LT, the prevalence of metabolic alterations increased steeply , as alsoA consistent number of subjects also presented with issues of overweight and obesity. Weight gain after transplantation may occur due to the modification of dietary habits, increased appetite, and the effect of immunosuppressive drugs  . In addiDuring the study period, 63 CVEs were observed. The most frequently occurring of these were stroke and myocardial infarction.CVEs were associated with sex, age, arterial hypertension, dyslipidemia, diabetes, and the presence of the metabolic syndrome. Our multivariate analysis selected the male sex, older age when transplanted, and patients presenting with diabetes and dyslipidemia. The results of other studies are consistent with ours, thus confirming how important it is to focus on these factors during patient follow-up , 15, 32.One of the most frequently acknowledged CVE risk factors in liver transplant patients is diabetes. Two broad-scale studies reported in the literature have, however, provided conflicting results on this particular point , 34.Our single-center study found results similar to those of Kuo et al. . Indeed,The duration of DM is included in the calculation of CV risk , and theIn patients with cirrhosis of nonmetabolic origin, the coexistence of low cholesterol levels (following reduced liver synthesis) and reduced mean arterial pressure (as a consequence of vasodilatation) may decrease the rate of cardiovascular complications but the interaction between diabetes and liver disease is likely to be more complex. Some longitudinal studies reported that the low CV complications in cirrhotic patients with diabetes was explained by the higher mortality, mostly related to cirrhosis-related complications, in these patients .A second recognized risk factor for CVE is the presence of metabolic syndrome (MS). The diagnosis of MS should therefore be sought and promptly addressed with multidisciplinary approaches. However, the diagnosis of MS can be complex and fluctuating after liver transplantation, influenced by collateral effects due to immunosuppressive therapies and the trend to increase body weight.In this regard, the concept of MAFLD has also emerged, as it has broader diagnostic criteria and allows for identifying a segment of the population at risk that is not included in the definition of MS. Unfortunately, only a subgroup of patients in our cohort had complete data for the assessment of MAFLD. Therefore, the number of patients with MAFLD could have been underestimated. Despite this bias, in contrast to the normal population , we founFinally, there may be additional factors that have an impact on the onset of metabolic comorbidities and cardiovascular risk. Our study did not reveal any clear association between the type of immunosuppressive therapy and its possible role. Even those patients who were taking everolimus (a non-CNI that may increase the risk of hyperlipidemia) did not exhibit a higher risk of cardiovascular events.The strengths of the present study are the rather large sample size, the long median follow-up (118.5 months), and the possibility of direct access to patient information.The limitations of this study are related to its observational and retrospective nature. Some data were missing, as shown in Tables Additionally, important risk factors  have not been considered, as this information is not always available for each patient.From a clinical point of view, the present results suggest that special attention should be given to preexisting diabetes, as these patients are at increased risk of cardiovascular morbidity. Patients with preexisting DM could benefit from tighter treatment of diabetes and, at the same time, a strict control of other risk factors for cardiovascular disease, such as hyperlipidemia, arterial hypertension, and overweight. A multidisciplinary approach involving a cardiologist and a diabetic center is recommended.Patients with de novo metabolic syndrome have an increased risk of CVe; thus, even if de novo DM as a single risk factor is not significantly correlated with CV, it deserves attention in the context of de novo metabolic syndrome.Future prospective studies on large cohorts of liver transplant recipients are needed to better stratify cardiovascular risk after LT and how to act on it: what are the therapeutic interventions and whether they should differ from those applied in the general population. This becomes even more relevant considering the increasing survival of liver transplant patients, the increasing age at transplantation, and the increasing frequency of metabolic etiology of liver disease. For all of these reasons, it is necessary to continue research in this area to identify the best strategy for follow-up and therapy of these patients.


P < 0.001). In the validation group, the results were consistent with those of the training group, with a C-index value of 0.726 . This study constructed a practical nomogram to predict 1-, 3-, and 5-year OS for patients with colorectal mucinous adenocarcinoma based on the SEER data.A nomogram was constructed to predict the survival of patients with colorectal mucinous adenocarcinoma based on data extracted from the Surveillance, Epidemiology and End Result (SEER) database. Data collected between 2010 and 2018 were obtained from the SEER database. The log-rank test and multivariate Cox regression were performed to identify the independent prognostic factors for overall survival, which were further used to construct a nomogram model to predict 1-, 3-, and 5-year overall survival. In total, 10,846 patients diagnosed with colorectal mucinous adenocarcinoma were enrolled in the study. The following 11 variables were associated with survival and were further incorporated into the nomogram: age at diagnosis, primary site, grade, tumour size, lymph node dissection, T stage, N stage, M stage, surgery for primary site, chemotherapy, and household income. The concordance index (C-index) value was 0.725 , and the receiver operating characteristic curves and calibration curves showed satisfactory predictive accuracy. Both the C-index and time-independent area under the curve values were greater than those of the American Joint Committee on Cancer 7th TNM classification system (both  Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer-related death worldwide . The majDespite advances in treatment strategies, such as targeted therapy and immunotherapy, the 5-year survival of locally advanced and metastatic CRC patients is still unsatisfactory . EffectiA nomogram is a simple visual predictive model that is widely used for prognosis prediction. It is more comprehensive in terms of the inclusion of all effective prognostic factors and more intuitive because the estimated survival of each patient can be conveniently calculated by combining the score of each parameter and matching the corresponding percentage . To the Eligible patients were extracted from the database “Incidence-SEER Research Data, 18 Registries, Nov 2020 Sub(2000–2018)” using the SEER*Stat program (v8.4.0). The inclusion criteria were as follows: (1) patients who were diagnosed with primary colorectal adenocarcinoma according to the third version of the International Classification of Disease for Oncology (ICD-O-3) and (2) patients who had histologically confirmed MAC subtype (ICD-O-3 coded as 8480/8481). The exclusion criteria were as follows: (1) patients with more than one primary cancer; (2) patients diagnosed by clinical criteria or based on autopsy or the death certificate; (3) patients with incomplete survival information; (4) patients with a follow-up of less than 1 month; and (5) patients with incomplete clinicopathological and treatment information . Eligible patients were randomly divided into a training group and a validation group in a 70:30 ratio.In this study, 14 variables were collected from the database, including sex, age at diagnosis, race, primary site, grade, tumour size, lymph node dissection, T stage, N stage, M stage, surgery, radiotherapy, chemotherapy, and median household income. All tumours were staged according to the TNM staging system of the AJCC . The primary tumour site was divided into the caecum-ascending colon , transverse colon, descending colon-sigmoid , and rectum. For continuous variables, patients were divided into two groups, with the age of 65 years as the cutoff. The cutoff value for tumour size was 3 cm. The endpoint of this study was overall survival (OS), which was defined as the duration between diagnosis and death due to all causes.All patient data were obtained from the SEER database, which records cancer data for approximately 30% of the American population across different regions . A SEER P value ≤ 0.1 during univariate analysis were further examined by multivariate backward stepwise Cox proportional hazard regression analysis. Statistically significant variables in the multivariate Cox regression analysis (P ≤ 0.05) were determined to be independent prognostic factors to predict the survival outcome. Additionally, variance inflation factor examinations of the effective prognostic factors were evaluated to exclude multicollinearity problems. Then, these selected factors were used to establish a nomogram model to predict 1-, 3-, and 5-year OS in the training and validation groups performed by the rms package in R (version 4.1.2).Categorical variables are shown as proportions and frequencies and were compared by the Chi-square test or Fisher’s exact test. The associations between each variable and survival were first evaluated with univariate analysis using the log-rank test. Variables with a P < 0.05 (two-tailed) was considered statistically significant.The performance of the nomogram was evaluated by detecting its discrimination and calibration abilities both internally (in the training group) and externally . The bootstrapping resampling approach (1000 repetitions) was applied to interval validation. The concordance index (C-index) and the receiver operating characteristic (ROC) curve were used to validate discrimination performance. A higher C-index value and a larger area under the curve (AUC) in the ROC curve represented better discrimination ability. In addition, we calculated the C-index and the ROC curve using the AJCC 7th TNM classification system and then compared the results with our nomogram to identify differences. The calibration curves were used to evaluate the calibration ability of the nomogram. A 45-degree plot represented an optimal model. All statistical analyses were conducted using SPSS 26.0  and R . A difference of n ═ 7527) or the validation group (n ═ 3319). A detailed flowchart of patient selection is presented in n ═ 5639, 52.0%), aged ≥ 65 years  and white . A total of 71.7% of patients had a household income higher than 75,000 dollars. The most common primary site was the right colon , followed by the left colon , rectum , and transverse colon . The tumour grade proportions of all patients were well , moderate , poor , and undifferentiated . More than 80% of the patients had a tumour larger than 3 cm . Stages T1-4 accounted for 3.8%, 11.2%, 55.8%, and 29.2% of the population, respectively. Regarding the treatment methods, only 1.2% of the patients did not undergo surgery, and 92.7% of the patients had more than 4 lymph node dissections. Radiotherapy and chemotherapy were administered to 9.3% and 44.8% of the patients, respectively. The 1-, 3-, and 5-year OS rates of all enrolled patients were 87.4%, 68.3%, and 57.2%, respectively. The detailed clinicopathological features of the two groups are listed in In total, 10,846 patients diagnosed with colorectal MAC were enrolled in the study and were assigned to the training group (P < 0.05). The variance inflation factor exam suggested that multicollinearity issues did not exist , as shown in The nomogram was validated internally using data from the training group and externally using data from the validation group. The C-indices of the nomogram in the training group and the validation group were 0.725  and 0.726 (95% CI 0.713–0.739), respectively. In this study, we also calculated the C-index for both groups using the AJCC 7th TNM classification system to compare the values with our nomogram. The results showed that the prognosis-predicting ability of the nomogram was significantly better than that of the AJCC 7th TNM classification system (P < 0.001), reflecting the superiority of this nomogram as a predictive tool for the prognosis of colorectal MAC patients.In this study, we analyzed 10,846 US colorectal MAC patients from the SEER database who were diagnosed during 2010–2018. After randomly dividing the patients into the training group and the validation group in a 70:30 ratio, we established an effective nomogram to predict the 1-, 3-, and 5-year OS in the training group and then internally and externally validated the discrimination and calibration of the model. Additionally, we compared the predictive capacity of our nomogram to the TNM staging system by the C-index as well as ROC values, and both presented a significant increase . The relationship between molecular differences and clinical features is still undefined , 14, 15.The TNM stages are the most important and generally acknowledged prognostic factors for patients with malignant solid tumours. Our study indicated a consistent result: TNM stages, especially the M stage, were the most valuable variables among all factors. There was another pivotal variable, surgery, which remarkably impacted the prognosis according to our nomogram. Additionally, four or more regional lymph node dissections during surgery were found to be a beneficial factor for colorectal MAC patients. These results have been proven in numerous studies and illustrate the importance of standard surgical treatment, which is the only chance for a radical cure or long-term survival , 24.P ═ 0.026) [Whether MAC is related to poor outcomes in CRC patients is controversial. Unlike signet ring cell CRC, MAC is not identified as an independent factor for worse prognosis by the AJCC, and neither the National Comprehensive Cancer Network (NCCN) nor the European Society for Medical Oncology (ESMO) guidelines suggest disparate standard treatments particularly designed for MAC , 26. Sev═ 0.026) . A meta-═ 0.026) .Despite the ambiguity in MAC prognosis, a consensus has been reached regarding the impaired response to chemotherapy. It has been observed that compared with NMAC, MAC is less responsive to neoadjuvant chemotherapy, adjuvant chemotherapy, and palliative chemotherapy, expressed by lower disease-free survival rates and survival rates , 30, 31.There was a nomogram for the prognosis of colorectal MAC patients constructed by Lian et al. Compared to our nomogram study, this previous study was mainly focused on the pattern of distant metastases, and external validation was absent . Our stuKRAS, NRAS, BRAF mutations, and MSI status, details of treatments, such as regimens of chemotherapy, target therapy, and immunotherapy, family history of disease, and comprehensive pathology descriptions, which may influence the effectiveness of our nomogram [Certain limitations existed in this research. First, CRC is a heterogeneous disease, and the prognosis of CRC is affected by many factors. In addition to the characteristics mentioned in our study, clinical information was not reported or was reported incompletely in the SEER database, for instance, gene status, such as nomogram . FurtherIn conclusion, this study constructed a practical and user-friendly nomogram based on the SEER database to predict 1-, 3-, and 5-year OS in patients with colorectal MAC. The nomogram was validated both internally and externally and demonstrated a significant advantage in predictive accuracy compared to TNM stage classification. Due to the retrospective design and the absence of more clinical and gene information, prospective studies are required in the future.


Supplemental Digital Content is available in the text The results of how matrix metalloproteinases (MMPs) polymorphisms affect esophageal cancer (EC) risk are not consistent, especially for MMP1,2,7 and 9. A meta-analysis focused on the impact of MMPs to digestive cancers, but not a precise analysis to EC, therefore, we designed the current study to make a clear understanding of the association between MMPs polymorphisms and EC.Q test and I2 statistics are used to estimate heterogeneity. Sensitivity analysis, Egger test, and Begg's funnel plot were employed to assess the results. In-silico analysis was performed to study the association between the polymorphism and mRNA expression.Up to March 2020, we searched several databases to find case-control cohorts concerned about the risk of MMPs polymorphisms to EC risk. Odds ratios with 95% confidence intervals under five genetic models to generate the risk predicted value. The 19 case-control studies were enrolled, including 8371 EC patients and 12041 health controls. We observed the increased risk in BA vs. AA and BB + BA vs. AA models of MMP1-rs1799750 polymorphism. The protective effectiveness of EC was found in the MMP2 rs243865 polymorphism in B vs. A, BA vs. AA, and BB + BA vs. AA models. Meanwhile, the risk effect was also observed in the MMP7 rs11568818 polymorphism in most genetic models. In the furthermore bioinformatics analysis, we found that MMP1, MMP3, MMP7, MMP9, MMP12, MMP13 all increased in the tumor tissues, and the genetic alteration in the polymorphisms could impact the mRNA expression of the above MMPs.MMP1 rs1799705 and MMP7 rs1156818 polymorphisms will take part in the tumorigenesis of EC, while MMP2 rs243865 acts as a protective role to decrease the risk of EC. Several researchers reported that EC development is dependent upon several factors, such as genetic susceptibility, environment, alcohol consumption, cigarette smoking.,7Esophageal cancer (EC), consisting of two major histological types, esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC), is the seventh most frequent cancer globally, and causes the sixth cancer-specific death, based on the data from World Health Organization, and Eastern Asia accounts for the highest rate of both incidence and mortality. reported that EC patients from hospital-based studies showed a higher survival rate while comparing with the population-based surveillance, Chen et al also drew out that individuals aged 40 to 69 years in high risk areas of EC showed a significant decrease incidence and mortality. Therefore, it is necessary to find the biomarkers to distinguish the different susceptibility of EC. Zhang et al reported that Matrix metalloproteinase 2 (MMP2) -1306 (rs243865) had a decreased incidence of ESCC, this result was also confirmed by Yu et al 's study. However, Haniehsadat et al revealed that No statistically significant differences were observed in the genotype and allele frequencies of MMP2 (-1306C/T) and MMP9 (-1562C/T) between patients and controls. Li et al made a meta-analysis among the digestive cancers in 2013, and they reported that MMP1 and MMP7 are the risk factors for digestive cancers, while MMP2 and MMP9 play protect roles, however the precision role of EC patients were not conducted. Due to this kind of consistent, we designed a study to enroll all the publications mentioning the matrix metalloproteinases (MMPs) polymorphisms and susceptibility of EC, to try to generate a more convincing conclusion. And we also used bioinformatics analysis to find the potential reason for the effect of MMPs polymorphisms on EC.Hou et al22.1Up to March 2020, two investigators of our team searched through the Web of Science, Google Scholar, Wanfang database, PubMed, Embase, and CNKI databases to find the case-control cohorts which meet our criteria. The following search strategy is used for the searching of relevant citations:  AND  AND (SNP OR variant OR mutation OR polymorphism OR genotype). This study is a meta-analysis and does not require institutional review board approval or patient consent.2.2Articles included in our research must meet the following conditions: (1) study the relationship between EC and MMPs; (2) provide sufficient data of each patient for extraction and calculation; (3) subjects are human patients; (4) the case-control study included control group and EC patients case group. In contrast, studies would be removed if they were: (1) lack of genotype frequency data; (2) case only study, review and meta-analysis, repetitive publications; (3) animal or cell line based studies. When duplicate data appear in different articles, our meta-analysis only includes the latest data for calculation.2.3http://www.ohri.ca/programs/clinical_epidemiology/oxford.asp).These following items are extracted from all the enrolled studies, including disease type, first author, ethnicity, source of control, genotype distribution in cases, and controls, publication year. Any differences were resolved through group discussions until all consensus was reached. The Newcastle–Ottawa Scale (NOS) was applied to assess the quality of all the enrolled studies (2.4http://gepia.cancer-pku.cn/), which contains the data of gene expression from the TCGA and the GTEx projects.The mRNA expression of MMPs among normal esophagus tissues and EC tissues were compared with the GEPIA (2.5I2 statistics are used to estimate heterogeneity. The random-effects model is applicable when existing heterogeneity (P < .1); otherwise, the fixed effect model is used when P > .1. Chi-square test was employed to calculate the Hardy–Weinberg equilibrium of the control group to compare the expected and actual genotype frequencies. Furthermore, the method of evaluating the stability of the overall analysis is sensitivity analysis, and the method of evaluating publication bias is through the Egger test and the Begg's funnel plot. A statistically significant result is a P value of less than .05 after adjusting by Bonferroni correction.The meta-statistical analysis were performed using STATA 12.0. The relationship between EC and MMPs was assessed using pooled odds ratios with 95% confidence intervals under B vs. A, BB vs. AA, BA vs. AA, BB + BA vs. AA, and BB vs. BA + AA models . Additionally, we performed a stratified analysis based on disease type, race, source of control, and quality score. The Q test and 33.1–12,17–29 The details of demographic characters and genotype frequencies are shown in Table http://links.lww.com/MD2/A462 which illustrates the quality of the enrolled studies).The search and enrolled process are shown in Figure 3.23.2.1P = .001]) and subgroup analysis of PB . In addition, we found an important association between MMP1 rs1799750 polymorphism and EAC in the subgroup analysis of Caucasian. .Table 3.2.2P < .001); BA vs. AA ; BB + BA vs. AA ]. We got the same results as the overall analysis in the subgroup analysis of PB  and Y  .In regard to rs2285053 polymorphism, the risk of ESCC and rs2285053 polymorphism is irrelevant . Additionally, we did not find any important association between rs3025058 polymorphism and EC in the subgroup analysis of Asians, Caucasian, ESCC, EAC, Y, and N.According to our calculations, rs3025058 polymorphism was not associated with the risk of EC in the overall analysis. .As for rs11568818 polymorphism, the study strongly showed that rs11568818 polymorphism can increase the risk of ESCC among the overall analysis , and the results of this meta-analysis of rs2250889 polymorphism and rs17576 polymorphism showed that these two genetic polymorphisms do not have a relationship with the risk of ESCC .As for rs2276109 polymorphism, the result of subgroup analysis of Caucasian showed that rs2276109 polymorphism increased the risk of EAC .However, the analysis results for rs652438 polymorphism and the risk of EAC are negative .In regard to rs2252070 polymorphism, the overall 3.3http://links.lww.com/MD2/A463 which illustrates the details of sensitivity analysis). What's more, Begg test was used to evaluate the publication bias for the selected literature, the symmetric channel distribution plan shows that there is no prejudice in the publication .To test the robustness of the results we obtained, we delete individual studies one by one when conducting sensitivity analysis. We have not observed any significant changes in OR and the corresponding 95% CI, which supported that the results of the current study are stable and representative (see Supplementary Digital Content Table S2, 3.4Based on the above results, we already knew that the polymorphism of some MMPs could alter the risk score of EC. Based on the TCGA and GTEx data, we found that MMP1, MMP3, MMP7, MMP9, MMP12, MMP13 all increased in the tumor as compared with the normal esophageal tissues Fig. .4,31 There are 28 members in the MMP family, and 23 of them are expressed in human tissues, typically MMPs share common structures, including a ∼80 amino acid propeptide, a ∼170 amino acid catalytic metalloproteinase domain, a hinge region with variable size, and a ∼200 amino acids hemopexin domain.,33 Based on the different structures of MMPs, they share different functions. MMP1, MMP8, and MMP13 are human collagenases, MMP3 and MMP10 are tromelysins, plus MMP12, 19, 20, and 27 are all archetypal MMPs; MMP7 and 26 are matrilysins because of the lack of hemopexin domain; MMP2 and MMP9 incorporate three fibronectin type II modules that provide a compact collagen binding domain as the gelatinases; And the three secreted MMP11, 21 and 28 proteins and two unusual transmembrane MMP23A and MMP23B all share the Furin-like cleavage site. In the past decades, increasing evidence confirmed the functions of MMPs in pathological processes, including inflammation, vascular disease, emphysema, liver fibrosis, as well as tumors.,35–38The zinc-dependent MMPs are endopeptidases known for their ability to cleave the conditions of the extracellular matrix, of which could help the tumor cells to penetrate and infiltrate into the subjacent stromal matrix, and finally lead to tumor invasion and metastasis. reported that the polymorphism rs11568818 in MMP7 gene was also associated with a 2.39-fold increased prostate cancer risk in Poland, while Meng et al also, compared the different effects of MMP polymorphisms in urinary cancer, they revealed that the rs3918241, rs2250889, rs17576, and rs17577 polymorphisms of MMP9 are not associated with altered risk of urinary cancer. As to MMP8, Shen et al reported that the rs11225395, rs34009635, and rs35866072 polymorphisms are not associated with the increased risk of lung cancer in Taiwan district, however, González-Arriaga et al found that the rs215502 of MMP8 acted as a protective role and decreased the lung cancer risk in Spanish. In breast cancer, Wang et al reporting the increased frequency of MMP8 rs11225394 polymorphism, while the decreased frequency of MMP8 rs3787268 polymorphism, these results indicated the internal conflict of MMP8 gene with the risk of breast cancer. Numerous studies also evaluated the predicted value of MMPs polymorphisms in EC risk, however the results are not consistent, therefore, we designed the current study to remove the cloud.What's more, the polymorphisms of MMPs were also concerned to predict the risk of tumorigenesis in several studies. Białkowska et al reported that the altered allele 2G/2G of MMP-1 rs1799750 leads the significantly higher levels of MMP-1 expression than the major 1G/1G, and we also observed the higher level of MMP-1 in EC than normal tissues. For the MMP-7 rs1156881, Gerger et al also reported that the altered G allele can lead to a higher promoter activity. Therefore, we can derive the correlation of polymorphism, MMPs expression and tumorigenesis.In the current study, we performed a comprehensive search to enroll all the available data about the polymorphisms of MMPs and EC risk. Finally, 19 case-control researches, including 8371 EC patients and 12041 health controls were recorded for the subsequent analysis. The odds ratios and 95% confidence intervals in five genetic models were conducted to evaluate the risk of EC. We observed the increased risk in the heterozygote comparison model and the dominant comparison model in MMP1-rs1799750 polymorphism, as well as in the EAC/Caucasian subgroups. The shielding effectiveness of EC was found in the MMP2 rs243865 polymorphism in B vs. A, BA vs. AA, and BB + BA vs. AA models. Meanwhile, the risk effect was also observed in the MMP7 rs11568818 polymorphism in most genetic models. And the polymorphisms in other MMPs showed no function in the tumorigenesis of EC. In the furthermore bioinformatics analysis, we found that MMP1, MMP3, MMP7, MMP9, MMP12, MMP13 all increased in the tumor as compared with the normal esophageal tissues. In a prior study, Przybylowska et alThe current study concerned all the applicable studies to analyze the association between MMPs polymorphisms and EC, and showing some advantages. First, we enrolled all eligible studies based on the language of English and Chinese, the race of Asian and Caucasian to conduct a comprehensive study to obtain stable results. Second, the NOS method is used to evaluate the quality of each case-control study. At the same time, we eliminated low-quality studies to ensure the reliability of the combined results. Third,The stability of the results was evaluated by sensitivity analysis, and the results of Egger test and Begg's funnel chart confirmed that the research results were not biased. On the contrary, there are several disadvantages for the current study. First of all, due to the lack of environmental factors that may affect the phenotype, unreliable results may be obtained. What's more, in about half of the concerned polymorphisms, there are only two or three eligible studies enrolled, therefore, the results of these polymorphisms might be inconvincible. Finally, although we conducted the meta-analysis using the Der Simonian and Laird methods, the heterogeneity between recorded publications may affect the results.5All in all, based on the results in the current study, we can draw out that MMP1 rs1799705 and MMP7 rs1156818 polymorphisms will take part in the tumorigenesis of EC, while MMP2 rs243865 acts as a protective role to decrease the risk of EC. More studies are needed to confirm the predicted value of MMPs polymorphisms to the risk of EC.Conceived and designed the study: Hai Chen; Performed the literature search and data extraction: Hai Chen, Zaicheng Yu, Congshu Hua, Tengfei Ge; Analyzed the data: Heng Zhang, Junling Jian, Jianfeng Xie; Drafted the manuscript: Hai Chen, Xianquan Xu.Conceptualization: Hai Chen.Data curation: Congshu Hua, Heng Zhang, Junling Jian, Tengfei Ge, Jianfeng Xie.Formal analysis: Junling Jian, Jianfeng Xie.Investigation: Congshu Hua, Heng Zhang, Tengfei Ge.Project administration: Zaicheng Yu.Resources: Congshu Hua, Heng Zhang, Tengfei Ge.Writing – original draft: Hai Chen, Xianquan Xu.Writing – review & editing: Zaicheng Yu.


Ischemic stroke is the second-leading cause of death. Hyperglycemia, which is characteristic of diabetes mellitus, contributes to the development of endothelial dysfunction and increases the risk of stroke. Isoxsuprine is an efficient beta-adrenergic agonist that improves blood flow to the ischemic aria and stops the infarct core from growing. However, low bioavailability, a short biological half-life, and first-pass hepatic metabolism reduce the therapeutic efficacy of oral isoxsuprine. Therefore, the authors focused on developing isoxsuprine-loaded liposomes containing ethanol and propylene glycol (ILEP) formulation as nasal drops for the treatment of ischemic stroke in diabetic patients. Different ILEP formulations were optimized using Design Expert software, and the selected formulation was examined in vivo for its anti-stroke effect using a rat model of diabetes and stroke. The optimized ILEP, composed of 15% propylene glycol, 0.16% cholesterol, 10% ethanol, and 3.29% phospholipid, improved the sustainability, permeation, and targeting of isoxsuprine. Furthermore, the in vivo studies verified the improved neurological behavior and decreased dead shrunken neurons and vascular congestion of the rats treated with the optimized ILEP formulation, demonstrating its anti-stroke activity. In conclusion, our study found that treatment with an optimized ILEP formulation prevented the initiation and severity of stroke, especially in diabetic patients. Ischemic stroke occurs when oxygen and nutrients are denied to brain tissue because of a lack of blood flow to that area of the brain ,2. AmongThe nasal route holds promise for the direct delivery of drugs to the brain and is an appealing alternative to other administration routes ,17. The Isoxsuprine was kindly donated by the Egyptian branch of Abbott Pharmaceuticals . Cholesterol and phospholipids were purchased from the Egyptian branch of Sigma-Aldrich . Methanol, ethanol, propylene glycol, and chloroform were purchased from Cornell Lab .®  was used to generate a 33 Box–Behnken design for the preparation of 15 formulations of ISP-loaded liposomes containing ethanol and propylene glycol (ILEP), with X1 representing the concentration of propylene glycol, X2 representing the concentration of phospholipid, and X3 representing the concentration of ethanol  formulations as a blank formulation at a wavelength of 274 nm. Different ILEP and PLEP formulations were developed using the thin-film hydration technique . A solutCt is the total amount of ISP, and Cf is the amount of free ISP.Unentrapped ISP was separated by spinning the ILEP formulation at 15,000 rpm for 1 h in a cooling centrifuge . After collecting the ILEP pellets, they were mixed with phosphate buffer (pH 6.8) and placed in the refrigerator until analysis. We ensured that all of the ILEP pellets were successfully separated by recentrifuging the unentrapped ISP solution at 15,000 rpm for 2 h at 4 °C till a clear solution resulted ,25,26. UTo determine the vesicle size of different ILEP formulations, a sample was diluted and investigated using dynamic light scattering on a piece of zeta sizer equipment  .p-value utilized to establish the optimal model [Analysis of variance (ANOVA) in Design Expert software was used to analyze the results for each dependent variable, with criteria including lack of fit, F-value, and al model . ApplyinThe interaction between ISP and the ILEP-producing components was evaluated by analyzing the thermograms of ISP, cholesterol, phospholipid, and optimized ILEP with a differential scanning calorimeter  . The DSCTo examine the morphological characteristics of the optimized ILEP formulation, a transmission electron microscope  was used . On a caThe stability of the ILEP can be determined by measuring the zeta potential, which is a quantitative evaluation of the strength of repulsive forces between vesicles . The polUtilizing a diffusion method with dialysis bags , we compared the drug release from the optimized ILEP to that from free ISP . To achiUsing a diffusion membrane made from an epidermal layer of abdominal rat skin, we investigated the ex vivo permeation of free ISP and the optimized ILEP formulation. The experiment was approved by the ethics committee at Beni-Suef University in Egypt. Here, phosphate buffer  was used as the receptor medium inside a Hanson dissolution apparatus at 37 °C and 100 rpm . Over a Twenty-four mature male albino rats (200–250 g body weight) were housed in an air-conditioned room with free access to food and water and a humidity range of 30–70%. After a week of eating an adapted diet, the rats were weighed and divided into five groups of six (n = 6). Before the study began, the mean body weight, blood glucose level, red blood cells (RBCs), white blood cells (WBCs), alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea, and creatinine were all recorded for all groups. The in vivo experiment was approved by the ethics committee at Beni-Suef University in Egypt (BSU-IACUC: 022-413).Twenty-four adult male albino rats were divided into two groups. A control negative group (n = 6) was given intraperitoneal injections of 0.9% saline solution and fed a normal diet. The remaining rats (n = 18) were fed a high-fat diet consisting of 6% cholesterol, 1% sodium cholate, 0.2% propyl thiouracil, and 5% refined sugar for 12 weeks to induce the f diabetes and atherosclerosis . Blood gEach group’s rats were trained in the respective neurobehavioral task both before the start of the experiment and again after 22 h after reperfusion. We used a point system to quantify the severity of the behavioral issues ,38. By making use of the rat’s natural instinct to grasp a vertical metal grid while dangling by its tail, the grip strength test is used to quantify rat muscle force in vivo . The flexion test is an evaluation used to detect neurological abnormalities in rats by watching their forelimb postures while suspended by their tails . According to the method provided by Tabassum et al., spontaneous motor activity can be utilized to infer a rat’s neurological status . The Morris water maze test, as reported by Ashafaq et al., is used to assess the cognitive–behavioral abilities of rats by watching how well they remember their location in the maze . At the end of the study, rats were euthanized by cervical dislocation after receiving an intraperitoneal injection of a 0.1 mg/100 gm concentration of ketamine (90 mg/kg) and xylazine (5 mg/kg). Blood was centrifuged to separate serum, and the levels of cholesterol, TG, and glucose were measured in accordance with the manufacturer ,35.Brain tissue samples (4–6 µm) were preserved by fixing them in 10% neutral buffer formalin, dehydrating them in ethyl alcohol, cleaning them in xylene, and finally embedding them in paraffin. These specimens were stained with hematoxylin and eosin for histological examination .The rats’ weights were recorded from the beginning of the experiment to the day of the sacrifice . Alteratp-value of 0.05.SPSS  was used for statistical analysis, and the results were summarized as the mean ± SD (standard deviation), with significance determined by Tukey’s post hoc test at a p-value > 0.05) impact on the determination of the entrapment efficacy of ILEP formulations. p-value, and high F-value. Furthermore, the model’s predicted R2 of 0.9981 and 0.9951 and adjusted R2 of 0.9986 and 0.9964 indicate that it may be used to interpolate EE% and vesicle size data, respectively, with high confidence. The model’s signal is strong enough for optimization, and it may be used to direct the design process with an adequate precision of 188.948 for EE% and 107.067 for vesicle size, respectively.This study used a Box–Behnken analysis design to successfully prepare and optimize ILEP formulations. Various PLEP formulations were prepared as blanks, and the findings indicated that the absorbance of all PLEP formulations was minimal and had an insignificant  impact on EE%. Propylene glycol and ethanol have a negative  impact on particle size, while phospholipid has a positive  impact on particle size.After applying the criteria of smallest vesicle size and highest EE%, the Design Expert program suggested the optimized formulation. Because it has the highest desirability index (0.762), the formulation with 15% propylene glycol, 0.16% cholesterol, 10% ethanol, and 3.29% phospholipid was chosen as the optimum one. In vitro experiments of the optimized ILEP formulation showed that the predicted values of responses were accurate, with an average EE% of 88.56 ± 0.61% and a vesicle size of 189.62 ± 7.05 nm.The TEM image  revealedWith a PDI of 0.281, the optimized ILEP formulation showed that uniform vesicles were formed. With a zeta potential of −41.5 mV, the optimized ILEP formulation showed increased vesicle stability and decreased aggregation propensity of vesicles.t test, the total amount of ISP released from the free ISP solution was significantly  greater than the total amount of ISP released from the optimized ILEP formulation. A sustained release of 55.59% of ISP from the optimized ILEP formulation was observed at 24 h compared to that of free ISP with 97.42% at 8 h.The in vitro drug release profile from the optimized ILEP formulation compared to the free ISP is shown in 2) was significantly  greater than the total amount of ISP permeated from the free ISP solution (1071 ± 70.63 µg/cm2). In addition, the flux (Jss) of the optimized ILEP formulation (58.47 ± 3.24 µg/cm2/h) was significantly  higher than the flux of ISP from the free ISP solution (27.91 ± 2.47 µg/cm2/h) with an enhancement ratio of 2.094 after 24 h.The ex vivo drug permeation profile from the optimized ILEP formulation compared to the free ISP is shown in p-value < 0.05) greater than those in the control negative group  lower than that of the control positive group, suggesting their antidiabetic and anti-atherosclerotic activities. Among treatment groups, the serum glucose, cholesterol, and TG levels of the optimized ILEP formulation group were the lowest with 68.18%, 89.09%, and 49.54%, respectively. Compared to the free ISP group, serum glucose, cholesterol, and triglyceride levels were shown to be significantly  lower in the optimized ILEP formulation group by ratios of 1.15, 1.06, and 1.36, respectively.Blood glucose, cholesterol, and triglyceride levels in the control positive group were significantly  higher activities compared to the control negative group by 4.04- and 2.33-fold, respectively. In the grip strength test, the control positive group had significantly  lower activity compared to the control negative group by 2.45-fold. In the Morris water maze test, the control positive group took significantly  longer time to locate the target quadrant compared to the control negative group by 8.69-fold. Upon treatment with free ISP and optimized ILEP formulation, flexion, spontaneous motor activities, grip strength, and time required to locate the target quadrant were all found to be significantly  restored compared to that of the control positive group, suggesting their anti-ischemic stroke activity. Among treatment groups, the flexion, spontaneous motor activity, and time spent in the target quadrant of the optimized ILEP formulation group were the lowest with 84.21%, 67.48%, and 84.85%, respectively, and the grip strength of the optimized ILEP formulation was the highest by a ratio of 2.62%. Compared to the free ISP group, flexion, spontaneous motor activity, and time spent in the target quadrant were shown to be significantly  lower in the optimized ILEP formulation group by a ratio of 26, 1.96, and 1.07, respectively, and the grip strength of the optimized ILEP formulation was shown to be significantly  higher by a ratio of 2.16%.Ischemic stroke was successfully induced in rats, as shown in Ischemic strokes were successfully induced in rats, as shown in p > 0.05) rise in body weight compared to the control negative group, indicating healthy growth. The hematological parameters such as Hb, RBC, MCV, MCH, MCHC, WBCs, neutrophil, lymphocyte, and monocyte and biochemical parameters such as creatinine, urea, AST, and ALT were not significantly different  between the optimized ILEP formulation group and the control negative group.Daily nasal treatment of an optimized ILEP formulation resulted in minimal toxicity or mortality in rats, and there were no behavioral changes compared to the control positive group. In the optimized ILEP formulation group, measurements showed a nonsignificant (p-value, lack of fit, F-value, predicted R2, adjusted R2, and adequate precision, Design Expert software determined which model gave the best fit [p-value, insignificant lack of fit, and highest values of F-value, predicted R2, and adjusted R2. Since the model’s appropriate precision is greater than 4, it is judged to be accurate enough for optimization. With this study, the authors focused on developing an ILEP formulation for the treatment of ischemic stroke in diabetic patients. ILEP formulations are lipid vesicles composed of phospholipids, cholesterol, propylene glycol, and ethanol ,21,41. Pbest fit . The datAnalysis of the impact of each variable can be carried out with the help of the program’s coded polynomial equations and 3D surface graphs ,44. TherUsing DSC, we can measure the crystallinity of ISP and keep track of any interactions between ISP and the other ingredients in our optimized ILEP formulation . The disA stroke occurs when there is a blockage in the blood supply to the brain, usually caused by thrombosis or embolism ,3. HyperToxicology studies on animals were conducted to better understand the potential hazards of nasal administration of the optimized ILEP formulation and to identify any noticeable adverse effects. Compared to the control negative group, all rats administered the optimized ILEP formulation showed no signs of illness or death and no significant differences in body weight, demonstrating its normal growth. Due to the lack of significant fluctuation in RBC and WBC, it was determined that the nasal delivery of the optimized ILEP formulation had no effect on the immune system, erythropoiesis, or the morphology or osmotic fragility of red blood cells. Liver and kidney functions were unaffected by nasal delivery of the optimized ILEP formulation since there was no significant difference in the levels of serum ALT, AST, creatinine, and urea. These results indicated that the optimized ILEP formulation had no detectable influence on normal growth, indicated no organs were negatively affected, and showed no evidence of toxicity when administered via the nasal route.With this study, the authors focused on developing an ILEP formulation for the treatment of ischemic stroke in diabetic patients. The release studies verified the sustained release potential of the optimized ILEP formulation, demonstrating its ability to increase the biological half-life and slow the elimination of ISP. Furthermore, the in vivo studies verified the improved neurological behavior and decreased edema, dead shrunken neurons, and vascular congestion of the rats treated with the optimized ILEP formulation, demonstrating its anti-stroke activity. In conclusion, our study found that treatment with an optimized ILEP formulation prevented the initiation and severity of stroke, especially in diabetic patients.