ObjectiveTo explore the protective effect and underlying mechanism of Gegen Qinliantang on cognitive function in db/db mice with diabetic cognitive impairment （DCI）. MethodsThirty-two 8-week-old male db/db mice were randomly assigned to the model group， dapagliflozin group （1.0 mg·kg^-1·d^-1）， low-dose Gegen Qinliantang group （6.24 g·kg^-1·d^-1）， and high-dose Gegen Qinliantang group （24.96 g·kg^-1·d^-1）. Eight db/m mice served as the normal group. All mice were administered the corresponding treatment once daily by gavage for 10 consecutive weeks. Body weight and fasting blood glucose （FBG） were dynamically monitored. The Morris water maze test was used to evaluate cognitive function. Hematoxylin-eosin （HE） staining and Nissl staining were used to observe pathological changes in the hippocampus. Enzyme-linked immunosorbent assay （ELISA） was employed to measure the levels of tumor necrosis factor-α （TNF-α） and interleukin-1β （IL-1β） in hippocampal tissue. Immunofluorescence double staining was used to detect the co-expression of M1 microglial marker CD16/32 and ionized calcium-binding adapter molecule 1 （IBA1） in the hippocampus. Western blot analysis was performed to detect the protein expression of postsynaptic density protein 95 （PSD-95）， synapsin （SYN）， nuclear factor-κB p65 （NF-κB p65）， and phosphorylated NF-κB p65 （p-NF-κB p65） in the hippocampus. ResultsCompared with the normal group， the model group showed significantly increased body weight and FBG levels （P<0.01）， significantly prolonged escape latency and reduced platform crossings in the Morris water maze test （P<0.01）， disordered arrangement of hippocampal neurons， nuclear pyknosis， increased neuronal necrosis， reduced Nissl bodies， decreased expression of synaptic proteins PSD-95 and SYN （P<0.01）， increased CD16/32⁺ /IBA1⁺ positive rate， elevated levels of TNF-α and IL-1β， and an increased p-NF-κB p65/NF-κB p65 ratio （P<0.01）. Compared with the model group， the dapagliflozin group exhibited significantly reduced FBG levels at weeks 5 and 10 （P<0.05， P<0.01） and increased body weight. The high-dose Gegen Qinliantang group showed significantly reduced FBG at week 10 （P<0.05）. Escape latency was significantly reduced on days 3 and 5 of the water maze test in the dapagliflozin group and on day 5 in the high-dose Gegen Qinliantang group （P<0.05）. Platform crossings were significantly increased in both the dapagliflozin group and the high-dose Gegen Qinliantang group （P<0.05）. Hippocampal pathological damage was alleviated to varying degrees in the dapagliflozin group and the low- and high-dose Gegen Qinliantang groups， with significantly increased expression of PSD-95 and SYN （P<0.01）. Further studies revealed that both low- and high-dose Gegen Qinliantang reduced hippocampal IL-1β levels and the CD16/32⁺/IBA1⁺ positive rate of microglia， while the high-dose group also significantly reduced hippocampal TNF-α levels and the p-NF-κB p65/NF-κB p65 （P<0.05， P<0.01）. ConclusionGegen Qinliantang can improve hyperglycemia， cognitive dysfunction， and synaptic damage in DCI， inhibit M1 polarization of microglia and neuroinflammation， and its mechanism may be related to the inhibition of NF-κB activation.

Purpose: Erectile dysfunction (ED) is a common male sexual dysfunction. Gut microbiota plays an important role in various diseases. To investigate the effects and mechanisms of intestinal flora dysregulation induced by high-fat diet (HFD) on erectile function. Materials and Methods: Male Sprague–Dawley rats aged 8 weeks were randomly divided into the normal diet (ND) and HFD groups. After 24 weeks, a measurement of erectile function was performed. We performed 16S rRNA sequencing of stool samples. Then, we established fecal microbiota transplantation (FMT) rat models by transplanting fecal microbiota from rats of ND group and HFD group to two new groups of rats respectively. After 24 weeks, erectile function of the rats was evaluated and 16S rRNA sequencing was performed, and serum samples were collected for the untargeted metabolomics detection. Results: The erectile function of rats and the species diversity of intestinal microbiota in the HFD group was significantly lower, and the characteristics of the intestinal microbiota community structure were also significantly different between the two groups. The erectile function of rats in the HFD-FMT group was significantly lower than that of rats in the ND-FMT group. The characteristics of the intestinal microbiota community structure were significantly different. In the HFD-FMT group, 27 metabolites were significantly different and they were mainly involved in the several inflammation-related pathways. Conclusions Intestinal microbiota disorders induced by HFD can damage the intestinal barrier of rats, change the serum metabolic profile, induce low-grade inflammation and apoptosis in the corpus cavernosum of the penis, and lead to ED.

Purpose: Erectile dysfunction (ED) is a common male sexual dysfunction. Gut microbiota plays an important role in various diseases. To investigate the effects and mechanisms of intestinal flora dysregulation induced by high-fat diet (HFD) on erectile function. Materials and Methods: Male Sprague–Dawley rats aged 8 weeks were randomly divided into the normal diet (ND) and HFD groups. After 24 weeks, a measurement of erectile function was performed. We performed 16S rRNA sequencing of stool samples. Then, we established fecal microbiota transplantation (FMT) rat models by transplanting fecal microbiota from rats of ND group and HFD group to two new groups of rats respectively. After 24 weeks, erectile function of the rats was evaluated and 16S rRNA sequencing was performed, and serum samples were collected for the untargeted metabolomics detection. Results: The erectile function of rats and the species diversity of intestinal microbiota in the HFD group was significantly lower, and the characteristics of the intestinal microbiota community structure were also significantly different between the two groups. The erectile function of rats in the HFD-FMT group was significantly lower than that of rats in the ND-FMT group. The characteristics of the intestinal microbiota community structure were significantly different. In the HFD-FMT group, 27 metabolites were significantly different and they were mainly involved in the several inflammation-related pathways. Conclusions Intestinal microbiota disorders induced by HFD can damage the intestinal barrier of rats, change the serum metabolic profile, induce low-grade inflammation and apoptosis in the corpus cavernosum of the penis, and lead to ED.

ObjectiveTo explore the protective effect and underlying mechanism of Gegen Qinliantang on cognitive function in db/db mice with diabetic cognitive impairment （DCI）. MethodsThirty-two 8-week-old male db/db mice were randomly assigned to the model group， dapagliflozin group （1.0 mg·kg^-1·d^-1）， low-dose Gegen Qinliantang group （6.24 g·kg^-1·d^-1）， and high-dose Gegen Qinliantang group （24.96 g·kg^-1·d^-1）. Eight db/m mice served as the normal group. All mice were administered the corresponding treatment once daily by gavage for 10 consecutive weeks. Body weight and fasting blood glucose （FBG） were dynamically monitored. The Morris water maze test was used to evaluate cognitive function. Hematoxylin-eosin （HE） staining and Nissl staining were used to observe pathological changes in the hippocampus. Enzyme-linked immunosorbent assay （ELISA） was employed to measure the levels of tumor necrosis factor-α （TNF-α） and interleukin-1β （IL-1β） in hippocampal tissue. Immunofluorescence double staining was used to detect the co-expression of M1 microglial marker CD16/32 and ionized calcium-binding adapter molecule 1 （IBA1） in the hippocampus. Western blot analysis was performed to detect the protein expression of postsynaptic density protein 95 （PSD-95）， synapsin （SYN）， nuclear factor-κB p65 （NF-κB p65）， and phosphorylated NF-κB p65 （p-NF-κB p65） in the hippocampus. ResultsCompared with the normal group， the model group showed significantly increased body weight and FBG levels （P<0.01）， significantly prolonged escape latency and reduced platform crossings in the Morris water maze test （P<0.01）， disordered arrangement of hippocampal neurons， nuclear pyknosis， increased neuronal necrosis， reduced Nissl bodies， decreased expression of synaptic proteins PSD-95 and SYN （P<0.01）， increased CD16/32⁺ /IBA1⁺ positive rate， elevated levels of TNF-α and IL-1β， and an increased p-NF-κB p65/NF-κB p65 ratio （P<0.01）. Compared with the model group， the dapagliflozin group exhibited significantly reduced FBG levels at weeks 5 and 10 （P<0.05， P<0.01） and increased body weight. The high-dose Gegen Qinliantang group showed significantly reduced FBG at week 10 （P<0.05）. Escape latency was significantly reduced on days 3 and 5 of the water maze test in the dapagliflozin group and on day 5 in the high-dose Gegen Qinliantang group （P<0.05）. Platform crossings were significantly increased in both the dapagliflozin group and the high-dose Gegen Qinliantang group （P<0.05）. Hippocampal pathological damage was alleviated to varying degrees in the dapagliflozin group and the low- and high-dose Gegen Qinliantang groups， with significantly increased expression of PSD-95 and SYN （P<0.01）. Further studies revealed that both low- and high-dose Gegen Qinliantang reduced hippocampal IL-1β levels and the CD16/32⁺/IBA1⁺ positive rate of microglia， while the high-dose group also significantly reduced hippocampal TNF-α levels and the p-NF-κB p65/NF-κB p65 （P<0.05， P<0.01）. ConclusionGegen Qinliantang can improve hyperglycemia， cognitive dysfunction， and synaptic damage in DCI， inhibit M1 polarization of microglia and neuroinflammation， and its mechanism may be related to the inhibition of NF-κB activation.

Purpose: Erectile dysfunction (ED) is a common male sexual dysfunction. Gut microbiota plays an important role in various diseases. To investigate the effects and mechanisms of intestinal flora dysregulation induced by high-fat diet (HFD) on erectile function. Materials and Methods: Male Sprague–Dawley rats aged 8 weeks were randomly divided into the normal diet (ND) and HFD groups. After 24 weeks, a measurement of erectile function was performed. We performed 16S rRNA sequencing of stool samples. Then, we established fecal microbiota transplantation (FMT) rat models by transplanting fecal microbiota from rats of ND group and HFD group to two new groups of rats respectively. After 24 weeks, erectile function of the rats was evaluated and 16S rRNA sequencing was performed, and serum samples were collected for the untargeted metabolomics detection. Results: The erectile function of rats and the species diversity of intestinal microbiota in the HFD group was significantly lower, and the characteristics of the intestinal microbiota community structure were also significantly different between the two groups. The erectile function of rats in the HFD-FMT group was significantly lower than that of rats in the ND-FMT group. The characteristics of the intestinal microbiota community structure were significantly different. In the HFD-FMT group, 27 metabolites were significantly different and they were mainly involved in the several inflammation-related pathways. Conclusions Intestinal microbiota disorders induced by HFD can damage the intestinal barrier of rats, change the serum metabolic profile, induce low-grade inflammation and apoptosis in the corpus cavernosum of the penis, and lead to ED.

Animal experiments are an essential component of life sciences and medical research. However, the external validity and reliability of individual animal studies are frequently challenged by inherent limitations such as small sample sizes, high design heterogeneity, and poor reproducibility, which impede the effective translation of research findings into clinical practice. Systematic reviews and meta-analysis represent a key methodology for integrating existing evidence and enhancing the robustness of conclusions. Currently, however, the application of systematic reviews and meta-analysis in the field of animal experiments lacks standardized guidelines for their conduct and reporting, resulting in inconsistent quality and, to some extent, diminishing their evidence value. To address this issue, this paper aims to systematically delineate the reporting process for systematic reviews and meta-analysis of animal experiments and to propose a set of standardized recommendations that are both scientific and practical. The article's scope encompasses the entire process, from the preliminary preparatory phase [including formulating the population， intervention， comparison and outcome （PICO） question, assessing feasibility, and protocol pre-registration] to the key writing points for each section of the main report. In the core methods section, the paper elaborates on how to implement literature searches, establish eligibility criteria, perform data extraction, and assess the risk of bias, based on the Preferred Reporting Items for Systematic Reviews and Meta-analysis （PRISMA) statement, in conjunction with relevant guidelines and tools such as Animal Research： Reporting of in Vivo Experiments （ARRIVE) and a risk of bias assessment tool developed by the Systematic Review Centre for Laboratory Animal Experimentation (SYRCLE). For the presentation of results, strategies are proposed for clear and transparent display using flow diagrams and tables of characteristics. The discussion section places particular emphasis on how to scientifically interpret pooled effects, thoroughly analyze sources of heterogeneity, evaluate the impact of publication bias, and cautiously discuss the validity and limitations of extrapolating findings from animal studies to clinical settings. Furthermore, this paper recommends adopting the Grading of Recommendations Assessment, Development and Evaluation (GRADE) methodology to comprehensively grade the quality of evidence. Through a modular analysis of the entire reporting process, this paper aims to provide researchers in the field with a clear and practical guide, thereby promoting the standardized development of systematic reviews and meta-analysis of animal experiments and enhancing their application value in scientific decision-making and translational medicine.

BACKGROUND:Meningeal lymphatic vessels can drain cerebral spinal fluid and amyloid β-protein,promoting T lymphocyte to transport and home to deep cervical lymph nodes.A simple,quick and definite method of dural separation and accurate localization of deep cervical lymph nodes can provide strong support for the study of neurodegenerative diseases.OBJECTIVE:To establish a convenient and practical method for exfoliating dural and deep cervical lymph nodes.METHODS:ICR mice,3 months old,were taken,anesthetized and injected with Evans blue and tracer in the occipital pool for localizing deep cervical lymph nodes.A midline incision of about 3 cm in length was made about 5 mm above the clavicle,the superficial fat and fascia were bluntly separated,and the lateral sternocleidomastoid muscle was pulled to expose the deep cervical lymph nodes,which were removed under a stereomicroscope and frozen at-80℃.Subsequently,the mouse head was cut and the skin and muscles of the head were separated to expose the entire skull structure.The skull and brain tissue were separated from the foramen magnum along the lower parietal bone with scissors,and the complete skull top was obtained.The skull was sequentially fixed in 40 g/L paraformaldehyde solution for 24 hours,120 g/L paraformaldehyde for 24 hours,and 120 g/L paraformaldehyde for 10,20,30,and 40 minutes,and the dural structure was stripped.The drainage capacity of meningeal lymphatic vessels and deep cervical lymphatic vessels was verified by tracer,and the meningeal lymphatic vessels were identified by the lymphatic vessel endothelial hyaluronan receptor 1 using the immunofluorescence method.RESULTS AND CONCLUSION:(1)Obvious blue staining was observed in deep cervical lymph nodes 15 minutes after Evans blue staining.(2)The skull was sampled and fixed in 120 g/L paraformaldehyde for 24 hours,resulting in a less tight connection between the dura mater and the skull,and easier stripping of the dural structures with an intact shape.The dura mater fixed at 120 g/L concentration was more resilient and remained more intact during peeling compared with the conventional 40 g/L concentration;120 g/L paraformaldehyde fixed meninges for a short time,and 30-40 minutes was preferred.(3)The frozen section of deep cervical lymph nodes showed the presence of the tracer,complete meningeal lymphatic vessels were visible in the dura mater,and the tracer was observed at the tail of lymphatic vessels.Immunofluorescence staining for endothelial hyaluronan receptor 1 was positive in the deep cervical lymph nodes and dural lymphatics.In summary,the best peeling concentration and time is 120 g/L paraformaldehyde fixed for 24 hours.At this concentration,the dura mater has a stretched morphology,a better toughness,and is more intact after peeling,which is conducive to later use.Verified by Evans blue,tracers and immunofluorescence,deep cervical lymph nodes are located accurately,which can be used as a basis for the study of various neurodegenerative diseases.

Objective To investigate the protective mechanism of ergothioneine against oxidative stress and inflammatory damage in vascular dementia(VaD)neurons through activation of the nuclear factor erythroid 2-related factor 2/heme oxygenase-1(Nrf2/HO-1)signaling pathway.Methods A total of 15 VaD patients(VaD group)admitted in our hospital from December 2023 to April 2024 were prospectively recruited,and another 15 healthy individuals taking physical examination during same period were subjected and served as health group.Then,plasma ergothioneine levels in the two groups were quantified by liquid chromatography-tandem mass spectrometry(LC-MS/MS).In in vitro experiments,hippocampal neurons were isolated from neonatal rats,and then divided into control,oxygen-glucose deprivation model,ergothioneine,and ergothioneine+ML385 groups.The expression levels of key molecules of Nrf2/HO-1 signaling pathway,oxidative stress markers,inflammatory cytokines,and apoptosis-related proteins were detected with RT-qPCR and Western blotting.Results The VaD patients exhibited significantly reduced plasma ergothioneine level(694.71±106.87 vs 1268.84±152.87)and down-regulated mRNA levels of Nrf2(0.49±0.14 vs 1.00±0.17),HO-1(0.35±0.23 vs 1.00±0.14)and NQO1(0.39±0.16 vs 1.00±0.12)than the health group(P＜0.05,P＜0.01).Compared with the control group,the model group showed significant increases in inflammatory factor levels.Ergothioneine treatment resulted in significant decreases in malondialdehyde and reactive oxygen species contents,and neuronal apoptosis rate.The ergothioneine+ML385 group showed a significant increase in hippocampal neuronal apoptotic rate,when compared with the ergothionein group.Conclusion Ergothioneine protects VaD neurons against oxidative stress and inflammatory damage by activating the Nrf2/HO-1 signaling pathway.

Objective:To explore the lifestyle pattern of the physical examination population and analyze its association with metabolic associated fatty liver disease (MAFLD).Methods:This was a cross-sectional study. Based on the data of 196 515 physical examination individuals from the Health Management Center of the Third Xiangya Hospital of Central South University from January 2016 to December 2020, the subjects were grouped and characterized by principal component analysis and cluster analysis. Among them, 137 277 cases with MAFLD diagnosis information were included in the association analysis between lifestyle pattern and MAFLD. The differences in lifestyle pattern choice among different age, sex, education level, marital status, occupational category and medical insurance type and their differences with the risk of MAFLD were analyzed. The generalized linear mixed model was used to control confounding factors and then association analysis was conducted.Results:There were 6 types of lifestyle patterns in the physical examination population, which were respectively: indulgent type-both physical and mental damage, remedial type-excessive diet, giving type-unique intensity, comfortable type-natural health, heavy smoking type-sedentary injury, heavy drinking type-attempting to make up, accounting for 7.29%, 9.62%, 7.43%, 52.16%, 9.77%, 13.73% in the population. Among them, the male lifestyle pattern was mainly the indulgent type, the remedial type, the heavy smoking type and the heavy drinking type, showing the characteristics of unhealthy lifestyle pattern; Women tended to have healthier lifestyle patterns. After association analysis with MAFLD, it was found that the prevalence of MAFLD was more than 50% in the people who belonged to the indulgent type, remedial type, the heavy smoking type and the heavy drinking type (53.62%, 57.06%, 51.25% and 50.50%, respectively), and the prevalence of MAFLD in the giving type group was 40.17%. The risk of MAFLD in comfortable group was relatively low (28.25%), and the difference in risk of MAFLD among all modes was statistically significant after controlling for confounding factors ( P<0.001). Conclusion:According to cluster mining, there are 6 types of lifestyle patterns in the physical examination population, and the healthier lifestyle pattern has a lower risk of MAFLD.

Purpose: Erectile dysfunction (ED) is a common male sexual dysfunction. Gut microbiota plays an important role in various diseases. To investigate the effects and mechanisms of intestinal flora dysregulation induced by high-fat diet (HFD) on erectile function. Materials and Methods: Male Sprague–Dawley rats aged 8 weeks were randomly divided into the normal diet (ND) and HFD groups. After 24 weeks, a measurement of erectile function was performed. We performed 16S rRNA sequencing of stool samples. Then, we established fecal microbiota transplantation (FMT) rat models by transplanting fecal microbiota from rats of ND group and HFD group to two new groups of rats respectively. After 24 weeks, erectile function of the rats was evaluated and 16S rRNA sequencing was performed, and serum samples were collected for the untargeted metabolomics detection. Results: The erectile function of rats and the species diversity of intestinal microbiota in the HFD group was significantly lower, and the characteristics of the intestinal microbiota community structure were also significantly different between the two groups. The erectile function of rats in the HFD-FMT group was significantly lower than that of rats in the ND-FMT group. The characteristics of the intestinal microbiota community structure were significantly different. In the HFD-FMT group, 27 metabolites were significantly different and they were mainly involved in the several inflammation-related pathways. Conclusions Intestinal microbiota disorders induced by HFD can damage the intestinal barrier of rats, change the serum metabolic profile, induce low-grade inflammation and apoptosis in the corpus cavernosum of the penis, and lead to ED.