Animal experiments are essential tools in biomedical research, serving as a bridge between basic research and clinical trials. Systematic reviews and meta-analyses (SRs/MAs) of animal experiments are crucial methods for integrating evidence from animal experiment, which can facilitate the translation of findings into clinical research, reduce translational risks, and promote resource integration in basic research. With the continuous development of the Grading of Recommendations, Assessment, Development, and Evaluation (GRADE) methodology, its application in SRs/MAs of animal experiments has gained increasing attention. This article first outlines the principles and specific applications of the GRADE methodology in SRs/MAs of animal experiments, including qualitative descriptive systematic reviews, meta-analyses, and network meta-analyses. It then deeply analyzes the misuse of the GRADE methodology in practice, including incorrect evidence grading, improper classification of evidence, misapplication in qualitative systematic reviews, inconsistencies between the documentation of the upgrading and downgrading process and results, and inappropriate use for making recommendations. Furthermore, this article comprehensively discusses the factors influencing the grading of evidence certainty in SRs/MAs of animal experiments, including the impact of bias risk, indirectness, inconsistency, imprecision, and publication bias on evidence downgrading, as well as the role of large effect sizes and cross-species consistency in evidence upgrading. Finally, in response to the issues discussed, improvement strategies are proposed, including further research and optimization of the GRADE methodology for SRs/MAs of animal experiments, the development of reporting guidelines tailored to the characteristics of SRs/MAs in animal experiment research, and enhanced professional training for researchers in the GRADE methodology. This article aims to improve the quality of evidence in SRs/MAs of animal experiments, strengthen their reliability in clinical decision-making, and promote the more efficient translation of findings from animal experiment research into clinical practice.

ObjectiveTo investigate the recurrence of ischemic stroke (IS) and to analyze the association between four indices of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and triglycerides (TG) and the risk of IS recurrence by analyzing the follow-up data related to IS in the community-based natural population of Songjiang District, Shanghai, so as to provide a scientific basis for improving the prognosis of stroke patients in the community and controlling IS recurrence. MethodsA prospective follow-up study was conducted among the IS patients in the community-based cohort population, collecting data about patient’s age, gender, disease history, biochemical indicators, and etc. Cox regression model and restricted cubic spline model were used to analyze the relationship between different levels of plasma lipids and the recurrence of IS in these patients. ResultsA total of 1 368 patients with IS were included. The total follow-up duration was 7 171.46 person-years, with a median follow-up time of 6.24 years. There were 420 cases of IS recurrence, resulting in a cumulative recurrence rate of 30.70%. The results of multivariate Cox regression analysis showed that the recurrence risk of IS was reduced when the baseline TC and LDL-C levels of IS patients were in the ranges of 4.65‒5.67 mmol·L^-1 and 2.52‒3.46 mmol·L^-1, respectively. The results of restricted cubic spline analysis showed a U-shaped relationship between baseline TC and LDL-C levels and the recurrence risk in IS patients. ConclusionThe cumulative recurrence rate of patients with IS in the community of Songjiang District in Shanghai is high, and the levels of TC and LDL-C at baseline survey are correlated with the recurrence of IS in these patients. It is suggested to pay more attention to the levels of LDL-C and TC in patients with IS, so as to improve the prognosis.

The Ehlers-Danlos syndromes(EDS)are a group of rare hereditary connective tissue disorders characterized by joint hypermobility,skin hyperextensibility,and tissue fragility.The clinical and genetic hetero-geneity of EDS frequently leads to underdiagnosis and misdiagnosis.Genetic testing is an essential approach to clarify the underlying diagnosis.Recent research has preliminarily established genotype-phenotype correlations and introduced the novel concept of"disease spectrum"in some subtypes.These studies deepen our under-standing of EDS etiology and provide important insights into clinical management.Published in 2023,the Chinese Guidelines for Diagnosis and Treatment of the Ehlers-Danlos Syndromes(the Guidelines)recommend performing genetic testing with deep phenotyping for patients who meet the clinical diagnostic criteria or are sus-pected of having EDS.However,it should be noted that the clinical diagnosis might differ from the molecular diagnosis.Furthermore,cutting-edge approaches such as periodic data reanalysis,integration of RNA sequen-cing into family-based whole-genome sequencing,and third-generation sequencing may facilitate the reclassifi-cation of variants of uncertain significance or resolve undiagnosed cases.This article summarizes recent progress in the genetics research of EDS,with the hope of offering a valuable resource for clinical diagnosis,treatment and scientific research to optimize the quality of life of patients with EDS.

Objective To investigate the liver disease phenotypes and clinical features of patients with different genotypes of Wilson's disease(WD).Methods A retrospective analysis was performed for 163 patients with WD who were diagnosed and underwent genetic testing in The Fifth Medical Center of Chinese PLA General Hospital from August 2008 to June 2023,and clinical manifestations,laboratory examination,pathological examination,imaging examination,and ATP7B genetic testing results were collected.According to ATP7B gene mutation,the patients were divided into groups as follows:R778L mutation group and non-R778L mutation group;P992L mutation group and non-P992L mutation group;truncation mutation group and non-truncation mutation group.Liver disease phenotypes and clinical features were analyzed for the patients with c.2333G>T/p.R778L mutation(R778L mutation),c.2975C>T/p.P992L mutation(P992L mutation),and truncation mutation of the ATP7B gene.The Mann-Whitney U test or the Kruskal-Wallis H test was used for comparison of continuous data between groups,and the chi-square test or the Fisher's exact test was used for comparison of categorical data between groups.Results The 163 patients with WD had varying severities of liver disease phenotypes,among whom 121(74.23%)were diagnosed with chronic liver disease,36(22.09%)were diagnosed with decompensated cirrhosis,and 6(3.68%)were diagnosed with fulminant WD,and in addition,there were 5 patients(2 with chronic liver disease and 3 with decompensated cirrhosis)with neurological abnormalities.For the 163 patients with WD,R778L mutation(with an allele frequency of 28.2%)was the most common mutation in the ATP7B gene,followed by P992L mutation(with an allele frequency of 12.6%),and truncation mutation showed an allele frequency of 11.0%.There was no significant difference in the distribution of the three mutations across different liver disease phenotypes(P>0.05).The R778L mutation group had a significantly lower level of ceruloplasmin(CP)than the non-R778L mutation group[0.04(0.02-0.08)g/L vs 0.08(0.03-0.13)g/L,Z=-2.889,P=0.004].Compared with the non-P992L mutation group,the P992L mutation group had significantly higher levels of alanine aminotransferase[135.0(80.5-237.0)U/L vs 80.5(36.0-173.3)U/L,Z=2.684,P=0.007]and aspartate aminotransferase[121.4(77.0-195.0)U/L vs 84.0(39.0-123.3)U/L,Z=3.388,P<0.001].Compared with the non-truncation mutation group,the truncation mutation group had significantly lower levels of CP[0.03(0.02-0.08)g/L vs 0.06(0.03-0.11)g/L,Z=-3.136,P=0.002]and serum copper[3.20(2.15-5.00)mg/L vs 4.20(2.60-7.50)mg/L,Z=-2.296,P=0.025].Conclusion R778L mutation,P992L mutation and truncation mutation are not associated with liver disease phenotype in WD patients;however,R778L mutation is associated with a lower level of CP,P992L mutation is associated with higher levels of ALT and AST,and truncation mutation is associated with lower levels of CP and serum copper.

Integrating evidence from animal experiments is a critical component of biomedical research, providing essential prior information for in-depth investigations of disease mechanisms and new drug development. Animal models have played an irreplaceable role in simulating human diseases. However, the integration of evidence from animal experiments has faced numerous challenges, including insufficient emphasis, significant heterogeneity in study designs, high publication bias, and discrepancies with clinical research practices. This paper first identifies existing issues in the original research evidence from animal experiments, such as the selection and applicability of animal models, considerations in the design of experimental studies, and factors influencing the translation of animal experimental evidence. It then discusses various methods for integrating this evidence, including systematic review and meta-analysis, overview of systematic review/umbrella review, scoping review, and evidence mapping, while highlighting recent advancements in their application. Finally, the paper addresses the main challenges currently encountered in the integration of evidence from animal experiments and proposes targeted improvement strategies aimed at enhancing the efficiency of translating research outcomes into clinical practice and promoting the advancement of evidence-based medicine. By continuously optimizing original experimental research protocols and evidence integration practices, this work aims to establish a more efficient and scientific environment for the synthesis of evidence from animal experiments, ultimately contributing to clinical trials and human health.

Objective To investigate the mechanism underlying the neuroprotective effect of linarin(LIN)against microglia activation-mediated inflammation and neuronal apoptosis following spinal cord injury(SCI).Methods Fifty C57BL/6J mice(8-10 weeks old)were randomized to receive sham operation,SCI and linarin treatment at 12.5,25,and 50 mg/kg following SCI(n=10).Locomotor function recovery of the SCI mice was assessed using the Basso Mouse Scale,inclined plane test,and footprint analysis,and spinal cord tissue damage and myelination were evaluated using HE and LFB staining.Nissl staining,immunofluorescence assay and Western blotting were used to observe surviving anterior horn motor neurons in injured spinal cord tissue.In cultured BV2 cells,the effects of linarin against lipopolysaccharide(LPS)-induced microglia activation,inflammatory factor release and signaling pathway changes were assessed with immunofluorescence staining,Western blotting,RT-qPCR,and ELISA.In a BV2 and HT22 cell co-culture system,Western blotting was performed to examine the effect of linarin against HT22 cell apoptosis mediated by LPS-induced microglia activation.Results Linarin treatment significantly improved locomotor function(P<0.05),reduced spinal cord damage area,increased spinal cord myelination,and increased the number of motor neurons in the anterior horn of the SCI mice(P<0.05).In both SCI mice and cultured BV2 cells,linarin effectively inhibited glial cell activation and suppressed the release of iNOS,COX-2,TNF-α,IL-6,and IL-1β,resulting also in reduced neuronal apoptosis in SCI mice(P<0.05).Western blotting suggested that linarin-induced microglial activation inhibition was mediated by inhibition of the TLR4/NF-κB signaling pathway.In the cell co-culture experiments,linarin treatment significantly decreased inflammation-mediated apoptosis of HT22 cells(P<0.05).Conclusion The neuroprotective effect of linarin is medicated by inhibition of microglia activation via suppressing the TLR4/NF-κB signaling pathway,which mitigates neural inflammation and reduce neuronal apoptosis to enhance motor function of the SCI mice.

Objective:To describe the epidemiological characteristics and trends of leukemia incidence in Qidong between 1972 and 2021, and provide guidelines for prevention and control measures and strategies.Methods:The cancer registry data was collected and analyzed on leukemia incidence during 1972—2021 in Qidong by sex, age and time. Crude incidence rate (CR), China age-standardized rate (ASRC), world age-standardized rate (ASRW), and average annual change percentage (AAPC) was calculated by Joinpoint software. Age-period-cohort (APC) model was used to analyze the influence of age, period and birth cohort on the changes in the incidence trend of leukemia patients.Results:From 1972 to 2021, there were 2 948 patients with leukemia in Qidong, accounting for 2.00% of all cancer new cases, CR of leukemia was 5.26/10 5, ASRC was 4.34/10 5, ASRW was 4.35/10 5. The truncated incidence of 35—64 years old was 5.29/10 5, the cumulative incidence rate between the ages of 0 and 74 years old was 0.40%, the cumulative risk was 0.40%. There were 1 608 male patients, the CR, ASRC, and the ASRW were 5.81/10 5, 4.88/10 5 and 4.85/10 5. The number of female patients were 1 340, and the CR, ASRC, and the ASRW were 4.71/10 5, 3.86/10 5 and 3.91/10 5, respectively. Temporal trends indicated significant upward trends in ASRC among both gender, males and females with AAPC values of 1.41% ( P＜0.001), 1.15% ( P＜0.001), and 1.73% ( P＜0.001), respectively. The results of the APC model showed that the average net drift value of leukemia incidence in all age groups was 1.57% (95% CI, 1.24%-1.89%), and the highest value of local drift was 3.20% (95% CI, 1.63%-4.78%) in the 80~ years old group. The incidence of leukemia increased with age. With the passage of time, the risk of leukemia incidence increased gradually compared with the rate ratio of leukemia incidence (risk ratio [ RR], 1.00) in 1992—1996, the RR of leukemia incidence increased from 0.70 during 1972—1976 to 1.57 during 2017—2021. The later the cohort was born, the greater the risk of leukemia incidence compared with the relative risk of leukemia incidence ( RR, 1.00) in 1952—1956 cohort, the RR of leukemia incidence increased from 0.24 in the 1892—1896 cohort to 2.73 in the 2017—2021 cohort. Conclusions:The incidence of the leukemia has presented a rising trend in the past fifty years. Leukemia incidence increased with age, and the period and cohort effects on the risk of incidence increase. Further research is needed to investigate the risk factors related to leukemia.

Objective : To investigate the protective effect of melatonin (MT) on formaldehyde (FA) inhalation-in- duced acute lung injury (ALI) in rats and its mechanism through the regulation of nuclear factor E2-related factor 2 (Nrf2) signaling pathway． Methods : Fifty female Wistar rats were randomly divided into Control group ，FA group，FA + MT 5 mg / kg group，FA + MT 10 mg / kg group and FA + MT 20 mg / kg group，with 10 rats in each group．Except for the Control group，all other groups inhaled 3 mg / m3 FA daily for 21 d consecutively to construct the tainted model，and then treated with different MT doses for 14 d．The tainting was continued during the MT treatment．Hematoxylin-eosin (HE) staining was used to observe the histopathological changes in lung tissue，lung water content and lung coefficient were weighed and measured，glutathione ( GSH) ，superoxide dismutase (SOD) and 8-hydroxydeoxyguanosine ( 8-OHdG) levels were measured by absorbance photometric method ，and enzyme linked immunosorbent assay(ELISA) was used to measure the levels of tumor necrosis factor-alpha (TNF-α) ，in- terleukin (IL) -6，and IL-1 β concentrations，Western blot to detect the protein expression levels of Nrf2，heme ox- ygenase-1 (HO-1) ，nuclear factor-κB ( NF-κB) ，and phosphorylated nuclear factor-κB ( p-NF-κB) in lung tis- sues，and quantitative polymerase chain reaction(qPCR) to detect the Nrf2，HO-1，and Kelch-like ECH-associated protein 1 (Keap1) mRNA expression levels. Results : Compared with the control group，lung injury was obvious in rats in the FA group ; lung tissue GSH and SOD levels were reduced ，and 8-OHdG levels were elevated ( P < 0. 05) ; alveolar lavage fluid TNF-α , IL-6，and IL-1 β levels were elevated (P＜0. 05) ; Nrf 2 and HO-1 protein expression levels were reduced in the lung tissue (P＜0. 05) ，and p-NF-κB protein expression levels were was ele- vated (P＜0. 05) ; the relative mRNA expression of Nrf2 and HO-1 in lung tissue was decreased，and the relative mRNA expression of Keap1 was elevated (P＜0. 05) ．Compared with the FA group，the lung injury of rats in the MT group was improved ; the levels of GSH and SOD in the lung tissue were increased (P＜0. 05) ，and the level of 8-OHdG was decreased (P＜0. 05) ; the levels of TNF-α , IL-6，and IL-1 β in the alveolar lavage fluid were de- creased (P＜0. 05) ; and the expression levels of the Nrf2 and HO-1 proteins in the lung tissue were increased (P ＜0. 05) ．p-NF-κB protein expression level was decreased (P ＜0. 05) ; the relative mRNA expression levels of Nrf2 and HO-1 in lung tissues were increased (P＜0. 05) ，and the relative mRNA expression level of Keap1 was decreased (P＜0. 05) in lung tissues，and all of them were in a dose-dependent manner． Conclusion MT can al- leviate oxidative stress and inflammatory responses and mitigate FA exposure-induced acute lung injury by regula- ting the Nrf2 / Keap1 / HO-1 signaling pathway.

Objectives:To investigate the early related factors for hepatic insufficiency after hemihepatectomy and to construct and validate a nomogram model.Methods:This is a retrospective cohort study.There were 207 patients with liver tumor who underwent hemihepatectomy in the Department of Hepatobiliary Surgery, Cancer Hospital, Chinese Academy of Medical Sciences from October 2016 to December 2022. Using the random number method, patients were randomly divided into a model group( n=166) and a validation group( n=41) according to an 4∶1 ratio. There were 118 males and 48 females in the modeling group,with an age ( M(IQR)) of 59.0(13.3) years (range: 22.0 to 81.0 years),42 patients in the group with postoperative liver insufficiency and 124 patients in the group without postoperative liver insufficiency. There were 32 males and 9 females in the validation group, with an age of 54.0(19.0) years (range: 25.0 to 81.0 years). The first results of the peripheral blood test of patients within 24 hours after surgery were collected,and the independent related factors for incomplete postoperative liver function were determined by multivariate Logistic regression analysis,and related factors of postoperative incomplete liver function were screened by best subset selection. A nomogram model of the related factors of postoperative hepatic insufficiency after hemihepatectomy was constructed using R software,validated by internal and external validation of the model. Results:Multivariate logistic regression analysis showed that elevated D-dimer level and decreased antithrombin Ⅲ (AT-Ⅲ) activity within 24 hours after surgery were independent related factors for the development of postoperative hepatic insufficiency in hemihepatectomized patients. The results of the best subset selection showed that ALT, D-dimer, and AT-Ⅲ activity levels within 24 hours postoperatively were the most relevant factors for postoperative hepatic insufficiency. The R software was applied to build a nomogram prediction model based on the above three indicators in the model set, and the receiver operating characteristic(ROC) curve of the model showed an area under the curve of 0.803 and the calibration curve showed a U-index of -0.012 for the model( P=0.977). The results of the clinical decision analysis and the clinical impact curve indicated that the model had good clinical utility. The internal validation results of the Bootstrap method suggested that the model had reasonable consistency. The area under the ROC curve of the validation group model was 0.806, suggesting that the model had a good generalization prediction ability. Conclusions:The levels of ALT, D-dimer, and AT-Ⅲ activity within 24 hours after hemihepatectomy are valuable indicators for predicting liver insufficiency after hemihepatectomy. The nomogram model is reliable and can be used as an indicator for close postoperative monitoring.

Objective:To assess the value of serum alpha-fetoprotein(AFP),protein induced by vitamin K absence or antagonist-Ⅱ(PIVKA-Ⅱ)and glypican-3(GPC-3)in the diagnosis of hepatocellular carcinoma(HCC).Methods:Studies of AFP,PIVKA-Ⅱ,GPC-3 or in combination for the diagnosis of HCC since 2002 were searched in PubMed,Web of Science and Embase databases.The literature was screened according to the inclusion and exclusion criteria,the quality of the included articles was evaluated by QUADAS checklist,and relevant data were extracted by Meta DiSc,Review Manager 5.4 and Stata 15.1.The diagnostic values of AFP,PIVKA-Ⅱ and GPC-3 alone or in combination for HCC were assessed with receiver operating characteristic(ROC)curve.Results:A total of 32 articles were included in the study.Meta-analysis showed that when a single marker was used to diagnose HCC,the area under the ROC curve(AUC)of PIVKA-Ⅱ was the highest(0.88,95%CI:0.85-0.91),followed by GPC-3 and AFP.The AUC of combination of serum markers was higher than that of a single marker,and the AUC of PIVKA-Ⅱ combined with GPC-3 was the highest(0.90,95%CI:0.87-0.92).When a single marker was used for diagnosis,the sensitivity of PIVKA-Ⅱ and GPC-3 were relatively high(0.75 and 0.76),while the specificity of PIVKA-Ⅱ(0.88)and AFP(0.87)were higher than that of GPC-3(0.81).The sensitivity of the combination of serum markers was higher than that of a single marker,while the specificity was not significantly improved.When a single marker is used to diagnose HCC,the diagnostic odds ratio(DOR)of PIVKA-Ⅱ was the highest(22,95%CI:13-36),followed by GPC-3 and AFP.The DOR of the combination of two markers in the diagnosis of HCC was higher than that of a single marker,and the DOR of AFP combined with GPC-3 was the highest(25,95%CI:9-67).The DOR of the combination of the three markers was significantly reduced to 10(95%CI:7-45).Conclusions:When a single marker is used,PIVKA-Ⅱhas a higher diagnostic value for HCC.The combination of two markers can significantly improve the diagnostic sensitivity,and AFP combined with PIVKA-Ⅱ is recommended for the diagnosis of HCC.The combination of all three markers failed to further improve the diagnostic value.