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.

BACKGROUND:Deep muscle stimulation has the effects of releasing muscle adhesion,relieving muscle spasm,improving and restoring muscle compliance and elasticity.Electromyographic biofeedback therapy can promote nerve recovery and improve lower limb motor function and gait. OBJECTIVE:To observe the effect of the effect of deep muscle stimulation combined with electromyographic biofeedback therapy on the spasm of the triceps surae and gait changes after stroke by using a digital muscle detector and three-dimensional gait analysis system. METHODS:A total of 72 patients who met the inclusion criteria were selected from the Rehabilitation Department of the First Affiliated Hospital of Guangdong Pharmaceutical University from October 2020 to October 2023.And they were enrolled and randomly divided into two groups(n=36 per group):a control group and a combined group.The control group received routine rehabilitation therapies,electromyographic biofeedback and pseudo deep muscle stimulation,while the combined group received true deep muscle stimulation treatment on the basis of the control group,five times per week,for 4 consecutive weeks.The oscillation frequency and dynamic stiffness of the affected gastrocnemius muscle,active range of motion of the ankle dorsiflexion muscle,electromyographic signal of the tibialis anterior muscle,Fugl-Meyer assessment of the lower limbs,and three-dimensional gait analysis parameters were statistically analyzed before and after treatment in two groups. RESULTS AND CONCLUSION:After treatment,oscillation frequency and dynamic stiffness values of the inner and outer sides of the affected gastrocnemius muscle in both groups of patients were significantly reduced compared with before treatment(P<0.05),and the combined group showed a more significant decrease compared with the control group(P<0.05).The active range of motion of the ankle dorsiflexion muscle,electromyographic signal of the tibialis anterior muscle,and Fugl-Meyer scores after treatment were significantly increased or improved compared with before treatment(P<0.05),while the combined group showed a more significant increase or improvement compared with the control group(P<0.05).In terms of gait parameters,the walking speed,frequency,and stride in both groups of patients were significantly increased compared with before treatment(P<0.05),while the combined group showed a more significant increase compared with the control group(P<0.05).The percentage time of support phase on the healthy side was shortened compared with before treatment(P<0.05),while the combined group showed a more significant decrease compared with the control group(P<0.05).In addition,there was no significant difference between the two groups except for the percentage of healthy side support(P>0.05).To conclude,the combination of deep muscle stimulation and electromyographic biofeedback can effectively alleviate triceps spasm in the short term after stroke,improve ankle dorsiflexion function,enhance lower limb motor function,and improve gait.The treatment effect is significant and worthy of clinical promotion and application.

Objective: To investigate the effect of auricular therapy on sleep improvement and the GABAergic system pathway in a rat model of insomnia and to explore its possible mechanism. Methods: According to the random number table, 60 male SD rats were randomly divided into blank control, model, auricular point sticking, auricular bloodletting, and auricular bloodletting combined with sticking groups, with 12 rats per group. Insomnia was induced by intraperitoneal injection of p-chlorophenylalanine. After establishing the insomnia model, 36 rats were treated once a day with auricular point sticking or bloodletting for 5 consecutive days. After the intervention, the general condition and body weight of rats were observed; the righting reflex test was used to detect the sleep latency and duration; HE staining was used to observe the morphology of hypothalamic neuron cells; and an enzyme-linked immunosorbent assay was used to detect the GABA and glutamate content in rat serum. Immunohistochemistry(IHC) and real-time fluorescence quantitative PCR were used to detect GABA ARα1 and GABA ARγ2 protein and mRNA expression in the hypothalamus of rats, and Western blotting(WB) was used to detect GABA ARα1, GABA ARγ2, GAD65/67, GAT-1, and GABA-T protein expression in the hypothalamus of rats. Results: Compared with the blank control group, the model group had a lower body weight, a significantly shorter sleep duration (P<0.05), severe damage to the morphological structure of hypothalamic neurons with disordered cell arrangement, larger intercellular gaps, enlarged cell bodies, and a vacuolated appearance. All the intervention groups had significantly higher body weight and longer sleep duration than the model group (P<0.05). Compared with the other intervention groups, the auricular point sticking group had a longer sleep duration (P<0.05), and the hypothalamic neuron cells in all intervention groups improved, with the auricular point sticking group showing more apparent improvement. The model group had a lower GABA and higher glutamate contents, and GABA ARα1, GABA ARγ2, and GAD65/67 protein expression in the hypothalamus were lower than in the blank control group. In contrast, GAT-1 and GABA-T protein expression was higher, and GABA ARα1 and GABA ARγ2 mRNA expression was lower (P<0.05). The serum GABA content in the auricular point sticking and auricular bloodletting groups was higher, and the serum glutamate content in the auricular point sticking and auricular bloodletting combined sticking groups was lower than in the model group. GABA ARα1 mRNA expression in the hypothalamus of each intervention group was significantly increased, and GABA ARγ2 mRNA expression in the hypothalamus of the auricular point sticking and auricular bloodletting combined sticking groups increased. GABA ARα1(IHC, WB), GABA ARγ2(WB), and GAD65/67 protein expression in the hypothalamus of the auricular point sticking group increased, whereas GAT-1 and GABA-T protein expression decreased. GABA ARα1 and GABA ARγ2 protein expression(IHC, WB) in the hypothalamus of the auricular bloodletting group increased, whereas GABA-T protein expression decreased. GABA ARγ1(IHC) and GABA ARγ2(WB) protein expression in the hypothalamus of the auricular bloodletting combined sticking group increased, whereas GAT-1 and GABA-T protein expression decreased (P<0.05). Compared with in the inventation groups, the serum GABA content in the auricular point sticking group increased, the serum glutamate content decreased, GABA ARα1 and GABA ARγ2 mRNA expression in the hypothalamus increased, and GABA ARα1(IHC), GAD65/67 protein expression increased. In contrast, GABA-T protein expression decreased (P<0.05), and GABA ARγ2 protein expression(IHC) in the hypothalamus of the auricular bloodletting group increased (P<0.05). Conclusion Auricular therapy, particularly auricular point sticking, may have modulated the GABAergic system pathway by upregulating hypothalamic GABA ARα1, GABA ARγ2, and GAD65/67 protein expression while downregulating GAT-1 and GABA-T protein expression to alleviate symptoms in an insomnia rat model.

ObjectiveBy comparing the composition and content of volatile components in raw products， wine-washed products and wine-fried products of Ligusticum sinense cv. Chaxiong rhizome（LSCR）， to investigate the influence of wine processing on the volatile components of LSCR， in order to provide a basis for the development of quality standards for LSCR and its processed products. MethodsElectronic nose was used to identify the odors of LSCR， wine-washed and wine-fried LSCR， and their volatile components were detected by headspace gas chromatography-mass spectrometry（HS-GC-MS）， and the relative mass fractions of these components were determined by peak area normalization method. Principal component analysis（PCA） and orthogonal partial least squares-discriminant analysis（OPLS-DA） were performed on the obtained sample data by SIMCA 14.1 software， and the differential components of LSCR， wine-washed and wine-fried LSCR were screened according to the variable importance in the projection（VIP） value>1. Pearson correlation analysis was used to explore the relationship between volatile differential flavor components and electronic nose sensors. ResultsElectronic nose detection results showed that there were significant differences in the odors of LSCR， wine-washed and wine-fried LSCR， mainly reflected in the sensors S2， S4， S5， S6， S11， S12， S13. And a total of 62 compounds were identified from LSCR and its wine-processed products， among which 46， 50 and 51 compounds were identified from LSCR， wine-fried and wine-washed LSCR， respectively. There were 21 differential components between the raw products and wine-fried products， of which 10 components were increased and 11 were decreased after processing. There were 20 differential components between the raw products and wine-washed products， of which 11 constituents increased and 9 decreased after processing. There were 17 differential components between the wine-wash products and wine-fried products. Compared with the wine-washed products， the contents of 13 components in the wine-fried products increased， and the contents of 4 components decreased. The increasing trend of the content of phthalides in the wine-washed products was more obvious than that in the wine-fried products， but the content of total volatile components was higher in the wine-fried products than the wine-washed products. Correlation analysis showed that there were different degrees of correlation between the 7 differential sensors of electronic nose and 24 differential volatile components， mainly phthalides and olefins. ConclusionThe odor and the content of volatile components in LSCR changed obviously after wine processing， and n-butylphthalide， Z-butylidenephthalide and E-ligustilide can be used as the candidate differential markers of volatile components in LSCR before and after wine processing.

Methods: This study enrolled 40 patients with AIS, 20 patients with congenital scoliosis (CS), and 20 patients with spinal degenerative disease (SDD). All patients underwent open posterior surgery in our hospital, and a paravertebral muscle (multifidus muscle) biopsy was performed intraoperatively. This study included many indexes that describe muscle, especially dystrophin staining. The above pathological results were compared among the AIS, CS, and SDD groups. The correlation between the Cobb angle and Nash–Moe classification and the above pathological results was analyzed in patients with AIS. Results: Significant reductions in the dystrophin staining of dystrophin-1 (p<0.001), dystrophin-2 (p<0.001), and dystrophin-3 (p<0.001) were observed in the AIS group than in the CS and SDD groups. The higher the Nash–Moe classification in the AIS group, the more significant the loss of dystrophin-2 (p=0.042) in the convex paraspinal muscles. Additionally, a significantly positive correlation was observed between the reductions of dystrophin-2 on the concave side of the AIS group and Cobb angle (p=0.011). Conclusions Dystrophin protein deficiency in the paraspinal muscles plays a crucial role in AIS formation and progression. The severity of scoliosis in patients with AIS is correlated with the extent of dystrophin loss in the paravertebral muscles. Therefore, dystrophin dysfunction may be relevant to AIS occurrence and development.

Metabolites produced as intermediaries or end-products of microbial metabolism provide crucial signals for health and diseases, such as metabolic dysfunction-associated steatotic liver disease (MASLD). These metabolites include products of the bacterial metabolism of dietary substrates, modification of host molecules (such as bile acids [BAs], trimethylamine-N-oxide, and short-chain fatty acids), or products directly derived from bacteria. Recent studies have provided new insights into the association between MASLD and the risk of developing chronic kidney disease (CKD). Furthermore, alterations in microbiota composition and metabolite profiles, notably altered BAs, have been described in studies investigating the association between MASLD and the risk of CKD. This narrative review discusses alterations of specific classes of metabolites, BAs, fructose, vitamin D, and microbiota composition that may be implicated in the link between MASLD and CKD.

Background/Aims: Liver cirrhosis involves chronic inflammation and progressive fibrosis.Among various immune cells, CD8+ T cells are considered a major contributor to hepatic inflammation and fibrosis. However, the exact molecular pathways governing CD8+ T-cell-mediated effects in cirrhosis remain unclear. Methods: This study analyzed transcriptomic and single-cell sequencing data to elucidate CD8+ T-cell heterogeneity and implications in cirrhosis. Results: Weighted gene co-expression analysis of bulk RNA-seq data revealed an association between cirrhosis severity and activated T-cell markers like HLA and chemokine genes. Furthermore, single-cell profiling uncovered eight CD8+ T-cell subtypes, notably, effector memory (Tem) and exhausted (Tex) T cells. Tex cells, defined by PDCD1, LAG3, and CXCL13 expression, were increased in cirrhosis, while Tem cells were decreased. Lineage tracing and differential analysis highlighted CXCL13+ Tex cells as a terminal, exhausted subtype of cells with roles in PD-1 signaling, glycolysis, and T-cell regulation. CXCL13+ Tex cells displayed T-cell exhaustion markers like PDCD1, HAVCR2, TIGIT, and TNFRSF9. Functional analysis implicated potential roles of these cells in immunosuppression. Finally, a CXCL13+ Tex-cell gene signature was found that correlated with cirrhosis severity and poorer prognosis of liver cancer. Conclusions In summary, this comprehensive study defines specialized CD8+ T-cell subpopulations in cirrhosis, with CXCL13+ Tex cells displaying an exhausted phenotype associated with immune dysregulation and advanced disease. Key genes and pathways regulating these cells present potential therapeutic targets.

Objective: Accurate evaluation of inflammation severity in ulcerative colitis (UC) can guide treatment strategy selection. The potential value of the pericolic fat attenuation index (FAI) on CT as an indicator of disease severity remains unknown.This study aimed to assess the diagnostic accuracy of pericolic FAI in predicting UC severity. Materials and Methods: This retrospective study enrolled 148 patients (mean age 48 years; 87 males). The fat attenuation on CT was measured in four different locations: the mesocolic vascular side (MS) and opposite side of MS (OMS) around the most severe bowel lesion, the retroperitoneal space (RS), and the subcutaneous area. The fat attenuation indices (FAI MS, FAI OMS, and FAI RS) were calculated as the fat attenuation measured in MS, OMS, and RS, respectively, minus that of the subcutaneous area, and were obtained in the non-enhanced, arterial, and delayed phases. Correlations between the FAI and UC Endoscopic Index of Severity (UCEIS) were assessed using Spearman’s correlation. Predictors of severe UC (UCEIS ≥7) were selected by univariable analysis. The performance of FAI in predicting severe UC was evaluated using the area under the receiver operating characteristic curve (AUC). Results: The FAIMS and FAI OMS scores were significantly higher than FAI RS in three phases (all P < 0.001). The FAIMS and FAI OMS scores moderately correlated with the UCEIS score (r = 0.474–0.649 among the three phases). Additionally, FAI MS and FAI OMS identified severe UC, with AUC varying from 0.77 to 0.85. Conclusion Increased CT attenuation of pericolic adipose tissue could serve as a noninvasive marker for evaluating UC severity. FAI MS and FAI OMS of three phases showed similar prediction accuracies for severe UC identification.

Objective: To assess the dynamic changes of microcirculation at acupoints in patients with primary dysmenorrhea and cold congelation and blood stasis syndrome using laser speckle blood flow imaging. Methods: Patients with primary dysmenorrhea and cold coagulation and blood stasis syndrome (primary dysmenorrhea group, n=53) and healthy female college students(control group, n=57) who met the inclusion and exclusion criteria from October 2020 to July 2022 were enrolled at Hebei University of Chinese Medicine. On the premenstrual and first day of menstruation, a laser speckle blood flow imaging system was used to measure the microcirculation blood flow perfusion on the surface of acupoints related to the conception, thoroughfare, and governor vessels, and stomach, spleen, and bladder meridians in the abdomen and lumbosacral regions. The dynamic changes in microcirculation were calculated based on the difference in average blood flow perfusion at each acupoint before and after menstruation. Receiver operating curve (ROC) analysis was used to analyze the diagnostic efficacy of dynamic changes in microcirculation on the surface of each acupoint. The microcirculation sensitization rate of acupoints was calculated. Results: Compared with the control group, the dynamic changes in microcirculation at the following acupoints in the primary dysmenorrhea group were increased (P<0.05): conception vessel (Yinjiao[CV7], Qihai[CV6], Shimen[CV5], Guanyuan[CV4]); left thoroughfare vessel (left Huangshu[KI16], left Zhongzhu[KI15], left Siman[KI14], left Qixue[KI13], left Dahe[KI12], left Henggu[KI11]); left stomach meridian (left Tianshu[ST25], left Wailing[ST26], left Qichong[ST30]); left spleen meridian (left Daheng[SP15], left Fujie[SP14]); right thoroughfare vessel (right Huangshu[KI16], right Zhongzhu[KI15], right Siman[KI14], right Qixue[KI13], right Dahe[KI12], right Henggu[KI11]); right stomach meridian (right Wailing[ST26], right Daju[ST27], right Shuidao[ST28], right Guilai[ST29], right Qichong[ST30]); and right spleen meridian (right Fujie[SP14]). The area under the ROC curve of conception vessel (Yinjiao[CV7], Qihai[CV6], Shimen[CV5], Guanyuan[CV4]), thoroughfare vessel (right Siman[KI14], left Huangshu[KI16], right Qixue[KI13], right Zhongzhu[KI15], right Dahe[KI12], left Zhongzhu[KI15], left Siman[KI14], right Huangshu[KI16], left Qixue[KI13], right Henggu[KI11], left Henggu[KI11], left Dahe[KI12]); stomach meridian (left Tianshu[ST25], right Guilai[ST29], left Wailing[ST26], right Shuidao[ST28], right Daju[ST27], right Wailing[ST26], right Qichong[ST30], left Qichong[ST30]), and spleen meridian (left Daheng[SP15], left Fujie[SP14], right Fujie[SP14]) was 0.610-0.682 (P<0.05). Compared with the control group, the sensitization rate of some acupoints in the primary dysmenorrhea group increased (P<0.05). Conclusion With the onset of menstruation, the blood flow perfusion of some acupoints in the abdomen (thoroughfare, and conception vessels, and stomach and spleen meridians) of patients with primary dysmenorrhea and cold blood coagulation and blood stasis syndrome increased, and the status of acupoints changed from a resting state to an active state. These acupoints are sensitive in patients with primary dysmenorrhea and cold blood coagulation and blood stasis syndrome and have a certain diagnostic efficacy, providing a basis for further analyzing the efficacy and mechanism of acupuncture and moxibustion to treat primary dysmenorrhea with cold blood coagulation and blood stasis syndrome.

Objective: To investigate the trends in mortality and life loss of gastric cancer in Wenzhou City, Zhejiang Province from 2014 to 2023, so as to provide the evidence for formulating the prevention and control strategy for gastric cancer. Methods: The surveillance on causes of death data of permanent residents in Wenzhou City were collected through the Wenzhou Chronic Disease Monitoring and Management Information System from 2014 to 2023. The crude mortality of gastric cancer was calculated, and standardized by the data from the Sixth Chinese National Population Census in 2010. The life loss were measured using potential years of life lost (PYLL) and rate of potential years of life lost (PYLLR). The characteristics of mortality and life loss of gastric cancer in different genders and age groups were described. The trends in mortality and PYLLR of gastric cancer were analyzed using the average annual percent change (AAPC). Results: Totally 17 080 deaths were reported due to gastric cancer in Wenzhou City from 2014 to 2023, accounting for 12.58% and ranking third in the order of malignant tumor deaths. The crude mortality of gastric cancer was 20.73/105, and the standardized mortality was 15.22/105, showing decreasing trends (AAPC=-3.311%, -6.470%, both P<0.05). The crude mortality of gastric cancer was 29.22/105 in men and 11.61/105 in women, with standardized mortality rates of 20.81/105 and 8.74/105 (both P<0.05). The crude mortality of gastric cancer appeared a tendency towards a rise with increasing age (P<0.05), reaching the highest rate of 225.88/105 in the group aged 80 to <85 years. The PYLL and PYLLR of gastric cancer were 107 607.50 person-years and 1.37‰. The PYLLR appeared a tendency towards a decline from 2014 to 2023, with AAPC of -6.667% (P<0.05). Conclusions The mortality and PYLLR of gastric cancer in Wenzhou City appeared a tendency towards a decline from 2014 to 2023. Men and the elderly populations were the key groups for the prevention and treatment of gastric cancer.