文献类型：期刊文献

中文题名：基于肝脾相关探讨四君子汤治疗脾气虚证大鼠肝损伤的效应机制

英文题名：Mechanism of Si Junzitang in Treatment of Liver Injury in Rats with Spleen Qi Deficiency Syndrome Based on Liver and Spleen Correlation

作者：彭鹏[1,2];白敏[1,2];金锦[1,2];袁启慧[1,2];杨晓轶[3];杜娟[3];段永强[1,4]

第一作者：彭鹏

机构：[1]宁夏医科大学中医学院,银川750004;[2]宁夏医科大学中医燥证教育部重点实验室,银川750004;[3]甘肃中医药大学,兰州730000;[4]宁夏区域高发病中西医结合防治研究重点实验室,银川750004

第一机构：宁夏医科大学中医学院,银川750004

年份：2025

卷号：31

期号：24

起止页码：11

中文期刊名：中国实验方剂学杂志

外文期刊名：Chinese Journal of Experimental Traditional Medical Formulae

收录：;北大核心:【北大核心2023】；

基金：国家自然科学基金地区项目(82460896,81660758,81160420)。

语种：中文

中文关键词：四君子汤;脾气虚证;转录组学;NOD信号通路;NOD样受体蛋白3(NLRP3)炎症小体;肝脏炎症损伤

外文关键词：Si Junzitang;spleen Qi deficiency syndrome;transcriptomics;NOD signaling pathway;NOD-like receptor protein3(NLRP3)inflammasome;liver inflammatory injury

摘要：目的:基于转录组学探讨四君子汤治疗脾气虚证大鼠肝损伤的作用机制并进行实验验证。方法:将60只雄性SD大鼠随机分为空白组,模型组,四君子汤低、中、高剂量组(6、12、24 g·kg^(-1)·d(-1)),以及自然恢复组,每组10只,采用复合多因素造模法(强迫游泳+小承气汤灌胃+饮食失节)建立脾气虚证大鼠模型,持续干预30 d后,测定体质量、3 h进食量,评价脾气虚证宏观证候积分。检测各组大鼠血清天门冬氨酸氨基转移酶(AST)和丙氨酸氨基转移酶(ALT)的变化,苏木素-伊红(HE)染色观察各组大鼠肝组织病理学改变。转录组测序(RNA-Seq)分析空白组、模型组和四君子汤高剂量组的差异基因表达,并对差异基因进行基因本体(GO)和京都基因与基因组百科全书(KEGG)富集分析,采用免疫荧光法(IF)和蛋白免疫印迹法(Western blot)对富集分析到的NOD样受体蛋白3(NLRP3)、凋亡相关斑点样蛋白(ASC)、胱天蛋白酶-1(Caspase-1)和Gasdermin D的N端结构域(GSDMD-N)进行测定,免疫组织化学(IHC)染色法检测通路下游炎症因子白细胞介素(IL)-1β、IL-6、IL-18的表达情况。结果:与空白组比较,模型组大鼠体质量、3 h进食量显著降低(P<0.01),脾气虚证宏观证候积分,血清AST、ALT水平显著升高(P<0.01),肝组织病理呈轻度炎症损伤;与模型组比较,四君子汤各剂量组显著改善上述指标及肝损伤,呈剂量依赖性(P<0.05,P<0.01)。RNA-Seq分析显示,与空白组比较,模型组差异表达基因(DEGs)有1254个;相对于模型组,四君子汤高剂量组治疗后改变的相关基因有842个,基于GO和KEGG富集分析结果,提示脾气虚证大鼠肝损伤中NOD样受体信号通路被激活,NLRP3炎症小体可能是关键靶点。IF、IHC和Western blot结果显示,与空白组比较,模型组NLRP3、ASC、Caspase-1、GSDMD-N蛋白及下游炎症因子IL-1β、IL-6、IL-18表达显著升高(P<0.01);四君子汤高剂量组上述蛋白表达显著降低(P<0.01),而且能显著降低IL-1β、IL-6、IL-18炎症因子水平(P<0.01)。结论:四君子汤能够有效改善脾气虚证大鼠肝脏轻度炎症损伤,且呈现剂量依赖性,其机制可能与抑制NLRP3/ASC/Caspase-1通路活化,下调焦亡执行蛋白GSDMD-N的表达,并减少焦亡相关炎症因子的释放有关。
Objective:To investigate the mechanism of Si Junzitang in treating liver injury in rats with spleen Qi deficiency syndrome based on transcriptomics and to experimentally verify its effects.Methods:Sixty male SD rats were randomly divided into blank group,model group,low-dose Si Junzitang(6 g·kg^(-1)·d(-1)),medium-dose Si Junzitang group(12 g·kg^(-1)·d(-1)),high-dose Si Junzitang group(24 g·kg^(-1)·d(-1)),and natural recovery group,with 10 rats in each group.A composite multifactorial modeling method(forced swimming+intragastric administration of Xiao Chengqitang+irregular diet)was used to establish a spleen Qi deficiency model.After 30 days of continuous intervention,body weight and 3-hour food intake were measured,and macroscopic symptom scores for spleen Qi deficiency syndrome were evaluated.Serum levels of aspartate aminotransferase(AST)and alanine aminotransferase(ALT)in each group were detected,and hematoxylin and eosin(HE)staining was used to observe histopathological changes in liver tissue.Transcriptome sequencing(RNA-Seq)was used to identify differentially expressed genes(DEGs)among the blank,model,and high-dose Si Junzitang groups.Gene ontology(GO)and Kyoto encyclopedia of genes and genome(KEGG)enrichment analyses were performed on the DEGs.Immunofluorescence(IF)and Western blot were used to detect NOD-like receptor protein 3(NLRP3),apoptosis-associated speck-like protein(ASC),Caspase-1,and the N-terminal domain of gasdermin D(GSDMD-N).Immunohistochemistry(IHC)was used to detect the expression of downstream inflammatory cytokines interleukin-1β(IL-1β),interleukin-6(IL-6),and interleukin-18(IL-18).Results:Compared with the blank group,the model group showed significantly reduced body weight and 3-hour food intake,significantly increased macroscopic symptom scores,and elevated serum AST and ALT levels(P<0.01),with mild inflammatory liver injury observed histologically.Compared with the model group,Si Junzitang at all doses significantly improved these parameters and alleviated liver injury in a dose-dependent manner(P<0.05,P<0.01).RNA-Seq analysis revealed 1254 DEGs between the blank and model groups,and 842 DEGs between the model and high-dose Si Junzitang groups.GO and KEGG enrichment analyses indicated that the NOD-like receptor signaling pathway was activated in liver injury associated with spleen Qi deficiency,suggesting that the NLRP3 inflammasome may be a key target.Results from IF,IHC,and Westernblot showed that compared with the blank group,the expression of NLRP3,ASC,Caspase-1,GSDMD-N,and the downstream inflammatory cytokines IL-1β,IL-6,and IL-18 were significantly increased in the model group(P<0.01),while these levels were markedly decreased in the high-dose Si Junzitang group(P<0.01).Conclusion:Si Junzitang effectively improves mild inflammatory liver injury in rats with spleen Qi deficiency syndrome in a dose-dependent manner.Its mechanism may be associated with inhibition of the NLRP3/ASC/Caspase-1 signaling pathway,downregulation of the pyroptosis executioner protein GSDMD-N,and reduction of pyroptosis-related inflammatory cytokine release.