灵芝酸A对刀豆球蛋白A诱导的急性免疫性肝损伤小鼠模型的影响及其作用机制  

Effect of ganoderic acid A on a mouse model of concanavalin A-induced acute immune liver injury and its mechanism

作　　者：崔怡 乔凤杰 邱嘉昊 刘羽飞 高竺君 尚志 高月求[1,2] CUI Yi;QIAO Fengjie;QIU Jiahao;LIU Yufei;GAO Zhujun;SHANG Zhi;GAO Yueqiu(Laboratory of Cell Immunity,Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;Shuguang School of Clinical Medicine,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China)

机构地区：[1]上海中医药大学附属曙光医院细胞免疫实验室,上海201203 [2]上海中医药大学曙光临床医学院,上海201203

出　　处：《临床肝胆病杂志》2024年第12期2415-2423,共9页Journal of Clinical Hepatology

基　　金：国家自然科学基金(82104606,81874436)。

摘　　要：目的观察灵芝酸A(GA-A)对刀豆球蛋白A(ConA)诱导的小鼠自身免疫性肝炎(AIH)模型的治疗作用。方法将35只小鼠随机分为空白组(NC组)、模型组(ConA组)和GA-A低、中、高剂量治疗组(GA-A-L组、GA-A-M组、GA-A-H组),每组各7只小鼠。通过尾静脉注射ConA建立经典AIH小鼠模型,1 h后通过腹腔注射不同剂量GA-A治疗。应用蛋白质组学技术探究GA-A对肝细胞的保护机制,另外在体外用全反式维甲酸(ATRA)将HL-60细胞分化为dHL-60中性粒细胞来验证GA-A的作用机制。检测炎症(血清ALT和AST活性、HE染色及炎症相关基因)、凋亡(TUNEL染色)、中性粒细胞和中性粒细胞胞外陷阱(NET)标志物[髓过氧化物酶(MPO)、瓜氨酸化组蛋白3(CitH3)、Ly6G、游离双链DNA(dsDNA)]及p38磷酸化指标。计量资料两组间比较采用成组t检验;多组间比较采用单因素方差分析,进一步两两比较采用LSD-t检验。结果与NC组相比,ConA组小鼠血清ALT和AST水平显著增加(P值均<0.001)。与ConA组相比,GA-A治疗显著降低ALT和AST水平(P值均<0.01)。HE染色结果表明,ConA组小鼠肝脏发生明显坏死。GA-A治疗后显著减少肝坏死面积和TUNEL阳性肝细胞的数量(P值均<0.05)。另外,与ConA组相比,GA-A治疗后血清和肝组织中炎症因子IL-6、TNF-α和IFN-γ表达水平显著降低(P值均<0.05)。蛋白质组学分析提示,GA-A通过抑制NET的释放和p38 MAPK通路来减轻ConA诱导的急性免疫性肝损伤。小鼠肝组织免疫荧光染色结果显示,与ConA组相比,GA-A治疗组MPO阳性中性粒细胞的数量及Ly6G和CitH3阳性细胞的数量显著降低(P值均<0.01)。Western Blot及dsDNA结果显示,GA-A显著抑制小鼠肝组织及dHL-60细胞中的NET标志物dsDNA、CitH3以及p38磷酸化水平(P值均<0.05)。结论GA-A抑制p38 MAPK通路和NET释放减轻肝脏炎症反应和肝细胞死亡,从而减轻ConA诱导的急性免疫性肝损伤。本研究为GA-A通过调节嗜中性粒细胞功能治疗免疫Objective To investigate the therapeutic effect of ganoderic acid A(GA-A)on a mouse model of concanavalin A(ConA)-induced autoimmune hepatitis(AIH).Methods A total of 35 mice were randomly divided into control group(NC group),model group(ConA group),and low-,middle-,and high-dose GA-A treatment groups(GA-A-L,GA-A-M,and GA-A-H groups,respectively),with 7 mice in each group.ConA was injected via the caudal vein of mice to establish a classic mouse model of AIH,and different doses of GA-A were administered via intraperitoneal injection 1 hour later for treatment.Proteomic techniques were used to investigate the protective mechanism of GA-A on hepatocytes,and HL-60 cells were differentiated into dHL-60 neutrophils by all-trans retinoic acid in vitro to validate the mechanism of action of GA-A.Related indicators were measured,including inflammatory markers(the activities of serum alanine aminotransferase[ALT]and aspartate aminotransferase[AST],HE staining,and inflammation-related genes),apoptosis markers(TUNEL staining),neutrophils,and neutrophil extracellular trap(NET)markers(myeloperoxidase[MPO],citrullinated histone H3[CitH3],Ly6G,and free double-stranded DNA[dsDNA]),and p38 phosphorylation markers.The independent samples t-test was used for comparison of continuous data between two groups;a one-way analysis of variance was used for comparison between multiple groups,and the least significant difference ttest was used for further comparison between two groups.Results Compared with the NC group,the ConA group had significant increases in the serum levels of ALT and AST(both P<0.001),and compared with the ConA group,GA-A treatment significantly reduced the levels of ALT and AST(both P<0.01).HE staining showed that the mice in the ConA group had significant liver necrosis,while GA-A treatment significantly reduced the area of liver necrosis and the number of TUNEL-positive cells(both P<0.05).Compared with the ConA group,the GA-A group had significant reductions in the expression levels of the inflammatory factors inte

关 键 词：灵芝酸A 伴刀豆球蛋白A 肝炎 自身免疫性 胞外诱捕网 蛋白质组学 小鼠 近交C57BL 

分 类 号：R285.5[医药卫生—中药学]