青蒿琥酯通过PI3K/Akt/mTOR信号通路改善DDC诱导的胆汁淤积性肝纤维化  

Artesunate improves DDC-induced cholestatic liver fibrosis through PI3K/Akt/mTOR signaling pathway in mice

作　　者：唐菀 赵楠 张晓珣 柴进 TANG Wan;ZHAO Nan;ZHANG Xiaoxun;CHAI Jin(Department of Gastroenterology,Cholestatic Liver Diseases Center,Center for Metabolic Associated Fatty Liver Disease,First Affiliated Hospital,Army Medical University(Third Military Medical University),Chongqing,400038,China)

机构地区：[1]陆军军医大学(第三军医大学)第一附属医院消化内科,胆汁淤积肝病中心和代谢性肝病中心,重庆400038

出　　处：《陆军军医大学学报》2023年第10期1001-1009,共9页Journal of Army Medical University

基　　金：国家自然科学基金优秀青年科学基金(81922012);重庆市杰出青年科学基金(cstc2021jcyj-jqX0005)。

摘　　要：目的探究青蒿琥酯(artesunate,ART)对3,5-二乙氧羰基-1,4-二氢-2,4,6-三甲吡啶(3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-collidine,DDC)诱导的胆汁淤积性肝纤维化的作用及分子机制。方法将24只8周龄雄性C57BL/6J小鼠按随机数字表法分为3组:对照组(n=6)、0.1%DDC组(n=9)和0.1%DDC+ART组(n=9)。喂养含0.1%DDC的饲料2周,构建胆汁淤积小鼠模型,同时给予0.1%DDC+ART组小鼠青蒿琥酯(30.0 mg/kg,1次/d)肌肉注射。检测小鼠血清中肝损伤相关指标的水平;进行HE染色和天狼星红染色,根据Scheuer系统进行肝组织病理学评分。采用实时荧光定量PCR检测小鼠肝脏内α-SMA、Col1a1、Col1a2和Tgfβ1等表达水平,同时检测小鼠肝组织内羟脯氨酸(hydroxyproline,HYP)含量。在细胞实验中进一步探索青蒿琥酯减轻肝纤维化的机制,使用TGF-β联合ART处理LX2细胞,采用Western blot检测PI3K/Akt/mTOR信号通路相关蛋白的表达。使用ART和通路抑制剂共同处理LX2细胞后,检测肝星状细胞(hepatic stellate cell,HSC)活化标志物α-SMA的表达。结果青蒿琥酯给药明显减轻DDC诱导的胆汁淤积性肝损伤和肝纤维化(P<0.05),显著降低了胆汁淤积小鼠血清中ALT、AST、ALP和TBA的表达水平(P<0.05)。青蒿琥酯抑制小鼠肝脏内纤维化相关指标α-SMA、Col1a1、Col1a2和Tgfβ1 mRNA的表达,并减少肝组织内HYP含量(P<0.05)。青蒿琥酯可以抑制TGF-β诱导的LX2细胞内PI3K/Akt/mTOR信号通路的激活(P<0.05)。PI3K/Akt/mTOR信号通路抑制剂增强青蒿琥酯对TGF-β诱导的HSC活化的抑制作用(P<0.05)。结论青蒿琥酯能够明显改善DDC诱导的胆汁淤积性肝纤维化,它可通过调控PI3K/Akt/mTOR信号通路抑制HSC的活化,从而发挥其抗纤维化的作用。Objective To explore the effect and molecular mechanism of artesunate(ART)on cholestatic liver fibrosis induced by 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-collidine(DDC).Methods 8-week-old male C57BL/6J mice(n=24)were randomly divided into 3 groups:the control group(n=6),the 0.1%DDC group(n=9)and the 0.1%DDC+ART group(n=9).Mice have been fed with diet containing 0.1%DDC for 2 weeks to establish cholestatic mouse model,and ART was given intramuscular injection(30.0 mg/kg)once a day in the 0.1%DDC+ART group.The indicators of liver injury in serum of mice were detected.HE staining and Sirius red staining were performed,and histopathological scores were obtained according to Scheuer scoring system.RT-qPCR was used to detect the levels ofα-SMA,Col1a1,Col1a2 and Tgfβ1 in mouse liver.Hydroxyproline(HYP)content was detected with HYP detection kit.LX2 cells were treated with TGF-βcombined with ART,and the proteins related to PI3K/Akt/mTOR pathway were detected by Western blot assay.The expression ofα-SMA,hepatic stellate cell(HSC)activation marker,was detected after ART and pathway inhibitors were used to treated with LX2 cells.Results ART significantly ameliorated DDC-induced cholestatic liver injury and liver fibrosis(P<0.05).ART markedly decreased the levels of ALT,AST,ALP and TBA in serum of mice(P<0.05).ART inhibited the mRNA expression ofα-SMA,Col1a1,Col1a2 and Tgfβ1,and reduced the content of HYP in liver tissues of mice with cholestasis(P<0.05).In addition,ART obviously inhibited TGF-β-induced activation of PI3K/Akt/mTOR pathway in LX2 cells(P<0.05).PI3K/Akt/mTOR signaling pathway inhibitors enhanced the inhibitory effect of HSC activation induced by TGF-β(P<0.05).Conclusion ART observably improves DDC-induced cholestatic liver fibrosis,and it can inhibit HSC activation by regulating PI3K/Akt/mTOR signaling pathway,thus exerting anti-fibrosis effect.

关 键 词：肝纤维化 胆汁淤积 青蒿琥酯 PI3K/Akt/mTOR信号通路 

分 类 号：R282.71[医药卫生—中药学] R285.5[医药卫生—中医学] R575.22