甜菜碱对二甲基亚硝胺致肝硬化大鼠的作用及机制研究  

Research on the Effect and Mechanism of Betaine on Dimethylnitrosamine Induced Cirrhosis in Rats

作　　者：程天宇 梁悦 陈高峰[1,2] 刘伟[1,2] 慕永平[1,2] 陈佳美[1,2] 刘平[1,2,3] CHENG Tianyu;LIANG Yue;CHEN Gaofeng;LIU Wei;MU Yongping;CHEN Jiamei;LIU Ping(Institute of Liver Diseases,Shanghai Academy of Traditional Chinese Medicine,Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;Key Laboratory of Liver and Kidney Diseases(Ministry of Education),Shanghai Key Laboratory of Traditional Chinese Medicine,Shanghai 201203,China;Institute of Interdisciplinary Integrative Medicine Research,Shanghai University of Traditional Chinese Medicine,Shanghai Chemical Biology of Frontier Base 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,上海市中医药研究院肝病研究所,上海201203 [2]肝肾疾病病证教育部重点实验室,上海市中医临床重点实验室,上海201203 [3]上海中医药大学交叉科学研究院,上海市中医药化学生物学前沿基地,上海201203

出　　处：《中药新药与临床药理》2024年第12期1799-1807,共9页Traditional Chinese Drug Research and Clinical Pharmacology

基　　金：国家自然科学基金重点项目(82130120)。

摘　　要：目的探讨甜菜碱对二甲基亚硝胺(DMN)致肝硬化大鼠的作用及机制。方法将40只雄性Wistar大鼠随机分为正常组、模型组、易善复组(141.8 mg·kg^(-1))及甜菜碱低、高剂量组(100、400 mg·kg^(-1)),每组8只。给予0.5%DMN溶液(2 mL·kg^(-1))腹腔注射复制肝硬化大鼠模型。灌胃给药,每天1次,持续2周。采用全自动生化分析仪检测血清丙氨酸氨基转移酶(ALT)活性;HE、天狼星红(SR)染色法观察肝脏组织病理变化;免疫组化法检测肝组织中α-SMA、F4/80、CD68表达情况;碱水解法测定肝组织中羟脯氨酸(Hyp)含量;Western Blot法检测肝组织中α-SMA、NLRP3、pro-Caspase1、IL-18蛋白表达水平;Real-time qPCR法检测肝组织中纤维化及炎症相关基因的表达水平。结果与正常组比较,模型组大鼠肝小叶结构破坏,肝细胞肿胀坏死,伴有大量炎性细胞浸润,血清ALT水平显著升高(P<0.01);肝组织有大量胶原沉积,胶原阳性面积比及Hyp含量显著升高(P<0.01);肝组织中F4/80、CD68阳性面积比及F4/80、CD68、TNF-αmRNA表达水平均显著升高(P<0.01);肝组织纤维间隔有大量α-SMA阳性表达,α-SMA阳性面积比及蛋白表达水平显著升高(P<0.01),Acta2、Col1a1、TGF-β1mRNA表达显著上调(P<0.01);肝组织中NLRP3、pro-Caspase1、IL-18蛋白表达水平及NLRP3、IL-1β、IL-18 mRNA表达水平均显著升高(P<0.01)。与模型组比较,甜菜碱低、高剂量组大鼠肝组织损伤及炎性细胞浸润均有不同程度改善,且甜菜碱高剂量组的血清ALT水平明显降低(P<0.05);甜菜碱低、高剂量组肝组织胶原沉积有不同程度减轻,胶原阳性面积比及Hyp含量显著下降(P<0.05,P<0.01);肝组织中F4/80、CD68阳性面积比及F4/80、CD68、TNF-αmRNA表达水平均显著降低(P<0.05,P<0.01);肝组织α-SMA阳性面积比及蛋白表达水平显著降低(P<0.05,P<0.01),Acta2、Col1a1、TGF-β1 mRNA表达显著下调(P<0.05,P<0.01);肝组织中NLRP3、pro-Caspase1、IL-18蛋白表达水平�Objective To investigate the effect and mechanism of betaine on dimethylnitrosamine(DMN)-induced liver cirrhosis in rats.Methods Forty male Wistar rats were randomly divided into normal group,model group,Yishanfu(141.8 mg·kg^(-1))group,low-and high-dose(100,400 mg·kg^(-1))betaine groups,with eight rats in each group.A rat model of liver cirrhosis was replicated by intraperitoneally injected with 0.5%DMN solution(2 mg·kg^(-1)).Then the corresponding drugs were given orally once a day for two weeks.The activity of alanine aminotransferase(ALT)in serum was detected by biochemical analyzer.The pathological changes of liver tissue were observed by hematoxylin-eosin(HE)staining and Sirius red(SR)staining.The positive expressions ofα-SMA,F4/80 and CD68 in liver tissue were determined by immunohistochemistry.The content of hydroxyproline(Hyp)in liver tissue was determined by alkaline hydrolysis method.Western Blot was used to determine the protein expressions ofα-SMA,NLRP3,pro-Caspase1 and IL-18.The mRNA expression levels of fibrosis-related and inflammation-related genes in liver tissue were detected by real-time fluorescence quantitative polymerase chain reaction(qPCR).Results Compared with the normal group,the structure of hepatic lobule of the rats in the model group was damaged.Also,the liver cells were swollen and necrotic,with a large amount of inflammatory cell infiltration.Serum ALT level was significantly increased(P<0.01).A large amount of collagen deposition in liver tissue was observed,Hyp content and percentage of collagen positive area were significantly increased(P<0.01).The percentage of positive area of F4/80 and CD68,as well as the mRNA expressions of F4/80,CD68 and TNF-αin liver tissue were significantly increased(P<0.01).Large amounts ofα-SMA positive expression were found in fibrous septum of liver tissue.The percentage of positive area and protein expression ofα-SMA were obviously increased(P<0.01).The mRNA levels of Acta2,Col1a1,TGF-β1 were significantly up-regulated(P<0.01).Moreover,the

关 键 词：甜菜碱 二甲基亚硝胺 肝硬化 NLRP3炎症小体信号通路 巨噬细胞 肝星状细胞 肝纤维化 大鼠 

分 类 号：R285.5[医药卫生—中药学]