扶正化瘀复方抑制小鼠自噬的抗纤维化机制研究  

Study on anti- fibrotic mechanism of Fuzheng -Huayu formula to suppress autophagy in mice

作　　者：黄兰蔚[1,3] 姜娜 平键[2,4,5,6] 张晶[2,3] 徐列明 Huang Lanwei;Jiang Na;Ping Jian;Zhang Jing;Xu Lieming(Department of Gastroenterology, Baoshan Branch, Shuguang Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 201999, China;Shuguang Hospital affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China;Institute of Liver Diseases, Shanghai University of TCM, Shanghai 201203, China;Key Laboratory of Diseases and Syndromes of Liver and Kidney Diseases, Ministry of Education, Shanghai 201203, China;Shanghai Key Clinical Laboratory of Traditional Chinese Medicine, Shanghai 201203, China;Key Laboratory of State Administration of Traditional Chinese Medicine (Chronic Liver Disease Deficiency and Damage), Shanghai 201203, China)

机构地区：[1]上海中医药大学附属曙光医院宝山分院消化科,201999 [2]上海中医药大学附属曙光医院,201203 [3]上海中医药大学肝病研究所,201203 [4]肝肾疾病病证教育部重点实验室,上海201203 [5]上海市中医临床重点实验室,201203 [6]国家中医药管理局重点研究室(慢性肝病虚损),上海201203

出　　处：《中华肝脏病杂志》2019年第8期621-627,共7页Chinese Journal of Hepatology

基　　金：国家自然科学基金(81373859);上海宝山区科委项目(13-E-19);上海宝山区特色专科建设计划(BSZK-2018-A03).

摘　　要：目的明确扶正化瘀复方抗肝纤维化的作用是否与其抑制自噬有关。方法C57小鼠随机分为正常组(N组)和造模组,造模组采用四氯化碳腹腔注射诱导小鼠肝纤维化模型,正常组注射等剂量橄榄油,1周后将造模组随机分为模型(M)组、雷帕霉素(Rapa)组、雷帕霉素加氯喹(Rapa+CQ)组、雷帕霉素加丹参酚酸B(Rapa+SalB)组、雷帕霉素加扶正化瘀方(Rapa+FZ)组,各药物组每日相应药物灌胃,N组、M组等剂量饮用水灌胃。5周后处死小鼠,HE和天狼猩红染色分别观察各组肝组织炎症和胶原沉积情况,碱水解法测定羟脯氨酸含量。蛋白质印迹法检测肝组织中自噬的表达变化和微管相关蛋白1轻链3Ⅱ/Ⅰ(LC3II/I)、p62、α-平滑肌肌动蛋白(ɑ-SMA)和Ⅰ型胶原蛋白表达,冰冻切片免疫荧光染色观察各组肝组织ɑ-SMA及LC3B免疫荧光定位。两独立样本间的比较用t检验,多个样本均数比较采用LSD检验或DunnettT3检验。结果N组与M组小鼠体质量差异无统计学意义,各药物组小鼠体质量均明显下降(F=14.041,P<0.001)。M组和各药物组小鼠的肝质量/体质量比和脾质量/体质量比较N组均有明显增加(F值分别为26.992、6.589,P值均<0.001)。与M组比较,各用药组小鼠肝组织p62蛋白表达降低,其中Rapa组和Rapa+SalB组差异有统计学意义(F=3.085,P值分别为0.039、0.003),Rapa+SalB组则更低;与M组比较,Rapa组LC3BⅡ的表达显著升高(F=7.514,P=0.01)。免疫荧光染色显示N组小鼠肝脏内未见LC3B与α-SMA共染细胞,M组小鼠肝内见到共染细胞,各药物组小鼠肝内共染细胞明显多于M组,其中Rapa+FZ组的共染细胞较少。与N组相比,M组和各药物组胶原显著增加,各药物组胶原沉积较M组减少,各药物组间差异无统计学意义。与N组(值为77.75±48.79)相比,M组小鼠的肝组织羟脯氨酸明显升高(值为293.48±84.43)(F=3.015,P=0.005),各药物组小鼠肝组织羟脯氨酸含量均较M组减少,但差异无统计Objective To determine whether the anti-hepatic fibrosis effect of Fuzheng-Huayu formula is related to suppress autophagy in mice. Methods C57 mice were randomly divided into normal group (N group) and model group. The model group was induced by intraperitoneal injection of carbon tetrachloride to induce liver fibrosis in mice, and the normal group was injected with equal volume of olive oil. After 1 week, the model group was randomly divided into model (M) group, rapamycin (Rapa) group, rapamycin plus chloroquine (Rapa+CQ) group, rapamycin plus salvianolic acid B (Rapa+Sal B) group, rapamycin plus Fuzheng -Huayu formula (Rapa+FZ) group. Each drug group was administered corresponding drugs by gavage on a daily basis, and N group and M group were given the equal amount of drinking water by gavage. After 5 weeks, the mice were sacrificed, and HE and Sirius red staining were used to observe the inflammation and collagen deposition on liver tissue in each group. The hydroxyproline content was determined by alkaline hydrolysis method. Western blotting was used to detect changes in the expression of autophagy in liver tissue and microtubule-associated protein 1 light chain 3II/I (LC3II/I), p62,α-smooth muscle actin (ɑ-SMA) and type I collagen expression. Immunofluorescence staining was used to observe the immunofluorescence localization of ɑ-SMA and LC3B in liver tissues of each group.). A t-test was used to compare the two independent samples. LSD or Dunnett’s T3 test were used to compare the mean of multiple samples. Results There was no significant difference in N and M groups in terms of body weight. The body weight of the mice in each drug group decreased significantly (F = 14.041, P < 0.001). The liver/spleen /body weight ratios of each drug group and M group were significantly higher than the N group (F = 26.992, 6.589, P < 0.001). The expression of p62 protein in the liver tissue of mice in each drug group was lower than M group, and the difference between Rapa group and Rapa+Sal B group (F = 3.085, P = 0.

关 键 词：肝纤维化 肝星状细胞 自噬 扶正化瘀复方 丹参酚酸B 

分 类 号：R285.5[医药卫生—中药学]