扶正化瘀方通过改变急性肝损伤小鼠模型肝脏CD8^(+)T淋巴细胞表型功能预防肝纤维化的价值分析

Value of Fuzheng Huayu prescription in preventing liver fibrosis by altering the phenotypic function of CD8+T lymphocytes in the liver of mice with acute liver injury

目的观察应用扶正化瘀方后急性肝损伤小鼠模型肝脏CD8^(+)T淋巴细胞对共培养的肝星状细胞的影响,探讨扶正化瘀方预防肝纤维化的作用机制。方法18只SPF级雄性C57BL/6NCrl Vr小鼠随机分为正常组、模型组、扶正化瘀方组,每组6只。扶正化瘀方组提前给予扶正化瘀方灌胃5 d。实验前12 h以2 mL/kg体质量的剂量腹腔注射10%的CCl_(4),腹主静脉取血,留取血清并检测ALT、AST,留取部分肝脏做病理观察;应用流式细胞术分选小鼠肝脏CD8^(+)T淋巴细胞,预先小鼠体内用药,再与小鼠肝星状细胞株(JS 1)以2∶1比例共培养于96孔板中24 h、48 h,使用qPCR方法检测各组Col.ⅠmRNA和α-SMA mRNA表达变化。符合正态分布的计量资料多组间比较采用方差分析,进一步两两比较采用SNK-q或LSD-t检验。结果模型组的ALT和AST水平均显著高于正常组(P值均<0.0001)。扶正化瘀方组小鼠的ALT升高的幅度明显小于模型组(P<0.001)。HE染色显示,扶正化瘀方组小鼠肝细胞变性、坏死程度较模型组有所减轻。与正常组比较,模型组总淋巴细胞、CD45、CD4-CD8^(+)T、CD8^(+)CD28-T显著上升,而扶正化瘀方组与模型组相比较,上述淋巴细胞比例显著降低(P值均<0.001)。自各组小鼠肝脏分离的CD8^(+)T淋巴细胞与JS 1共培养48 h,模型组与对照组(JS 1单培养)和正常组相比α-SMA mRNA的表达明显增加(P值均<0.01);Col.ⅠmRNA表达也明显高于对照组和正常组(正常小鼠肝脏CD8^(+)T淋巴细胞与JS 1共培养)(P值均<0.001)。扶正化瘀方组α-SMA mRNA和Col.ⅠmRNA表达显著低于模型组(P值均<0.01)。结论扶正化瘀方能通过改变小鼠肝脏CD8^(+)T淋巴细胞功能表型间接抑制活化的肝星状细胞。

Objective To investigate the effect of liver CD8^(+)T lymphocytes on co-cultured hepatic stellate cells(HSCs)after the application of Fuzheng Huayu prescription in a moues model of acute liver injury,as well as the mechanism of action of Fuzheng Huayu prescription in preventing liver fibrosis.Methods A total of 18 specific pathogen-free male C57BL/6NCrl Vr mice were randomly divided into normal group,model group,and Fuzheng Huayu prescription group,with 6 mice in each group.The mice in the Fuzheng Huayu prescription group were given Fuzheng Huayu prescription for 5 days in advance.At 12 hours before the experiment,10%CCl_(4) was injected intraperitoneally at a dose of 2 mL/kg body weight.Blood was collected from the main abdominal vein,and the serum was separated to measure the levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST).Part of the liver was used for pathological observation.After the mice were pretreated with medication in vivo,flow cytometry was used for the sorting of mouse liver CD8^(+)T lymphocytes,which were then co-cultured with the mouse HSC cell line(JS 1)in a 96-well plate at a ratio of 2∶1,and after co-culture for 24 and 48 hours,qPCR was used to measure the changes in the mRNA expression of Col.I andα-SMA.An analysis of variance was used for comparison of normally distributed continuous data between multiple groups,and the SNK-q test or the least significant difference t-test was used for further comparison between two groups.Results The model group had significantly higher activities of ALT and AST than the normal group(both P<0.0001),and compared with the model group,the Fuzheng Huayu prescription group had a significantly lower degree of increase in ALT activity(P<0.001).HE staining showed that the Fuzheng Huayu prescription group had a significantly lower degree of hepatocyte degeneration and necrosis compared with the model group.Compared with the normal group,the total lymphocytes,CD45,CD4-CD8^(+)T and CD8^(+)CD28-T in the model group increased significantly,while the proportion of the above lymphocytes in the Fuzheng Huayu formula group decreased significantly compared with the model group(P<0.001).CD8^(+)T lymphocytes isolated from the liver of mice in each group were co-cultured with JS 1 for 48 hours,and compared with the control group(JS 1 cultured alone)and the normal group,the model group had a significant increase in the mRNA expression ofα-SMA(both P<0.01)and significantly higher mRNA expression of Col.I than the control group and the normal group(normal mouse liver CD8^(+)T lymphocytes co-cultured with JS 1)(both P<0.001).The Fuzheng Huayu prescription group had significantly lower mRNA expression levels ofα-SMA and Col.I than the model group(both P<0.01).Conclusion Fuzheng Huayu prescription can indirectly inhibit activated HSCs by altering the functional phenotype of CD8^(+)T lymphocytes in mouse liver.