清热解毒凉血方治疗慢加急性肝衰竭小鼠模型的效果及机制

Clinical effect of Qingre Jiedu Liangxue prescription in treatment of mice with acute-on-chronic liver failure and related mechanism

目的探究清热解毒凉血方对慢加急性肝衰竭(ACLF)小鼠模型的作用机制。方法将36只雄性C57BL/6小鼠随机分为正常组(n=6)、中药组(n=6)、西药组(n=6)和模型组(n=18)。除正常组外,其他组均腹腔注射10%CCl4,3次/周,共8周,于最后一次给予LPS 0.5 mg/kg联合D-gal 400 mg/kg。模型组给予D-gal后于3 h、12 h、24 h分别处死小鼠,每次6只。中药组及西药组在小鼠腹腔注射10%CCl44周后,开始灌胃中药/西药,持续4周,予D-gal后3 h处死小鼠,同时处死正常组小鼠。检测血清ALT、AST以及炎症因子[粒细胞-巨噬细胞集落刺激因子(GM-CSF)、TNF、IFN、单核细胞趋化因子(MCP)1、IL];HE及天狼猩红染色观察肝组织病理变化;免疫荧光法观察F4/80、CD11b;免疫组化法观察髓过氧化物酶(MPO)的表达;Western Blot测定CD44、黏附分子(ICAM)1、高迁移率族蛋白(HMGB)1的表达情况。计量资料多组间比较采用单因素方差分析,进一步两两比较采用SNK-q检验。结果与正常组相比,模型组-3h ALT、AST水平明显升高(χ^2值分别为51.41、71、71.22,P值均<0.001);与模型组-3h相比,中药组小鼠血清中IL-17A升高明显(P<0.01),TNFα、GM-CSF、IL-10、IL-1β、MCP-1、IFNγ表达均降低(P值均<0.05)。与模型组相比,西药组小鼠血清中GM-CSF、IFNγ、IFNβ均降低(P值均<0.05)。HE染色提示与正常组相比,模型组小鼠肝细胞损伤严重,炎性细胞浸润明显,给予中药/西药后,小鼠肝脏病理示均有所改善。模型组-3 h中MPO表达量明显较正常组升高,模型组-12 h与模型组-24 h的MPO的表达量逐渐降低(P值均<0.05),但高于正常组;中药组MPO表达量与模型组-3 h相比降低(P<0.05)。模型组中F4/80、CD11b表达量与正常组相比逐渐升高;中药组F4/80、CD11b表达量与模型组-3 h相比升高。模型组中CD44、ICAM-1表达量与正常组相比均明显升高(P值均<0.05);中药组HMGB1、CD44、ICAM-1表达量与模型组-3h相比均降低(P值均<0.05)。结论清热解毒凉血方能够降低ACLF小鼠模型血清中ALT、AST,改善肝损伤,同时升高IL-17A,降低TNFα、GM-CSF、IL-10、IL-1β、MCP-1、IFNγ的表达,另外,清热解毒凉血方可降低小鼠肝组织中MPO、CD44、ICAM-1、HMGB1的表达,升高F4/80、CD11b的表达,表明清热解毒凉血方可能通过调节中性粒细胞与Kupffer细胞改善肝损伤。

Objective To investigate the mechanism of action of Qingre Jiedu Liangxue prescription in the treatment of mice with acute-on-chronic liver failure(ACLF).Methods A total of 36 male C57BL/6 mice were randomly divided into normal group(n=6),traditional Chinese medicine(TCM)group(n=6),Western medicine group(n=6),and model group(n=18).All mice except those in the normal group were given intraperitoneal injection of 10%CCl4 three times a week for 8 weeks,and LPS 0.5 mg/kg and D-gal 400 mg/kg were given after last injection.The mice in the model group were sacrificed at 3,12,and 24 hours after D-gal administration.After 4 weeks of intraperitoneal injection of 10%CCl4,the mice in the TCM group and the Western medicine group were given TCM or Western medicine by gavage for 4 weeks and were sacrificed at 3 hours after D-gal administration.Serum levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST),and inflammatory factors were measured;HE staining and picrosirius red staining were used to observe liver pathological changes;immunofluorescence assay was used to observe F4/80 and CD11b;immunohistochemistry was used to measure the expression of myeloperoxidase(MPO);Western blot was used to measure the expression of CD44,intercellular adhesion molecule-1(ICAM-1),and high-mobility group box 1(HMGB1).A one-way analysis of variance was used for comparison of continuous data between multiple groups,and the SNK-q test was used for further comparison between two groups.Results Compared with the normal group,the 3-hour model group had significant increases in the serum levels of ALT and ASTχ^2=51.41 and 71.22,(both P<0.001),and compared with the 3-hour model group,the TCM group had a significant increase in the serum level of interleukin-17A(IL-17A)and significant reductions in tumor necrosis factor-α(TNFα),granulocyte-macrophage colony-stimulating factor(GM-CSF),and interleukin-10(IL-10)(all P<0.01),as well as significant reductions in the expression of interleukin-1β(IL-1β),monocyte chemotactic protein 1(MCP-1),and interferon gamma(IFNγ)(all P<0.05).Compared with the model group,the Western medicine group had significant reductions in the serum levels of GM-CSF,IFNγ,and interferon beta(all P<0.05).HE staining showed that compared with the normal group,the model group had severe hepatocyte injury and significant inflammatory cell infiltration.Liver pathological changes were improved after the administration of TCM and Western medicine.The 3-hour model group had a significant increase in the expression of MPO compared with the normal group(P<0.05),and the expression of MPO gradually decreased in the 12-and 24-hour model groups(P<0.05);the TCM group had a significant reduction in the expression of MPO compared with the 3-hour model group(P<0.05).The model group had significant increases in the expression of F4/80 and CD11b compared with the normal group,and the TCM group had significant increases in the expression of F4/80 and CD11b compared with the 3-hour model group.The model group had significant increases in the expression of CD44 and ICAM-1 compared with the normal group(both P<0.05),and the TCM and Western medicine groups had significant reductions in the expression of HMGB1,CD44,and ICAM-1 compared with the 3-hour model group(all P<0.05).Conclusion In mice with ACLF,Qingre Jiedu Liangxue prescription can reduce the serum levels of ALT and AST,improve liver injury,increase IL-17A,and reduce TNFα,GM-CSF,IL-10,IL-1β,MCP-1,and IFNγ.In addition,Qingre Jiedu Liangxue prescription can reduce the expression of MPO,CD44,ICAM-1,and HMGB1 and increase the expression of F4/80 and CD11b in liver tissue.It is suggested that Qingre Jiedu Liangxue prescription can improve liver injury by regulating neutrophils and Kupffer cells,and further studies are needed to investigate the specific mechanism.