复方保肝宁减轻小鼠肝纤维化的机制:抑制肝脏组织中的IDO1进而促进树突状细胞表型成熟

Baoganning formula alleviates liver fibrosis in mice by inhibiting hepatic IDO1 expression and promoting phenotypic maturation of dendritic cells

目的观察中药复方保肝宁对肝纤维化模型小鼠的保护作用并探讨其作用机制。方法本研究分为两部分,第1部分:按0.6 mL/(kg·d)予以腹腔注射25%CCl_(4)(CCl_(4)∶橄榄油=1∶3)的方式诱导野生型小鼠建立肝纤维化病理模型,随机分为对照组、模型组、阳性对照组及保肝宁低、中、高浓度组,10只/组。造模给药8周后处死小鼠,取小鼠血清检测AST、ALT水平。另取肝组织做HE、天狼星红染色及α-平滑肌肌动蛋白(α-SMA)免疫组化染色并检测吲哚胺2,3-双加氧酶1(IDO1)的表达水平。流式细胞仪检测小鼠肝脏树突状细胞(DCs)及T细胞的数量和表型变化。第2部分:按3×10^(11)v.g/只尾静脉注射IDO1过表达腺相关病毒,取正常野生型小鼠随机分为腺相关病毒阴性对照(AAV9-NC)干预组、IDO1过表达腺相关病毒(AAV9-IDO1)干预组及高浓度保肝宁+IDO1过表达腺相关病毒联合干预组,6只/组。干预4周后,取肝组织做IDO1免疫组化染色并检测小鼠肝脏树突状细胞(DCs)及T细胞的表型变化。结果与模型组比较,保肝宁高浓度组能有效降低肝纤维化小鼠血清中AST、ALT水平(P<0.01);改善肝组织病理损伤,减少肝纤维组织的形成及α-SMA和IDO1的沉淀(P<0.05)。保肝宁高浓度治疗组肝纤维化小鼠肝脏中CD11C^(+)DCs、CD11C^(+)CD80^(+)DCs、CD11C^(+)CD86^(+)DCs、CD11C^(+)CD40^(+)DCs、CD11C^(+)MHCII^(+)DCs细胞和CD3^(+)T、CD3^(+)CD4^(+)T细胞比例与模型组相比均呈不同程度的升高(P<0.05)。高浓度保肝宁干预后可显著降低IDO1过表达腺相关病毒干预小鼠肝脏中IDO1的表达水平及上调CD11C^(+)CD40^(+)DCs、CD11C+MHCII^(+)DCs和CD3^(+)CD4^(+)T细胞比例(P<0.05)。结论保肝宁对CCl_(4)所致小鼠肝纤维化具有一定的治疗作用,其机制可能与降低肝组织中IDO1的表达水平继而促进肝脏中DCs表型成熟使DCs刺激T细胞增殖能力增强有关。

Objective To investigate the protective effect of Bao Gan Ning(BGN),a traditional Chinese medicinal formula,against CCl_(4)-induced liver fibrosis in mice and explore the mechanism.Methods C57 BL/6 mice were randomly divided into control group,liver fibrosis model group,positive control group and the low-,middle-,and high-dose BGN groups(n=10).In all but the control group,the mice were subjected to daily intraperitoneal injection of 25%CCl_(4)(in olive oil)to induce liver fibrosis and intragastric gavage of corresponding drugs.After 8 weeks,serum levels of ALT and AST were detected.Pathological examination of the liver was performed using HE and Sirius Red staining andα-SMA immunohistochemistry.The expression level of indoleamine 2,3-dioxygenase 1(IDO1)and phenotypic changes of hepatic DCs in the liver were measured.Another 18 mice were randomly divided into AAV9-NC,AAV9-IDO1 and high-dose BGN+AAV-IDO1 groups(n=6)for corresponding treatment,and 4 weeks later the deposit of hepatic IDO1 and phenotypic changes of the hepatic DCs were analyzed.Results Compared with those in the model group,serum AST and ALT levels decreased significantly in BGN group(P<0.01).Obvious liver fibrosis was observed in the model group,while the mice treated with BGN showed obviously reduced cell necrosis and collagen proliferation in the liver with significantly lowered the expression levels of hepaticα-SMA and IDO1(P<0.05).The percentages of CD11 C^(+)DCs,CD11 C^(+)CD80^(+)DCs,CD11 C^(+)CD86^(+)DCs,CD11 C^(+)CD40^(+)DCs,CD11 C^(+)MHCII^(+)DCs,CD3^(+)T cells,and CD3^(+)CD4^(+)T cells all increased significantly in BGN group as compared with the model group(P<0.05).In mice with adenovirus-mediated IDO1 overexpression in the liver,BGN treatment significantly lowered the expression level of IDO1(P=0.000)and increased the percentages of hepatic CD11 C^(+)CD40^(+)DCs,CD11 C^(+)MHCII^(+)DCs and CD3^(+)CD4^(+)T cells(P<0.05).Conclusion BGN can effectively inhibit liver fibrosis in mice possibly by lowering the expression level of IDO1 in the liver,thus improving the function of hepatic DCs and subsequently promoting proliferation of T cells.