二十五味松石丸调节巨噬细胞表型改善CCL_(4)诱导的大鼠肝纤维化

Ershiwuwei Songshi Pills(二十五味松石丸) Improve CCL_4-Induced Liver Fibrosis in Rats by Regulating Macrophage Phenotype

目的:探讨二十五味松石丸通过调节肝巨噬细胞表型改善CCL_(4)诱导大鼠肝纤维化的作用及可能机制。方法:将50只雄性SD大鼠随机分为正常对照组、模型对照组、二十五味松石丸88、176、352 mg/kg组,除正常对照组外,其余大鼠采用皮下注射CCL_(4)橄榄油建立肝纤维化模型,造模的同时按分组给予相应药物治疗。4 w后检测大鼠肝组织中病理变化和胶原纤维沉积;生化检测血清丙氨酸氨基转移酶(ALT)和天门冬氨酸氨基转移酶(AST)活力;ELISA法检测肿瘤坏死因子(TNF-α)、白介素-6(IL-6)、透明质酸(HA)和层粘连蛋白(LN)含量;免疫组化检测肝组织中α-平滑肌肌动蛋白(α-SMA)、转化生长因子β1(TGF-β1)、CD68和CD163阳性表达;免疫印迹检测肝组织中α-SMA、TGF-β1、CD68、CD163、p-STAT1和p-STAT3蛋白表达;实时荧光定量PCR法检测肝组织中Cd68、Cd163 mRNA表达;免疫荧光双标检测CD68、CD163在肝组织中比例情况。结果:与正常对照组相比,模型对照组大鼠血清中ALT、AST活力显著升高(P<0.01),TNF-α、IL-6、HA和LN含量显著升高(P<0.01),病理损伤区域较大,胶原沉积明显增多;TGF-β1、α-SMA的阳性面积和蛋白表达显著升高(P<0.01),CD68^(+)阳性细胞、mRNA和蛋白表达显著升高(P<0.01),CD163^(+)阳性细胞、mRNA和蛋白表达显著下降(P<0.01);p-STAT1蛋白表达显著增加(P<0.01),p-STAT3蛋白表达显著降低(P<0.01);与模型对照组相比,二十五味松石丸治疗各组大鼠血清的ALT、AST活力显著降低(P<0.01),TNF-α、IL-6、HA和LN含量明显降低(P<0.05或P<0.01),病理损伤区域减少,胶原沉积明显降低;TGF-β1、α-SMA的阳性面积和蛋白表达显著降低(P<0.01),CD68^(+)阳性细胞、mRNA和蛋白表达显著降低(P<0.01),CD163^(+)阳性细胞、mRNA和蛋白表达显著增加(P<0.01);p-STAT1蛋白表达明显减少(P<0.05或P<0.01),p-STAT3蛋白表达明显增加(P<0.05或P<0.01)。结论:二十五味松石丸能改善CCl_(4)诱导的大鼠肝纤维化,可能通过STAT信号通路抑制CD68^(+)巨噬细胞表型、促进CD163^(+)巨噬细胞表型,降低组织炎症反应。

Objective:To explore the effect and underlying mechanism of Ershiwuwei Songshi Pills(二十五味松石丸) in improving liver fibrosis in carbon tetrachloride(CCl_(4))-induced rats by regulating the phenotype of hepatic macrophages.Methods:Fifty male SD rats were randomly divided into a normal control group,a model control group,and low-,medium-,and high-dose Ershiwuwei Songshi Pills groups(88,176,and 352 mg/kg).The liver fibrosis model was induced by subcutaneous injection of CCl_(4) olive oil in rats except for those in the normal control group.The corresponding drugs were administered to the rats in each group simultaneously.After four weeks,the pathological changes and collagen fiber deposition in the rat liver tissues were evaluated.Biochemical detection was conducted to measure serum alanine aminotransferase(ALT) and aspartate aminotransferase(AST) levels.ELISA was performed to measure the levels of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),hyaluronic acid(HA),and laminin(LN).Immunohistochemistry was used to determine the expression of α-smooth muscle actin(α-SMA),transforming growth factor-β1(TGF-β1),CD68,and CD163 in the liver tissues.Western blot was performed to detect the protein expression of α-SMA,TGF-β1,CD68,CD163,phosphorylated signal transducer and activator of transcription 1(p-STAT1),and p-STAT3 in the liver tissues.Real-time fluorescence quantitative PCR was used to measure the mRNA expression of CD68 and CD163 in the liver tissues.Immunofluorescence double staining was performed to assess the proportions of CD68 and CD163-positive cells in the liver tissues.Results:Compared with the normal control group,the model control group showed potentiated serum ALT and AST activities(P<0.01),elevated levels of TNF-α,IL-6,HA,and LN(P<0.01),larger areas of pathological damage,increased collagen deposition,increased positive areas and protein expression of TGF-β1 and α-SMA(P<0.01),increased expression of CD68^(+)-positive cells,mRNA,and protein(P<0.01),decreased expression of CD163^(+)-positive cells,mRNA,and protein(P<0.01),elevated protein expression of p-STAT1(P<0.01),and reduced p-STAT3 protein expression(P<0.01).Compared with the model control group,the Ershiwuwei Songshi Pills groups showed blunted serum ALT and AST activities(P<0.01),decreased levels of TNF-α,IL-6,HA,and LN(P<0.05 or P<0.01),reduced areas of pathological damage,decreased collagen deposition,reduced positive areas and protein expression of TGF-β1 and α-SMA(P<0.01),decreased expression of CD68^(+)-positive cells,mRNA,and protein(P<0.01),increased expression of CD163^(+)-positive cells,mRNA,and protein(P<0.01),decreased protein expression of p-STAT1(P<0.05 or P<0.01),and increased p-STAT3 protein expression(P<0.05 or P<0.01).Conclusion:Ershiwuwei Songshi Pills can improve CCl_(4)-induced liver fibrosis in rats possibly by inhibiting the CD68^(+) macrophage phenotype and promoting the CD163^(+) macrophage phenotype through the STAT signaling pathway,thus reducing tissue inflammation response.