扶正化瘀方对肝纤维化小鼠肝癌发生的影响与机制

Effect and mechanism of Fuzheng Huayu Decoction on carcinogenesis in mice with liver fibrosis

目的:观察扶正化瘀方对肝纤维化背景小鼠模型肝癌发生的影响,及其调节肝脏衰老肿瘤相关成纤维细胞与肝星状细胞的相关作用机制。方法:采用二乙基亚硝胺(DEN)联合四氯化碳(CCl4)诱导24周建立C57BL/6小鼠肝癌模型,自造模12周肝纤维化明显时给予扶正化瘀方灌胃至24周。观察模型组18周、24周和扶正化瘀方组小鼠肝组织形态变化、肿瘤数量;免疫组织化学法检测Ki67表达,荧光定量PCR测定Afp、Gpc3基因水平;天狼猩红染色观察肝组织胶原沉积;免疫组织化学染色检测α-SMA、成纤维细胞活化蛋白FAP表达;半乳糖苷酶染色、γ-H2AX、FOXO4免疫组织化学染色检测细胞衰老;蛋白印迹法检测肝组织α-SMA、FAP、γ-H2AX、FOXO4蛋白表达。结果:随造模时间延长,小鼠肝癌发生率增加,模型组24周肝癌发生率为100%;肝组织胶原沉积增多,α-SMA表达逐渐增加;汇管区及胶原沉积周围可见FAP阳性表达的肿瘤相关成纤维细胞,同时该处SA-β-Gal、γ-H2AX、FOXO4阳性表达显著增多(P<0.05),且FAP、γ-H2AX及FOXO4蛋白表达量随造模时间延长而增加。模型组24周肿瘤组织周围衰老的肿瘤相关成纤维细胞明显增多(P<0.05)。与模型组比较,扶正化瘀方组肿瘤发生明显减少(P<0.05),肝组织胶原沉积及α-SMA阳性染色及蛋白表达量减少;SA-β-Gal、FAP、γ-H2AX、FOXO4阳性表达减少(P<0.05),FAP、γ-H2AX及FOXO4蛋白表达水平显著降低,肿瘤周围衰老的成纤维细胞明显减少(P<0.05)。结论:DEN复合CCl4诱导小鼠模型具有从肝纤维化到肝癌的病变特点,肿瘤周围衰老成纤维细胞增多;扶正化瘀方可减少肝纤维化背景小鼠肝癌的发生,其机制与抑制肝星状细胞活化和肝纤维化、减少肿瘤周围衰老成纤维细胞相关。

Objective:To observe the effect of Fuzheng Huayu Decoction on carcinogenesis of fibrosis-dependent hepatocellular carcinoma(HCC)model and explore the change of hepatic stellate cell activation and senescent cancer-associated fibroblasts(CAFs).Methods:C57BL/6 male mice,intraperitoneally injected diethylnitrosamine(DEN)combined with CCl4,were induced fibrosis-dependent HCC.The morphological changes of liver tissue and the number of tumors were observed at 18 and 24 weeks.The relative expression levels of Afp and Gpc3 gene in tumor and adjacent tissues were determined by fluorescence quantitative PCR.Senescence-associatedβ-galactosidase staining and immunohistochemical staining were performed to observe cell senescence markers,such asγ-H2AX,FOXO4,of FAP+fibroblasts around the tumor tissue.Western blotting was applied to detect expression level ofα-SMA,FAP,γ-H2AX and FOXO4 in liver tissues.The model was given Fuzheng Huayu Decoction by gavage from 12 weeks onwards.After 12 weeks of administration,the incidence of tumor,the degree of liver fibrosis and the positive staining of fibroblast senescence markers in the liver and adjacent tumor tissue were evaluated.Results:The collagen deposition and activation of hepatic stellate cells in the liver increased with model time.At 24 weeks,the incidence of liver cancer in the model group was 100%.In the model group,SA-β-Gal andγ-H2AX+fibroblasts could be seen in the portal area and around the collagen deposition.Compared with 18 weeks,the positive expression ofγ-H2AX in FAP+CAFs around the tumor tissue increased at 24 weeks.Compared to the control group,the expression of FAP,γ-H2AX and FOXO4 in the paracancerous tissues increased with the prolongation of the modeling time(P<0.05).Compared with the model group,the collagen deposition and hepatic stellate cell activation in Fuzheng Huayu Decoction group were reduced and the incidence of tumor was lower than that in the model group(71.4%vs 100.0%,P<0.05).The number of FAP+CAFs andγ-H2AX and FOXO4 positive staining around the cancer tissue decreased in the portal area and around the fibrous collagen in Fuzheng Huayu Decoction group(P<0.05).After application of Fuzheng Huayu Decoction,α-SMA,FAP,γ-H2AX and FOXO4 protein expressed in paraneoplastic tissues were significantly lower than model group at 24 weeks(P<0.05).Conclusion:In the dynamic model of carcinogenesis induced by DEN combined with CCl4,the senescent fibroblasts around the tumor increased with the modeling time and tumor development.With the prolongation of model time,senescent fibroblasts were also seen around the tumor,which were involved in tumorigenesis and development.Fuzheng Huayu Decoction can resist fibrosis and reduce the occurrence of HCC.Its mechanism is related to inhibiting hepatic stellate cell activation and reducing senescent fibroblasts around the tumor tissue.