【摘要】 目的:观察抗肝纤维化中药扶正化瘀方防治肝硬化小鼠模型肝癌发生的作用，并结合网络药理学筛选可能的作用靶点并进行验证，探讨其药理机制。方法:以DEN联合CCl₄诱导C57BL/6雄性小鼠肝硬化肝癌模型，设正常对照组、18周肝硬化模型组、24周肝癌模型组及扶正化瘀组。通过肝脏大体观察、肝体比、脾体比、肝功能、病理切片及免疫组化（AFP、Ki67）评价扶正化瘀方对肝硬化癌变的干预效果；结合网络药理学靶点，采用多色免疫荧光与蛋白印迹验证其药理机制。结果:（1）与18周和24周模型组相比，扶正化瘀组肝脏表面颗粒显著减小，肿瘤数量减少，脾体比、AST、ALT水平降低，肝组织病理提示肝细胞坏死及胶原沉积明显减少，癌旁组织中α-SMA的表达显著降低，肿瘤组织中Ki67表达水平和AFP阳性表达细胞显著减少；（2）网络药理学提示脂质信号通路在扶正化瘀方预防肝硬化肝癌发生中起关键作用；（3）CD36⁺活化的星状细胞在其中起调节作用，与模型组相比，扶正化瘀组癌旁组织中CD36⁺活化的HSC显著减少、α-SMA、CD36蛋白表达水平在癌旁和肿瘤组织中显著降低；（4）蛋白印迹和多色免疫荧光显示，与正常组比较，模型组中CD36⁺活化HSC中CPT1A的表达随造模时间延长上调，而扶正化瘀方可显著下调癌旁组织中CPT1A的表达。结论:扶正化瘀方可预防肝硬化小鼠肝癌的发生，CD36⁺活化HSC在其中起关键调节作用，其机制可能是通过抑制CPT1A酶的表达，减少CD36⁺HSC的活化，从而降低肝硬化肝癌的发生。更多还原

【Abstract】 Objective: To investigate the preventive effects and underlying mechanisms of the traditional Chinese medicine formula Fuzheng Huayu on the progression of liver cirrhosis to hepatocellular carcinoma（HCC） using a murine model, and to elucidate key therapeutic targets via network pharmacology-based analysis. Methods: Male C57BL/6 mice were subjected to the DEN+CCl₄ induction protocol to establish a cirrhosis-to-HCC progression model. Mice were randomized into four groups: a normal control group（N group）, an 18-week model group（18 w group, representing the cirrhosis stage）, a 24-week model group（24 w group, representing the HCC stage）, and an FZHY treatment group（FZHY group）. The efficacy of Fuzheng Huayu in preventing HCC was evaluated by assessing liver tumor burden（number and maximum diameter of nodules）, hepatosplenic indices, liver function, and histopathological analysis. The expression of proliferation markers, including AFP and Ki67, was assessed via immunohistochemistry. Subsequently, candidate targets identified by network pharmacology were validated in liver tissues using multicolor immunofluorescence and Western blotting to explore the molecular mechanisms of Fuzheng Huayu. Results: Compared with the 18 w and 24 w model groups, the FZHY group exhibited significantly reduced liver surface nodules, decreased tumor number, and lower spleen-to-body weight ratio. Serum levels of AST and ALT were markedly decreased. Histopathological examination revealed a significant reduction in hepatocyte necrosis（HE staining） and collagen deposition（Sirius Red staining）. The expression of α-SMA in adjacent tissues was significantly decreased, and immunohistochemistry demonstrated a substantial reduction in Ki67-positive cells within tumor tissues（P<0.01） and AFP-positive cells（P<0.000 1）. Network pharmacology analysis indicated that lipid signaling pathways play a pivotal role in Fuzheng Huayu’s prevention of liver cirrhosis and HCC progression. Key differentially expressed genes included carnitine palmitoyltransferase-1（CPT-1）. CD36⁺activated stromal cells were identified as regulatory components in this process. Compared to the model group, the FZHY group showed a significant reduction in CD36⁺activated hepatic stellate cells（HSCs） in adjacent tissues（P<0.000 1）, decreased α-SMA expression（P<0.000 1）, and markedly reduced CD36 protein levels in both adjacent and tumor tissues（P<0.000 1）. Notably, areas with fewer CD36⁺activated HSCs also contained fewer CD36⁺activated HSCs within tumor tissues. Western blot and multicolor immunofluorescence analyses revealed that CPT1A expression in CD36⁺activated HSCs increased progressively with modeling duration in the control group but was significantly downregulated in the FZHY group’s adjacent tissues（P<0.001）. Conclusion: Fuzheng Huayu can prevent the occurrence of liver cancer in mice with liver cirrhosis. CD36⁺activated HSC plays a key regulatory role in this process. Preliminary research suggests that the underlying mechanism may involve the inhibition of CPT1A enzyme expression, thereby reducing the activation of CD36⁺hepatic stellate cells and subsequently decreasing the incidence of hepatocellular carcinoma in the context of liver cirrhosis.更多还原