扶正化瘀方对非酒精性肝纤维化小鼠肝组织血管生成基因的调节作用及其对肝纤维化的影响*

目的 探讨血管生成基因在非酒精性脂肪性肝纤维化形成中的作用，以阐明扶正化瘀方治疗非酒精性脂肪性肝纤维化的作用机制。方法 选用7~8w龄健康雄性C57BL/6J小鼠，给予高脂、蛋氨酸-胆碱缺乏（MCD）饮食喂养8w，建立非酒精性脂肪性肝纤维化模型。采用扶正化瘀方进行干预，以蛋氨酸-胆碱充足饮食设立对照组。以HE和Masson染色观察小鼠肝组织脂肪变、炎症和纤维化程度；采用RT-PCR、免疫组化和Western blot法检测α-平滑肌肌动蛋白（α-SMA）、缺氧诱导因子-1α（HIF-1α）及其下游因子血管内皮生成因子（VEGF)和诱导型一氧化氮合酶（iNOs）mRNA及其蛋白水平。结果 模型组小鼠肝细胞出现大泡性为主的脂肪变性，小叶内可见点灶状肝细胞坏死及炎细胞浸润、窦周纤维化，汇管区纤维组织增生，纤维间隔形成；应用扶正化瘀方干预组动物肝损伤、炎症及纤维化程度显著改善，肝组织α-SMA、HIF-1α、VEGF和iNOs mRNA水平亦较模型组显著减低，依次为（3.83±0.53） 对 （8.33±2.32）、（3.02±0.55）对（4.50±0.78）、（3.23±0.94）对（7.40±1.54）和（3.60±0.96）对 （9.94±1.60），（P<0.01）；上述蛋白表达水平与基因水平呈一致变化趋势，扶正化瘀方干预组与模型组α-SMA、HIF-1α、VEGF和iNOs表达水平依次为（0.53±0.04） 对 （1.08±0.20）、（0.44±0.02）对（0.55±0.08）、（0.54±0.07） 对 （1.08±0.03）、和（1.61±0.21）对（3.30±0.88），（P<0.05）。结论 扶正化瘀方可通过抑制肝星状细胞活化，下调HIF-1α及其下游促血管生成因子VEGF和iNOs表达，阻止或减缓非酒精性脂肪性肝纤维化的进展。

Impact of Fuzheng Huayu recipe on the expression of angiogenic growth factor in mice with nonalcoholic fatty liver diseases

Objective To investigate the role of the angiogenic gene in the development of nonalcoholic fatty liver diseases（NAFLD） and to elucidate the mechanism of Fuzheng Huayu （FZHY） recipe in alleviating NAFLD related fibrosis in vivo. Methods Healthy male C57BL/6J mice aged 7~8 weeks were adopted to induce NAFLD related fibrosis by feeding methionine-choline deficient （MCD） diet for 8 weeks. Meanwhile， the intervention group was administrated with FZHY. Histological hepatic change in mice such as hepatic steatosis， inflammation and fibrosis were observed by hematoxylin & eosin and Masson trichrome staining. The expression of α-smooth muscle actin （α-SMA），hypoxia-inducible factor-1α （HIF-1α） and its downstream factors e.g. vascular endothelial growth factor （VEGF） and inducible nitrogen synthase （iNOs） were detected by either quantitative real-time PCR or Western blot or immunohistochemistry. Results The mice in model group were present with macrovesicular steatosis，inflammatory infiltration and spotty and focal necrosis in hepatic lobule，peri-sinusoid fibrosis，and proliferation of fibrous tissues and fibrous septums in portal area，while the hepatic damage，inflammation and fibrosis in the intervention group were ameliorated by FZHY administration；Meanwhile，significantly down-regulated mRNA levels of α-SMA，HIF-1α，VEGF and iNOs in the intervention group were observed （3.83±0.53） vs. （8.33±2.32），（3.02±0.55 ） vs. （4.50±0.78），（3.23±0.94） vs. （7.40±1.54），（3.60±0.96） vs. （9.94±1.60），respectively，（P<0.01） for all]，and the protein expression were consistent with their mRNA，e.g. （0.53±0.04） vs. （1.08±0.20），（0.44±0.02） vs. （0.55±0.08），（0.54±0.07）vs.（1.08±0.03），（1.61±0.21）vs.（3.30±0.88），respectively，P<0.05]. Conclusion FZHY recipe down-regulates the expression of HIF-1α and its downstream pro-angiogenic growth factors，VEGF and iNOs，by inhibiting the activation of hepatic stellate cells（HSCs），thus preventing or retarding the development of NAFLD related fibrosis in mice.