祛湿化瘀方抑制Wnt3a/β-catenin/VEGF信号通路保护肠道血管屏障治疗实验性NAFLD的机制研究

Mechanism study of Qushi Huayu Decoction inhibiting Wnt3a/β-catenin/VEGF signaling pathway to protect gut vascular barrier in treatment of experimental NAFLD

目的:通过观察祛湿化瘀方对非酒精性脂肪性肝病(NAFLD)小鼠回肠肠道血管屏障功能的影响,揭示祛湿化瘀方治疗NAFLD的机制。方法:(1)采用高脂高糖(HFHC)饮食模型,C57BL/6J小鼠随机分为正常组,HFHC饮食模型组,祛湿化瘀方低、高剂量组,12周末开始给药,直至18周末;(2)检测小鼠肝组织甘油三酯(TG),血清生化指标;HE染色观察肝组织、回肠病理;油红O染色观察肝组织脂质;免疫荧光观察回肠质膜相关蛋白1(PV-1)的表达;Western Blot检测肝脏LBP和回肠Wnt3a、p-β-catenin、VEGF的表达。结果:与正常组比较,模型组小鼠肝组织TG,血清ALT、AST活性,肝组织NAS评分,血清TG、TC、LDL-C、空腹血糖、空腹胰岛素、HOMA-IR显著升高(P<0.01),祛湿化瘀方干预后各项指标均显著降低(P<0.05,P<0.01)。模型组小鼠肝组织HE染色显示肝细胞轮廓肿胀、形态不规则,脂肪变性、气球样变及炎性细胞浸润多见;回肠HE染色显示回肠组织结构受到破坏,可见绒毛顶部少量细胞脱落;肝组织油红O染色显示肝细胞轮廓变大变圆,细胞质中可见大量脂滴;回肠PV1免疫荧光结果显示PV1表达显著升高(P<0.01);Western Blot结果显示肝脏LBP、回肠Wnt3a、p-β-catenin/β-catenin、VEGF表达显著升高(P<0.05,P<0.01);祛湿化瘀方干预后肝组织、回肠病理均改善,肝细胞中脂滴明显变小,回肠PV1表达显著降低(P<0.05),肝脏LBP、回肠Wnt3a、p-β-catenin/β-catenin、VEGF表达显著降低(P<0.05,P<0.01)。结论:祛湿化瘀方可通过抑制回肠中Wnt3a/β-catenin/VEGF信号通路,下调PV1的表达,保护肠道血管屏障的功能,改善NAFLD。

Objective:To reveal the mechanism of treatment of non-alcoholic fatty liver disease(NAFLD) by Qushi Huayu Decoction by observing the effect of Qushi Huayu Decoction on ileocecal gut vascular barrier function in NAFLD mice.Methods:(1)Using high-fat and high-carbohydrate(HFHC) diet model, C57BL/6J mice were randomly divided into normal group,HFHC model group, and Qushi Huayu Decoction low-dose and high-dose groups. The drug was administered at the end of 12weeks until the end of 18 weekends;(2)the mice were examined for TG of the liver tissues, serum biochemical indices;HE staining to observe the pathology of liver tissue and ileum;Lipid in liver tissue was observed by oil red O staining;Immunofluorescence to observe the expression of ileal PV-1;Western Blot to detect the expression of hepatic LBP and ileal Wnt3a, p-β-catenin,VEGF. Results: Compared with the normal group, liver TG, serum ALT, AST activity, liver NAS score, serum TG, TC, LDL-C,fasting blood glucose, fasting insulin and HOMA-IR were significantly elevated in the model group of mice(P<0.01), after the intervention of Qushi Huayu Decoction, it was significantly reduced(P<0.05, P<0.01). In the model group, HE staining of liver tissue showed that the outline of liver cells was swollen, irregular shape, steatosis, ballooning and inflammatory cell infiltration were common. HE staining of the ileum showed that the structure of the ileum was damaged and a small number of cells at the top of the villi were shed. Oil red O staining of liver tissue showed that the outline of liver cells became large and round, and a large number of lipid droplets could be seen in the cytoplasm. Immunofluorescence results of PV1 in ileum showed that PV1 expression was significantly increased(P<0.01). Western Blot results showed that the expressions of LBP in liver, Wnt3a in ileum, p-β-catenin/β-catenin and VEGF were significantly increased(P<0.05, P<0.01). After the intervention of Qushi Huayu Decoction, the liver tissue and ileum pathology were improved, the lipid droplets in hepatocytes were significantly smaller, the expression of PV1in ileum was significantly decreased(P<0.05), and the expressions of liver LBP, ileum Wnt3a, p-β-catenin/β-catenin and VEGF were significantly decreased(P<0.05, P<0.01). Conclusion: Qushi Huayu Decoction can protect the function of gut vascular barrier and improve NAFLD by inhibiting the Wnt3a/β-catenin/VEGF signaling pathway and down-regulating the expression of PV1 in the ileum.