mTOR信号通路在Omega-3多不饱和脂肪酸饮食干预小鼠肝纤维化进程中的作用及其机制

Intervention of mouse hepatic fibrosis by Omega-3 polyunsaturated fatty acid diet in mediation of mTOR signaling pathway

目的探讨mTOR信号通路在Omega-3多不饱和脂肪酸饮食干预四氯化碳(CCl_(4))诱导小鼠肝纤维化进程中的作用及其机制。方法将C57BL/6小鼠随机分为对照组,4周模型组,8周模型组及治疗组,对照组经腹腔注射玉米油;模型组及治疗组经腹腔注射含20% CCl_(4)的玉米油溶液,5 mL/kg,每周2次,除4周模型组注射4周外,其余3组持续注射8周;治疗组在造模的同时,从第4周开始进行二十碳五烯酸(eicosapentaenoic acid,EPA)/二十二碳六烯酸(docosahexaenoic acid,DHA)灌胃治疗,4 g/kg,每日1次,对照组及8周模型组灌胃等剂量生理盐水。在造模完成后24 h内剖解,观察肝脏大体形态;小鼠肝组织包埋切片,HE、Masson三色及天狼星红染色评估肝组织病理变化及纤维化程度;免疫组织化学染色分析肝组织中肝星状细胞(hepatic stellate cell,HSC)活化标志蛋白(α-SMA)、抗凋亡蛋白(Bcl-2、Bcl-xL)、细胞增殖核抗原蛋白(proliferating cell nuclear antigen,PCNA)的表达水平;Western blot分析促纤维化蛋白(Collagen1A1、Fibronectin及α-SMA)的表达水平,同时检测m TOR信号通路中m TOR及S6蛋白的磷酸化水平。结果与8周模型组比较,治疗组小鼠肝脏形态有显著改善,但肝组织表面仍有细小颗粒;小鼠肝组织中炎症细胞浸润、肝细胞坏死及胶原沉积显著减少,其炎症及纤维化程度接近4周模型组。经免疫组织化学分析,肝组织中α-SMA蛋白表达水平显著减少(P <0.01),Bcl-2、Bcl-xL及PCNA蛋白表达水平均显著增加(P均<0.01);经Western blot分析,α-SMA、Collagen1A1、Fibronectin蛋白表达水平均显著减少(P均<0.01);m TOR及S6蛋白的磷酸化水平显著下降(P均<0.01)。结论 Omega-3多不饱和脂肪酸饮食干预能抑制CCl4诱导的小鼠肝纤维化,其作用机制可能与抑制mTOR信号通路有关。

Objective To investigate the role of mTOR signaling pathway in intervention of carbon tetrachloride-induced hepatic fibrosis in mice by Omega-3 polyunsaturated fatty acid diet and the relevant mechanism.Methods C57BL/6 mice were randomly divided into control group,4-week model,8-week model and treatment groups.The mice in control group were injected i.p.with corn oil,while those in model and treatment groups with corn oil containing 20% carbon tetrachloride,twice a week at a dosage of 5 mL/kg.The mice in 4-week model group were injected for 4 weeks,while those in the other three groups for 8 weeks.However,the mice in treatment group were treated with eicosapentaenoic acid(EPA)/docosahexaenoic acid(DHA)by gavage since the 4 th week of modeling,once a day at a dosage of 4 g/kg,while those in control and 8-week model groups with an equal dosage of physiological saline.The mice were dissected within 24 h after establishment of model for gross observation of liver,of which the liver was prepared into section and observed for pathological change and fibrosis level by HE,Masson trichrome and sirius red staining.The expression levels of marker protein α-SMA of hepatic stellate cell activation,anti-apoptotic proteins Bcl-2 and Bcl-xL and proliferating cell nuclear antigen(PCNA)protein in liver tissue were determined by immunohistochemical staining,while those of fibrogenic proteins Collagen1 A1,Fibronectin and α-SMA as well as phosphorylation levels of mTOR and S6 protein in m TOR signaling pathway by Western blot.Results Compared with those in 8-week model group,the morphology of liver of mice in treatment group was improved significantly,while small particles were still observed on the surface of liver tissue;the inflammatory cell infiltration,necrosis of hepatocyte and collagen deposition in liver tissue decreased significantly,and the levels of inflammation and fibrosis were closed to those in 4-week model group.Immunohistochemical staining showed that,compared with those in 8-week model group,the expression level of α-SMA decreased significantly(P < 0.01),while those of Bcl-2,Bcl-xL and PCNA increased significantly(each P < 0.01).However,Western blot showed that the expression levels ofα-SMA,Collagen1 Aa and Fibronectin as well as the phosphorylation levels of mTOR and S6 protein decreased significantly(each P < 0.01).Conclusion Omega-3 polyunsaturated fatty acid diet inhibited the carbon tetrachloride-induced hepatic fibrosis in mice,of which the mechanism may be associated with the inhibition of mTOR signaling pathway.