天然纯鲜粉葛饮料通过TLR4/MyD88/NF-κB通路抑制小鼠酒精性肝损伤研究

Study on Natural Pure Fresh Pueraria Beverage Inhibited Alcoholic Liver Injury Via TLR4/MyD88/NF-κB Pathway in Mice

目的:探讨超高温瞬时灭菌全新工艺制备的无化学添加剂的天然纯鲜粉葛饮料对酒精性肝损伤小鼠的保护作用以及对TLR4/MyD88/NF-κB通路的影响。方法:采用高效液相色谱法对鲜粉葛饮料中异黄酮成分进行含量测定。采取43%酒精灌胃的方法建立小鼠酒精性肝损伤模型。检测血清中谷草转氨酶(AST)、谷丙转氨酶(ALT)、总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白(LDL)、高密度脂蛋白(HDL)及炎症因子水平;苏木素-伊红(HE)染色分析各组肝脏病理形态变化;免疫组化检测肝组织Toll样受体4(TLR4)、髓样分化分子88(MyD88)、核转录因子(NF-κB p65)的表达。结果:天然纯鲜粉葛饮料中检测到6种异黄酮成分,其中葛根素含量最高,达到622.6μg/mL。天然纯鲜粉葛饮料预处理能降低小鼠体内两种血清转氨酶含量水平(P<0.01);饮料高剂量组能降低血清中TG、TC、LDL、白细胞介素-6(IL-6)、白细胞介素-1β(IL-1β)和肿瘤坏死因子-α(TNF-α)含量水平(P<0.001),提高HDL含量水平(P<0.05)。病理切片结果显示天然纯鲜粉葛饮料能有效改善小鼠肝组织出现的脂肪空泡,胞质疏松,细胞核排列不规则等情况,并抑制小鼠肝组织中TLR4、MyD88、NF-κB p65蛋白的表达(P<0.01,P<0.001)。结论:无化学添加剂的天然纯鲜粉葛饮料可减少小鼠肝脏内脂质沉积和降低炎症因子水平,对酒精性肝病具有一定的预防保护作用,其机制可能与抑制TLR4/MyD88/NF-κB通路的激活有关。

Objective To investigate the protective effects of natural pure fresh pueraria beverage without chemical additives prepared by the brand new process of ultra-high temperature instantaneous sterilization on mice with alcoholic liver injury and its effects on TLR4/MyD88/NF-κB pathway.Methods The content of isoflavones in natural pure fresh pueraria beverage was determined by high performance liquid chromatography(HPLC).A mice model of alcoholic liver injury was established by intragastric gavage with 43%alcohol.The levels of aspartate aminotransferase(AST),alanine aminotransferase(ALT),total cholesterol(TC),triglyceride(TG),low-density lipoprotein(LDL),high-density lipoprotein(HDL),and inflammatory factors in serum were determined.And the pathological changes of the liver of each group were observed by(hematoxylin-eosin,HE)staining.In addition,the expression of Toll like receptors 4(TLR4),myeloid differentiation factor 88(MyD88)and nuclear factor kappa-B p65(NF-κB p65)in the liver were measured by immunohistochemical staining.Results Six isoflavones were detected in natural fresh Pueraria beverage,and the content of puerarin was the highest,reaching 622.6μg/mL.The experiments showed that the levels of AST and ALT in mice were reduced by the pretreatments with natural pure freshpueraria beverage(P<0.01).The serum TG,TC,LDL,the interleukin-6(IL-6),interleukin-1beta(IL-1β)and tumor necrosis factor-alpha(TNF-α)levels were reduced in the beverage high-dose group(P<0.001),while the content of HDL was increased(P<0.05).Pathological sections showed that the fatty vacuoles,loose cytoplasm and irregular arrangement of nuclei in the liver tissue of mice were improved effectively,while the expressions of TLR4,MyD88 and NF-κB p65 proteins in those of mice were inhibited with natural pure fresh pueraria beverage(P<0.01,P<0.001).Conclusion These results suggested that natural pure fresh pueraria beverage without chemical additives could reduce the fatty deposition and levels of inflammatory factors in the liver of mice,thus had a certain preventive and protective effect on alcoholic liver injury.Its mechanism may be related with inhibition of the activation of TLR4/MyD88/NF-κB pathway.