健脾化瘀解毒方调控PI3K/AMPK-mTOR-ULK1抑制ROS诱导GPL细胞自噬的机制研究

Study on the Mechanism of Jianpi Huayu Jiedu Formula on Autophage Induced by ROS of Gastric Precancerous Lesions Based on PI3K/AMPK-mTOR-ULK1 Pathway

目的:探讨健脾化瘀解毒方通过抑制氧化应激所致胃癌前病变(GPL)大鼠胃黏膜上皮细胞(GECs)自噬,保护胃黏膜、防治癌变的机制。方法:SD大鼠随机分为正常对照组、模型组、维酶素组及健脾化瘀解毒方高(9 g/kg)、低(4.5 g/kg)剂量组,复制GPL大鼠模型,于造模第12 w连续灌胃给药10 w,实验结束时麻醉大鼠后,取血分离血清检测ROS、SOD、MDA水平;取胃窦部胃黏膜,观察组织病理学(HE染色)和肠上皮化生(AB-PAS染色),检测GECs的PI3K、mTOR、AMPK、Raptor、ULK1、ATG13、Beclin1、ATG4B mRNA及miR182、miR30a、miR206和Beclin1、HMGB1、ATG5、MUC2、CDX2、ATG2B蛋白表达。结果:正常对照组大鼠胃黏膜无异常;模型组大鼠胃黏膜萎缩,空泡样变、组织异型性变,异型增生灶占胃黏膜厚度的1/3,各给药组胃黏膜病理学改变不同程度减轻;与模型组比较,健脾化瘀解毒方组PI3K、mTOR mRNA及miR30a、miR206显著升高,AMPK、Raptor、ULK1、ATG13、Beclin1、ATG4B mRNA及miR182和Beclin1、HMGB1、ATG5、MUC2、CDX2蛋白表达显著降低(P<0.01)。结论:健脾化瘀解毒方可通过影响miR182、miR30a、miR206等miRNAs活化,调控PI3K/AMPK-mTOR-ULK1通路,从而抑制氧化应激诱导自噬,逆转GPL大鼠GECs的肠化和损伤,防止GPL癌变。

Objective:To explore the intervention mechanism of Jianpi huayu jiedu formula to protect gastric mucosa and prevent carcinogenesis by inhibiting the autophagy of gastric mucosal epithelial cells(GECs)in rats with gastric precancerous lesions(GPL)induced by oxidative stress.Methods:SD rats were randomly divided into normal control group,model group,Vita group,low(4.5 g/kg)and high(9 g/kg)does groups of Jianpi huayu jiedu formula.The GPL rat model was replicated and given for 10 weeks by continuous gavage on the 12th week of modeling.At the end of the experiment,the rats were anesthetized,blood was collected and serum was separated to measure the lerels of ROS,SOD and MDA.The gastric mucosa in the antrum was taken and pathology(HE staining),intestinal metaplasia(AB-PAS staining)were observed.The expressions of PI3K,mTOR,AMPK,Raptor,ULK1,ATG13,Beclin1,ATG4B mRNA of GECs and miR182,miR30a,miR206 and Beclin1,HMGB1,ATG5,MUC2,CDX2,ATG2B proteins were measured.Results:There was no abnormal gastric mucosa in normal control group.In the model group,rats gastric mucosa was atrophic,vacuole-like and tissue atypia,and dysplasia lesions accounted for 1/3 of the thickness of gastric mucosa.The pathological changes of gastric mucosa could be alleviated in different degrees in all drug administration groups.Compared with the model group,the PI3K,mTOR mRNA,miR30a,miR206 in Jianpi huayu jiedu formula group were significantly increased,AMPK,Raptor,ULK1,ATG13,Beclin1,ATG4B mRNA and miR182,Beclin1,HMGB1,ATG5,MUC2 and CDX2 proteins expressions were significantly decreased(P<0.01).Conclusion:Jianpi huayu jiedu formula can regulate PI3K/AMPK-mTOR-ULK1 pathway by activating miRNAs such as miR182,miR30a and miR206,thereby inhibiting induce autophagy induced by oxidative,reversing intestinal metaplasia and damage of GECs in GPL rats,and preventing GPL carcinogenesis.