基于mTOR/Beclin1/LC3信号轴探讨制萎扶胃丸对胃癌前病变大鼠胃窦组织自噬的影响

Effect of Zhiwei Fuwei Pills on autophagy in gastric antrum tissue of rats with precancerous lesions of gastric cancer based on mTOR/Beclin1/LC3 signaling axis

目的:探讨制萎扶胃丸(ZWFW)对胃癌前病变(PLGC)大鼠胃窦组织哺乳动物雷帕霉素靶蛋白(mTOR)/自噬关键分子酵母Atg6同系物(Beclin1)/微管相关蛋白1轻链3(LC3)信号轴关键分子表达的影响。方法:SPF级SD大鼠随机分为正常组,模型组,叶酸组,ZWFW低、中、高剂量组,除正常组常规饲养外,模型组、叶酸组、ZWFW低、中、高剂量组,采用N-甲基-N’-硝基-N-亚硝基胍(MNNG)联合饥饱失常、乙醇灌胃、氨水自由饮用以及雷尼替丁饲料喂养五因素复合造模法建立PLGC大鼠模型后,分别用生理盐水、叶酸片水溶液(0.002 g/kg)、ZWFW低、中、高剂量水溶液(0.42,0.84,1.67 g/kg)予以治疗4周后剖腹取胃。采用苏木素-伊红(HE)染色观察大鼠胃窦组织病理学变化,采用实时荧光定量聚合酶链式反应(Real-time PCR)、蛋白免疫印迹法(Western blot)及免疫组化检测大鼠胃窦组织mTOR、Beclin1、微管相关蛋白1轻链3β(LC3B)的mRNA及蛋白表达。结果:与正常组比较,模型组大鼠胃窦组织胀大,胃壁变薄,胃黏膜色泽苍白,皱襞萎缩浅平,走行紊乱,可见结节及赘生物;HE染色示:与正常组比较,模型组胃黏膜腺体排列拥挤、紊乱,细胞形态不一,可见大量杯状细胞,细胞浆嗜碱性,细胞核大、深染、不规则,黏膜肌层浸润破坏;与模型组比较,ZWFW显著改善了胃黏膜腺体结构排列紊乱和细胞异型性等病理表现。与正常组比较,模型组大鼠胃窦组织m TOR mRNA和蛋白表达明显升高(P<0.05),Beclin1和LC3B mRNA及蛋白表达明显降低(P<0.05);与模型组比较,ZWFW中、高剂量组胃窦组织mTOR mRNA和蛋白表达降低(P<0.05),ZWFW低剂量组胃窦组织Beclin1、LC3B蛋白表达升高(P<0.05),中、高剂量组胃窦组织Beclin1、LC3B mRNA和蛋白表达升高(P<0.05)。结论:ZWFW可显著改善PLGC模型大鼠胃黏膜异常组织病理学表现,其机制可能与下调mTOR表达,上调Beclin1、LC3B表达进而促进自噬相关。

AIM:To investigate the effect of Zhiwei Fuwei Pills(ZWFW)on the expression of mammalian target of rapamycin(mTOR)/autophagy key molecule yeast Atg6 homologue(Beclin1)/microtubuleassociated protein 1 light chain 3(LC3)signaling axis key molecules in gastric antrum tissue of rats with precancerous gastric lesions(PLGC).METHODS:SPF SD rats were randomly divided into normal group,model group,folic acid group,ZWFW low-dose,medium-dose,high-dose group.In addition to the normal group,the model group,folic acid group,ZWFW low-dose,medium-dose and highdose groups,were used to establish the PLGC rat model by five factors compound modeling methods:N-methyl-N'-nitro-n-nitroguanidine(MNNG)combined with hunger and satiation,ethanol intragastric administration,free drinking of ammonia and ranitidine feed.The rats were treated with normal saline,folic acid tablet aqueous solution(0.002 g/kg),ZWFW low-dose, medium-dose, high-dose aqueoussolution (0.42, 0.84, 1.67 g/kg) for 4 weeks, and thestomach was removed by laparotomy. Hematoxylineosin(HE) staining was used to observe the histopathologicalchanges in the antrum of rats, and realtimepolymerase chain reaction (real-time PCR) ,Western blot (WB) and immunohistochemistry(IHC) were used to detect the expression of mammaliantarget of rapamycin mTOR, yeast Atg6 homologue1 (Beclin1), microtubule-associated protein 1light chain 3β (LC3B) mRNA and protein in the antrumof rats. RESULTS: Compared with the normalgroup, the Gastric antrum tissue of the modelgroup was distended, thinner gastric wall, palegastricmucosa, atrophic and flat folds, disorderedcourse and nodules and vegetations were visible.HE staining showed that compared with the normalgroup, the gastric mucosal glands in the modelgroup were crowded and disordered, and the cell morphology was different, including a large numberof goblet cells, basophilic cytoplasm, large, hyperchromaticand irregular nuclei, and mucosal muscleinfiltration and destruction. Compared with themodel group, treated by ZWFW can significantly improvethe pathological manifestations of gastric mucosalgland structure disorder and cell atypia. Comparedwith the normal group, mTOR mRNA and proteinexpression were significantly increased (P<0.05) and Beclin1 and LC3B mRNA and protein expressionwere significantly decreased (P<0.05) inthe antral tissue of rats in the model group;comparedwith the model group, mTOR mRNA and proteinexpression were decreased (P<0.05) in the mediumand high dose groups of ZWFW, Beclin1 and LC3B protein expression in the antral tissue of ratsin the low dose group of ZWFW and Beclin1 andLC3B mRNA and protein expression were increased(P<0.05) in the medium and high dose groups. CONCLUSION:Zhiwei Fuwei Pills can significantly improvethe abnormal histopathological findings ofgastric mucosa in PLGC model rats, and the mechanismmay be related to the down-regulation ofmTOR expression, up-regulation of Beclin1 andLC3B expression and then promoting autophagy.