【摘要】 目的 研究竹叶花椒甲醇提取物（ZADCM）对BRL 3A细胞自噬水平与活性氧（ROS）生成量的影响。方法 体外培养BRL 3A细胞，将其与ZADCM(70μg·mL^-1)处理3、6、12、24 h或不同浓度的ZADCM(30、50、70μg·mL^-1)孵育24 h,干预给药用3-甲基腺嘌呤(10 mmol·L^-1)、二甲双胍(2.5 mmol·L^-1)处理；采用CCK-8法检测BRL 3A细胞存活率，流式细胞术检测BRL 3A细胞内ROS生成量，免疫荧光法观察自噬标志性蛋白LC3B的荧光强度，Western blotting检测p-mTOR、mTOR、p-ULK1、ULK1、Beclin-1和LC3B蛋白的表达。结果 不同浓度ZADCM处理24 h后，与正常对照组比较，免疫荧光结果显示LC3B荧光强度降低；ZADCM(70μg·mL^-1)处理12或24 h后，Western blotting结果显示，LC3B和Beclin-1蛋白表达水平均降低（P<0.05或P<0.01）,p-mTOR²⁴⁴⁸/mTOR、p-ULK1⁷⁵⁷/ULK1均显著升高（P<0.05）。激光共聚焦结果显示ROS灶点增加，且流式细胞术结果显示，药物处理组的ROS水平均明显升高（P<0.05或P<0.01）;采用3-甲基腺嘌呤或二甲双胍干预后，与药物处理组比较，细胞内ROS升高或降低，自噬相关蛋白表达降低或升高，ZADCM组BRL 3A细胞存活率降低或升高。结论 ZADCM可能通过激活BRL 3A细胞内mTOR/ULK1信号通路，抑制细胞自噬，并伴随ROS累积，引起细胞活力下降。更多还原

【Abstract】 Objective To study the effects of the methanolic extract of Zanthoxylum armatum DC.（ZADCM） on autophagy levels and reactive oxygen species（ROS） production in BRL 3A cells. Methods BRL 3A cells were cultured in vitro and the cells were treated with ZADCM(70 μg·mL^-1) for 3,6,12 and 24 h or ZADCM at different concentrations(30,50 and 70 μg·mL^-1) for 24 h.The intervention was then carried out by using 3-methyladenine(10 mmol·L^-1) and metformin treatment(2.5 mmol·L^-1).The effect of ZADCM on the survival rate of BRL 3A cells was detected by CCK-8 assay, and ROS production in BRL 3A cells was detected by flow cytometry.The expression level of autophagy marker protein LC3B was detected by immunofluorescence, and the expression levels of p-mTOR,mTOR,p-ULK1,ULK1,Beclin-1 and LC3B proteins were detected by Western blotting. Results Compared with the normal control group, immunofluorescence results showed a decrease in LC3B fluorescence intensity after 24 h of treatment with different concentrations of ZADCM.Western Blotting results showed that the expression levels of autophagy related proteins LC3B and Beclin-1 were significantly decreased（P<0.05 or P<0.01） after 12 or 24 h of treatment with ZADCM(70 μg·mL^-1),and p-mTOR²⁴⁴⁸/mTOR and p-ULK1⁷⁵⁷/ULK1 were significantly increased（P<0.05）. ROS focal points were decreased detected by confocal laser scanning, and ROS levels were significantly increased detected by flow cytometry（P<0.05 or P<0.01）.In addition, after the intervention with 3-methyladenine or metformin, intracellular ROS increased or decreased, and the expression of autophagy related proteins decreased or increased compared with the drug treatment group. Conclusion ZADCM can activate the intracellular mTOR/ULK1 signaling pathway in BRL 3A cells and inhibit autophagy leading to decreased cell viability with ROS accumulation.更多还原