基于p53/AMPK/mTOR信号通路调控细胞自噬探讨益气扶正解毒汤抑制Lewis肺癌小鼠皮下肿瘤生长的作用机制

Study on the Mechanism of Yiqi Fuzheng Jiedu Decoction Inhibiting Subcutaneous Tumor Growth in Mice with Lewis Lung Cancer through Regulating Autophagy by p53/AMPK/mTOR Pathway

目的观察益气扶正解毒汤(YQT,黄芪、麦冬、黄芩等)对Lewis肺癌小鼠皮下肿瘤生长的影响,并基于p53/腺苷酸活化蛋白激酶(AMPK)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路调控细胞自噬探讨其可能的作用机制。方法采用Lewis肺癌细胞构建C57BL/6小鼠皮下肿瘤模型,随机分为模型组、顺铂组(3 mg·kg^(-1))及YQT低、中、高(8.7、17.4、34.8 g·kg^(-1))剂量组,每组8只。造模后第7天开始,YQT组予以相应剂量灌胃给药,每日1次,连续21 d。采用HE染色法观察肿瘤组织的病理变化;免疫组化法检测肿瘤组织Ki67的表达;透射电镜检测肿瘤组织自噬小体数量;RT-PCR法检测肿瘤组织自噬相关蛋白LC3、Beclin-1、Atg5、Atg7 mRNA表达水平;Western Blot法检测LC3、Beclin-1、p62、Atg5、Atg7和p53/AMPK/mTOR通路蛋白表达水平。结果与顺铂组比较,YQT高剂量组的抑瘤率无明显差异(P>0.05)。与模型组比较,YQT各剂量组小鼠皮下肿瘤的体积明显缩小(P<0.05),肿瘤质量均明显降低(P<0.05),病理切片显示肿瘤组织有较多的坏死区域,肿瘤组织中的自噬小体数量明显增加,LC3Ⅱ/Ⅰ、Beclin-1、Atg5蛋白表达明显上调(P<0.05);YQT中、高剂量组小鼠肿瘤组织中Ki67蛋白表达明显下调(P<0.05),Atg7、p53、磷酸化AMPK(p-AMPK)/AMPK蛋白表达明显上调(P<0.05),p62、磷酸化mTOR(p-mTOR)/mTOR蛋白表达明显下调(P<0.05);YQT高剂量组小鼠肿瘤组织的LC3Ⅱ/Ⅰ、Beclin-1、Atg5、Atg7 mRNA表达明显上调(P<0.05)。结论YQT可能通过p53/AMPK/mTOR信号通路上调细胞自噬水平,抑制Lewis肺癌小鼠皮下肿瘤生长及肿瘤细胞增殖。

Objective To observe the effect of Yiqi Fuzheng Jiedu Decoction(YQT;Astragali radix,Ophiopogonis radix,Scutellariae radix,et al)on subcutaneous tumour growth in Lewis lung cancer mice and to investigate its possible mechanism of action based on the p53/Adenosine monophosphate-activated protein kinase(AMPK)/mammalian target of rapamycin(mTOR)signalling pathway regulating cellular autophagy.Methods Lewis lung cancer cells were used to construct a subcutaneous tumor model in C57BL/6 mice,which were randomly divided into model group,cisplatin group(3 mg·kg^(-1))and YQT low-,medium-and high-(8.7,17.4 and 34.8 g·kg^(-1))dose groups,8 mice in each group.The tumor tissues were stained with HE to observe the pathological changes;the immunohistochemistry was used to detect the expression of Ki67;the transmission electron microscopy was used to detect the number of autophagic vesicles;RT-PCR method was used to detect the mRNA expression of LC3,Beclin-1,Atg5 and Atg7.The expression levels of LC3,Beclin-1,p62,Atg5,Atg7 and p53/AMPK/mTOR pathway proteins were detected by the Western Blot method.Results Compared with cisplatin group,there was no significant difference in tumor inhibition rate in YQT high-dose group(P>0.05).Compared with model group,subcutaneous tumor volume of mice in YQT dose groups was significantly smaller(P<0.05),and tumor mass was significantly decreased(P<0.05).Pathological sections showed that there were many necrotic areas in tumor tissues,and the number of autophagosomes in tumor tissues was significantly increased.The protein expressions of LC3Ⅱ/Ⅰ,Beclin-1 and Atg5 were significantly up-regulated(P<0.05).The protein expression of Ki67 was significantly down-regulated in YQT medium-and high-dose groups(P<0.05),and Atg7,p53,and the protein expression of phosphorylated AMPK(P-AMPK)/AMPK was significantly up-regulated(P<0.05).The protein expressions of p62,phosphorylated mTOR(P-MTOR)/mTOR was significantly down-regulated(P<0.05);The mRNA expressions of LC3Ⅱ/Ⅰ,Beclin-1,Atg5 and Atg7 in tumor tissues of mice in YQT high-dose group were significantly up-regulated(P<0.05).Conclusion YQT may inhibit subcutaneous tumor growth and tumor cell proliferation in Lewis lung cancer mice by upregulating cellular autophagy levels via the p53/AMPK/mTOR signaling pathway.