抑制AMPK观察清燥救肺汤对肺癌细胞自噬启动相关蛋白表达的影响

Effect of Qingzao Jiufeitang on Protein Expression Related to Autophagy Initiation in Lung Cancer Cells Through AMPK inhibition

目的:观察应用腺苷酸活化蛋白激酶(AMPK)抑制剂(compound C)后,清燥救肺汤对肺癌细胞自噬启动相关蛋白AMPK,哺乳动物雷帕霉素靶蛋白(mTOR),UNC-51样激酶1(ULK1)表达的影响。方法:雄性C57BL/6J小鼠随机分为模型组,环磷酰胺(CTX)组(50 mg·kg^(-1)),清燥救肺汤组(11 g·kg^(-1)),AMPK抑制剂组(10 mg·kg^(-1)),清燥救肺汤加AMPK抑制剂组(清燥加抑制剂组)(11 g·kg^(-1)+10 mg·kg^(-1))。小鼠右腋皮下注射Lewis肺癌细胞构建肺癌荷瘤模型。造模24 h后,CTX组腹腔注射给药,隔日1次,共7次,AMPK抑制剂组与清燥加抑制剂组腹腔注射compound C,每日1次,共14 d。清燥救肺汤组和清燥加抑制剂组,造模前后14 d均以中药设定剂量灌胃给药。实验结束后处死各组小鼠并取荷瘤组织,统计各组瘤质量;透射电镜(TEM)观察各组肺癌组织自噬溶酶体的生成情况;蛋白免疫印迹法(Western blot)检测AMPK,磷酸化腺苷酸活化蛋白激酶(p-AMPK),mTOR,磷酸化哺乳动物雷帕霉素靶蛋白(p-mTOR),ULK1,磷酸化UNC-51样激酶1(p-ULK1),微管相关蛋白1轻链3B(LC3B)和p62蛋白的表达;苏木素-伊红(HE)染色观察各组肺癌组织病理学变化。结果:与模型组比较,清燥救肺汤组瘤质量降低(P<0.01),电镜下发现自噬溶酶体生成,p-AMPK,p-ULK1,LC3B,LC3B-Ⅱ蛋白表达和p-AMPK/AMPK,p-ULK1/ULK1,LC3B-Ⅱ/LC3B-Ⅰ均升高(P<0.05,P<0.01),p-mTOR,p62蛋白表达和p-mTOR/mTOR均降低(P<0.05)。与清燥救肺汤组比较,清燥加抑制剂组电镜下未见自噬溶酶体生成,p-AMPK,p-ULK1,LC3B,LC3B-Ⅱ蛋白表达和p-AMPK/AMPK,p-ULK1/ULK1,LC3B-Ⅱ/LC3B-Ⅰ均明显降低(P<0.05,P<0.01),p62蛋白表达升高(P<0.05)。HE染色结果显示,各给药组肺癌组织与模型组比较,病理有明显改善。结论:清燥救肺汤能促进自噬发生标志蛋白LC3B-Ⅱ升高及p62蛋白表达降低从而诱导自噬发生,其自噬启动机制可能不是调控AMPK/mTOR/ULK1通路介导,而是通过AMPK/ULK1通路实现的。

Objective:To observe the effects of Qingzao Jiufeitang on the expression of adenosine 5′-monophosphate(AMP)-activated protein kinase(AMPK),mammalian target of rapamycin(mTOR),and UNC-51-like kinase 1(ULK1)in lung cancer cells after the application of AMPK inhibitor(compound C).Method: Male C57 BL/6 J mice were randomly divided into a model group,a cyclophosphamide(CTX)group(50 mg·kg^(-1)),a Qingzao Jiufeitang group(11 g·kg^(-1)),an AMPK inhibitor group(10 mg·kg^(-1)),and a Qingzao Jiufeitang combined with AMPK inhibitor group(combination group)(11 g·kg^(-1)+10 mg·kg^(-1)). Lewis lung cancer cells were subcutaneously injected into the right axilla to induce a tumor-bearing model. 24 hours after modeling,the mice in the CTX group were intraperitoneally injected once every other day for seven times in total. The mice in the AMPK inhibitor group and the combination group received intraperitoneal injection of compound C,once a day for 14 days. The mice in the Qingzao Jiufeitang group and the combination group were administered orally at the set dose for 14 days before and after modeling. At the end of the experiment,the mice in each group were sacrificed. The tumor-bearing tissues were collected,and the tumor weight of each group was counted. Transmission electron microscopy(TEM)was used to observe the formation of autolysosomes in lung cancer tissues of each group. Western blot was used to detect the protein expression of AMPK,phosphorylated AMPK(p-AMPK),mTOR,phosphorylated mTOR(p-mTOR),ULK1,phosphorylated ULK1(p-ULK1),microtubule-associated protein 1 light chain 3B(LC3B),and p62. Hematoxylin-eosin(HE)staining was used to observe the pathological changes of lung cancer in each group. Result: Compared with the model group,the Qingzao Jiufeitang group showed decreased tumor weight(P<0.01),the formation of autolysosomes under the electron microscope,increased protein expression of p-AMPK,p-ULK1,LC3B,LC3B-Ⅱ,and p-AMPK/AMPK,p-ULK1/ULK1,and LC3B-Ⅱ/LC3B-Ⅰ ratios(P<0.01,P<0.05),and reduced protein expression of pmTOR, p62, and p-mTOR/mTOR ratio(P<0.05). Compared with the Qingzao Jiufeitang group, the combination group showed no autolysosomes formation under the electron microscope, decreased protein expression of p-AMPK,p-ULK1,LC3B,LC3B-Ⅱ,and p-AMPK/AMPK,p-ULK1/ULK1,LC3B-Ⅱ/LC3B-Ⅰ ratios(P<0.05,P<0.01),and increased p62 protein expression(P<0.05). HE staining results showed that the pathological changes of lung cancer tissues in the groups with drug intervention were improved compared with those in the model group. Conclusion: Qingzao Jiufeitang can promote the elevation of LC3B-Ⅱ and decrease the expression of p62 protein,thus inducing autophagy. The mechanism of autophagy initiation may be achieved by the AMPK/ULK1 pathway instead of the mediation by the AMPK/mTOR/ULK1 pathway.