氮芥通过激活腺苷酸活化蛋白激酶/自噬通路抑制角质形成细胞增殖活力  

Nitrogen mustard inhibits keratinocyte proliferation by activating AMPK/autophagy pathway

作　　者：董训虎 陈明亮[1] 叶枫[1] 但国蓉[1] 邹仲敏[1] DONG Xunhu;CHEN Mingliang;YE Feng;DAN Guorong;ZOU Zhongmin(Department of Chemical Defense,Faculty of Military Preventive Medicine,Army Medical University (Third Military Medical University),Chongqing,400038,China)

机构地区：[1]陆军军医大学(第三军医大学)军事预防医学系防化医学教研室

出　　处：《第三军医大学学报》2019年第14期1295-1300,共6页Journal of Third Military Medical University

基　　金：国家自然科学基金青年科学基金(81703268,81803279)~~

摘　　要：目的探讨腺苷酸活化蛋白激酶(AMP-activated protein kinase,AMPK)/自噬通路在氮芥(nitrogen mustard,NM)诱导角质形成细胞增殖活力下降中的作用。方法用不同浓度(1、5、10、20μmol/L)的氮芥单独或加入5 mmol/L自噬特异性抑制剂(3-methyladenine, 3-MA)、20 nmol/L自噬特异性激动剂(rapamycin, RAPA)、10μmol/L AMPK特异性抑制剂(compound C, CC)处理人角质形成细胞株(HaCaT细胞)24 h后,CCK-8法检测细胞增殖活力,Western blot检测AMPK、pAMPK、LC3-B2/B1和p62的表达水平,共聚焦显微镜观察自噬体形成。结果 NM处理后HaCaT细胞增殖活力被显著抑制(P<0.05),且处理浓度越高抑制效应越明显;当NM浓度为20μmol/L时,角质形成细胞增殖活力约下降40%。同时,NM处理能力显著提高pAMPK、LC3-B2的表达水平并下调p62的表达,且随处理浓度的增加,表达变化越明显;同时,NM可明显促进HaCaT细胞自噬体的形成。加入3-MA或CC干预后能显著抑制NM的上述效应(P<0.05),而RAPA干预对NM处理的效应无明显影响(P>0.05)。结论氮芥能够激活AMPK/自噬通路诱导角质形成细胞的毒性损伤,抑制细胞的增殖活性。Objective To explore the role of AMP-activated protein kinase(AMPK)/autophagy signaling pathway in nitrogen mustard(NM)-induced cytotoxicity in cultured keratinocytes. Methods HaCaT cells were treated with different concentrations of NM(1, 5, 10 and 20 μmol/L) in the presence or absence of 5 mmol/L 3-methyladenine(3-MA, a specific inhibitor of autophagy), 20 nmol/L rapamycin(RAPA, a specific activator of autophagy), or 10 μmol/L compound C(a specific inhibitor of AMPK) for 24 h. CCK-8 assay was used to assess the cell viability, and the expression of p62, LC3, AMPK and pAMPK in the cells were detected using Western blotting;laser confocal microscopy was performed to detect the formation of autophagosomes in the cells after the treatments. Results Treatment with NM significantly decreased the viability of HaCaT cells in a dose-dependent manner, and NM at 20 μmol/L markedly decreased the cell viability to 60% of the control level. NM also dose-dependently up-regulated the phosphorylation of AMPK and LC3-B2, down-regulated the expression of p62, and significantly promoted the formation of autophagosomes in HaCaT cells. The application of 3-MA and compound C both abolished the effects of NM(P<0.05), whereas RAPA did not produce significant changes in the effect of NM(P>0.05). Conclusion NM induces cytotoxicity in cultured keratinocytes by activating AMPK/autophagy signaling pathway to inhibit the cell proliferation.

关 键 词：氮芥 自噬 腺苷酸活化蛋白激酶 角质形成细胞 细胞增殖 

分 类 号：R322.991[医药卫生—人体解剖和组织胚胎学] R827.12[医药卫生—基础医学]