Ghrelin通过AKT/ERK信号通路对LPS诱导的肺泡上皮细胞凋亡的影响

Effects of Ghrelin on human alveolar epithelial cell apoptosis in response to lipopolysaccharide through AKT/ERK signaling pathway

目的:研究Ghrelin对内毒素(Lipopolysaccharide,LPS)所致的肺泡Ⅱ型上皮细胞(A549)凋亡的影响及其机制。方法:CCK-8(Cell Counting Kit-8)法检测LPS刺激对A549的细胞毒性;原位末端标记法(TUNEL)检测细胞凋亡率;流式细胞术检测细胞内一氧化氮(NO)的产生;Western blot检测诱导型一氧化氮合成酶(iN OS)、AKT、ERK、p-AKT、p-ERK信号通路蛋白以及cleaved caspase-3、Bax、Bcl-2凋亡相关蛋白的表达。结果:CCK-8检测结果显示LPS可显著抑制A549细胞的增殖,降低细胞活力;TUNEL检测发现Ghrelin可显著抑制LPS导致的A549细胞的凋亡(P<0.05);LPS可以促进iN OS的表达,增加细胞内NO的产生,并同时抑制AKT、ERK通路的活性,上调下游促凋亡蛋白Bax以及终末凋亡蛋白cleaved caspase-3的表达,下调抗凋亡蛋白Bcl-2的表达,而应用Ghrelin预处理后可以逆转LPS对AKT、ERK通路活性的抑制,继而下调Bax以及cleaved caspase-3的表达,上调Bcl-2的表达,差异均具有统计学意义(P<0.05),但Ghrelin对细胞内NO的产生无明显影响。结论:Ghrelin可以通过上调AKT及ERK通路的活性抑制LPS诱导的肺泡上皮细胞凋亡,但不能降低iN OS诱导产生NO的水平。

Objective ： To investigate the protective effects of Ghrelin on LPS-induced apoptosis of human alveolar epithelial A549 cells, along with the possible molecular mechanisms. Methods ： CCK-8 assay was used to examine the cell viability of A549 treated by LPS. Apoptosis of A549 cells was measured by TUNEL. NO（Nitric oxide）production was detected by NO-specific fluorescent probe 3-Amino,4-aminomethyl-2＇, 7＇ -difluoresceindiac.etate（DAF-FM DA）. Western blot was also performed to examine the expressions of iNOS（inducible nitric oxide synthase） ,AKT,ERK,p-AKT,p-ERK and apoptotic proteins,such as Bcl-2,Bax,and cleaved caspase- 3. Results： LPS exposure impaired cell viability and increased apoptosis of A549 cells significantly in concentration-and time-dependent manners accompanied with increased Bax and cleaved caspase-3 production,coupled with decreased Bcl-2 levels. Meanwhile,LPS promoted iNOS expression and the production of NO. Ghrelin pretreatment ameliorated LPS-caused alterations in the ratio of Bax/Bcl-2 and cleaved caspase-3 expression. TUNEL analysis showed that Ghrelin could decrease the apoptosis induced by LPS in A549 （ P 〈0. 05）. Simultaneously,LPS remarkably decreased the expression of p-AKT and p-ERK in A549 cells,which was abrogated by Ghrelinpretreatment. However,Ghrelin had no significant effect on NO production induced by LPS. Conclusion： Ghrelin reduces LPS-induced apoptosis of human alveolar epithelial cells partly through activating the AKT and ERK pathway,but the level of iNOS derived NO couldnot be reduced.