慢性间歇性低氧通过Ang Ⅱ-AT1R激活PI3K/Akt促进LLC细胞迁移研究

Chronic intermittent hypoxia promotes the migration of LLC cells by activating the PI3K/Akt signaling pathway through Ang Ⅱ-AT1R

目的探究慢性间歇性低氧(CIH)通过血管紧张素Ⅱ(AngⅡ)-AngⅡ1型受体(AT1R)对小鼠Lewis肺癌(LLC)细胞迁移的影响以及相关机制。方法将20只小鼠随机分为常氧对照组(10只)和CIH组(10只)。用常氧和CIH处理后的LLC细胞皮下成瘤小鼠血清配制成完全培养基来培养LLC细胞,检测LLC细胞形态。Transwell检测不同处理方法对LLC细胞迁移的影响;蛋白质印迹法检测LLC细胞迁移相关信号蛋白水平的影响;酶联免疫吸附测定(ELISA)法检测小鼠血清与LLC细胞培养基上清中AngⅡ水平。结果ELISA结果显示,CIH组小鼠血清中AngⅡ的水平升高,t=13.948,P=0.008。不同处理方法小鼠血清培养LLC细胞结果显示,CIH组小鼠血清促进LLC细胞形态由上皮样转化为间充质样。Transwell结果显示,Losartan拮抗CIH组小鼠血清对LLC细胞迁移有促进作用,CIH的主效应为F=22.667,P=0.001,Los的主效应为F=29.706,P=0.001,CIH与Los二者间存在交互拮抗效应,F=10.303,P=0.012。用AngⅡ处理LLC细胞后发现,AngⅡ促进LLC细胞形态由上皮样转化为间充质样,Transwell结果显示,AngⅡ以浓度依赖性的方式促进LLC细胞迁移,F=42.497,P=0.003。AngⅡ以及AngⅡ联合使用AT1R拮抗剂Losartan处理LLC细胞,Transwell结果显示,Losartan拮抗AngⅡ对LLC细胞迁移有促进作用,F=53.432,P<0.001;蛋白质印迹结果显示,Losartan拮抗AngⅡ对vimentin(F=77.819,P=0.001)、MMP 2(F=49.363,P=0.001)、β-catenin(F=23.847,P<0.001)和p-Akt/Akt(F=58.608,P=0.007)水平有促进作用。AngⅡ以及AngⅡ联合使用PI3K阻断剂LY294002处理LLC细胞后,Transwell结果显示,LY294002拮抗AngⅡ对LLC细胞迁移有促进作用,F=52.792,P=0.002;蛋白质印迹结果显示,LY294002拮抗AngⅡ对vimentin(F=94.231,P=0.032)、MMP 2(F=48.612,P=0.020)、β-catenin(F=46.037,P<0.001)和p-Akt/Akt(F=19.644,P=0.002)水平有促进作用。进一步给予LLC细胞间歇性低氧(IH)以及IH联合Losartan处理,ELISA结果显示,IH提高LLC细胞培养基上清液中AngⅡ水平,t=5.022,P<0.001;Transwell结果显示,Losartan拮抗IH对LLC细胞迁移有促进作用,IH的主效应为F=84.587,P<0.001,Los的主效应为F=92.102,P<0.001,IH与Los二者的交互拮抗效应,F=70.097,P<0.001;蛋白质印迹结果显示,Losartan拮抗IH对vimentin(F=49.382,P=0.002)、β-catenin(F=61.919,P=0.002)、MMP2(F=14.132,P<0.001)和p-Akt/Akt(F=100.038,P=0.013)水平有促进作用。结论CIH通过AngⅡ-AT1R激活PI3K/Akt信号通路促进LLC细胞迁移。

Objective To investigate the effect of chronic inermittent hypoxia(CIH)on the migration of Lewis lung carcinoma(LLC)cells through AngiotensinⅡ(AngⅡ)-angiotensinⅡtype 1 receptor(AT1 R)and its related mechanism.Methods Totally twenty mice were randomly divided into normoxic control group(10 mice)and CIH group(10 mice).LLC cells were cultured in the complete culture medium with serum from LLC cells subcutaneously tumorigenic mice that were exposing to normoxia or CIH,then its morphology was detected.Transwell assay was used to detect the effect of different treatment methods on LLC cell migration.Western blot was used to investigate the effect of different treatments on the migration-related signaling proteins of LLC cells.ELISA was used to detect the level of AngⅡin mice serum and LLC cell culture medium supernatant.Results ELISA results showed that the level of AngⅡin mice serum increased the level of AngⅡin mice serum in CIH group(t=13.948,P=0.008);The results of LLC cells cultured in serum of mice with different treatment methods showed that the serum from the CIH group mice induced transformation of LLC cells from epithelioid to mesenchymal;Transwell results showed that Losartan antagonized the promoting effect of CIH group mice serum on LLC cell migration,the main effect of CIH was F=22.667,P=0.001,the main effect of Losartan was F=29.706,P=0.001,there was an antagonistic effect between CIH and Losartan(F=10.303,P=0.012).LLC cells were treated with different concentrations of AngⅡ,it was found that AngⅡpromoted the morphology of LLC cells to transform from epithelioid to mesenchymal;Transwell results showed that AngⅡpromoted the migration of LLC cells in a concentration-dependent manner(F=42.497,P=0.003).After treating LLC cells with AngⅡand AngⅡ+AT1 Rantagonist Losartan,Transwell results showed Losartan antagonized the auxo-action of AngⅡon LLC cell migration(F=53.432,P<0.001);Western blot showed that Losartan antagonized the upregulation of AngⅡon vimentin(F=77.819,P=0.001),MMP 2(F=49.363,P=0.001),β-catenin(F=23.847,P<0.001),p-Akt/Akt(F=58.608,P=0.007).After LLC cells were treated with AngⅡand AngⅡ+PI3Kblocker LY294002,Transwell results showed LY294002antagonized the effect of AngⅡ-induced LLC cell migration(F=52.792,P=0.002);Western blot showed LY294002antagonized the upregulation of AngⅡon vimentin(F=94.231,P=0.032),MMP 2(F=48.612,P=0.020),β-catenin(F=46.037,P<0.001)and p-Akt/Akt(F=19.644,P=0.002).LLC cells were further treated with IH and IH+Losartan,ELISA results showed that IH upregulated the level of AngⅡin LLC cells culture medium supernatant(t=5.022,P<0.001);Transwell results showed that Losartan antagonized the effect of IH-induced cell migration of LLC cells,the main effect of IH was F=84.587,P<0.001,the main effect of Losartan was F=92.102,P<0.001,there was an antagonistic effect between IH and Losartan(F=70.097,P<0.001);Western blot showed that Losartan antagonized the upregulation of IH on vimentin(F=49.382,P=0.002),β-catenin(F=61.919,P=0.002),MMP2(F=14.132,P<0.001)and p-Akt/Akt(F=100.038,P=0.013).Conclusion Chronic intermittent hypoxia promotes the migration of LLC cells by activating PI3K/Akt signaling pathway through AngⅡ-AT1R.