CTGF诱导的人胚肺成纤维细胞转分化中PI3K/Akt与p38MAPK磷酸化的关系

目的 探讨结缔组织生长因子(CTGF)诱导的人胚肺成纤维细胞(HFL-I)转分化中磷脂酰肌醇3-激酶/蛋白激酶B(PI3K/Akt)和丝裂原活化蛋白激酶(p38MAPK)通路的活动状态以及相关性的研究.方法 将体外培养的HFL-I分成4组:(1)CTGF处理组(20 ng/ml);(2) LY294002(10 μmol/L)预处理60min,再行CTGF刺激(20 ng/ml):(3)SB203580 (10 μmol/L)预处理60 min,再行CTGF刺激(20 ng/ml); (4)LY294002(10 μmol/L)和SB203580(10 μmol/L)联合作用60 min,再加入CTGF(20 ng/ml).选取不同时相点,Western blot测定平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、p-Akt和Akt的蛋白表达:RT-PCR检测PI3K和p38MAPKmRNA表达.结果 与对照组相比,CTGF诱导15 min后p-Akt水平升高,30 min时达至顶峰(P＜0.01).CTGF诱导24h后α-SMA表达显著升高(P＜0.01),可被LY294002阻断.LY294002和SB203580单独或联合预处理后,显著抑制CTGF诱导的p-Akt活化(P＜0.01).结论 CTGF诱导的成纤维细胞-肌成纤维细胞转分化中有PI3K/Akt通路的激活,PI3K和p38MAPK均可以调节Akt的活性,其特异性抑制剂均可阻断Akt的磷酸化.

Relevance of PI3K/Akt and p38MAPK pathways to induction of lung fibroblast-myofibroblast transdifferentiation by CTGF

Objective To investigate the effects of CTGF on the transdifferentiation process of human fetal lung fibroblast,and observe the relationship between phosphatidylinositol 3-kinase(PI3K/Akt)and p38 mitogen-activated protein kinases(p38MAPK) signal pathways.MethodsTCultured human fibroblasts were divided into four groups :(1)CTGF(20ng/ml) treatment;(2) preincubated cells with LY2940002(10υmol/L) for 60 minutes followed by CTGF(20ng/ml);(3)preincubated cells with SB203580(10υmol/L)for 60 minutes followed by CTGF(20ng/ml);(4) preincubated cells with LY2940002(10υmol/L) and SB203580(10υmol/L)for 60 minutes followed by CTGF(20ng/ml).The expression levels of total-Akt,phosphor-Akt andα-SMA in each group were detected by Western blot.PI3K and p38MAPK mRNA in each group were evaluated by RT-PCR.ResultsTCompared to the control group,p-Akt level rose 15 minutes after CTGF induction,and 30 minutes got to the summit(P<0.01).α-SMA increased markedly 24 hours after the treatment of CTGF(P<0.01).LY294002 and/or SB203580 were shown to largely down-regulate the expression of p-Akt in response to CTGF(P<0.01).ConclusionsPI3K/Akt is a upstream mediator in the lung fibroblast-myofibroblast process promoted by CTGF,and PI3K/Akt inhibitor LY294002 and/or p38MAPK inhibitor SB203580 can significantly abrogated the effect of CTGF.The results showed PI3K and p38MAPK had a feedback regulation for phosphorylation of Akt.