GSK3β/eEF2K信号通过调控自噬参与肺成纤维细胞诱导分化

doi:10.6040/j.issn.1671-7554.0.2021.0878

摘要/Abstract

摘要： 目的探讨糖原合成酶激酶3β(GSK3β)/真核延伸因子2 激酶(eEF2K)在肺成纤维细胞转化为肌成纤维细胞过程中的表达,以及对细胞自噬和纤维化的影响。方法将L-929细胞(对照组)于适宜条件下培养48 h,加入5 ng/mL TGF-β1诱导其表型转化为肌成纤维细胞(TGF-β1组)。将空质粒、si-eEF2K及si-GSK3β转染肌成纤维细胞,培养48 h后采用免疫荧光染色法检测p-eEF2K和α-SMA蛋白表达情况,蛋白质印迹法检测各组eEF2K、p-eEF2K、GSK3β、P62及LC3-Ⅱ/Ⅰ蛋白表达情况。结果与对照组相比,TGF-β1组细胞p-eEF2K、GSK3β 蛋白和α-SMA蛋白表达显著增加(P<0.05);转染si-eEF2K及si-GSK3β后,L-929细胞p-eEF2K/eEF2K表达下降(P<0.05),P62和LC3-Ⅱ/Ⅰ表达水平增加(P<0.05),α-SMA蛋白表达降低(P<0.05)。结论 GSK3β/eEF2K信号可能通过降低自噬活性促进肺成纤维细胞向肌纤维细胞的表型转化,促进细胞纤维化进程。

关键词: L-929细胞, 成纤维细胞, 肌纤维细胞, 糖原合成酶激酶3β/真核延伸因子2 激酶, siRNA转染

Abstract: Objective To explore the expression of glycogen syntheses kinases 3β(GSK3β)/eukaryotic elongation factor 2 kinases(eEF2K)during the conversion of lung fibroblasts into myofibroblasts, and to investigate its effects on cellular fibrosis and autophagy. Methods L-929 cells(control group)were cultured under appropriate conditions for 48 h, and transformed into myofibroblasts(TGF-β1 group)by 5ng/mL TGF-β1. Myofibroblasts were transfected with empty plasmid, si-eEF2K and si-GSK3β. The protein expressions of p-eEF2K and α-SMA were detected with immunofluorescence staining after 48 hours of culture, and the protein expressions of eEF2K, p-eEF2K, GSK3β, P62 and LC3-Ⅱ/Ⅰ were detected with Western blotting. Results Compared with the control group, the TGF-β1 group had increased expression of p-eEF2K, GSK3β and α-SMA(P<0.05). After transfection with si-eEF2K and si-GSK3β, the expressions of p-eEF2K and α-SMA decreased, while the expressions of P62 and LC3-Ⅱ/Ⅰ increased(P<0.05). Conclusion GSK3β/eEF2K signalling may promote the phenotypic transformation of lung fibroblasts to myofibroblasts by reducing autophagic activity and facilitating the process of cellular fibrosis.

Key words: L-929 cells, Fibroblasts, Myofiber cells, Glycogen syntheses kinases 3β/eukaryotic Elongation factor 2 kinases, siRNA transfection

中图分类号:

R563.9

覃超群,黄斌,阳芳,王昌明,肖影,黄汉灿,李丽英,高枫. GSK3β/eEF2K信号通过调控自噬参与肺成纤维细胞诱导分化[J]. 山东大学学报 (医学版), 2022, 60(5): 8-15.

QIN Chaoqun, HUANG Bin, YANG Fang, WANG Changming, XIAO Ying, HUANG Hancan, LI Liying, GAO Feng. GSK3β/eEF2K signaling involves in the induced differentiation of lung fibroblasts by regulating autophagy[J]. Journal of Shandong University (Health Sciences), 2022, 60(5): 8-15.

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