自噬通过GATA4调控人牙髓细胞衰老反应

Autophagy Regulates Senescence in Human Dental Pulp Cells via GATA4

目的:探究脂多糖(lipopolyasccharide,LPS)诱导的炎症微环境下,自噬对人牙髓细胞(human dental pulp cells,hDPCs)衰老反应的调控作用。方法:体外分离培养hDPCs,10μg/L LPS连续刺激hDPCs 6 d,并分别利用自噬抑制剂3-甲基腺嘌呤(3-methyladenine,3-MA)与GATA结合蛋白4(GATA binding protein 4,GATA4)小干扰RNA(small interfering RNA,siRNA)进一步处理hDPCs。Western blot检测不同处理条件下hDPCs内GATA4、微管相关蛋白轻链3(microtubule-associated protein light chain 3,LC3)、衰老相关蛋白p53及p16的表达变化。细胞免疫荧光染色检测不同处理条件下LC3的表达,并对衰老相关β-半乳糖苷酶(senescence-associatedβ-galactosidase,SA-β-Gal)进行染色。结果:LPS连续刺激hDPCs后,LC3、GATA4、p53、p16的表达及SA-β-Gal阳性细胞数增加;3-MA抑制hDPCs自噬后,GATA4、p53及p16的表达及SA-β-Gal阳性细胞数进一步增加;敲低GATA4后,LC3表达无变化,而p53、p16的表达及SA-β-Gal阳性细胞数明显降低。结论:在LPS诱导的炎症微环境下,自噬通过GATA4负性调节hDPCs的衰老反应。

Objective:To investigate how autophagy acts on senescence in human dental pulp cells(hDPCs)in lipopolysaccharide(LPS)-induced inflammatory microenvironment.Methods:hDPCs were isolated and cultured in vitro and continuously stimulated by 10 ng/L LPS for 6 days.Autophagy inhibitor 3-methyladenine(3-MA)and GATA binding protein 4(GATA4)siRNA were respectively applied to treat hDPCs.Western blotting was used to detect the expression of GATA4,microtubule-associated protein light chain 3(LC3),and senescence-related proteins p53 and p16 in hDPCs under different treatments.Cell immunofluorescent assay was utilized to evaluate the expression of LC3.β-galactosidase staining kit was used to detect the expression of senescence-associatedβ-galactosidase(SA-β-Gal)in hDPCs.Results:After LPS stimulation,the expressions of LC3,GATA4,p53,and p16 as well as SA-β-Gal positive cells in hDPCs significantly increased compared with those in control group.After inhibition of autophagy by 3-MA,the expression of GATA4,p53,p16,and SA-β-Gal positive cells further increased.Compared to Nc group,GATA4 siRNA group experienced a dramatic decrease in p53 and p16 expression and SA-β-Gal positive cells,however,there was no significant change in LC3 expression.Conclusion:Under a LPS-induced inflammatory microenvironment,autophagy negatively regulates the senescence in hDPCs via GATA4.