内源性硫化氢对硫酸铍诱导人支气管上皮细胞自噬的影响

Effect of endogenous hydrogen sulfide on autophagy induced by beryllium sulfate in human bronchial epithelial cells

目的探讨内源性硫化氢(H2S)对硫酸铍(BeSO4)诱导人支气管上皮细胞(16HBE)自噬的影响。方法用0、100、150、200、250和300μmol/L BeSO4处理16HBE细胞48 h,MTT法测定细胞活力,亚甲基蓝分光光度法测定细胞内H2S含量,Western blot测定自噬标志蛋白LC3-B、Beclin-1及P62表达;用300μmol/L硫氢化钠(NaHS)或10 mmol/L DL-炔丙基甘氨酸(PPG)预处理16HBE细胞6 h后,用BeSO4处理细胞,研究H2S含量对上述指标的影响。结果 (1)BeSO4作用16HBE细胞48 h的半数致死浓度为249μmol/L;与对照组相比,BeSO4组自噬蛋白LC3-II/LC3-I和Beclin-1表达升高,细胞活力、H2S含量和P62蛋白表达降低,当BeSO4浓度>100μmol/L时,差异均有统计学意义(P<0.05);(2)与BeSO4组比较,NaHS+BeSO4组细胞活力、H2S水平和P62蛋白表达均升高(P<0.05),LC3-II/LC3-I和Beclin-1蛋白表达有降低趋势,但差异无统计学意义;(3)与BeSO4组比较,PPG+BeSO4组细胞活力、H2S水平及P62蛋白表达降低,LC3-II/LC3-I和Beclin-1蛋白表达升高(P<0.05)。结论在本试验条件下,BeSO4染毒能致16HBE细胞活力和内源性H2S含量下降,并引起细胞自噬;内源性H2S能抑制BeSO4诱导的16HBE细胞自噬、减轻细胞毒性,发挥保护功能。

Objective To investigate the effect of endogenous hydrogen sulfide(H2S)on Beryllium sulfate(BeSO4)-induced autophagy in human bronchial epithelial cells(16HBE).Methods 16HBE cells were dealt with different concentrations of BeSO4(0,100,150,200,250 and 300μmol/L)for 48 hours.MTT assays were used to detect the viability of 16HBE cells.The contents of hydrogen sulfide were evaluated with methylene blue assay.Western blot was used to determine the expression of autophagy-associated protein LC3-B,Beclin-1 and P62.16HBE cells were pretreated with 300μmol/L sodium hydride(NaHS)or 10 mmol/L DL-Propargylglycine(PPG)for 6 hours,and then,were stimulated with BeSO4 for 48 hours.Result(1)The median lethal concentration(IC50)of BeSO4 treated 16HBE cells for 48 h was 249μmol/L.The ratio of LC3-II/LC3-I and expression of Beclin-1 in BeSO4 group was more elevated than the control group(P<0.05).The viability of cells,content of H2S and the expression of P62 in BeSO4 group were significantly lower than control group(P<0.05).(2)Compared with BeSO4 group,the viability of cells,the content of H2S and expression of P62 were increased in NaHS+BeSO4 group(P<0.05).The ratio of LC3-II/LC3-I and expression of Beclin-1 were down-regulated in NaHS+BeSO4 but the difference was not significant.(3)The viability of 16HBE cells,the content of H2S and expression of P62 were decreased in PPG+BeSO4 group than BeSO4 group(P<0.05).The ratio of LC3-II/LC3-I and expression of Beclin-1 were higher in PPG+BeSO4 group than BeSO4 group(P<0.05).Conclusion BeSO4 can decrease the viability,reduce the content of H2S and up-regulates the level of autophagy in 16HBE cells.Elevating the content of endogenous hydrogen sulfide in 16HBE cells can down-regulates the level of autophagy and reduce the injury by BeSO4.