Effects Of Hydrogen Sulfide On Autophagy Induced By Beryllium Sulfate In 16HBE Cell And Its Regulatory Mechanism

Objective:To investigate the effects of Hydrogen Sulfide（H₂S）on autophagy level of Human Bronchial Epithelial Cells（16HBE）induced by Beryllium Sulfate（BeSO₄）and its related regulatory mechanisms,and provide a basis for exploration of the toxicity mechanism of BeSO₄.Methods:1.After treatment of 16HBE cells with different concentrations of BeSO₄（0,100,150,200,250 and 300μmol/L）for 48 h.The MTT assay was used to detect the cell viability.The morphology of the cells was observed under microscope.The content of intracellular hydrogen sulfide was detected by methylene spectrophotometry.The expression levels of the autophagy-related proteins LC3,Beclin-1,and P62 were detected by Western Blot.The distribution of LC3-B fluorescence spote was observed by indirect immunofluorescence.The intracellular ultrastructure was observed by transmission electron microscopy.2.The 16HBE cells were pretreated with 0.1μmol/L Rapamycin（Autophagy Activator）and 50μmol/L Chloroquine（Autophagy Inhibitor）for 2 h,and then co-treated with 150μmol/L BeSO₄ for 48 h.The cell viability detected by MTT assay,and the autophagy related proteins expression were detected by Western Blot.3.16HBE cells were pretreated with 300μmol/L Sodium Hydrosulfide（NaHS）and 10 mmol/L DL-Propargyl Glycine（PPG）for 6h,replacement of fresh medium,and then treated with 150μmol/L BeSO₄for 48 h.The cell viability,the cell morphology and the content of hydrogen sulfide were detected.The expression levels of LC3,Beclin-1,P62,PI3K,t-Akt,p-Akt,mTOR,and CSE proteins were detected by Western Blot.The distribution of LC3-B fluorescence spots was observed by indirect immunofluorescence.Results:1.Compared with the control group,the activity of 16HBE cells,the content of hydrogen sulfide and the expression of P62 protein were decreased with the increase of BeSO₄（P<0.05）,and the expression of LC3-II and Beclin-1 were increased（P<0.05）.The half-inhibition concentration of 16HBE cells treated with BeSO₄ for 48 h was 249μmol/L.The morphology of cells treated with BeSO₄ was changed under microscope,the morphology of cells was irregular,and the original epithelial morphology was lost.The morphological changes of 16HBE cells were significantly increased with the dose of BeSO₄.The LC3-B in the cytoplasm of 16HBE cells in the BeSO₄ group were significantly increased,and some of the fusions were plaque-like.The number of autophagosomes in 16HBE cells was observed by transmission electron microscopy.2.Compared with the BeSO₄ group,the expression of autophagy proteins LC3-II and Beclin-1 was down-regulated in 16HBE cells of CQ+BeSO₄ group,the expression of P62 and the cell viability were increased（P<0.05）.The expression of LC3-II and Beclin-1 proteins were up-regulated,the expression of P62 was down-regulated,and the cell viability was decreased in 16HBE cells of Rapa+BeSO₄ group（P<0.05）.3.Compared with the BeSO₄ group,the viability of cells,the expression of P62 were increased in NaHS+BeSO₄ group（P<0.05）,the cell morphology changed significantly.The expression of LC3 and Beclin-1 although the difference was not statistically significant.The fluorescence spots of LC3-B was decreased.Compared with the BeSO₄group,the viability of cells,the expression of P62 were decreased in PPG+BeSO₄ group（P<0.05）,the cell morphology changed more obviously.The expression of LC3-II and Beclin-1 protein was up-regulated than BeSO₄ group（P<0.05）,and the phenomenon of LC3-B fluorescence spots was obvious increase.4.Compared with the control group,the level of CSE,PI3K,p-Akt,and mTOR proteins in 16HBE cells of BeSO₄ group were down-regulated（P<0.05）.Compared with the BeSO₄ group,the expression of CSE,PI3K,p-Akt,and mTOR protein was significantly up-regulated in the NaHS+BeSO₄ group（P<0.05）.The CSE,PI3K,p-Akt,and mTOR proteins in the PPG+BeSO₄ group were down-regulated than BeSO₄ group（P<0.05）.Conclusions:1.After treatment with BeSO₄,16HBE cells can be damaged,the content of intracellular hydrogen sulfide was reduced,and excessive autophagy will occur.2.Chloquine can reduce the damage effect of BeSO₄ on 16HBE cells by inhibition autophagy.3.Hydrogen sulfide can play the role of signal molecules to inhibit BeSO₄ induced autophagy of 16HBE cells and reduced cells damage by activate PI3K/Akt/mTOR signal pathway.