Effect And Possible Mechanisms Of Exogenous Hydrogen Sulfide On The TGF-β₁-induced Human Embryonic Lung Fibroblasts Transformation

Objectives: To investigate the effects and possible mechanisms of exogenous hydrogen sulfide on human embryonic lung fibroblast transformation(proliferation and collagen production).Methods: Vitro HFL-F MRC-5 cell to the exponential growth phase,to join the TGF-β1(10μg /1) for 12 h, then added different concentrations of sodium hydrosulfide(H2S donor) for a certain time, MTT assay was used to detect the cell proliferation, and draw the growthcurve of 12 h,24h,48 h time point,the determination of NaHS in fibroblastproliferation,pick out the most suitable concentration of Na HS.The test cells were randomly divided into four groups: group1(control): cells cultured with MEM/NE- AA;TGF-β1 group: the normal cultured HFL-F MRC-5 cells into TGF-β1(10μg/1) function of 24h;NaHS group(H2S donor):with the optimal concentration of sodium hydrosulfide joined HFL-F MRC-5,stimulation of 24h; NaHS+ TGF-β1 group: TGF-β1(10μg/l) pretreated HFL-F MRC-5 12 h,then add the optimum concentration of sodium hydrosulfide were incubated 24 h.Above each group cells,the expression level by Western blot assay for the detection of ρ-Smad2/3 and Smad3 protein in 0min,15 min,30min and 60 min,the expression level of α-SMA,type I collagen was detected after 24 h. Application of inverted phase contrast microscope to observe the normal cells and join the TGF-β1(10μg/l) the morphological changes of the cells after stimulation,and in0 h,12h,24 h,48h photographic recording.Results:1.By inverted phase contrast microscope can see, after the TGF-β1 stimulated, lung fibroblasts from fusiform or star shaped structure,gradually transformed into the structure of flat fiber myofibroblasts, along with the extension of time, the cell proliferation.2.Compared with the control group, TGF-β1 stimulates lung fibroblasts 12 h, adding different concentrations of sodium hydrosulfide(H2S donor), cell proliferationappears to have weakened in different degree.Compared with 12 h, 48 h group, 24 h group, the survival rate is maximum,suggesting that sodium hydrosulfide at 50μmol/L~1200μmol/L concentration of TGF-β1 in vitro induced lung fibrolast proliferation inhibition and in a time and dose related.3.Compared with TGF-β1 group, NaHS group and TGF-β1+NaHS lung fibroblasts α-SMA, I collagen was significantly weakened, while Na HS group of α-SMA, I collagen expression minimum. Description of exogenous hydrogen sulfide can downregulate the expression of human fetal lung fibroblast α-SMA and collagen type I have a downward effect,can inhibit lung fibroblasts into myofibroblasts.4.Compared with normal control group, NaHS group lung fibrob-lasts ρ-Smad2/3 have no difference, and TGF-β1 in lung fibroblasts ρ-Smad2/3 protein compared with NaHS+TGF-β1 group was significantly increased; compared with 15 min, 60 min, 30 min time point expression in pulmonary fibroblast ρ-Smad2/3 protein was more obvious.5.Compared with the control group, Na HS group, TGF-β1 group and Na HS+TGF-β1 groups of lung fibroblasts Smad3 expression were upregulated, TGF-β1 group of Smad3 protein expression increase more obviously. In the same test group, 15 min time point compared with30 min 、60min time point, lung fibroblasts Smad3 protein expression increased more significantly. In the 30 min time point, NaHS+TGF-β1group and NaHS group was significantly lower than that TGF-β1 group Smad3 protein expression levels. Show that sodium hydrosulfide can cut TGF-β1/Smad signaling pathway in ρ-Smad2/3, Smad3 protein expression,and for ρ-Smad2/3, Smad3 protein expression regulation has certain temporal correlation.Conclusions: Exogenous hydrogen sulfide can inhibit lung fibroblast proliferation and collagen synthesis.which may be related to blocking the TGF-β/Smad signaling pathway.