The Role Of Hydrogen Sulfide On SB203580Influence Apoptosis Of Hepatic Stellate Cell

Objective:Discuss the influence of Hydrogen sulfide (H2S) and SB203580on rat hepatic stellate cells (HSC)proliferation and apoptosis; research the role of P38MAPK signaling pathway that in hydrogen sulfideinfluence the hepatic stellate cells proliferation and apoptosis. Explore whether H2S through P38MAPKsignaling pathway to postpone the onset of liver fibrosis.Methods:（1）Culture of HSC-T6cells: HSC-T6cells were cultured in DMEM/H containing10%FBS. Thecells were adherent after6-8h, the medium was aspirated after24h-48h, the cells were subcultured whenthe proliferation of cells was up to90%of the growth area in72h, and the cells only were not used in theexperiment until characteristics of the cells was stable;(2) HSC-T6cell proliferation detected by MTTassay: The cells were divided in control group and experimental group, the experimental groupingaccording to the dosing concentration gradient. The NaHS concentration control at25umol/L、50umol/L、75umol/L、100umol/L、200umol/L, the concentration of SB203580control at10umol/L、25umol/L、50umol/L、75umol/L、100umol/L. Drugs intervention for48hours then affected by MTT20μl response4hours in the dark and affected by DMSO150μl to terminatied the reaction. Use microplate reader todetection opticaldelnsity(A) at570nm wavelength;（3）Using flow cytometry to detect the influence of H2Sand SB203580on HSC-T6cell apoptosis: The cells were divided in5groups randomly, which were normalcontrol group(N), DMSO group, NaHS50umol/L group, SB20358075umol/L group(SB), SB20358075umol/L+NaHS50umol/L group(SB+NaHS), using flow cytometry by Annexin V-FITC/PI amphophilcells to detect the HSC apoptosis rate. At the same experimental conditions, cells cultivate for48h,colouration by Hoechst33342, then apoptosis rate observed and counted by Leica DMIRB;(4) Using PCRmethod to detect collagen type and collagen type Ⅲ mRNA expression in HSC: The cells were alsodivided in5groups randomly: which were group N, DMSO, NaHS, SB, SB+NaHS, total RNA was isolatedfrom HSC-T6cells, PCR method to detect collagen type and collagen type Ⅲ mRNA expression inHSC;(5) Western blot detection the expression of P-P38and Caspase-3protein: The group was the same asPCR, total protein were extracted and then changes of the expression of P-P38、Caspase-3protein levelwas detected by Western blot.Results:（1）HSC cells of rat grew very well and the characteristics of the cells was stable after subcultured;the rate of overall mortality was less than5%; the cell morphology observed under inverted microscope,cell density was increased in NaHS group, the apoptotic cells visible and cells size were shrinkage in SBgroup,(2) After different intervention treatment of HSC T6cell culture after48h, compared with thecontrol group, NaHS in low concentration(25umol/L、50umol/L、75umol/L) can promote HSC T6cellproliferation, NaHS50umol/L group’s effect on promoting proliferation significantly, the differences havestatistically significant(P<0.05); SB203580inhibit HSC T6cell proliferation, and also induced cellapoptosis, the differences have statistically significant(P<0.05).(3) NaHS has no significant difference oncell apoptosis compared with the normal control group, SB203580can significantly induce HSC-T6cellapoptosis, and join with NaHS can increase the effect on promating apoptosis, the difference wasstatistically significant(P<0.05).(4) NaHS make collagen typeⅠ、Ⅲ mRNA expression enhancement inHSC T6cells, SB203580prompted collagen typeⅠand collagen type Ⅲ mRNA expression leveldecreased. The joint of NaHS and SB203580made collagen typeⅠand collagen type Ⅲ mRNAexpression significant reduced, the difference was statistically significant(P<0.05).(5) NaHS can increasethe level of P-P38and caspase-3positive expression in HSC-T6cells. The joint of NaHS and SB203580 made P-P38protein expression reduced and caspase-3protein expression enhancement.Conclusions:(1) H2S through activating P38MAPK signaling pathway promote the proliferation of rat hepaticstellate cells.(2) P38MAPK signaling pathway regulation of hepatic stellate cell apoptosis after it wasblocked, and delays the development of liver fibrosis.(3) After P38MAPK signaling pathway is blockedby SB203580, hydrogen sulfide, made activated hepatic stellate cells proliferation inhibited and apoptosispromoted. H2S is involved in regulating expression of collagen typeⅠ,collagen type Ⅲ mRNA andP-P38、Caspase-3protein level in hepatic stellate cells when P38MAPK signaling pathway was blocked.