Effects Of The Hydrogen Sulfide On Proliferation And Apoptosis Of Hepatocytes From Hepatic Fibrosis Rats Via P38MAPK Signal Pathway

Objective:To investigate the regulating effects of the hydrogen sulfide on proliferation, apoptosis and P-p38MAPK protein expression of hepatocytes from hepatic fibrosis rats.Objective:To investigate the regulating effects of the hydrogen sulfide on proliferation, apoptosis and P-p38MAPK protein expression of hepatocytes from hepatic fibrosis rats.Methods:①Crbon tetrachioride induced rat liver fibrosis model. Pancreatic enzymes the two-step perfusion method was used to extract hepatic fibrosis rat liver cells. Trypan blue staining to detect cell activity, and cell activity> 90%, cell yield> 107/rat, can continue to the following study.②Clls proliferation was determined by MTT method. The new extraction of liver cell placed in 96-well plates with 2×104/well. When there is big island connected cells, serum-free medium WME synchronization processing for 12 h, and then divided into the test group and the control group. The test group was sub-divided into H2S group (administrated by 25,50,75,100,200 μmol/L) and SB group (administrated by 12.5,25,50,100,200 μmol/L). the experiment was terminated after the aforementioned drugs administration for 48h in 5% CO2. MTT (5 g/L) 20 μL was added to each well for 4h in 5% CO2, and ceased with 150 μL DMSO. Absorbance value (A) was determined by microplate reader under 490 nm wavelength, so as to get the proliferation and inhibition rate. ③Flow cytometry technology detection of liver fibrosis rats liver cell apoptosis rate. Cell preparation as the MTT assay. Cells were grouped to normal control, H2S 50μmol/L, SB203580 25μmol/L, SB203580 25 umol/L+H2S 50 umol/L, and cells were collected and added binding buffer and Annexin V-FITC/PI after 48 h incubation in 5% CO2. Flow cytometry was used to detect the apoptosis rate.④ Western blot was to determine the expression of P-p38 of cells in the control group, H2S, SB, SB+H2S.Objective:To investigate the regulating effects of the hydrogen sulfide on proliferation, apoptosis and P-p38MAPK protein expression of hepatocytes from hepatic fibrosis rats.Methods:①Crbon tetrachioride induced rat liver fibrosis model. Pancreatic enzymes the two-step perfusion method was used to extract hepatic fibrosis rat liver cells. Trypan blue staining to detect cell activity, and cell activity> 90%, cell yield> 107/rat, can continue to the following study.②Clls proliferation was determined by MTT method. The new extraction of liver cell placed in 96-well plates with 2×104/well. When there is big island connected cells, serum-free medium WME synchronization processing for 12 h, and then divided into the test group and the control group. The test group was sub-divided into H2S group (administrated by 25,50,75,100,200 μmol/L) and SB group (administrated by 12.5,25,50,100,200 μmol/L). the experiment was terminated after the aforementioned drugs administration for 48h in 5% CO2. MTT (5 g/L) 20 μL was added to each well for 4h in 5% CO2, and ceased with 150 μL DMSO. Absorbance value (A) was determined by microplate reader under 490 nm wavelength, so as to get the proliferation and inhibition rate. ③Flow cytometry technology detection of liver fibrosis rats liver cell apoptosis rate. Cell preparation as the MTT assay. Cells were grouped to normal control, H2S 50μmol/L, SB203580 25μmol/L, SB203580 25 umol/L+H2S 50 umol/L, and cells were collected and added binding buffer and Annexin V-FITC/PI after 48 h incubation in 5% CO2. Flow cytometry was used to detect the apoptosis rate.④ Western blot was to determine the expression of P-p38 of cells in the control group, H2S, SB, SB+H2S.Results:①Pancreatic enzymes the two-step perfusion method can acquire enough hepatic fibrosis rat liver cells with viability 90% and production of (1-3)×107, which satisfied the experiment. ② cells can form island after 48 h culture and can perform the following experiment. ③H2S (50 μmol/L) promoted the promotion of liver cells, and the difference had statistical significance (P<0.05), while it had no effect on cell apoptosis. SB203580 inhibited the promotion of liver cells, and which showed dose-dependent (P<0.05), and meanwhile, the apoptosis was induced (P<0.05). P-p38MAPK protein high expressed in each groups, the expression of H2S group was higher than the control, which had statistical significance (P<0.05). The P-p38MAPK expression in SB, SB+H2S groups was decreased compared to that in the control and H2S group, the differences possessed significance (P<0.05).Objective:To investigate the regulating effects of the hydrogen sulfide on proliferation, apoptosis and P-p38MAPK protein expression of hepatocytes from hepatic fibrosis rats.Methods:①Crbon tetrachioride induced rat liver fibrosis model. Pancreatic enzymes the two-step perfusion method was used to extract hepatic fibrosis rat liver cells. Trypan blue staining to detect cell activity, and cell activity> 90%, cell yield> 107/rat, can continue to the following study.②Clls proliferation was determined by MTT method. The new extraction of liver cell placed in 96-well plates with 2×104/well. When there is big island connected cells, serum-free medium WME synchronization processing for 12 h, and then divided into the test group and the control group. The test group was sub-divided into H2S group (administrated by 25,50,75,100,200 μmol/L) and SB group (administrated by 12.5,25,50,100,200 μmol/L). the experiment was terminated after the aforementioned drugs administration for 48h in 5% CO2. MTT (5 g/L) 20 μL was added to each well for 4h in 5% CO2, and ceased with 150 μL DMSO. Absorbance value (A) was determined by microplate reader under 490 nm wavelength, so as to get the proliferation and inhibition rate. ③Flow cytometry technology detection of liver fibrosis rats liver cell apoptosis rate. Cell preparation as the MTT assay. Cells were grouped to normal control, H2S 50μmol/L, SB203580 25μmol/L, SB203580 25 umol/L+H2S 50 umol/L, and cells were collected and added binding buffer and Annexin V-FITC/PI after 48 h incubation in 5% CO2. Flow cytometry was used to detect the apoptosis rate.④ Western blot was to determine the expression of P-p38 of cells in the control group, H2S, SB, SB+H2S.Results:①Pancreatic enzymes the two-step perfusion method can acquire enough hepatic fibrosis rat liver cells with viability 90% and production of (1-3)×107, which satisfied the experiment. ② cells can form island after 48 h culture and can perform the following experiment. ③H2S (50 μmol/L) promoted the promotion of liver cells, and the difference had statistical significance (P<0.05), while it had no effect on cell apoptosis. SB203580 inhibited the promotion of liver cells, and which showed dose-dependent (P<0.05), and meanwhile, the apoptosis was induced (P<0.05). P-p38MAPK protein high expressed in each groups, the expression of H2S group was higher than the control, which had statistical significance (P<0.05). The P-p38MAPK expression in SB, SB+H2S groups was decreased compared to that in the control and H2S group, the differences possessed significance (P<0.05).Conclusion: ①Improvement of pancreatic enzyme two-step perfusion method is a practical and efficient separation method for liver fibrosis in rats liver cells. ②Low concentration of H2S had no apoptosis effect on hepatocytes of hepatic fibrosis rat, however, it promoted hepatocytes’ proliferation may through activate p38MAPK signal pathway.