Effect Of Reduced Glutathione On Proliferation And Activation Of Rat Hepatic Stellate Cells And Signaling Pathway Of Nrf2/Ho-1

ObjectiveTo explore the effects of reduced glutathione (GSH) on the proliferation and activation of rat hepatic stellate cell-T6(HSC-T6) and signaling pathway of Nrf2/HO-1.Methods1. Choose a rat hepatic stellate cell strain (HSC-T6) as the research object. Rat HSC-T6were incubated with various concentration of LPS(1、0.1、0.01、0.001μg/ml). After24h,48h,72h of reaction, MTT colorimetric assay was employed to detect the Proliferation of HSC-T6so that we can confirm the optimal concentration of lipopolysc-charide(LPS) used to activate HSC-T6and proper reaction time;2. Rat HSC-T6activated by lipopolysc-charide(LPS)(0.1μg/ml were incubated with various concentration of GSH(0、1、2、5、10mmol/L).After24h,MTT colorimetric assay and chemiluminescence immunoassay was respectively used to detect the proliferation of HSC-T6, the content of collagen IV and hyaluronic acid in supernatant of HSC-T6;The expression of a-SMA was analysed by immunocytochemical staining Image. The expression of Nrf2and HO-1was observed by immunohistochemistry in HSC-T6;The expression of Nrf2mRNAs and HO-1mRNAs was assessed by semi-quantitive reverse transcriptase polymerase chain reaction (RT-PCR).Results1. After rat HSC-T6were incubated with various concentration of LPS for24h, the cell proliferation(106.83±8.17、106.96±8.12、125.39±7.58) rised with the increase of concentration (0.001、0.01、0.1ug/ml),but cell proliferation (109.78±5.03)was reduced when the concentrati-on of LPS reached up to1.0ug/ml,the cell proliferation(98.67±5.68、93.63±4.54、95.34±5.91、93.76±5.36)、(94.15±4.48、93.51±8.72、90.62±9.10、92.13±8.84) were falling with the extension of time (48h,72h),the differences between each group of24h was statistically significant (P<0.05).2. Adverse reaction was not seen after HSC-T6were incubated with GSH,cell proliferation (0.79±0.02、0.74±0.03、0.70±0.02、0.62±0.01) was significantly reduced as GSH concentration(1、2、5、10mmol/L)increasing,compared with Ommol/LGSH group(0.88±0.03).(P<0.05). The proliferation of HSC-T6and the concentration of GSH present the index relationship. 3. The expression of HA and Ⅳ collagen (415.74±14.52、251.47±14.06) was increased significantly in the Ommol/L GSH group compared with the blank group(199.27±10.89、77.92±9.67)(P<0.05).As concentration of GSH(1、2、5、10mmol/L)increasing,HA(372.98±11.01、320.76±6.37、284.46±13.17、239.08±16.95) and Ⅳ collagen (191.27±17.49、163.85±16.26、133.03±13.14、103.31±12.52) reduced gradually compared with Ommol/L GSH group(415.74±14.52、251.47±14.06)(P<0.05). The expression of HA and Ⅳ collagen by HSC-T6and the concentration of GSH present the index relationship.4. The expression of a-SMA (3.52±0.06) was increased significantly in the Ommol/L GSH group compared with the blank group (0.90±0.02)(P<0.05). As concentration of GSH (1、2、5、10mmol/L)increasing, a-SMA (2.97±0.09、2.23±0.06、1.57±0.05、1.22±0.07) reduced gradually compared with Ommol/L GSH group (3.52±0.06)(P<0.05). The expression of a-SMA by HSC-T6and the concentration of GSH present the index relationship.5. Each group of GSH had positive staining in the nucleus (Nrf2) and cytoplasm (HO-1) showing some brownish red particle, Nrf2and HO-1protein were increased significantly compared with the Ommol/L GSH group.6. The expression of Nrf2mRNA、HO-1mRNA(1.21±0.11、1.25±0.09) were increased in the Ommol/L GSH group compared with the blank group (1.00±0.00、1.00±0.00)(P<0.05). As concentration of GSH (1、2、5、10mmol/L)increasing, Nrf2mRNA、HO-1mRNA were increased significantly compared with the Ommol/L GSH group(1.21±0.11、1.25±0.09)(P<0.05). The expression of Nrf2mRNA and HO-1mRNA by HSC-T6and the concentration of GSH present the index relationship.Conclusion1. GSH can inhibit the proliferation and activation of HSC-T6.2. GSH can reduce the secretion of HA and IVcollagen (ECM) in HSC-T6.3. GSH can inhibit the proliferation and activation of HSC-T6and reduce the secretion of HA and Ⅳcollagen (ECM) in HSC-T6, the mechanism of which may be related to the Nrf2/HO-1signaling pathways that regulate HSC-T6.