Experimental Research On Effects Of Anti-liver Fibrosis Of Baoganning Targeting At Hepatic Stellate Cells

Objective: To investigate the main serapharcological mechanisms of anti-fibrotic action of Chinese herb compound Baoganning. Methods: HSC-T6 were introduced into our laboratory and subcultured. Normal rats were medicated with compound Baoganning and the serum was collected to prepare the medicated serum, which was incubated with the subcultured HSC-T6. Then, observed the proliferation of the HSC-T6 by MTT colorimetric assay, tested the content of collagen I in the supernatant by using the ELISA, detected the cell apoptosis rate by using the acridine orange staining and flow cytometer, and then, measured the activity of gross TGF P i in the supernatant by mink lung epithelial cell(Mvllu) proliferation inhibition MTT assay, and tested immunocytochemical stain of TGF P i by means of ABC immunoenzymatic method. To further explore the effect on HSC function of oxidative stress, the content variation of F^Ch, MDA, GSH, GSH-Px, during the cell lesion damaged by FeNTA, were observed. In addition, the protective effects of autocrine oxidative stress of hepatic stellate cell by using Baoganning were studied.Result: (1) Compound Baoganning could significantly inhibit HSC-T6 proliferation in vitro, and the cell survival rate at low-dose and mediate-dose is 85.6% and 67.7%, separately. The normal nuclei DNA of HSC-T6, cultured in the normal rat serum, are even fluorescent yellow or Kelly. And the apoptosis rate (%) itself of HSC-T6 was 9.67±0.57. While the nuclei or cytoplasm, cultured in the medicated serum, were pykno-stained Kelly, even could see the Kelly fragment. And the apoptosis rate(%) were 22.73±0.78 and 23.97±2.50, respectively, which were significantly higher than that of the blank-control group (P<0.01=.(2) After dealt with the Baoganningmedicated serum at low-dose and mediate-dose group, the content of collagen I in the cell supernatant were 93.39\4.41ng/mg and 84.48 + 3.01ng/mg, which were significantly lower than that of normal group. The TGFpi expression intensity of HSC-T6, cultured 48h in the mediated-dose Baoganning medicated serum, is significantly decreased (P < 0.01). Compared with the normal group, the medicated serum could significantly inhibit the TGFpi content in the supernatant. (3) When HSC-T6 cultured with the FeNTA, lipid peroxidation product (H2O2, MDA) increased, and the antioxidant capacity (GSH-Px, GSH) decreased at 1h, 3h, 5h and 24h, in which that of 5h was the most significant. The compound Baoganning medicated serum could inhibit the H2O2 MDA content of HSC in oxidative stress. At the same time, it could also, promote the GSH-Px , GSH level to some extent. Above all, the compound Baoganning medicated serum has some antioxidant ability.Conclusions: Compound Baoganning could significantly inhibit the proliferation of HSC and the yield of TGFβ1, reduce the production of collagen I , and decrease the lipid peroxidation level of HSC in oxidative stress, when HSC-T6 were damaged by FeNTA. The protection to HSC in oxidative stress and the inhibition to the amplified autocrine effect may be the main mechanism of Baoganning on anti-hepatic fibrosis actions.