Study On Mechanisms Of Anti-hepatofibrotic Effects Of Astragalosides

Hepatic fibrosis is a kind of common disease that is associated with a significant morbidity and mortality, which is a major worldwide health care burden. For it is the common pathological feature in the development of chronic liver diseases, the key to control these diseases would be preventing, treating and reversing hepatic fibrosis. Current therapies are largely ineffective. Therefore, it is necessary to seek new effective anti-hepafibrtic drugs. Astragalosides (AST) is the active compound extract from the root of Astragalus membranaceus. Previous study from our laboratory showed that AST has significant anti-inflammatory, anti-oxidative, anti-hepatic injury, anti-hepatofibrotic and immunomodulatory effects without evident toxic and side effects. Based on the previous study in our laboratory, this article was designed to explore the mechanisms of anti-hepatic fibrosis effects of AST at gross, cellular and molecular levels in vivo and in vitro. The main contents are divided into the following sections:1. Inhibitory effect of AST on HSC functionIn vitro models for proliferation and collagen production of a cell strain namedHSC-T6 stimulated with serum, Kuppfer cell conditioned medium (KCCM) and cytokines (PDGF-BB,TGF-B1) respectively were established, which were measured by 3H-TdR and 3H-proline incorporation. The results of concentration-inhibition and time-effect relationships of AST on the proliferation and collagen production in vitro showed as the followings: (1) AST (32~256 mg.L-1) inhibited the proliferation of HSC-T6 cells, stimulated by mixed sera including 10%NBS and 3% rat serum, concentration and time dependently. So did AST (16~256 mg.L-1) on collagen production; (2) AST (8, 16, 32, 64 and 128 mg L-1) concentration-dependently inhibited KCCM(1: 4) or PDGF-B (10ug L-1) -driven proliferation of HSC-T6 cells; (3) AST (8~256mg L-1) concentration-dependently inhibited KCCM(1: 4) or TGF-B1 (2ug L-1) -driven collagen production of HSC-T6 cells. The results suggested that the inhibitory effects of AST on HSC functions are related to its direct inhibition on PDGF-driven proliferation and TGF-B1-driven collagen production. In addition, the half inhibitory concentrations (IC50) of AST were 62 mg. L-1 for HSC-T6 cells and 117 mg. L-1 for NIH3T3 cells stimulated with serum. This suggested that AST selectively inhibits HSC-T6 cells to a certain extent.2. The inhibitory effects of AST on PDGF and TGF-B1 are related to the reductions in PDGF, TGF-B1 and their receptor expression in liver tissue and in HSCsOn the basis of anti-hepatofibrotic effect of AST at three doses (20, 40, 80mg.kg-1.d-1, ig, 4~13wk) or a single dose of AST (80mg.kg-1.d-1 ,ig, 0~10wk,4~ 10wk or 8~10wk) on liver fibrosis in rats inducing by of CCl4, the PicTure two-step immunohistochemical (IHC) and immunocytochenmical (ICC) stainings were performed on fibrotic liver tissue chips. The results showed that AST could inhibit the elevated expression of PDGF, TGF- 0 land their receptors, PDGFR- B and TGF B RI, inliver tissues. Co-incubation of AST (16 64 and 128 mg.L-1) with HSC-T6 cells for 48hrs, the expression of PDGF, TGF-B land their receptors decreased concentration-dependently. The results indicated that the inhibition of AST on HSCs promoted by PDGF and TGF- B1 might be related to its reductions of the expression of PDGF, TGF- B land their receptors in liver tissues and in HSCs.3. Effect of AST on the cell cycle and apoptosis of HSC-T6 cellsIn combination of morphology including light and electron microscope observation, TUNEL stain and FACS assay, the effects of AST on the cell cycle and apoptosis of HSC-T6 cells were measured. The results showed that apoptotic index of AST (16, 64 and 128 mg.L-1) treated group was significant higher then that of control and typical morphological changes of apoptosis in HSC-T6 cells was observed.Flow cytometry analysis showed that the peaks of cell cycle histogram moved to left after treatment with AST (16, 64 and 128 mg.L-1 )for 48-72 hrs, showing with the increase of concentratio...