EFFECT OF ASTRAGALOSIDES ON SCHISTOSOMAL HEPATIC FIBROSIS IN VITRO

Abstract

Purpose of Study: The activation and proliferation of hepatic stellate cells (HSCs) and the increased extracelluar matrix synthesis of the cells may result in schistosomal hepatic fibrosis. One of the strategies to anti-hepatofibrosis is to inhibit HSCs proliferation. Astrogalosides (AST), which is the active compound extract from the root of Astragalus membranaceus has been known to have significant anti-hepatofibrotic effect without evident toxic and side effects. This study was designed to observe the effect of astragalosides on proliferation and collagen synthesis of HSC-T6 cells driven by Schistosoma japonicum soluble egg antigen-activated macrophage conditioned medium (SEA-MCM) and to investigate its possible mechanisms. Methods: SEA-MCM was prepared by injecting soluble egg antigen of Schistosoma japonicum into mice peritoneal for the use of establishing an in vitro model for proliferation and collagen production of a cell strain named HSC-T6, which was measured by MTT colorimetric assay and 3H-proline incorporation respectively. After the treatment of AST (32.5, 65 and 130 mg· L-1) for 48 and 72 hours, the cells were then harvested for the detection of the proliferation and collagen production respectively. The cells were coincubated with MCM (1:4 v/v) and AST (32.5 and 130 mg· L-1) for 48 hours to detect apoptosis, which was analyzed using hematoxylin and eosin staining and an in situ cell death detection kit based on the terminal TUNEL technique. HSC-T6 cells were incubated with SEA-MCM in the presence or absence of AST(32.5, 130 mg/L) for 6, 12 and 24 h to extract nuclear protein for the detection of NFkappaB by western-blotting. Results: The proliferation and collagen production of HSC-T6 cells were significantly promoted by SEA-MCM. The proliferation of HSC-T6 cells after exposure to AST for 48 hours was significantly repressed with the inhibition rates from 8.7% to 47.8% showing a concentration-dependent manner. The collagen production of the cells after exposure to AST for 72 hours was significantly repressed with the inhibition rates from 7.8% to 71.2% showing a concentration-dependent manner. The apoptotic indexes of the cells treated with AST(32.5 and 130 mg/L-1) were 11.71% and 25.41% respectively, which were significantly higher than that of control (5.26%). After six hours' incubation with SEA-MCM, nuclear NFkappaB level increased significantly. It was depressed after 12 hours' exposure to AST. Conclusion: Astragalosides may inhibit the proliferation and collagen production of hepatic stellate cells stimulated by macrophage conditioned medium that is activated by soluable egg antigen of Schistosoma japonicum. Induction of apoptosis of HSCs via NFkappaB pathway might be one of its mechanisms.