TNF-α Secreted By Macrophages Stimulated By Soluble Egg Antigen Of Schistosoma Japonicum Activates Hepatic Stellate Cells In Vitro And The Effect Of Astragalosides On Them

Schistosomiasis is a kind of parasitic disease, which severely imperils human health and widely prevails in some tropical countries,and hepatic fibrosis subsequently. The course of liver fibrosis is dynamically regulated by cytokines, which are produced by macrophages, lymphocytes, fibroblasts and others. TNF-αas an important mediator in inflammation directly participates the activation of hepatic stellate cells (HSC), the key cells in liver fibrosis. Astragalosides (AST) has been proved to have anti-inflammatory and anti-hepatic-fibrotic effects. In this article, macrophage activation and TNF-αexpression, after stimulated by soluble egg antigen (SEA) of Schistosoma japonicum, were investigated. And the proliferation and collagen production of HSC, driven by solube egg antigen induced macrophage conditioned medium were observed as well. Basing on the above knowledge, the effects of astragalosides (AST), which has been proved to have anti-inflammatory and anti-hepatic-fibrotic effects, on them were studied. The main contents are divided into the following sections:1 Activation and TNF-αexpression of cultured rat peritoneal macrophages driven by SEA and the effect of AST on themSEA of Schistosoma japonicum was prepared. Macrophages were incubated with SEA and AST for 3, 6 and 9 hours. The cells were harvested for hematoxylin-eosin staining, immunocytochemical staining, reverse transcription polymerase chain reaction (RT-PCR) and radioimmunoassay. The results were showed as the followings.(1)After exposure to SEA (10 mg·L-1) for 3, 6 and 9 hours, rat peritoneal macrophages were activated and TNF-αexpression was significant higher than that of the control. (2)After coincubation with AST(10, 20 mg·L-1)for 6 hours, TNF-αexpression of macrophage cells stimulated by SEA(10 mg·L-1)was inhibited. These results suggested that SEA may activate rat peritoneal macrophages and increase their TNF-αexpression in vitro, which may be inhibited by AST.2 HSC function driven by SEA-MCM and inhibitory effect of AST onSEA-MCM was prepared by injection of Schistosoma japonicum SEA via mice peritoneal. The proliferation and collagen synthesis of HSC-T6 cells stimulated with SEA-MCM were measured by MTT colorimetric assay and 3H-proline incorporation respectively. The result of AST on proliferation and collagen production in vitro showed as the followings: (1)The proliferation and collagen production of HSC-T6 cells were significantly promoted by SEA-MCM (1:2,1:4,1:8,1:16); (2)AST(10,20,40,80 and 160 mg·L-1) inhibited the proliferation of HSC-T6 cells, stimulated by SEA-MCM (1:4); concentration dependently, so did AST on collagen production. These results suggested that SEA may promote HSC-T6 cells proliferation and collagen production that was driven by SEA -MCM in vitro and inhibitory effect of AST on them.3. Effect of anti-TNF-αantibody on the proliferation and collagen systhesis of HSC-T6 cells stimulated with SEA-MCMAdd different dilute strength (1:100 and 1:200) TNF-αantibody in SEA-MCM (1:4), the proliferation and collagen synthesis of HSC-T6 cells stimulated with SEA-MCM were measured by MTT colorimetric assay and western blotting. The result of TNF-αantibody on proliferation and collagen production in vitro showed as the followings: The proliferation and collagen production of HSC-T6 cells were significantly inhibited by TNF-αantibody, concentration dependently. These results suggested that that TNF-αpromote HSC-T6 cells proliferation and collagen production that was driven by SEA in vitro, it was possible one of the important mechanism of Schistosoma japonicum hepatic fibrosis.