The Effects Of Danshensu On NF-κB Signal Transduction Pathway In Rat Hepatic Stellate Cells

Objectives:To investigate the effects of Danshensu on proliferation,apoptosis,synthesis and secretion of collagen tapeⅢand expression of p-IκBαand NF-κB p65 induced by IL-1βin rat hepatic stellate cells(HSCs), and to elucidate the molecular mechanisms of Danshensu against hepatic fibrosis.Methods:The rat HSC was isolated with improved collagenase digested in situ-liver recirculating perfusion and cultured in vitro. It was identified byα-SMA immunocytochemical stain .Then it was treated with different concentration of Danshensu. MTT colorimetric assay was used to detect proliferation of HSC. AO/EB immunoflurorescence micropy and combination Annexin-V-FITC/PI double-labelimmunofluorescence with flow cytometry were used to examine apoptosis of HSC. Synthesis and secretion of collagen tapeⅢwere detected by the quantitative immunocytochemical assay and ELISA method espectively. The amounts of cytoplasm p-IκBα,NF-κB p65 and of nuclear NF-κB p65 in HSC were determined by Western blot. Immunocytochemical stain and Western blot were used to observe nuclear translocation of NF-κB p65.Results: 1. The yield of HSC was about 3.5×107 per rat.The purity of HSC exceed 95%,and the survival rate of HSC exceed 96%. 2. Compared with normal control group,IL-1βcould increase proliferation of HSC (P<0.01),reduce apoptosis of HSC(P<0.05), and facilitate synthesis and secretion of collagen tapeⅢin HSC(P<0.01). After treated with different concentration of Danshensu, HSC's proliferation prominently reduced(P<0.01) and its apoptosis increased(P<0.01), especially of early apoptosis. Danshensu could also degrade synthesis and secretion of collagen tapeⅢin HSC (P<0.05 or P<0.01). Danshensu had a concentration-dependent effect on avove parts (P<0.05 or P<0.01), especially in 0.25mmol/L Danshensu treated group.Western Blot results showed that IL-1βelevated amounts of cytoplasm p-IκBαand nuclear NF-κBp65(P<0.01), decreased amounts of cytoplasm p65 markedly (P<0.05), then facilitated nuclear translocation of p65. Immunocytochemical stain results signed that the number of nuclear NF-κB p65 positive HSC increased and the colour deepened. It mained that IL-1βcould induce NF-κB activation in rat HSC. Danshensu had a concentration-dependent effect on reducing amounts of cytoplasm p-IκBαand nuclear NF-κB p65(P<0.05 or P<0.01), and 0.25mmol/L Danshensu could decrease nuclear translocation of p65 markedly(P<0.05). In above group,immunocytochemical stain results showed that the number of nuclear NF-κB p65 positive HSC decreased and the colour grew pallid. Above results indicated that Danshensu could inhibit IκBαphosphorylation,reduce expresstion of nucear NF-κB p65, and prevent nuclear translocation of NF-κB,and then inhibit NF-κB activation in rat HSC.Conclusions: 1.Improved isolation method of HSC has the character of high productin and high purity. Primary identified and cultured rat HSC has biological character of activated HSC and it was an important experimental object to investigate liver fibrosis in vitro.2.Danshensu could inhibit the proliferation of HSC, facilitate the apoptosis of HSC, and reduce the synthesis and secretion of collagen tapeⅢ. Meanwhile, Danshensu could inhibit IκBαphosphorylation,reduce expresstion of NF-κB p65, and prevent nuclear translocation of NF-κB,and then inhibit NF-κB activation in rat HSC. So Danshensu could inhibit Rel/NF-κB/IκB signal transduction pathway .It might be the mechanism of Danshensu against hepatic fibrosis.