Antifibrotic effects of Salvia miltiorrhiza on dimethylnitrosamine-intoxicated rats

Abstract

Excessive oxidative stress is implicated in hepatic fibrogenesis. Extracts of Salvia miltiorrhiza (Sm) have been shown to protect cells against oxidative stress. In this study we investigated the in vitro and in vivo effects of Sm on hepatic fibrosis. A cell line of rat hepatic stellate cells (HSC-T6) was stimulated with transforming growth factor-beta1 (TGF-beta1). The inhibitory effects of Sm (50-400 microg/ml) on TGF-beta1-induced alpha-smooth muscle actin (alpha-SMA) secretion and the mRNA expressions of fibrosis-related genes, including alpha-SMA, connective tissue growth factor (CTGF), and tissue inhibitor of metalloproteinase-1 (TIMP-1), were assessed. Fibrosis was induced by dimethylnitrosamine (DMN) administration in rats. DMN-treated rats were randomly assigned to 1 of 4 groups: saline, Sm (20 mg/kg), Sm (100 mg/kg), or silymarin (100 mg/kg), each given by gavage twice daily for 5 weeks starting from the onset of DMN administration. Sm (200 and 400 microg/ml) significantly inhibited TGF-beta1-stimulated alpha-SMA secretion and the mRNA expressions of alpha-SMA, CTGF, and TIMP-1 in HSC-T6 cells. Fibrosis scores of livers from DMN-treated rats with either a low (1.8 +/- 0.2) or high (1.8 +/- 0.1) dose of Sm, or silymarin (1.4 +/- 0.2) were significantly reduced in comparison with DMN-treated rats receiving saline (3.1 +/- 0.1). Hepatic collagen contents were also significantly reduced by either Sm or silymarin treatment. The mRNA expression levels of alpha-SMA, TGF-beta1, and procollagen I were all attenuated in Sm- and silymarin-treated rats. Moreover, levels of plasma aspartate transaminase activities were reduced by Sm and silymarin treatment. In conclusion, our results show that Sm exerted antifibrotic effects in both HSC-T6 cells and in rats with DMN-induced fibrosis.

Anti-Fibrotic Effects of Thalidomide on Hepatic Stellate Cells and Dimethylnitrosamine-Intoxicated Rats

“…The cultures were passaged by trypsinization every 4th day and cells were plated in 150-mm culture dishes at a density of 1Â 10 7 cells per dish in Waymouth medium and incubated under 5% CO 2 in air at 37°C. Bioassay systems of the HSC-T6 cell line have been established in our laboratory [29].…”

“…The cell pellet was resuspended gently and the cell suspension centrifuged at 250 g for 5 min at 4°C. The supernatant was collected as cytoplasmic fraction for the analysis of a-SMA (as previously reported by us) [29], IKKa expression and IjBa phosphorylation, according to the methods of Alpert and Vile`ek [31] and Yang et al [32]. Twenty lg of proteins in the cytoplasmic or nuclear fraction were separated on a 10% SDS-PAGE and transferred onto Immobilon-PVDF (Millipore, Bedford, MA, USA) in a transfer buffer (6.2 mM boric acid, pH 8.0).…”

“…The fraction of insoluble collagen after acid extraction, composed of crosslinked collagen, was then heated at 80°C for 60 min for conversion into soluble gelatin. The gelatin contents of the acid extracts were assayed using the Sircol collagen assay kit (Biocolor, Belfast, Nothern Ireland) according to the manufacturer's instructions and the method described by Shiba et al [34] and in our previous paper [29].…”

“…Hepatic fibrosis was induced by DMN administration in male Sprague-Dawley rats and we have recently documented changes in molecular and cell biological parameters related to fibrosis in these rats [29,36]. DMN (5 mg/kg) was injected intraperitoneally for 3 consecutive days per week for 4 weeks, according to the method of Jezequel et al [37].…”

“…The sections were then stained with picro-sirius red for collagen distribution [38]. A numerical scoring system for histologically assessing the extent of fibrosis was adapted from the formula of Scheuer [39], with minor modification [29]. Briefly, fibrosis was staged as: 0: no fibrosis; stage 1: enlarged, fibrous portal tracts; stage 2: periportal or portalportal septa, but intact architecture; stage 3: fibrosis with architectural distortion; stage 4: probable or definite cirrhosis.…”