Compound 861 On Human Hepatic Stellate Cells In The Mechanism

OBJECT To investigate the effects of herbal compound 861 (Cpd861) on cellproliferation, activation, collagen synthesis and degradation in human hepaticstellate cells (LX-2).METHODS1. Cell proliferation assay LX-2 cells were incubated with various concentrations of Cpd 861. Cellproliferation was analyzed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. The optical density was analyzed at 490 nm.2. Real-time quantitative PCR using SYBR Green I LX-2 cells were treated with Cpd 861, total RNA was extracted from cellsusing TRIzol reagent as the lysis buffer. The complementary DNA (cDNA) wassynthesized using oligo (dT)15 primers and Moloney Murine Leukemia VirusReverse Transcriptase (M-MLV RT). After the reverse-transcription reaction, the cDNA templates were amplified byquantitative real-time PCR with SYBR Green I. Primers were designed usingPrimer 3 software. After PCR, a melting curve was obtained. The melting curves ofall final PCR products were analyzed.3. Gelatin zymography Matrix metalloproteinases (MMPs) activity was analyzed by gelatinzymography. Conditioned media from LX-2 cells treated by Cpd 861 were collected.Samples were electrophoresed in a 10% polyacrylamide gel embedded in 1 mg·ml-1of gelatin. After electrophoresis, the gels were washed,incubated, and subsequentlystained and destained. The gels were scanned and the intensity of each lysis zonewas calculated.4. ELISA assay Conditioned media from LX-2 cells treated by Cpd 861 for 24, 48, 72 h werecollected. TIMP1 protein concentrations were measured with a sandwich ELISAKit.RESULTS1.Effect of Cpd861 on LX-2 cell proliferation The effects of Cpd 861 on LX-2 cell proliferation seemed to correlate with thedose. Significant proliferation inhibition was observed when Cpd 861 concentration英文摘要 - V -was over 0.03mg·mL-1. However, Cpd861, at 0.01mg·mL-1 and 0.003mg·mL-1, didnot inhibit LX-2 proliferation evidently.2.Effect of Cpd861 on LX-2 cell activation α-?smooth muscle actin (α-SMA) is the marker of activated hepatic stellate cells.The effects of Cpd 861 on the expression of α-SMA mRNA in LX-2 cells weremeasured by real-time quantitative PCR method using SYBR Green I technology.The expression levels of α-SMA mRNA decreased significantly when LX-2 cellswere exposed to the Cpd 861 for 48h (59% decrease relative to control, p<0.05) or72 h (60% decrease relative to control, p<0.01).3.Effects of Cpd861 on collagen typeⅠand Ⅲ expression in LX-2 cells when LX-2 cells were exposed to the Cpd 861 for 48 or 72 h, the mRNA levelsof collagen typeⅠand Ⅲ decreased significantly (p<0.05).4.Effects of Cpd861 on matrix metalloproteinase 1 and 2 expression andprotease activity in LX-2 cells The expression of matrix metalloproteinase 1 (MMP1) was significantlyincreased by Cpd 861. Compare to the controls, the mRNA levels increased 95% at24h (p<0.01), 65% at 48h (p<0.01) and 156% at 72h (p<0.001). However, whenLX-2 cells were exposed to the Cpd861 for 48 or 72 h, the mRNA levels of matrixmetalloproteinase 2 (MMP2) decreased. Cpd 861 also enhanced the activity ofmatrix metalloproteinases in LX-2 cells.5.Effects of Cpd861 on tissue inhibitors of metalloproteinase 1 expression inLX-2 cells After treated with Cpd 861 for 72h, the mRNA levels of tissue inhibitor ofmetalloproteinase 1(TIMP1) in LX-2 cells decreased significantly (p<0.05).Meanwhile, TIMP1 protein levels decreased.CONCLUSION In conclusion, Cpd861 could significantly inhibit LX-2 cell proliferation,activation and collagen synthesis. Meanwhile, Cpd861 could improve collagendegradation by enhancing MMP1 expression and inhibiting TIMP1 expression.However, Cpd861 could prevent normal basement membrane degradation byreducing MMP2 expression.