The role of extracellular signal-regulated kinase in induction of apoptosis with salvia miltiorrhiza monomer IH764-3 in hepatic stellate cells / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To explore the effect of Salvia miltiorrhiza monomer IH764-3 on apoptosis in hydrogen peroxide (H2O2)-stimulated hepatic stellate cells (HSCs).</p><p><b>METHODS</b>HSCs were cultured in medium with different IH764-3 doses (10 mg/L, 20 mg/L, 30 mg/L, 40 mg/L) and without IH764-3. Direct cell count, 3H-thymidine incorporation, Annexin-V/Propidium Iodide double-labeled flow cytometry, TUNEL and transmission electron microscopy were employed to estimate the influence of IH764-3 on proliferation and apoptosis of HSCs. The expression of extracellular signal-regulated kinase 1 (ERK1) mRNA and protein in HSCs were detected using RT-PCR and Western blot respectively.</p><p><b>RESULTS</b>It was showed that H2O2 could promote HSC proliferation. In contrast, IH764-3 at concentrations of 10 mg/L, 20 mg/L, 30 mg/L and 40 mg/L inhibited its proliferation. The inhibition rates were 7.13%, 28.36%, 53.80% and 73.10% (P < 0.01). And the inhibition rates of IH764-3 at concentrations of 30 mg/L at 12 h, 24 h and 48 h were 22.24%, 40.51% and 61.65%. Furthermore, IH764-3 could also induce the HSC apoptosis in dose-dependent an dtime-dependent manners (P < 0.01). In addition, after exposed of HSCs to IH764-3 for 24 h, ERK production decreased and ERK1 mRNA was down-regulated earlier about 2 h after exposure to IH764-3.</p><p><b>CONCLUSION</b>IH764-3 may inhibit the proliferation and induce apoptosis of HSCs in both dose-dependent and time-dependent manners, which may be related to down-regulation of ERK expression.</p>

The influence of down-regulation of focal adhesion kinase by RNA interference on the adhesion and migration of rat hepatic stellate cells in vitro / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the role of focal adhesion kinase (FAK) in adhesion and migration of hepatic stellate cells (HSC).</p><p><b>METHODS</b>Two recombinant plasmids expressing short hairpin RNAs (shRNAs) targeting FAK were constructed and one plasmid substantially suppressing FAK expression in HSC was selected. Real-time PCR and Western blot were used to detect the knockdown effects of FAK gene. After 48-hour treatment with FAK shRNA, toluidine blue colorimetric assay was used to detect the cell adhesion. Wound-healing assay and improved Boyden double-chamber were used to detect the cell migration induced by FN.</p><p><b>RESULTS</b>The recombinant plasmid expressing FAK shRNA was successfully constructed and transfected into HSC. Compared with the controls, the expression of FAK mRNA and protein in HSC treated with FAK shRNA was markedly down-regulated by 76.82% and 72.53%, respectively. The expression of p-FAK (Tyr397) protein was also decreased by 62.71% 48 h posttransfection. The adhesion of HSC was inhibited by 58.69% at 48 h after shRNA transfection. FAK gene silencing could also dramatically inhibit FN-stimulated HSC migration, and the cell migration distance and the cell number of crossing membrane were decreased by 58.27% and 83.70%, respectively.</p><p><b>CONCLUSIONS</b>FAK gene silencing suppresses adhesion and migration of HSC, and FAK may be a potential target for novel anti-fibrosis therapies.</p>

The effects of FRNK on expressions of MMP-2 mRNA and TIMP-2 mRNA in hepatic stellate cells / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To investigate the effects of FAK-related non-kinase (FRNK) on expressions of type I collagen and matrix metalloproteinase-2 (MMP-2) mRNA and tissue inhibitor of metalloproteinase-2 (TIMP-2) mRNA in rat hepatic stellate cells (HSC).</p><p><b>METHODS</b>Using in vitro cell culture technique, FRNK plasmids were transfected into HSC mediated by cationic liposome. Type I collagen synthesis capability in HSC was examined by 3H-Pro incorporation assay. The levels of FRNK in HSC were assayed by Western blot, and the expressions of MMP-2 and TIMP-2 were assayed by RT-PCR on mRNA levels.</p><p><b>RESULTS</b>The exposure of HSC to FRNK caused the expression of FRNK protein to be up-regulated, and the FRNK protein contents reached the highest point at 48 h after the transfection, P less than 0.05. The expressions of MMP-2 mRNA were up-regulated by FRNK; the expressions of TIMP-2 mRNA were down-regulated by FRNK; the ratios of MMP-2 mRNA/TIMP-2 mRNA were enhanced by FRNK.</p><p><b>CONCLUSION</b>After FRNK was transfected, the capability of type I collagen synthesis in HSC was inhibited, which may be related to the up-regulation of MMP-2 mRNA/TIMP-2 mRNA.</p>