| Objective: Based on the Chinese traditional theory of latent-qi and toxinretention in liver collaterals,we establish shenzhang particles,which has theeffect of reinforcing healthy qi,resolving stasis and dredging collaterals.Liver fibrosis is a major complication of various chronic hepatic diseases andresults from increased production and decreased degradation of theextra-cellular matrix. Activation of hepatic stellate cells (HSC) is an importantcommon disease driver. We then have employed the human hepatic stellatecell line to study the role of shenzhang particles on liver fibrosis and explorethe anti-fibrosis mechanism related to the Rho kinase signaling. Rho kinasesare the most widely studied mediators of the small G-protein RhoA. Abnormalactivation of this pathway has been shown to play a role in liver fibrosis.Additionally, Rho kinase signaling has also been linked to HSC activation andcontraction. In this study, we investigated the effects of shenzhang particles onRho-signaling pathways in activated human HSC cells, such as, contraction,migration, adhesion, proliferation, survival, and the transcriptional regulationof gene expression. The results described in this paper can be thereforeemployed shenzhang particles as anti-fibrosis relevant in vitro assay to helpidentify and optimize novel Rho kinase inhibitors.Methods:①Based on Serum pharmacology, HSC were treated with different concentrations of serum in the ShenZhang granules, which wererandomly divided into the normal control group (A), the5times group (B), the10times group (C) and20times group (D). Then，the level of α-SMA mRNAwas detected by RT-PCR; Changes of HSC by different concentration ofserum of ShenZhang granules on the cell cycle were test using flow cytometry;the effects of different concentrations of ShenZhang granules-serum on themigration of HSC were examined through cell scratch test.②Based on the first part of experiment, HSC were pre-treated with LPA,promoter of Rho/Rho kinase pathway or Y27632, inhibitor of Rho/Rho kinasepathway; then, the pre-treated cells were treated with10times concentrationof ShenZhang granules-serum for24hours, which divided into the normalcontrol group(C), the control pretreated with LPA(CL), the control pretreatedwith Y27632(CY) group,treated with ShenZhang granules-serum (T) andpretreated with LPA T group (TL). The effects of ShenZhang granules-serumon the migration of HSC were examined through cell scratch test; the cellcycle was test using flow cytometry. Then，the level of α-SMA protein wasdetected by fluorescence immunoassay.③Based on the second part of experiment, HSC were pre-treated withLPA, promoter of Rho/Rho kinase pathway or Y27632, inhibitor of Rho/Rhokinase pathway; then, the pre-treated cells were treated with10timesconcentration of ShenZhang granules-serum for24hours, which divided intothe normal control group(C), the control pretreated with LPA(CL), the controlpretreated with Y27632(CY) group,treated with ShenZhang granules-serum(T) and pretreated with LPA T group (TL). Then, the secretion levels of ET-1and TGF-β1were detected through Elisa.④Based on the second part of experiment, HSC were pre-treated withLPA, promoter of Rho/Rho kinase pathway or Y27632, inhibitor of Rho/Rhokinase pathway; then, the pre-treated cells were treated with10times concentration of ShenZhang granules-serum for24hours, which divided intothe normal control group(C), the control pretreated with LPA(CL), the controlpretreated with Y27632(CY) group,treated with ShenZhang granules-serum(T) and pretreated with LPA T group (TL). Through immunoprecipitation andWestern blotting, the level of the levels of GTP-Rho and p160ROCK wasdeteced.Results:①Cell cycle tests showed G2was obvious blocked, andproliferation of HSC was inhibited with10times the concentration group ofShenZhang granules-serum, the difference was statistically significant,compared with the normal control group (P <0.01); RT-PCR results alsoshowed that α-SMA mRNA level was the lowest in HSC by the treatment with10times Shenzhang granules-serum, the difference was statistically significant,compared with the normal control group (P <0.01);Scratch test showed10times of the concentration ShenZhang granules-serum treatment HSCmigration was inhibited, the difference was statistically significant, comparedwith the normal control group (P <0.01).②C ell cycle tests showed G2was obvious blocked, proliferation of HSCwas inhibited in T and TL group,which treated with ShenZhang granules-serum, the difference was statistically significant, compared with the normalcontrol group (P <0.01);Scratch test showed the migration of T and TL groupcells were inhibited, the difference was statistically significant, compared withthe normal control group (P <0.01); Fluorescence immunoassay result showedthe α-SMA expression of T and TL group cells was inhibited, the differencewas statistically significant, compared with the normal control group (P<0.01).③Elisa showed secretion levels of ET-1and TGF-β1were reduced bytreatment with ShenZhang granules serum in T and TL group cells, thedifference was statistically significant, compared with the normal control group (P <0.01);④I mmunoprecipitationand Western blotting showed that ShenZhanggranules-serum decreased the levels of GTP-Rho and p160ROCK in T and TLgroup cells, the difference was statistically significant, compared with thenormal control group (P <0.01).Conclusion:①From these results, it was concluded that Shenzhangparticles-serum inhibited activation of HSC via the reduction of α-SMAexpression and the level of α-SMA mRNA.②Shenzhang particles-serum inhibited activation of HSC via reducingthe level of secretion of ET-1and TGF-β1; Shenzhang granules-seruminhibited proliferation of the activated HSC.③Shenzhang granules-serum decreased the levels of GTP-Rho andp160ROCK; We hypothesize that shenzhang particles might inhibit theactivation of HSC through the Rho/Rho kinase signal pathway, as anti-fibrosisrelevant in vitro assay to help identify and optimize novel Rho kinaseinhibitors. |
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