【作者】 段妍君；

【导师】 吕文亮；

【作者基本信息】 湖北中医药大学 ， 中医临床基础， 2014， 博士

【摘要】 目的：本课题研究依据温病学“伏气学说”及络病学“毒伏肝络”理论，组方参杖颗粒，功效益气养阴、清热化湿解毒、活血通络。在前期研究揭示该方抗肝纤维化机制与调控肝星状细胞（Hepatic stellatecell，HSC）活化相关的基础之上，探讨参杖颗粒对Rho/Rho激酶途径的上游激活、中间传导及下游效应的调节作用，以明确该药抗肝纤维化作用的新靶点，为揭示其抗纤维化的机制提供新的实验依据。方法:①运用血清药理学方法制备不同浓度的参杖颗粒含药血清（对大鼠的给药剂量等同于临床剂量的5倍、10倍和20倍）及正常血清分组培养HSC，并利用流式细胞术检测HSC细胞周期、利用RT-PCR检测a-SMAmRNA、利用细胞迁移实验检测细胞迁移能力。最终筛选出最佳的用于实验研究的参杖颗粒含药血清浓度。②根据实验一筛选出的最佳实验浓度参杖颗粒含药血清干预培养HSC，应用Rho/Rho激酶途径的激动剂溶血磷脂酸（Lysophosphatidic Acid,LPA）及抑制剂Y-27632作为工具要，分别设立正常对照组（C）、激活对照组（CL）、抑制对照组（CY）、药物血清处理组（T）、激活后药物处理组（TL），运用荧光免疫检测各组HSC中a-SMA蛋白的表达；运用流式细胞术检测各组HSC细胞周期；应用细胞迁移实验测定各组HSC的迁移能力。③在实验二的基础之上，进一步利用Elisa试剂盒检测参杖颗粒含药血清处理前后各组HSC分泌ET-1、TGF-β1的表达水平。④在实验二的基础之上，应用免疫印迹法检测参杖颗粒含药血清处理前后p160ROCK（Rho的靶蛋白）磷酸化水平，应用免疫沉淀检测含药血清处理前后RhoGTP活化水平。结果：①细胞周期检测结果显示细胞明显被阻滞于G2期,以10×浓度组抑制细胞增殖的效果最明显，与正常对照组相比有显著统计学意义（P＜0.01）；RT-PCR结果显示a-SMA mRNA水平在10×浓度组血清处理的HSC中最低，与正常对照组相比有显著统计学意义（P＜0.01）；细胞迁移实验显示10×浓度组血清处理对HSC迁移能力抑制最明显，与正常对照组相比有显著统计学意义（P＜0.01）。②荧光免疫结果显示正常静息状态下HSC表达α-SMA较少(C组)，LPA作用于HSC后(CL组)导致α-SMA的表达增加，抑制剂Y-27632（CY组）则导致α-SMA表达下降，参杖颗粒含药血清处理静息HSC后（T组）α-SMA的表达更少，预先被LPA活化HSC加入含药血清处理后（TL组）HSC中α-SMA的表达被抑制，荧光密度分析T组较C组荧光密度下降明显（P<0.05）,TL组较CL组荧光密度显著下降（P＜0.01）；细胞周期检测结果显示T组、TL组细胞明显被阻滞于G2期, T组与正常对照组相比有显著统计学意义（P＜0.05），TL组较CL组相比有显著统计学意义（P＜0.01）；细胞迁移实验显示T组、TL组细胞迁移能力明显被抑制，与正常对照组相比有显著统计学意义（P＜0.05），TL组与CL组比较有显著性差异（P＜0.01）。③Elisa检测显示T组、TL组细胞分泌ET-1、TGF-β1明显减少，T组与正常对照组相比有显著统计学差异（P＜0.01），而TL组与CL组比较有显著性差异（P＜0.01）。④免疫沉淀实验结果显示T组、TL组细胞中Rho-GTP的活化水平明显降低，含药血清处理的T组中Rho活化水平几乎完全被抑制，与C组比较具有显著差异（P<0.05）；TL组与CL组比较，Rho的活化水平被显著抑制，差异具有统计学意义（P<0.01）。免疫印迹T组、TL组细胞中p160ROCK磷酸化水平显著被抑制，其中T组与C组相比有统计学差异（P＜0.05），TL组与CL组比较有显著性差异（P＜0.01）。结论：①参杖颗粒制备成5.25g/ml溶液，按1ml/100g的剂量给大鼠灌胃后所制备的含药血清具有最佳的抑制HSC活化的作用，因而被确定为最符合本实验要求的参杖颗粒含药血清浓度。②参杖颗粒通过减少HSC合成α-SMA蛋白、抑制HSC分泌ET-1和TGF-β1、抑制HSC增殖和迁移，从而抑制HSC的活化；参杖颗粒对HSC在合成、增殖、收缩、迁移、分泌等方面的抑制性调节作用是其发挥抗肝纤维化作用的可能机制。③参杖颗粒可以抑制活化的HSC中RhoGTP的活化水平及p160ROCK的磷酸化水平；参杖颗粒能够通过抑制Rho/Rho激酶途径，从而抑制HSC的增殖、收缩、迁移、分泌及活化；Rho/Rho激酶途径是参杖颗粒抗肝纤维化作用的重要细胞转导途径。更多还原

【Abstract】 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.更多还原