软肝冲剂对肝细胞影响及星状细胞活化机制的干预作用

目的：观察软肝冲剂抗肝纤维化的作用及机理。方法：体外培养四氯化碳（CCL4）肝纤维化大鼠肝细胞（HC）、星状细胞（HSC），观察软肝冲剂含药血清对其影响。结果：软肝冲剂有保护肝细胞、减轻CCl4对肝细胞损伤，抑制HSC活化，促肝细胞再生的作用。结论：软肝冲剂具有抗脂质过氧化和抑制HSC活化的作用，从而抑制肝纤维化的形成。     

软肝冲剂对肝细胞影响及星状细胞活化机制的干预作用

目的:观察软肝冲剂抗肝纤维化的作用及机理。方法:体外培养四氯化碳(CCL4)肝纤维化大鼠肝细胞(HC)、星状细胞(HSC),观察软肝冲剂含药血清对其影响。结果:软肝冲剂有保护肝细胞、减轻CC l4对肝细胞损伤,抑制HSC活化,促肝细胞再生的作用。结论:软肝冲剂具有抗脂质过氧化和抑制HSC活化的作用,从而抑制肝纤维化的形成。     

玉米须对大白鼠肝星状细胞增殖分化的影响

目的:观察玉米须(Stigma maydis or corn silk)对大鼠肝星状细胞(hepatic stellate cells)的增殖,并探讨其抗肝纤维化作用机制。方法:用链霉蛋白酶和胶原酶原位灌流,Metrizamide密度梯度离心分离大鼠肝星状细胞,用MTT比色法和ELISA法分别检测大鼠肝星状细胞增殖及Ⅰ型胶原的合成。结果:玉米须能明显抑制肝星状细胞的增殖及Ⅰ型胶原的合成,随着玉米须用量增加,抑制作用增强,呈明显的剂量效应关系(P0.05)。结论:玉米须可通过抑制大鼠肝星状细胞增殖及Ⅰ型胶原合成而起到抗肝纤维化的作用。     

化肝煎对肝细胞和活化肝星状细胞功能的影响

目的探讨化肝煎含药血清对肝细胞和活化肝星状细胞HSC-T6的影响及机制。方法体外构建肝细胞及CCl_4肝细胞损伤模型,加入不同体积浓度的化肝煎含药血清,通过MTS法检测肝细胞存活率;采用MTS法检测化肝煎干预肝星状细胞HSC-T6后的细胞存活率,同时采用实时荧光定量核酸扩增检测系统(qPCR)和蛋白印迹法(Western blot)检测eIF3(eukaryotic initiation factor 3)家族相关因子的表达。结果在1～8%体积浓度范围内化肝煎含药血清对肝细胞无细胞毒性,并可改善CCl_4所致肝细胞存活率下降;当化肝煎含药血清体积浓度为10%、30%、50%和60%时显著抑制活化肝星状细胞的增殖,此外,化肝煎含药血清对eIF3a mRNA及蛋白表达均显著抑制。结论化肝煎含药血清能保护肝细胞,并抑制活化肝星状细胞HSC-T6的增殖,可能跟抑制eIF3a而抗肝纤维化有关。     

中药血清与肝星状细胞研究进展

肝星状细胞（hepatic stellate cells,HSCs）的活化、增殖是肝纤维化形成和发展的中心环节。利用中药血清药理学研究方法发现,中药血清可干预肝纤维化形成和发展。综述了中药血清与肝星状细胞的研究进展,指出中药血清从抑制HSCs活化、HSCs增殖、HSCs收缩,促进HSCs凋亡等多靶点作用于肝星状细胞。可能是一种有效的抗肝纤维化治疗方法。     

川芎含药血清对大鼠肝星状细胞大麻素受体1相关信号通路的影响

目的观察川芎含药血清对血小板源生长因子(PDGF-BB)活化的大鼠肝星状细胞(HSCs)大麻素受体1(CB1)、黏着斑激酶(FAK)及磷酸化丝氨酸/苏氨酸蛋白激酶(p-Akt)蛋白表达的影响,探讨其抗纤维化的作用机制。方法制备川芎含药血清,MTT法检测川芎含药血清对肝星状细胞增殖情况,Western blot法分析肝星状细胞CB1、FAK、p-Akt蛋白的表达。结果与正常对照组相比,PDGF-BB作用24 h能刺激HSCs增殖,上调CB1、FAK、p-Akt蛋白的表达;川芎含药血清、秋水仙碱及大麻素受体1抑制剂AM251均能明显抑制上述效应;且加入AM251后,川芎含药血清对上述效应的抑制效果增强,且作用呈剂量依赖性。结论川芎含药血清可能通过大麻素相关信号通路抑制HSCs增殖,从而发挥防治肝纤维化的作用。     

蒙药肝乐康胶囊抗肝纤维化的实验研究

目的：研究肝乐康胶囊抗肝纤维化的作用。方法：采用CCl4和乙醇复合因素致大鼠肝纤维化模型，给予肝乐康胶囊水溶液灌胃，观察血清学、形态学变化。结果：肝乐康胶囊大剂量组能显著降低大鼠谷丙转氨酶、谷草转氨酶、脯氨酸肽酶、血清透明质酸的水平；减轻肝脏炎症及纤维化程度；肝组织胶原面积明显减少。结论：肝乐康胶囊具有保肝和抗肝纤维化作用，其作用机制可能与抗炎、抑制肝脏星状细胞的活化和增殖有关。     

中医药抗肝纤维化实验研究的现状与展望

目的：探讨中医药抗肝纤维化的实验研究现状。方法：对近10年来发表的中医药抗肝纤维化的实验研究方面的论文进行整理、研究。结果：中医药可减轻肝细胞坏死，促进肝细胞再生；抑制肝星状细胞活化增殖，诱导肝星状细胞凋亡；促进ECM及胶原纤维的降解。结论：实验研究证明，中药有良好的抗肝纤维化作用。     

虎金颗粒对肝功能及肝星状细胞增殖的影响

目的:探讨虎金颗粒对大鼠血清中血清酶成分含量的影响;探讨肝星状细胞(HSC-T6)的增殖情况,并观察虎金颗粒对HSC-T6细胞增殖的影响。方法:异种血清(猪血清)大鼠腹腔注射制作免疫性肝纤维化模型,以血清丙氨酸氨基转移酶(ALT)、血清谷草转氨酶(AST)含量检测为主要内容,随机将大鼠分为正常组、模型组、秋水仙碱组以及虎金颗粒大、中、小剂量组做比较分析;血清药理学方法培养肝星状细胞,用酶标测定仪测定OD值以测定其增殖活性。结果:模型组大鼠血清中ALT、AST含量较正常组显著上升,秋水仙碱组及虎金颗粒不同剂量组均能使上述指标发生不同程度的下调,其中中药中剂量组和大剂量组与西药组比较具有显著意义,但仍以大剂量组作用最为明显,小剂量组与西药组比较则无显著意义;当培养至24 h和48 h时,HSC-T6自身的增殖比较明显,虎金颗粒含药血清对其增殖表现出较显著的抑制作用,且随着含药血清浓度的增加,其抑制作用也有所增强,说明虎金颗粒含药血清对肝星状细胞的抑制作用有一定的量效依赖关系。结论:虎金颗粒可以改善肝功能,其抗肝纤维化的作用与其抑制肝星状细胞的增殖有关。     

复方中药抗肝纤维化的研究进展

近年来对肝纤维化的病因病机、治法方药及中药抗肝纤维化的作用机制的研究取得了丰硕成果。肝纤维化病机主要是正气虚损而邪气未清,淤血阻络是病理基础;临床治法以益气化淤、活血化痰、扶正化淤、软坚散结或滋补肝肾为主。复方中药抗肝纤维化的机理包括抑制胶原的生成,促进胶原降解;通过抑制细胞增殖相关的胞内信号转导,抑制肝星状细胞激活、增殖;通过调节细胞凋亡相关基因和细胞因子的活性,促进活化肝星状细胞凋亡;同时可以下调模型大鼠差异表达蛋白的表达,促进正常蛋白质合成。     

抗纤软肝冲剂药物血清对肝星状细胞增殖的影响

目的从细胞学水平探讨抗纤软肝冲剂抗肝纤维化的作用机制.方法采用酶消化法、密度梯度离心法,分离正常大鼠肝星状细胞(HSC),培养、鉴定及传代.制备抗纤软肝冲剂药物血清,温育传一代HSC,并设生理盐水组和秋水仙碱组为对照.3H-proline掺入法测定细胞活力;3H-TdR掺入法和MTT比色法测定细胞增殖.结果抗纤软肝冲剂组药物血清对细胞形态无影响,还能明显提高细胞的3H-proline掺入(P＜0.01),明显抑制细胞的3H-TdR掺入量和MTT转化(P＜0.01),且对MTT的抑制作用呈浓度依赖性递增趋势.结论抗纤软肝冲剂能提高细胞活力,无细胞毒性作用;能抑制HSC的增殖,这可能是该方抗肝纤维化的细胞学机制之.     

扶正化瘀复方药物血清对大鼠肝贮脂细胞增殖及胶原合成的影响

采用正常大鼠灌胃给药、分离血清而体外作用培养细胞的血清药理学方法，探讨扶正化瘀方对培养大鼠贮脂细胞功能的影响。结果表明该复方血清无明显细胞毒性，抑制细胞增殖及胶原合成，而且该作用与复方的体内给药方式、给药剂量、取血时间、药物血清作用浓度等有一定关系。     

扶正化瘀方对骨髓移植肝纤维化小鼠骨髓细胞肝脏迁移的影响

目的: 探讨扶正化瘀方(FZHY)对肝纤维化小鼠骨髓细胞肝脏归巢的作用。 方法: BABL/C雌性受鼠性别交叉法骨髓移植模型基础上,四氯化碳(CCl₄)诱导肝纤维化模型;成模后,治疗组以FZHY方4.6 g ·kg^-1 ·d^-1 ig 4周。 PCR,病理及肝功能检测成模情况;通过FISH法标记Y染色体,观察雌性受鼠肝脏内骨髓源性细胞含量;Western blot法检测雌性受鼠肝组织基质细胞衍生因子-1(SDF-1)蛋白相对表达量。 结果: 与模型组比较,FZHY组小鼠,肝组织损伤明显减轻。FISH法标记Y染色体观察显示,FZHY组小鼠较模型组肝内骨髓源性细胞减少(P<0.05)。Western blot结果,FZHY组小鼠较模型组肝脏SDF-1蛋白相对表达量明显减少(P<0.01)。 结论: 在肝脏损伤时,FZHY可能通过抑制肝内SDF-1蛋白的表达来阻止骨髓源性细胞迁移入肝,从而一定程度上抑制骨髓源性细胞入肝后促进肝纤维化。     

Selective apoptosis in hepatic stellate cells mediates the antifibrotic effect of phenanthrenes from Dendrobium nobile

Regardless of the etiology, cellular death of the liver parenchymal hepatocyte seems to be a primary event of hepatic fibrogenesis, which ultimately results in hepatic stellate cell (HSC) activation and the synthesis of extracellular matrix proteins. Recently it has been demonstrated that hepatic fibrosis can be a reversible process when the stimulus is properly eliminated. Apoptotic removal of active HSC is considered an essential part of the resolution. By employing the HSC cell line, HSC-T6, it was found that the methanol extract of Dendrobium nobile stem significantly inhibited the proliferation of HSC-T6 cells. Three phenanthrenes, denbinobin, fimbriol B and 2,3,5-trihydroxy-4,9-dimethoxyphenanthrene isolated from D. nobile were proven to inhibit HSC proliferation. Growth arrest of HSCs by these compounds was accompanied by cellular loss via autophagy-linked apoptosis. The maximal dose of these compounds, however, had little effect on primary cultured hepatocytes in rats. Collagen deposition in HSC-T6 cells was attenuated by these phenanthrenes. Collectively, the above results demonstrated that denbinobin, fimbriol B and 2,3,5-trihydroxy-4,9-dimethoxyphenanthrene exhibited antifibrotic activities possibly by the induction of selective cell death in HSCs but not in hepatocytes, implying that these compounds may be useful candidates for developing therapeutic agents for the prevention and treatment of hepatic fibrosis.     

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??OBJECTIVE To investigate the effects of the primary bioactive component diallyltrisulfide (DATS) contained in garlic on the activation of hepatic stellate cells (HSCs) induced by oxidative stress in vitro. METHODS Rat HSCs were cultured in vitro, and MTT assay was used to screen the effective dose of hydrogen peroxide (H₂O₂) for stimulating HSC proliferation. Then lactate dehydrogenase assay was used to examine the toxic effect of DATS on HSCs, and MTT assay to evaluate the anti-proliferative effects of DATS at non-toxic concentrations on HSCs. Flow cytometry was used to determine DATS effects on cell cycle. Hoechst33258 staining and flow cytometry were used to analyze apoptosis. Western blot assays were used to examine the expression of relevant proteins and fibrotic markers. RESULTS H₂O₂ at 5 ??mol??L^-1 significantly promoted HSC proliferation, and this concentration was selected to establish the oxidative stress-induced HSC activation model. MTT assay showed that DATS dose-dependently inhibited HSC proliferation in the presence of H₂O₂. DATS arrested the H₂O₂-treated HSC at G₂/M check point associated with downregulation of Cyclin B1 and CDK1. DATS also dose-dependently stimulated H₂O₂-treated HSCs to undergo apoptosis, which was associated with modulation of apoptosis-related molecules including Bax, Bcl-2, Caspase-9 and Caspase-3. Furthermore, DATS was found to reduce the protein abundance of a series of fibrotic marker proteins. CONCLUSION DATS effectively inhibits oxidative stress-induced HSC activation in vitro demonstrated by suppressed proliferation, arrests cell cycle, increases apoptosis and reduces fibrotic marker expression. These findings provide novel insights into DATS as a potential antifibrotic candidate for further development.     

Investigation of the absorbed and metabolized components of Danshen from Fuzheng Huayu recipe and study on the anti-hepatic fibrosis effects of these components

Ethnopharmacology

Fuzheng Huayu recipe (FZHY) was formulated on the basis of Chinese medicine theory in treating liver fibrosis. It has a significant efficacy against liver fibrosis caused by chronic hepatitis B, with the action mechanisms of inhibition of hepatic stellate cell activation, protection of hepatocyte oxidative injury and regulations of hepatic matrix remodeling etc.

Aim of the study

To identify the absorbed components and metabolites of Danshen in FZHY in rat serum, and find their active components for anti-liver fibrosis.

Material and methods

A valid high performance liquid chromatography–electrospray ionization ion trap mass spectrometry (HPLC-ESI/MSⁿ) method was established to investigate the absorbed and metabolized compounds of Danshen in FZHY in rat serum after oral administration. Mass spectra were acquired in both negative and positive modes. Otherwise, to evaluate the anti-hepatic fibrosis efficacies of absorbed and metabolized compounds, the LX-2 cell line of hepatic stellate cell (HSC), which was crucial cellular basis of fibrogenesis, was cultured and incubated with absorbed compounds, the cytotoxicity was determined with the cellomics Multiparameter Cytotoxicity Kit 1 by High Content Screening (HCS), the cell proliferation was assayed with EdU-DNA incorporation, and the cell activation was analyzed through α-smooth muscle actin (α–SMA) expression with high content screening technology.

Results

More than 11 compounds and 2 metabolites from Danshen were identified in the serum after oral administration of FZHY by comparing their mass spectra and retention behavior with reference compounds or literature data. Among these compounds, there were no obvious changes in nuclear morphology, membrane permeability with blow 96 μM of six polar compounds treatment in comparison with control cells, respectively. And the salvianolic acid B (6 μM, 48 μM), caffeic acid (6 μM, 48 μM) and rosmarinic acid (48 μM) could obviously inhibit LX-2 cells proliferation, down-regulate α–SMA expression.

Conclusion

The results proved that the established method could be applied to analyze the absorbed into blood compounds of Danshen after oral administration FZHY. These absorbed compounds included 11 compounds and 2 metabolites of Danshen. Among them, the salvianolic acid B, caffeic acid and rosmarinic acid were the effective components of FZHY to anti-hepatic fibrosis effects.     

雄芍汤保肝降酶及抗氧化有效部位的研究

目的：研究雄芍汤抗肝纤维化的药效物质基础,分别筛选其保肝降酶及抗氧化的有效部位。方法：Wistar 雄性大鼠随机分为正常组、模型组、扶正组、雄芍组、多糖组、总碱组和总苷组,采用DMN 腹腔注射法制备大鼠肝纤维化模型,造模成功后灌胃给药,治疗组分别给予扶正化瘀胶囊（0.105 g·mL-1）、雄芍汤药液（1.610 g·mL-1）、雄芍汤粗多糖提取物（35.420 mg·mL-1）、雄芍汤总苷提取物（25.725 mg·mL-1）、雄芍汤总生物碱提取物（0.196 mg·mL-1）,正常组与模型组灌服等量生理盐水,每日1 次,疗程4 周。4 周后处死取材,全自动生化分析仪检测血清肝功能指标ALT、AST、TIBL 和ALB;黄嘌呤氧化酶法检测血清SOD活力;DTNB 还原法检测血清GSH-PX 活力;TBA 法检测血清MDA 含量;HE 染色及Masson 染色观察肝组织病理学改变。结果：与模型组比较,除总碱组无显著性差异外,其余各治疗组ALT、AST 及TBIL 均显著降低（P<0.05 或P<0.01）,ALB 均显著升高（P<0.05 或P<0.01）。与模型组比较,扶正组、雄芍组和总碱组SOD 及GSH-PX 均显著升高（P<0.01）,MDA 均显著降低（P<0.05 或P<0.01）,多糖组、总苷组无显著性意义。结论：粗多糖部位和总苷部位为雄芍汤保肝降酶的有效部位,总生物碱部位为雄芍汤抗氧化的有效部位。     

常见中药复方制剂抗肝纤维化机制研究进展

中药复方制剂具有多层次、多途径、多靶点抗肝纤维化的整体治疗优势,目前对中药复方制剂抗肝纤维化机制的研究仍然集中在通过各种途径抑制HSC增殖和活化、减少ECM的合成、加快细胞凋亡、抗脂质过氧化等方面。如何获得中药复方制剂抗肝纤维化的最大效益,需要运用细胞分子生物学、基因技术等手段对其抗肝纤维化机制进行进一步研究。     

补阳还五汤含药血清对缺氧缺糖损伤PC12细胞形态和活力的影响

目的:探讨补阳还五汤含药血清对缺氧缺糖(oxygen-glucose deprivation,OGD)损伤PC12细胞形态和活力的影响.方法:采用中药血清药理学方法制备补阳还五汤含药血清,以5％,10％,20％的含药血清作用于OGD损伤PC12细胞,于倒置显微镜下观察细胞形态,用四甲基偶氮唑盐法检测细胞活力.结果:PC12细胞OGD损伤后形态发生改变、细胞活力降低;5％,10％补阳还五汤含药血清作用于OGD损伤PC12细胞12,18,24 h,均可减轻细胞形态损伤、提高细胞活力(P＜0.01);而20％补阳还五汤含药血清随作用时间的延长对OGD损伤PC12细胞的形态和活力具有双向调节作用.结论:一定浓度的补阳还五汤含药血清对OGD损伤PC12细胞的形态和活力具有保护作用.