Protective effects of Ampelopsis brevipedunculata against in vitro hepatic stellate cells system and thioacetamide-induced liver fibrosis rat model

Context: Ampelopsis brevipedunculata Maxim (Vitaceae) is a traditional medicinal herb used for treating liver disorders.

Objective: The hepatoprotective effects of A. brevipedunculata ethanol extract (ABE) was investigated in experimental models of fibrosis.

Materials and methods: Hepatic stellate cells (HSCs) system in vitro and thioacetamide (TAA)-induced liver fibrosis rat model in vivo were used. Sprague–Dawley rats were divided into five groups of eight each (control, TAA, TAA with ABE 10?mg/kg, ABE 100?mg/kg and silymarin 50?mg/kg groups, respectively). Fibrosis was induced except to the control group by TAA (200?mg/kg, i.p.) twice per week for 13?weeks. ABE and silymarin was administered orally six times per week from the 7th week to the 13th week.

Results: In HSC-T6 cells, ABE (0.1?mg/mL) and silymarin (0.05?mg/mL) significantly (p?p?in vivo studies, ABE (10 and 100?mg/kg) treatment ameliorated the altered levels of serum biomarkers significantly (p?p?p?Conclusion: These results collectively indicate that ABE can potentially be developed as a therapeutic agent in the treatment of hepatic fibrosis.     

姜黄素衍生物对原代大鼠肝星状细胞作用的体外研究

目的：观察姜黄素衍生物（Curc-OEG）抑制原代肝星状细胞（HSC）增殖、活化以及诱导其凋亡的作用,探讨其可能的作用机制。方法：采用IV型胶原酶、DNA酶消化SD雄性大鼠肝脏,percoll梯度离心得到纯化的肝星状细胞。细胞分离后1d分别加入0、6.25、12.5μg/mL的姜黄素衍生物,作用7d后用RT-PCR及Western blot检测细胞α-SMA、TGF-β1、Smad2的mRNA水平和蛋白表达量。活化的肝星状细胞在第14天分别加入0、6.25、12.5、25、50、75μg/mL的姜黄素衍生物,作用24h后RT-PCR检测凋亡基因Bax、Bcl-2的mRNA水平和纤维化相关基因TGF-β1、collagen I、NF-κB及TIMP-1的mRNA水平。结果：药物作用7d后,6.25μg/mL和12.5μg/mL浓度药物处理组与对照组相比,细胞数目分别减少了56%和86%。在12.5μg/mL浓度药物作用下,原代肝星状细胞α-SMA、TGF-β1及Smad2的mRNA表达水平分别下调83%、85%及75%,蛋白表达水平分别下调94%、92%及73%（P〈0.05）。25μg/mL浓度药物作用24h后,细胞凋亡明显。在50μg/mL浓度药物作用下,活化的肝星状细胞Bax的mRNA表达水平上调约2.3倍,Bcl-2的mRNA表达水平下调约5.6倍;TGF-β1、collagen I、NF-κB及TIMP-1的mRNA表达水平分别下调90%、83%、74%、65%（P〈0.05）。结论：姜黄素衍生物可以明显抑制原代肝星状细胞的增殖和活化,促进活化的肝星状细胞凋亡及减少细胞外基质的沉积。     

去甲肾上腺素和酚妥拉明对大鼠肝星状细胞的影响

目的：探讨α-肾上腺素激动剂去甲肾上腺素和拮抗剂酚妥拉明对体外活化的肝星状细胞（HSC-T6）的影响。方法：体外对HSC-T6进行复苏和培养传代;MTT法检测HSC-T6的增殖;放射免疫法检测HSC-T6培养上清液中透明质酸(HA)和III型前胶原（PCIII）的浓度;流式细胞仪检测HSC-T6凋亡率;免疫细胞化学染色检测组织基质金属蛋白酶抑制因子-1(TIMP-1)和基质金属酶-13(MMP-13)蛋白的表达。结果：在体外培养的活化HSC-T6中,去甲肾上腺素（10-5,10-7,10-9 mol•L-1）能促进细胞增殖,升高分泌HA和PCIII水平,增强TIMP-1蛋白的阳性表达并降低其凋亡率;酚妥拉明（10-5,10-7,10-9 mol•L-1）能抑制细胞增殖,促进MMP-13的阳性表达并增加HSC-T6的凋亡率。结论：去甲肾上腺素具有促进肝纤维化的作用,酚妥拉明则具有抗纤维化的作用,该作用与其对HSC-T6增殖、凋亡及HA和PCIII分泌水平调控有关。     

丹皮酚体外对肝星状细胞增殖和凋亡的影响

目的探讨丹皮酚对大鼠肝星状细胞(HSC)增殖和凋亡的影响。方法 HSC加入丹皮酚1.66～49.8 mg·L-1作用48 h后,MTT比色法和锥虫蓝染色法检测HSC的存活率。HSC与丹皮酚33.2 mg·L-1分别作用6,12,24,36和48 h,锥虫蓝染色法检测HSC的存活率。HSC与丹皮酚1.66～69.7 mg·L-1作用48 h用细胞乳酸脱氢酶(LDH)活性测定试剂盒检测细胞上清中LDH活性。丹皮酚1.66～49.8 mg·L-1与HSC作用24 h分别用光学显微镜、Hoechst 33258染色和流式细胞术检测HSC细胞形态、细胞凋亡和凋亡率;丹皮酚1.66～49.8 mg·L-1作用48 h用流式细胞术检测细胞周期。结果 MTT实验结果表明,丹皮酚16.6～49.8 mg·L-1对HSC增殖具有明显抑制作用,且具有浓度效应关系(r=0.955,P<0.05),作用48 h时IC50值为105.4 mg·L-1。锥虫蓝染色实验结果表明,丹皮酚对HSC存活率的抑制作用不仅具有浓度效应关系(r=-0.936,P<0.05),浓度为33.2 mg·L-1时还具有时间效应关系(r=-0.965,P<0.05)。丹皮酚16.6～69.7 mg·L-1作用48 h无明显细胞毒性。流式细胞仪检测结果显示,丹皮酚8.3～49.8 mg·L-1作用24 h能显著促进HSC细胞凋亡(P<0.05),且具有浓度效应关系(r=0.920,P<0.05)。丹皮酚33.2和49.8 mg·L-1作用48 h可调节细胞周期,G0/G1期细胞增加(P<0.01),S期细胞减少(P<0.01),具有浓度依赖性(r=0.980,P<0.05)。结论丹皮酚具有促进HSC凋亡和抑制HSC增殖的作用,可能是其抗肝纤维化作用机制之一。     

血小板衍生生长因子体外对大鼠肝星状细胞表达细胞外基质的影响

目的研究体外大鼠肝星状细胞(HSC)中血小板衍生生长因子(PDGF)信号通路的激活对于HSC表达细胞外基质(ECM)的影响。方法分离、纯化大鼠原代HSC并体外培养;以MTT实验检测PDGF对HSC增殖的作用;以West-ern blot和Real-time PCR方法分别检测PDGF对PDGF-βR与CTGF、ECM成分α-SMA、α1(I)collagen和fibronectin,以及调控ECM降解平衡的MMP-2和TIMP-1表达的影响。结果 MTT实验结果表明PDGF以剂量依赖性和时间依赖性的方式促进HSC的增殖。PDGF可以促进其受体PDGF-βR和CTGF的蛋白与mRNA表达。PDGF还可明显上调ECM成分α-SMA、α1(I)collagen和fibronectin的表达。此外,PDGF影响ECM的降解平衡,表现为TIMP-1的表达上调,而MMP-2的表达下调。结论 PDGF/PDGF-βR信号转导途径可明显促进HSC活化与ECM生成,抑制ECM降解。所得结果可以为药物阻滞PDGF信号转导从而抑制肝纤维化提供依据。     

瞬时受体电位M7通道蛋白对大鼠肝星状细胞凋亡的影响

目的通过转染特异性siRNA,下调瞬时受体电位M7通道蛋白(TRPM7)在大鼠肝星状细胞系HSC-T6中的表达,研究其对HSC-T6凋亡的影响及内在机制。方法利用Li-pofectamineTM2000将特异性TRPM7-siRNA转染到HSC-T6内,分别运用流式细胞术检测HSCs凋亡变化;Western blot检测TRPM7、Caspase-3蛋白的表达;RT-PCR检测TRPM7,Caspase-3、Bid mRNA的表达。结果 TRPM7-siRNA明显抑制TRPM7 mRNA和蛋白的表达。与正常组或对照组相比,转染组细胞凋亡率、凋亡蛋白Caspase-3及凋亡基因Bid的表达均明显上调。结论特异性TRPM7-siRNA能明显抑制TRPM7表达,诱导HSCs凋亡,其机制可能与其诱导凋亡基因Bid表达上调有关。     

Strong antiproliferative effects of baicalein in cultured rat hepatic stellate cells.

Recently, antifibrogenetic effects of Sho-saiko-to, a traditional herbal medicine in Japan, have been shown in experimental hepatic fibrosis, and flavonoids in Sho-saiko-to are suspected as active ingredients. Thus, we evaluated the effects of baicalein, a major flavonoid in Sho-saiko-to, on proliferation and protein synthesis in cultured rat hepatic stellate cells. Baicalein decreased [3H]thymidine incorporation in cells stimulated with platelet-derived growth factor-B subunit homodimer (PDGF-BB) in a concentration-dependent manner (approximate ED50<10 microM, P<0.0001), and the decrease observed with 10 microM baicalein was greater than those observed with 5 microM retinol or 500 microM 3-isobutyl-1-methylxanthine (IBMX). Baicalein consistently decreased [3H]thymidine incorporation and cell number in cells stimulated with fetal calf serum (ED50<10 microM, P<0.0001), and moderately suppressed [3H]leucine and [3H]proline incorporation (P<0.0001). These results demonstrate the strong antiproliferative effect of baicalein in hepatic stellate cells, showing the possibility of baicalein as an antifibrogenetic drug for hepatic fibrosis.     

大鼠肝星状细胞的分离和培养

目的:本文参考Friedman等的不连续密度离心法并作改良,建立了一种经济、简便、可靠的分离大鼠肝星状细胞(HSC)的方法。方法:用链霉蛋白酶和胶原酶灌流大鼠肝脏,Nycodenz密度梯度离心分离HSC,并进行体外培养,结果:分离培养的HSC经台盼蓝拒染实验活细胞大于98％,细胞得率为3.95×10个/肝脏。结论:该方法简便、实用、稳定、可靠,而且本方法的建立为进一步研究HSC与肝纤维化的关系,尤其是细胞水平及分子生物学水平的研究奠定了基础。     

Effects and regulation of osteopontin in rat hepatic stellate cells

Using a cDNA microarray, we identified osteopontin (OPN) as one of the genes upregulated in cultured activated hepatic stellate cells (HSCs). Northern and western blot analyses showed that OPN was increasingly expressed during the progressive activation of cultured rat HSCs, and a significant increase in OPN was observed in carbon tetrachloride-induced rat liver fibrosis. In biliary atresia, OPN protein was predominantly expressed in Kupffer cells and HSCs in the necrotic areas. Incubation of HSCs with recombinant OPN-induced significant proliferative and migratory effects, and induced matrix metalloproteinase 2 production and activation. Moreover, OPN increased type I collagen production and type II transforming growth factor-beta receptor mRNA and protein. In conclusion, this study shows that OPN is expressed in activated HSCs and suggests that the upregulation of OPN might be a central pathway of HSC activation.     

6种黄酮化合物对大鼠肝星状细胞胶原合成的抑制作用

目的：研究黄颜木素、槲皮素、芹菜素、根皮素、橙皮素和查尔酮对血清、巨噬细胞培养上清液和转化生长因子β1刺激的大鼠肝星状细胞HSC—T6胶原合成的影响。方法：^3H-脯氨酸掺入法测定细胞胶原合成。结果：黄颜木素、槲皮素、芹菜素、根皮素、橙皮素和查尔酮（12．5～50μmol／L）以浓度依赖方式抑制血清、巨噬细胞上清和转化生长因子β1诱导的胶原合成。结论：黄颜木素、槲皮素、芹菜素、根皮素、橙皮素和查尔酮具有抑制肝星状细胞胶原合成的作用，可能具有潜在的肝纤维化治疗作用。     

维拉帕米对肝星状细胞凋亡和Bcl-2表达的影响

目的:观察维拉帕米对人肝星状细胞(hepatic stellate cells,HSCs)凋亡的影响以及凋亡相关蛋白Bcl-2表达的变化。方法:流式细胞仪AnnexinV/PI双染法检测HSCs细胞的凋亡率,免疫组化SABC法检测Bcl-2表达的变化。结果:AnnexinV/PI双染法检测显示,不同浓度的维拉帕米均可诱导HSCs细胞凋亡,且有剂量相关性,与阴性对照组比较,差异有统计学意义(P<0.05);免疫组化检测显示,与对照组比较,维拉帕米组Bcl-2表达减少(P<0.05)。结论:维拉帕米可诱导HSCs凋亡,减弱Bcl-2的表达。     

维拉帕米对肝星状细胞增殖和凋亡的影响

目的观察维拉帕米对人肝星状细胞(hepatic stellate cells,HSCs)增殖和凋亡的影响。方法采用不同浓度的维拉帕米对HSCs处理48 h,CCK-8试剂盒(cell counting Kit-8)检测细胞增殖抑制率,HE染色观察细胞形态超微结构的变化,流式细胞仪Annexin-V/PI双染法检测HSCs的凋亡率。结果维拉帕米可抑制HSCs的增殖,且有剂量相关性,其IC50值为(150±10)mmol·L-1;HE染色可见维拉帕米组出现不同程度的细胞体积变小、核浓缩深染等凋亡形态改变;Annexin-V/PI双染法检测显示,不同浓度的维拉帕米均可诱导HSCs凋亡,与阴性对照组比较,差异有统计学意义(P<0.05)。结论维拉帕米可抑制HSCs的增殖,诱导其凋亡。     

Honokiol induces apoptosis via cytochrome c release and caspase activation in activated rat hepatic stellate cells in vitro

The therapeutic goal in liver fibrosis is the reversal of fibrosis and the selective clearance of activated hepatic stellate cells (HSCs) by inducing apoptosis. Over the past several years, we have screened for natural products that mediate apoptosis in activated HSCs. Among the candidate compounds, honokiol, isolated from Magnoliae cortex, was found to induce apoptotic death in activated rat HSCs, while there was no cell viability change in hepatocytes, at concentrations of 12.5-50 microM. Apoptosis was identified by DNA fragmentation, activation of caspase-3 and -9, and the proteolytic cleavage of poly(ADP-ribose) polymerase, down-regulation of bcl-2 and the release of mitochondrial cytochrome c into the cytoplasm.     

6种黄酮类化合物对离体肝储脂细胞增殖的影响(英文)

目的:研究黄颜木素、槲皮素、芹菜素、根皮素、橙皮素和查耳酮对血清、巨噬细胞上清和血小板源生生长因子刺激的HSC-T6细胞增殖的影响。方法:结晶紫染色法测定细胞增殖。结果:黄颜木素、槲皮素、芹菜素、根皮素、橙皮素和查耳酮(6.25-50μmol/L)以剂量依赖方式显著抑制血清、巨噬细胞上清和血小板源生生长因子诱导的HSC-T6细胞增殖。结论:黄颜木素、槲皮素、芹菜素、根皮素、橙皮素和查耳酮具有抑制肝储脂细胞增殖的作用。     

白芍总苷治疗四氯化碳致大鼠肝纤维化的作用与其影响肝星状细胞功能的关系*

目的:研究白芍总苷(TGP)通过影响肝星状细胞(HSC)的功能发挥治疗四氯化碳(CCl_4)致大鼠肝纤维化的作用。方法:建立CCl_4诱导化学性肝纤维化模型,全自动生化仪检测血清中丙氨酸氯基转移酶(ALT)、天冬氨酸氨基转移酶(AST)和碱性磷酸酶(ALP)水平;放免法检测血清及体外HSC培养上清中透明质酸(HA)和Ⅲ型前胶原(PCⅢ)的含量;流式细胞仪检测HSC凋亡率。苏木素-伊红染色处理肝脏组织切片,光镜观察病理学改变。结果:在CCl_4诱导大鼠肝纤维化模型,TGP给药(40,80,160mg·kg~(-1),ig)均明显降低血清ALT,AST,ALP,HA和PCⅢ水平;升高白蛋白水平及白蛋白球蛋白比值(A/G)。TGP(60,120和240mg·L~(-1))能明显降低HSC体外分泌的HA和PCⅢ水平,促进HSC凋亡,明显改善肝脏病理组织状况。结论:TGP对CCl_4致大鼠肝纤维化具有治疗作用,该作用与其降低HSC分泌HA和PCⅢ、促进HSC凋亡有关。     

大鼠肝星状细胞中多种细胞色素P450亚型的表达

目的采用改良的肝星状细胞(HSCs)体外分离和纯化技术,观察刚分离的大鼠HSCs中细胞色素P450(CYP450)亚型的表达,初步探讨CYP450同工酶在HSCs生理功能和激活中的作用。方法采用链霉蛋白酶/胶原酶序贯灌流和Nycodenz密度梯度离心方法,分离和纯化大鼠HSCs,半定量RT-PCR法检测HSCs和肝细胞内多种与视黄醇代谢相关的CYP450亚型mRNA的表达。结果HSCs的得率为每肝(0.8～1.2)×107。刚分离的大鼠HSCs中均能检测到CYP1A1,CYP1B1,CYP2B1/2和CYP2E1mRNA表达,其中,HSCs中CYP1A1mRNA及CYP1B1mRNA表达量接近甚至高于同一个体肝细胞中的表达水平。但CYP3A1mRNA未检测到。结论刚分离的大鼠HSCs可较高水平地表达多种与视黄醇代谢相关CYP450亚型,提示HSCs中CYP450亚型可能参与了HSCs的生理功能调节和激活过程。     

蛇毒神经生长因子对大鼠肝星状细胞增殖、凋亡及肝纤维化相关蛋白质表达的影响

目的探讨眼镜蛇毒神经生长因子(NGF)对大鼠肝星状细胞(HSC-T6)增殖、凋亡以及蛋白质表达差异的影响,进一步为蛇毒NGF抗肝纤维化提供依据。方法实验分为对照组(单纯HSC-T6培养)和实验组(NGF进行干预)。应用MTT检测眼镜蛇毒NGF对HSC-T6细胞增殖抑制率;流式细胞技术检测眼镜蛇毒NGF对HSC-T6细胞凋亡的影响;双向电泳技术观察HSC-T6细胞蛋白质表达变化;质谱鉴定差异表达的蛋白。结果 MTT结果显示NGF对HSC-T6细胞增殖具有明显抑制作用(5 mg.L-1NGF时抑制率为48.2%±1.9%,P<0.01);流式细胞仪也有同样的发现,NGF(5 mg.L-1)干预组的凋亡率21.15%±3.31%明显高于对照组的2.7%±1.55%(P<0.05);比较分析空白对照组和NGF作用组的2-DE图谱,找到差异蛋白质点47个,其中与对照组相比在NGF作用组表达上调22个,下调25个;质谱鉴定出9个蛋白质,其中4个表达上调,5个表达下调。结论蛇毒NGF能抑制大鼠肝星状细胞(HSC-T6)的增殖,诱导其凋亡,并且影响HSC-T6细胞蛋白质的表达。     

黄芪总苷对肝星状细胞增殖和合成胶原的抑制作用

目的　探讨黄芪总苷 (AST)对肝星状细胞增殖和合成胶原的影响。方法　采用枯细胞条件培养基 (KCCM )及含新生牛血清 (NBS)和健康大鼠血清 (RS)的混合血清刺激大鼠肝星状细胞 (HSC)系HSC T6 ,用 3H TdR和3H 脯氨酸参入法检测HSC增殖活性和胶原合成状况。结果　AST(1 6、32、64、1 2 8和 2 56mg·L- 1 )对KCCM 1∶4刺激HSC T6细胞增殖和胶原合成均有明显的抑制作用 ;在含 1 0 %NBS- 3 %RS混合血清刺激的HSC T6细胞 ,AST(32、64、1 2 8和 2 56mg·L- 1 )作用 48h对HSC T6细胞增殖 ,及AST(1 6、32、64、1 2 8和 2 56mg·L- 1 )作用 72h对HSC T6细胞胶原合成均有明显的抑制作用 ,呈浓度依赖型趋势。结论　AST对体外激活肝星状细胞的增殖和产生胶原有明显抑制作用 ,该作用可能是AST抗肝纤维化的机制之一     

Effects of ischemia-reperfusion and pretreatment with mildronate on rat liver mitochondrial function

Mildronate (3-(2,2,2-trimethylhydrazinium) propionate), which is mostly used in cardiological practice and is considered an anti-ischemic drug, was designed to inhibit carnitine biosynthesis in order to prevent accumulation of cytotoxic intermediate products of fatty acid beta-oxidation. Recently it was shown that the mitochondrial respiratory chain may also be a target for mildronate action. In this study, we aimed to investigate whether mildronate can protect the liver against a 90-min normothermic ischemia/30-min reperfusion-induced mitochondrial dysfunction. Rats were pre-treated for one or two weeks with mildronate (100 mg/kg/day or 200 mg/kg/day) or Ringer solution and subjected to ischemia/reperfusion. We found that ischemia/reperfusion caused a decrease in mitochondrial State 3 respiration rate and in the respiratory control index (RCI), and an increase in State 2 respiration rate with succinate, glutamate + malate and palmitoyl-L-carnitine + malate. One or two weeks of pre-treatment of rats with different doses of mildronate did not reduce the ischemia/reperffusion-induced decrease in the State 3 respiration rate or RCI; however, a one week pre-treatment slightly diminished the increase in the State 2 respiration rate with glutamate + malate substrates. The leakage of the liver enzymes, aspartate aminotransferase, alanine aminotransferase and lactate dehydrogenase, was similar in both the untreated and pre-treated with mildronate groups. No steatotic livers were observed in any experimental groups after mildronate pre-treatment. In conclusion, 90 min of liver ischemia followed by a 30 min reperfusion has a deleterious effect on rat liver mitochondrial function. Mildronate pre-treatment of rats at doses of 100 or 200 mg/kg/day for one or two weeks did not prevent ischemia/reperfusion-induced mitochondrial dysfunction and liver injury.     

水飞蓟素抗肿瘤作用及其机制研究进展

水飞蓟素是从植物水飞蓟种子中提取得到的一类生物活性成分,含65%～80%黄酮木脂素(或称水飞蓟素混合物)、少许黄酮、20%～35%脂肪酸和一些多酚。水飞蓟素除用于治疗肝炎和肝硬化等肝脏疾病外,对前列腺癌、皮肤癌、膀胱癌、肺癌和结肠癌等具有抑制作用。近年研究发现,水飞蓟素抗肿瘤的作用机制与其调节细胞周期、诱导细胞凋亡及抑制新生血管形成等作用有关。另外,水飞蓟素具有抗炎、抗肿瘤转移及抗氧化活性,与抗肿瘤药物具有协同增效作用。