抗纤软肝颗粒对血小板源生长因子诱导的肝星状细胞增殖的影响

目的:研究抗纤软肝颗粒(KXR)对血小板源生长因子(PDGF)诱导的肝星状细胞(HSC)增殖的影响.方法:采用无血清培养,不同浓度的KXR温育HSC 24 h后,PDGF-BB(10 ng/ml)刺激24 h,再加入上述浓度的KXR 3 h后,又加入PDGF-BB(10 ng/ml)作用5 min,然后收集细胞.采用细胞计数法及流式细胞仪测定细胞增殖及细胞周期.结果:无血清培养显示该方对于PDGF诱导的细胞增殖具有抑制作用,并呈剂量依赖性(P<0.01).流式细胞术分析提示KXR能抑制PDGF诱导的HSC DNA合成期及合成后期和分裂期DNA百分含量,阻断细胞由静止期/DNA合成前期向DNA合成期转化,呈剂量依赖性(5 mg/ml组作用最显著,P<0.01).结论:KXR能抑制PDGF诱导的HSC增殖.     

抗纤软肝颗粒对肝星状细胞凋亡的影响

探讨抗纤软肝颗粒对肝星状细胞(HSC)凋亡的影响.方法:传代培养的大鼠肝星状细胞系HSC-T6与中药复方抗纤软肝颗粒(终浓度为5mg/ml、2.5mg/ml、1.25mg/ml)及药物血清(5%、10%、20%)共同培养48小时后,应用TUNEL法及流式细胞仪测定细胞凋亡、免疫组化检测Bcl-2/Bax.结果:TUNEL法及流式细胞仪测定结果显示抗纤软肝颗粒药物及含药血清均可诱导HSC凋亡(P＜0.05);并能下调凋亡抑制分子Bcl-2,上调促凋亡分子Bax(P＜0.01).结论:抗纤软肝颗粒可下调Bcl-2/Bax比率,以增加细胞对凋亡的敏感性,从而促进HSC凋亡.     

抗纤软肝颗粒对肝星状细胞增殖的影响

目的 :研究抗纤软肝颗粒对肝星状细胞增殖的影响。方法 :传代培养的大鼠肝星状细胞系 (HSC- T6 )与中药复方抗纤软肝颗粒 (终浓度为 5 m g/ ml、2 .5 mg/ m l、1.2 5 mg/ ml)及药物血清 (5 %、10 %、2 0 %)共同培养 48h后 ,应用 MTT法及流式细胞仪测定细胞增殖。结果 :抗纤软肝颗粒无论是药物还是含药血清 ,均能显著抑制 HSC-T6增殖 ,并使细胞周期阻滞于 G1 期 ,在安全浓度范围内 ,5 m g/ ml组和 10 %药物血清组作用最强 (P <0 .0 1,<0 .0 5 )。结论 :抗纤软肝颗粒抗肝纤维化的作用机制可能在于阻滞细胞周期的进行 ,从而抑制 HSC增殖。     

莪术提取物对PDGF诱导的肝星状细胞内Ca2+和PI3-K的影响

目的:探讨莪术提取物药物血清对PDGF诱导的肝星状细胞(HSC)内Ca2 、PI3-K的影响。方法:制备药物血清,不同浓度的莪术提取物药物血清温育肝星状细胞24小时后,PDGF-BB(10ng/ml)刺激24小时,再加入上述浓度的莪术提取物药物血清,3小时后,又加入PDGF-BB(10ng/ml)作用5分钟,然后收集细胞。采用钙指示剂Fura-2/Am测定细胞内Ca2 ,免疫印迹化学发光法检测PI3-K的表达。结果:不同浓度的莪术提取物药物血清与同浓度的生理盐水药物血清比较,能显著抑制PDGF诱导的胞内Ca2 浓度升高(P<0.01)。经10%莪术提取物药物血清处理的HSC,其PDGF诱导的PI3-K表达水平较10%生理盐水药物血清组显著降低(P<0.01)。结论:莪术提取物药物血清能抑制PDGF诱导的肝星状细胞内Ca2 内流及PI3-K的表达。     

抗纤软肝颗粒调控Hedgehog通路核转录因子Gli1抑制肝星状细胞活化的研究

目的:探讨抗纤软肝颗粒调控Hedgehog(Hh)信号通路抑制HSC活化的作用机制。方法:引进人肝星状细胞系HSCLX2,常规培养及传代,设立空白对照组、模型组和抗纤软肝颗粒中、低剂量组。药物作用24h后收集细胞,测定各组Hh信号通路相关信号分子Glil、Shh、Ptc、Smo的表达,转化生长因子-β1(TGF-β1)及PDGF-B的水平。结果:模型组HSC细胞Hh信号通路相关信号分子Shh、Ptc、Smo、Glil mRNA表达均增高,与空白对照组比较,差异具有显著性意义(P0.05);抗纤软肝颗粒中、低剂量组Shh、Ptc、Smo、Glil mRNA表达较模型组减少,差异具有显著性意义(P0.05),各剂量组之间比较,差异无显著性意义。HSC诱导活化后,模型组TGF-β1及PDGF-B合成增加,与空白对照组比较差异具有显著性意义(P0.05);抗纤软肝颗粒中、低剂量组TGF-β1及PDGF-B合成较模型组下降,差异具有显著性意义(P0.05),各剂量组之间比较,差异无显著性意义。结论:抗纤软肝颗粒能够下调Hh信号通路核转录因子Gl订及相关信号分子Shh、Ptc、Smo的表达,从而抑制HSC活化,减少TGF-β1、PDGF-B的合成。     

抗纤软肝颗粒对大鼠肝星状细胞增殖的影响

目的:探讨抗纤软肝颗粒(KXRG)对大鼠肝星状细胞(HSC)增殖的影响。方法:传代培养的HSC-T6(肝星状细胞系)与KXRG(1.25mg/ml、2.5mg/ml、5mg/ml)及其药物血清(5%、10%、20%)共同培养72小时后,应用MTT法测定细胞增殖、流式细胞仪测定HSC细胞周期各时相的DNA含量。结果:KXRG及药物血清均能显著抑制HSC的增殖,使细胞周期阻滞于S期,且2.5mg/ml药物和10%药物血清作用最强(P0.01)。结论:KXRG通过抑制HSC的活化,阻滞其细胞周期的正常进行,进而抑制HSC增殖,从而起到抗肝纤维化的作用。     

软肝煎药物血清对肝星状细胞/T6增殖及Ⅰ型胶原合成的影响

目的 :探讨软肝煎药物血清对肝星状细胞 (HSC) / T6的增殖、 型胶原合成的影响。方法 :实验分为大鼠血清组和药物血清两组 ,各组再分为 5 %、10 %及 2 0 % 3个浓度组 ,每组 4只大鼠。用 MTT比色法和 Alarmablue测定法评价软肝煎药物血清对 HSC/ T6的增殖抑制作用 ;用 Western blotting法和 EL ISA法评价软肝煎药物血清对 HSC/ T6合成 型胶原量的影响。结果 :在 MTT测定法中 ,与大鼠血清组相比 ,药物血清组的 5 %、10 %浓度差异有显著性意义 (P <0 .0 5 ) ,2 0 %浓度差异有非常显著性意义 (P <0 .0 1)。在 Alarma blue测定法中 ,2 4 h时与大鼠血清相比 ,药物血清组的 2 0 %浓度差异有显著性意义 (P<0 .0 5 ) ;30 h时与大鼠血清相比 ,药物血清组的2 0 %浓度差异有非常显著性意义 (P <0 .0 1)。 EL ISA法测定的细胞上清 型胶原含量 ,药物血清组与大鼠血清组相比差异有显著性意义 (P <0 .0 5 ) ,Western blottin法测定细胞内合成 型胶原的量 ,可知药物血清组 型胶原的含量明显少于大鼠血清组。结论 :软肝煎不仅能抑制 HSC的增殖 ,而且能抑制 HSC合成 型胶原的量 ,从而提示软肝煎有很好的抗肝纤维化作用。     

抗纤软肝颗粒对四氯化碳加乙醇诱导肝纤维化大鼠的影响

目的:观察中药抗纤软肝颗粒对四氯化碳(CCl_4)加乙醇诱导肝纤维化大鼠的影响。方法:SD雄性大鼠40只,随机分为3组。分别为模型对照组(M)15只,抗纤软肝颗粒组(K)15只,正常对照组(N)10只。M、K组大鼠分别以40%CCl_4橄榄油液腹腔注射,2坎/w,并以5%乙醇为唯一饮水;N组大鼠同期注射等剂量的橄榄油。造模同时,K组大鼠即予抗纤软肝颗粒2g/100g/d(中等剂量)灌胃,1次/d;M、N组大鼠同期予等量生理盐水灌胃,共7周。观察大鼠肝脏病理学变化及血清总蛋白(TP)、白蛋白(Alb)、球蛋白(Glo)、Alb/Glo比值、ALT、AST的变化。结果:K组大鼠肝细胞变性坏死及肝纤维化程度较M组明显减轻,结缔组织明显减少,纤维间隔显著变薄,未见明显假小叶;M组大鼠血清Alb显著降低,ALT、AST均有明显升高(P0.05);K组大鼠Alb回升,ALT及AST明显下降,与M组比较差异有显著性意义(P0.05)。结论:抗纤软肝颗粒可通过保护肝细胞,降解纤维组织起到抗肝纤维化的作用。     

软肝化坚颗粒调节肝星状细胞分泌胶原及相关细胞因子的研究

目的:观察软肝化坚颗粒药物血清对肝星状细胞(HSC)分泌Ⅰ、Ⅲ型胶原及肝纤维化相关细胞因子的影响.方法:通过灌胃制备小鼠软肝化坚颗粒及秋水仙碱药物血清.将HSC-T6细胞分为3组,分别加入含10％软肝化坚颗粒血清、10％秋水仙碱血清和10％正常小鼠血清的培养基,培养24小时后,收集培养上清.采用ELISA法检测Ⅰ型胶原、转化生长因子β1 (TGF-β1)、瘦素(LP)及血小板衍生生长因子(PDGF)的含量;采用放射免疫法检测Ⅲ型胶原含量.结果:与正常血清对照组相比,秋水仙碱血清组和软肝化坚颗粒血清组培养上清中Ⅰ型胶原含量均显著降低,P值均为0.000;Ⅲ型胶原的含量也均显著降低,P值分别为0.005和0.001.与正常血清对照组相比,秋水仙碱血清组培养上清中TGF-β1、LP和PDGF的含量均明显降低,P值均为0.000;软肝化坚血清组也均显著降低,P值均为0.000;软肝化坚颗粒血清组与秋水仙碱血清组相比,培养上清中TGF-β1、LP的含量均明显降低,P值分别为0.006和0.043.结论:软肝化坚颗粒可抑制活化的HSC产生TGF-β1、PDGF及LP等细胞因子,从而对HSC分泌胶原产生抑制作用.     

硫化氢对大鼠肝星状细胞增殖及Ca2+浓度的影响

目的 探讨硫化氢(H2S)对大鼠原代肝星状细胞(HsC)增殖及Ca2+浓度的影响及其作用机制.方法 大鼠肝星状原代细胞作为研究对象,将过氧化氢(H2O2)作用于大鼠原代HSC制造肝纤维化的氧化应激模型,用钙离子荧光探针Fluo-3/AM负载细胞,并在此基础上应用不同剂量的NaSH(H2S供体)和KATP通道抑制剂(格列本脲)对各组细胞进行干预,用激光扫描共聚焦显微镜(LSCM)和CCK-8的方法分别检测不同刺激条件对细胞内Ca2+浓度改变及细胞增殖情况的影响.结果 低浓度H2S(100μmo/L NaSH)明显降低HSC细胞内Ca2+浓度(P＜0.05),抑制细胞增殖;K离子通道阻断剂——格列本脲可阻断H2S的作用.高浓度H2S(1mmol/L NaSH)使HSC细胞内Ca2+浓度增加,促进细胞增殖.结论 低浓度H2S通过激活HSC细胞KATP通道,降低细胞内Ca2+浓度,从而抑制细胞增殖;高浓度H2S使HSC细胞内Ca2+浓度增加,促进细胞增殖.     

Intracellular pathways mediating Na+/H+ exchange activation by platelet-derived growth factor in rat hepatic stellate cells

BACKGROUND & AIMS: The Na+/H+ exchanger is the main intracellular pH regulator in hepatic stellate cells (HSCs), and its activity is increased by platelet-derived growth factor (PDGF). Amiloride, an Na+/H+ exchange inhibitor, reduces PDGF-induced HSC proliferation, suggesting that the Na+/H+ exchanger plays a role in regulating HSC proliferative response. The aim of this study was to characterize the intracellular pathways mediating activation of the Na+/H+ exchanger by PDGF in HSCs. METHODS: The activity of the Na+/H+ exchanger and HSC proliferation rate were evaluated under control condition and after incubation with PDGF in the absence or presence of specific inhibitors of the main intracellular pathways of signal transduction. Na+/H+ exchange protein expression was evaluated by means of Western blot. RESULTS: PDGF induced a significant increase in the activity of the Na+/H+ exchanger without modifying protein expression. Inhibition of the calcium/calmodulin- and protein kinase C-dependent pathways resulted in a significant inhibition of both Na+/H+ exchange activity and of PDGF-induced HSC proliferation. The involvement of the two pathways was confirmed by showing that incubation of HSCs with both phorbol-12-myristate-13-acetate, a potent protein kinase C activator, and thapsigargin, which increases intracellular calcium levels, significantly increased both the Na+/H+ exchanger activity and HSC proliferation rate. Inhibition of the protein kinase A pathway did not modify either PDGF-induced Na+/H+ exchange activation or PDGF-induced HSC proliferation. On the contrary, inhibition of the mitogen-activated protein kinase- and of phosphatidylinositol 3-kinase-dependent pathways significantly reduced PDGF-induced HSC proliferation without affecting the activity of the Na+/H+ exchanger. CONCLUSIONS: Activation of the Na+/H+ exchanger by PDGF in HSCs is mediated by calcium/calmodulin- and protein kinase C-dependent pathways. PDGF-induced HSC proliferation is mediated by Na+/H+ exchange-dependent and -independent pathways.     

Rearrangement of the cytoskeletal network induced by platelet-derived growth factor in rat hepatic stellate cells: role of different intracellular signalling pathways

BACKGROUND/AIMS: Cytoskeletal reorganization plays an important role in the regulation of different cell functions, such as proliferation and migration. Since platelet-derived growth factor (PDGF) stimulates both proliferation and chemotaxis of hepatic stellate cells (HSC), we investigated the effects of this cytokine on cytoskeletal components of cultured rat HSC. METHODS/RESULTS: Exposure of HSC to PDGF induced the formation of stress fibres and of a ruffled configuration of the plasma membrane, evaluated by both fluorescence and electron microscopy. These modifications were also induced by exposure to the protein kinase C (PKC) activator phorbol-12-myristate-13-acetate (PMA) and abolished by pretreatment with the PKC inhibitor calphostin C, with the Rho inhibitor C3 exoenzyme and with the intracellular calcium chelator MAPTAM, but not with the PI-3 kinase inhibitor wortmannin or with the mitogen-activated protein kinase kinase inhibitor PD 98059. PDGF induced a translocation of Rho from the cytosol to the membrane which was inhibited by C3 exoenzyme and by calpostin C, and which was also induced by PMA. Moreover, PDGF induced a rearrangement of vinculin which was prevented by C3 exoenzyme and calphostin C. CONCLUSIONS: PDGF-induced cytoskeletal reorganization in HSC is dependent on PKC and Rho, thus suggesting that these two pathways may play an important role in the response of liver to injury.     

Intracellular pH regulation and Na+/H+ exchange activity in human hepatic stellate cells: effect of platelet-derived growth factor, insulin-like growth factor 1 and insulin

BACKGROUND/AIMS: The Na+/H+ exchanger is involved in rat hepatic stellate cell (HSC) proliferation induced by platelet-derived growth factor (PDGF). We therefore evaluated in human HSC: (1) the mechanisms of intracellular pH regulation; (2) the relationship between Na+/H+ exchange activation and cell proliferation induced by PDGF, insulin-like growth factor 1 (IGF-1) and insulin. METHODS/RESULTS: pH(i) regulation was mainly dependent on the activity of the Na+/H+ exchanger, which was evaluated by measuring pH(i) recovery from an acute acid load. PDGF (25 ng/ml) gradually increased the activity of the Na+/H+ exchanger which peaked at 18 h and remained stable until the 24th h. IGF-1 (10 nmol/l), but not insulin (100 nmol/l), slightly but significantly increased the activity of the Na+/H+ exchanger. Amiloride (100 micromol/l) and 20 micromol/l 5-N-ethyl-N-isopropyl-amiloride completely inhibited HSC proliferation (evaluated by measurement of bromodeoxyuridine incorporation) induced by PDGF and IGF-1, but did not affect proliferation of HSC induced by insulin. Finally, IGF-1 did not modify the activity of the Na+/Ca2+ exchanger. CONCLUSIONS: The Na+/H+ exchanger is involved in HSC proliferation induced by PDGF and IGF-1, whereas the proliferative effect of insulin is mediated by intracellular pathways which are Na+/H+ exchange-independent.     

Proanthocyanidin derived from the leaves of Vaccinium virgatum suppresses platelet‐derived growth factor‐induced proliferation of the human hepatic stellate cell line LI90

Aim: Hepatic stellate cell (HSC) proliferation plays a pivotal role in liver fibrogenesis, and agents that suppress HSC activation, including platelet‐derived growth factor (PDGF)‐induced HSC proliferation, are good candidates for antifibrogenic therapies. In this report, we use the LI90 HSC line to elucidate the antifibrogenic effects of proanthocyanidin derived from the leaves of Vaccinium virgatum. Methods: Proanthocyanidin (PAC) was extracted from the leaves of blueberry V. virgatum (BB‐PAC), grape seeds (GS‐PAC) and Croton lechleri (CL‐PAC). These extracts were examined for their effects on PDGF‐BB‐induced LI90 cell proliferation and DNA synthesis. Extracellular signal‐regulated kinase (ERK) and Akt phosphorylation and PDGF receptor‐β (PDGFR‐β) expression were evaluated by western blot analysis. Results: BB‐PAC potently suppressed PDGF‐BB‐induced proliferation and DNA synthesis of LI90 cells. BB‐PAC also suppressed PDGF‐BB‐induced DNA synthesis in primary cultured rat HSC. Moreover, GS‐PAC and CL‐PAC suppressed PDGF‐BB‐induced DNA synthesis in LI90 cells. In contrast, the monomeric PAC catechin and epicatechin and dimeric PAC procyanidin B2 only slightly suppressed PDGF‐BB‐induced DNA synthesis. Western blot analysis showed that BB‐PAC completely or partially inhibited PDGF‐BB‐induced ERK and Akt phosphorylation, respectively. In addition, BB‐PAC partially inhibited the PDGF‐BB‐induced degradation of PDGFR‐β. Conclusion: Our results suggest that BB‐PAC suppresses activated HSC by inhibiting the PDGF signaling pathway. In addition, these results provide novel findings that may facilitate the development of antifibrogenic agents.     

Inhibition of the NA(+)/H(+) exchanger reduces rat hepatic stellate cell activity and liver fibrosis: an in vitro and in vivo study

BACKGROUND & AIMS: The Na(+)/H(+) exchanger is the main intracellular pH (pH(i)) regulator in hepatic stellate cells (HSCs) and plays a key role in regulating proliferation and gene expression. We evaluated the effect of specific inhibition of this exchanger on HSC proliferation and collagen synthesis in vivo and in vitro. METHODS: Rat HSCs were incubated in the presence of platelet-derived growth factor (PDGF), transforming growth factor (TGF)-beta1, iron ascorbate (FeAsc), and ferric nitrilotriacetate solution (FeNTA) with or without the Na(+)/H(+) exchanger inhibitor 5-N-ethyl-N-isopropyl-amiloride (EIPA). pH(i) and Na(+)/H(+) exchanger activity, cell proliferation, and type I collagen accumulation were measured by using the fluorescent dye 2',7'-bis-(carboxyethyl)-5(6)-carboxyfluorescein, by immunohistochemistry for bromodeoxyuridine, and by enzyme-linked immunosorbent assay, respectively. In vivo liver fibrosis was induced by dimethylnitrosamine administration and bile duct ligation (BDL) in rats treated or not treated with amiloride. RESULTS: PDGF, FeAsc, and FeNTA increased Na(+)/H(+) exchange activity and induced HSC proliferation. TGF-beta1 had no effect on the Na(+)/H(+) exchanger and was able, as for FeAsc and FeNTA, to induce type I collagen accumulation. EIPA inhibited all the effects determined by PDGF, FeAsc, and FeNTA and had no effect on TGF-beta1-induced collagen accumulation. In vivo, amiloride reduced HSC proliferation, activation, collagen deposition, and collagen synthesis. CONCLUSIONS: The Na(+)/H(+) exchanger can play a key role in the development of liver fibrosis and in HSC activation in vivo.     

苯扎贝特对血小板源生长因子诱导的血管平滑肌细胞增殖和小凹蛋白1表达的影响

目的 观察苯扎贝特对血小板源生长因子诱导的大鼠血管平滑肌细胞增殖及小凹蛋白1表达的影响并探讨其机制.方法 体外植块贴壁法培养大鼠胸主动脉血管平滑肌细胞,噻唑兰比色法检测各组细胞增殖情况,Western Blotting法观察各组小凹蛋白1的表达情况.结果 不同浓度的苯扎贝特对血小板源生长因子诱导的血管平滑肌细胞增殖均有一定的抑制作用,随浓度增加,其抑制作用增强(P<0.01).血小板源生长因子干预组小凹蛋白1的表达较对照组明显降低(P<0.01),而用不同浓度的苯扎贝特干预后小凹蛋白1的表达明显升高(P<0.01),且随着苯扎贝特浓度增加,小凹蛋白1的表述明显增加.结论 苯扎贝特抑制血小板源生长因子诱导的血管平滑肌细胞增殖的同时可以上调小凹蛋白1的表述.     

Dose dependent and divergent effects of superoxide anion on cell death, proliferation, and migration of activated human hepatic stellate cells

BACKGROUND AND AIMS: Activated myofibroblast-like cells, originating from hepatic stellate cells (HSC/MFs) or other cellular sources, play a key profibrogenic role in chronic liver diseases (CLDs) that, as suggested by studies in animal models or rat HSC/MFs, may be modulated by reactive oxygen intermediates (ROI). In this study, human HSC/MFs, exposed to different levels of superoxide anion (O(2)(.-)) and, for comparison, hydrogen peroxide (H(2)O(2)), were analysed in terms of cytotoxicity, proliferative response, and migration. METHODS: Cultured human HSC/MFs were exposed to controlled O(2)(.-) generation by hypoxanthine/xanthine oxidase systems or to a range of H(2)O(2) concentrations. Induction of cell death, proliferation, and migration were investigated using morphology, molecular biology, and biochemical techniques. RESULTS: Human HSC/MFs were shown to be extremely resistant to induction of cell death by O(2)(.-) and only high rates of O(2)(.-) generation induced either necrotic or apoptotic cell death. Non-cytotoxic low levels of O(2)(.-), able to upregulate procollagen type I expression (but not tissue inhibitor of metalloproteinase 1 and 2), stimulated migration of human HSC/MFs in a Ras/extracellular regulated kinase (ERK) dependent, antioxidant sensitive way, without affecting basal or platelet derived growth factor (PDGF) stimulated cell proliferation. Non-cytotoxic levels of H(2)O(2) did not affect Ras/ERK or proliferative response. A high rate of O(2)(.-) generation or elevated levels of H(2)O(2 )induced cytoskeletal alterations, block in motility, and inhibition of PDGF dependent DNA synthesis. CONCLUSIONS: Low non-cytotoxic levels of extracellularly generated O(2)(.-) may stimulate selected profibrogenic responses in human HSC/MFs without affecting proliferation.     

丹酚酸A抗肝细胞过氧化损伤对肝星状细胞增殖的影响

目的：观察丹酚酸A（SA－A）抗肝细胞过氧化损伤时在抑制肝星状细胞（HSC）增殖中的作用。方法：以不同浓度的丙二醛（MDA）刺激HSC,通过氚标胸腺嘧啶（^3H[TdR]）掺入法,观察对HSC增殖作用,以及SA－A对损伤肝细胞MDA含量及促HSC增殖的影响。结果：一定浓度的MDA有促进HSC增殖的作用;损伤肝细胞MDA含量明显增加;SA－A可抑制损伤肝细胞对HSC的促增殖作用,并可降低损伤肝细胞MDA的含量。结论：SA－A可通过抗肝细胞过氧化损伤抑制HSC的增殖。