肝星状细胞凋亡调控因素的研究进展

诱导HSC凋亡成为阻止肝纤维化进程的途径之一.生长因子、死亡受体配体(TRAIL、FAS)、细胞外基质(胶原、整合素)、信号转导蛋白和转录因子(NF-κB、IKKJNK)等多种因素参与调控HSC凋亡.     

肝星状细胞活化相关信号通路在肝纤维化中的研究进展

肝纤维化是多种慢性肝损伤造成的细胞外基质(extracellular matrix, ECM)过度累积及降解不足的病理结果,如不加以干预会逐渐进展为肝硬化,甚至肝细胞癌。肝星状细胞(hepatic stellate cell, HSC)是ECM的主要来源,并且HSC在肝纤维化的起始、发展和消退过程中发挥关键作用。近年来,HSC活化涉及的信号传导通路成为研究热点,本文总结了HSC活化过程中的重要信号通路。     

肝细胞生长因子与肝纤维化的研究进展

肝细胞生长因子是一种具有多种生物活性的细胞因子,其在促进肝细胞增殖、抑制肝星状细胞活化等方面有重要作用.肝纤维化进展过程中,各种因素引起肝细胞持续损伤,使损伤部位肝细胞再生,肝星状细胞激活,细胞外基质大量沉积,从而导致肝纤维化形成.此文就肝细胞生长因子的生物学特性及其在肝纤维化中发挥的作用作一综述.     

肝细胞生长因子的激活诱导肝星状细胞凋亡

目的 探讨大鼠骨髓间充质干细胞(BMSCs)与肝星状细胞(HSCs)共培养体系中肝细胞生长因子激活因子(HGFA)激活肝细胞生长因子(HGF)后对HSCs凋亡的影响.方法 用半透膜建立上下双层细胞共培养体系,各组培养24h、48 h及72h后,倒置相差显微镜观察细胞形态学变化;流式细胞仪检测BMSCs表面抗原及HSCs凋亡率;四甲基偶氮唑盐法检测HSCs增殖率;免疫组织化学法检测HSCs中α-平滑肌肌动蛋白表达;免疫荧光及组织化学染色法检测HGF的激活形式(即HGF-α链);酶联免疫吸附法检测HGF及HGFA浓度.计量资料采用单因素方差分析,组间均数的比较采用q检验. 结果 不同浓度HGF在各时段对HSCs无增殖抑制作用(P＞0.05),差异无统计学意义;HGFA在各时段对HSCs有明显增殖抑制作用,且呈浓度依赖性,在24h HGFA浓度为70 ng/ml时抑制作用为(0.26±0.00),较对照组的(0.13±0.04),明显增加,差异有统计学意义(P＜0.05);72h实验组HGF-α链相对表达量37.24±1.03低于HGFA干预组的40.44±0.77,差异有统计学意义(P＜ 0.05);两者在各时段与对照组比较,差异均有统计学意义(P＜ 0.01).实验组HSCs凋亡率在72h为40.77％±1.16％,均较HGFA干预组的33.35％±2.04高,差异有统计学意义(P＜ 0.05);两者在各时段与对照组比较,差异有统计学意义(P＜0.05).实验组及HGFA干预组中HGF浓度的降低呈时间依赖性,较HSCs空白对照组低;实验组HGFA的浓度在各时段均较对照组高.结论 BMSCs与HSCs共培养后促进HGFA的分泌,并激活HGF使其发挥促HSCs凋亡的作用.     

IGFBPrP1 siRNA诱导的大鼠肝星状细胞凋亡及其机制

目的研究IGFBPrP1 siRNA对大鼠肝星状细胞凋亡的影响,并探讨其可能的机制。方法体外培养大鼠肝星状细胞株（HSC-T6）,分别设立：正常对照组,阴性对照组,IGFBPrP1 siRNA转染组。IGFBPrP1 siRNA转染大鼠肝星状细胞,转染不同时间后,用CCK-8试剂盒检测肝星状细胞增殖的变化,AnnexinV/PI双标法流式细胞术检测肝星状细胞凋亡的变化,免疫细胞化学染色法检测P53及Bcl-2蛋白表达的变化。结果 （1）IGFBPrP1 siRNA转染大鼠肝星状细胞不同时间（24、48、72 h）后,转染组细胞增殖受到抑制且凋亡率明显增高,与正常对照组及阴性对照组相比,差异均有统计学意义（P〈0.01）;（2）IGFBPrP1 siRNA转染大鼠肝星状细胞48 h后,与正常对照组及阴性对照组相比,转染组P53蛋白的表达量显著增高（P〈0.01）;Bcl-2蛋白的表达明显降低（P〈0.01）。结论 IGFBPrP1 siRNA能显著抑制大鼠肝星状细胞增殖且能促进其凋亡;上调P53的表达,下调Bcl-2的表达可能是IGFBPrP1 siRNA诱导大鼠肝星状细胞凋亡的途径之一;推测抑制IGFBPrP1的表达有可能成为治疗肝纤维化的新靶点。     

Fasudil prevents liver fibrosis via activating natural killer cells and suppressing hepatic stellate cells

BACKGROUNDFasudil, as a Ras homology family member A (RhoA) kinase inhibitor, is used to improve brain microcirculation and promote nerve regeneration clinically. Increasing evidence shows that Rho-kinase inhibition could improve liver fibrosis.AIMTo evaluate the anti-fibrotic effects of Fasudil in a mouse model of liver fibrosis induced by thioacetamide (TAA). METHODSC57BL/6 mice were administered TAA once every 3 d for 12 times. At 1 wk after induction with TAA, Fasudil was intraperitoneally injected once a day for 3 wk, followed by hematoxylin and eosin staining, sirius red staining, western blotting, and quantitative polymerase chain reaction (qPCR), and immune cell activation was assayed by fluorescence-activated cell sorting. Furthermore, the effects of Fasudil on hepatic stellate cells and natural killer (NK) cells were assayed in vitro.RESULTSFirst, we found that TAA-induced liver injury was protected, and the positive area of sirius red staining and type I collagen deposition were significantly decreased by Fasudil treatment. Furthermore, western blot and qPCR assays showed that the levels of alpha smooth muscle actin (α-SMA), matrix metalloproteinase 2 (MMP-2), MMP-9, and transforming growth factor beta 1 (TGF-β1) were inhibited by Fasudil. Moreover, flow cytometry analysis revealed that NK cells were activated by Fasudil treatment in vivo and in vitro. Furthermore, Fasudil directly promoted the apoptosis and inhibited the proliferation of hepatic stellate cells by decreasing α-SMA and TGF-β1. CONCLUSIONFasudil inhibits liver fibrosis by activating NK cells and blocking hepatic stellate cell activation, thereby providing a feasible solution for the clinical treatment of liver fibrosis.     

MicroRNA-194 inactivates hepatic stellate cells and alleviates liver fibrosis by inhibiting AKT2

BACKGROUND Activation of hepatic stellate cells(HSCs)is a pivotal event in the onset and progression of liver fibrosis.Loss of microRNA-194(miR-194)has been reported in activated HSCs,but the actual role of miR-194 in liver fibrosis remains uncertain.AIM To explore the role and potential mechanism of miR-194-mediated regulation of liver fibrosis in vitro and in vivo.METHODS The expression of miR-194 was examined in human fibrotic liver tissues,activated HSCs,and a carbon tetrachloride(CCl4)mouse model by qPCR.The effects of AKT2 regulation by miR-194 on the activation and proliferation of HSCs were assessed in vitro.For in vivo experiments,we reintroduced miR-194 in mice using a miR-194 agomir to investigate the functions of miR-194 in liver fibrosis.RESULTS MiR-194 expression was notably lacking in activated HSCs from both humans and mice.Overexpression of miR-194(OV-miR-194)inhibitedα-smooth muscle actin(α-SMA)and type I collagen(Col I)expression and suppressed cell proliferation in HSCs by causing cell cycle arrest in G0/G1 phase.AKT2 was predicted to be a target of miR-194.Notably,the effects of miR-194 knockdown in HSCs were almost blocked by AKT2 deletion,indicating that miR-194 plays a role in HSCs via regulation of AKT2.Finally,miR-194 agomir treatment dramatically ameliorated liver fibrosis in CCl4-treated mice.CONCLUSION We revealed that miR-194 plays a protective role by inhibiting the activation and proliferation of HSCs via AKT2 suppression.Our results further propose miR-194 as a potential therapeutic target for liver fibrosis.     

肝细胞生长因子对大鼠原代肝星状细胞凋亡的影响及其机制

目的 探讨肝细胞生长因子(HGF)对血小板衍生生长因子BB(PDGF-BB)作用下大鼠原代肝星状细胞(HSC)凋亡的影响及其机制.方法 分离培养大鼠HSC,传代后使用PDGF-BB处理HSC,HGF进行干预,采用AO/EB染色、TUNEL法和流式细胞仪观察HGF对PDGF-BB作用下大鼠原代HSC凋亡的影响,免疫细胞化学分析P65的表达,电泳迁移率分析检测核因子κB DNA-蛋白结合活性. 结果对照组、PDGF-BB+HGF处理组和HGF处理组AO/EB染色法检测细胞凋亡率分别为1.98%±0.42%,5.10%±0.56%和8.43%±0.40%;流式细胞仪观察凋亡率分别为1.64%、3.22%和6.66%;TUNEL法凋亡率分别为2.35%±0.47%、4.89%±0.36%和7.34%±0.51%,各组间比较,差异均有统计学意义(t值分别为3.65、2.46和2.13,P值均＜0.05).对照组、PDGF-BB+HGF处理组和HGF处理组P65的表达依次减少,吸光度值分别为0.250±0.038、0.180±0.025和0.130±0.028,差异有统计学意义(t=2.08,P＜0.05).核因子κB DNA-蛋白结合活性相应减弱,吸光度值分别为283.97±43.20、129.93±15.54和61.56±15.14,差异有统计学意义(t=2.77,P＜0.05).结论 HGF能够诱导大鼠原代HSC和PDGF-BB作用下大鼠原代HSC的凋亡,抑制P65的表达和核因子κ B DNA-蛋白结合活性是HGF促进HSC凋亡的重要机制.     

Slight activation of nuclear factor kappa-B is associated with increased hepatic stellate cell apoptosis in human schistosomal fibrosis

To investigate the relationship between NF-κB activation and hepatic stellate cell (HSC) apoptosis in hepatosplenic schistosomiasis, hepatic biopsies from patients with Schistosoma mansoni-induced periportal fibrosis, hepatitis C virus-induced cirrhosis, and normal liver were submitted to α-smooth muscle actin (α-SMA) and NF-κB p65 immunohistochemistry, as well as to NF-κB Southwestern histochemistry and TUNEL assay. The numbers of α-SMA-positive cells and NF-κB- and NF-κB p65-positive HSC nuclei were reduced in schistosomal fibrosis relative to liver cirrhosis. In addition, increased HSC NF-κB p65 and TUNEL labeling was observed in schistosomiasis when compared to cirrhosis.These results suggest a possible relationship between the slight activation of the NF-κB complex and the increase of apoptotic HSC number in schistosome-induced fibrosis, taking place to a reduced HSC number in schistosomiasis in relation to liver cirrhosis. Therefore, the NF-κB pathway may constitute an important down-regulatory mechanism in the pathogenesis of human schistosomiasis mansoni, although further studies are needed to refine the understanding of this process.     

大鼠肝纤维化自发逆转过程中肝星状细胞凋亡与p75的表达

肝星状细胞（HSC）凋亡是肝纤维化逆转的主要机制之一。人和大鼠的HSC表达低亲和力神经生长因子受体p75，当p75与神经生长因子（NGF）结合时可引起细胞凋亡。本研究通过建立四氯化碳（CCl4）肝纤维化大鼠模型，对肝纤维化自发逆转过程中HSC凋亡和p75表达进行了研究。     

神经胶质成熟因子β对肝星状细胞活化和肝纤维化的影响

目的了解IFN β-1a抑制肝星状细胞活化新的作用分子，并进一步验证该分子与HSC活化和肝纤维化的关系。方法LX-2细胞经2000U／ml IFN β-1a处理48h后，采用二维电泳技术对IFN β-1a处理和未处理的LX-2细胞蛋白质进行分离，质谱鉴定IFN β-1a处理前后LX-2细胞中差异表达超过2倍的蛋白点。对于鉴定出的差异表达分子，通过Western blot和RT-PCR方法验证其在LX-2细胞中、肝组织中以及肺、脑、脾、肾、心组织中的表达情况。结果与对照组相比，IFN—β-1a处理组LX-2细胞中有31个差异蛋白点，其中1个特异表达、5个上调表达、25个下调表达。其中特异表达的蛋白经鉴定为神经胶质成熟因子β（GMFβ）；表达上调的5个点鉴定出2种蛋白，为组蛋白H2A和β-原肌球蛋白；表达下调的25个点鉴定出18种蛋白。进一步对GMFβ的表达情况进行验证发现，与对照组相比，经IFN β-1a作用后的LX-2的GMF β蛋白表达明显（t=1．81，P〈0．01）；大鼠正常肝组织中GMFβ的蛋白和mRNA相对表达量明显高于肝硬化肝组织（蛋白相对表达量为1．81对比0．10，mRNA相对表达量为0．85对比0．12，t值分别为2．53，2．13，P〈0．01）；GMFβ的蛋白和mRNA在大鼠的脑、脾、肝、肾组织中明显表达，而肺、心组织中无表达（t值分别为1．91、1．94，P〈0．01）。结论IFN β-1a处理后的LX-2的蛋白质表达存在差异，这些差异表达的蛋白质可能参与了IFN β-1a对HSC活化的抑制以及HSC的凋亡。IFN β-1a处理后的LX-2以及正常大鼠的肝组织中GMFβ有明显的表达，GMFβ可能抑制HSC的活化以及抑制肝纤维化的进展。     

Interaction between insulin-like growth factor binding protein-related protein 1 and trans-forming growth factor beta 1 in primary hepatic stellate cells

BACKGROUND:We previously showed that insulin-like growth factor binding protein-related protein 1(IGFBPrP1) is a novel mediator in liver fibrosis.Transforming growth factor beta 1(TGFβ1) is known as the strongest effector of liver fibrosis.Therefore,we aimed to investigate the detailed interaction between IGFBPrP1 and TGFβ1 in primary hepatic stellate cells(HSCs).METHODS:We overexpressed TGFβ1 or IGFBPrP1 and inhibited TGFβ1 expression in primary HSCs for 6,12,24,48,72,and 96 hours to investigate their interaction and observe the accompanying expressions of α-smooth muscle actin(α-SMA),collagen I,fibronectin,and phosphorylated-mothers against decapentaplegic homolog 2/3(p-Smad2/3).RESULTS:We found that the adenovirus vector encoding the TGFβ1 gene(Ad TGFβ1) induced IGFBPrP1 expression while that of α-SMA,collagen I,fibronectin,and TGFβ1 increased gradually.Concomitantly,Ad IGFBPrP1 upregulated TGFβ1,α-SMA,collagen I,fibronectin,and p-Smad2/3 in a time-dependent manner while IGFBPrP1 expression was decreased at 96 hours.Inhibition of TGFβ1 expression reduced the IGFBPrP1-stimulated expression of α-SMA,collagen I,fibronectin,and p-Smad2/3.CONCLUSIONS:These findings for the first time suggest the existence of a possible mutually regulation between IGFBPrP1 and TGFβ1,which likely accelerates liver fibrosis progression.Furthermore,IGFBPrP1 likely participates in liver fibrosis in a TGFβ1-depedent manner,and may act as an upstream regulatory factor of TGFβ1 in the Smad pathway.     

Tetrandrine inhibits activation of rat hepatic stellate cells in vitro via transforming growth factor-β signaling

AIM: To investigate the effect of various concentrations of tetrandrine on activation of quiescent rat hepatic stellate cells (HSCs) and transforming growth factor-β (TGF-β) signaling in vitro.METHODS: HSCs were isolated from rats by in situperfusion of liver and 18% Nycodenz gradient centrifugation, and primarily cultured on uncoated plastic plates for 24 hwith DMEM containing 20% fetal bovine serum (FBS/DMEM) before the culture medium was substituted with 2% FBS/DMEM for another 24 h. Then, the HSCs were cultured in 2% FBS/DMEM with tetrandrine (0.25, 0.5, 1,2 mg/L, respectively). Cell morphological features were observed under an inverted microscope, smooth muscleα-actin (α-SMA) was detected by immunocytochemistry and image analysis system, laminin (LN) and type Ⅲprocollagen (PCⅢ) in supernatants were determined byradioimmunoassay. TGF-β1 mRNA, Smad 7 mRNA and Smad 7 protein were analyzed with RT-PCR and Western blotting, respectively.RESULTS: Tetrandrine at the concentrations of 0.25-2 mg/L prevented morphological transformation of HSC from the quiescent state to the activated one, while α-SMA, LN and PCⅢ expressions were inhibited. As estimated by gray values, the expression of α-SMA in tetrandrine groups (0.25, 0.5, 1, 2 mg/L) was reduced from 21.3% to 42.2%(control: 0.67, tetrandrine groups: 0.82, 0.85, 0.96, or 0.96, respectively, which were statistically different from the control, P＜0.01), and the difference was more significant in tetrandrine at 1 and 2 mg/L. The content of LN in supernatants was significantly decreased in tetrandrine groups to 58.5%, 69.1%, 65.8% or 60.0% that of the control respectively, and that of PCⅢ to 84.6%, 81.5%,75.7% or 80.7% respectively (P＜0.05 vs control), with no significant difference among tetrandrine groups. RTPCR showed that TGF-β1 mRNA expression was reduced by tetrandrine treatments from 56.56% to 87.90% in comparison with the control, while Smad 7 mRNA was increased 1.4-4.8 times. The TGF-β1 mRNA and Smad 7 mRNA expression was in a significant negative correlation (r= -0.755, P＜0.01), and both were significantly correlated with α-SMA protein expression (r = -0.938, P＜0.01;r = 0.938, P＜0.01, respectively). The up-regulation of Smad 7 protein by tetrandrine (1 mg/L)was confirmed by Western blotting as well.CONCLUSION: Tetrandrine has a direct inhibiting effect on the activation of rat HSCs in culture. It up-regulates the expression of Smad 7 which in turn blocks TGF-β1 expression and signaling.     

肝纤维化血清学诊断标记物

肝纤维化是各种慢性肝病进展至肝硬化的必经阶段,其临床表现不典型,可进一步发展为肝癌,严重威胁患者生命健康。早期准确评估肝纤维化患者病情对疾病预后具有重要意义,血清学指标因其无创、操作方便、重复性好等优势一直是肝纤维化无创诊断的研究热点。本文简要介绍了传统血清学指标在肝纤维化辅助诊断中的应用,同时评述了指标转化生长因子β1、血清多花紫藤凝集素阳性巨噬细胞结合蛋白及YKL-40的临床应用价值及优缺点。此外,在本课题组长期研究的基础上建议将胰岛素样生长因子结合蛋白相关蛋白1作为进展期肝纤维化及肝硬化的实验室诊断指标。     

Tetrandrine inhibits activation of rat hepatic stellate cells in vitro via transforming growth factor-β signaling

AIM: To investigate the effect of various concentrations of tetrandrine on activation of quiescent rat hepatic stellate cells (HSCs) and transforming growth factor-β (TGF-β) signaling in vitro. METHODS: HSCs were isolated from rats by in situ perfusion of liver and 18% Nycodenz gradient centrifugugation, and primarily cultured on uncoated plastic plates for 24 h with DMEM containing 20% fetal bovine serum (FBS/DMEM) before the culture medium was substituted with 2% FBS/DMEM for another 24 h. Then, the HSCs were cultured in 2% FBS/DMEM with tetrandrine (0.25, 0.5, 1,2 mg/L, respectively). Cell morphological features were observed under an inverted microscope, smooth muscle-α-actin (α-SMA) was detected by immunocytochemistry and image analysis system, laminin (LN) and type Ⅲ procollagen (PCⅢ) in supernatants were determined by radioimmunoassay. TGF-β1 mRNA, Smad 7 mRNA and Smad 7 protein were analyzed with RT-PCR and Western blotting, respectively. RESULTS: Tetrandrine at the concentrations of 0.25-2 mg/L prevented morphological transformation of HSC from the quiescent state to the activated one, while α-SMA, LN and PCⅢ expressions were inhibited. As estimated by gray values, the expression of α-SMA in tetrandrine groups (0.25, 0.5, 1, 2 mg/L) was reduced from 21.3% to 42.2% (control: 0.67, tetrandrine groups: 0.82, 0.85, 0.96, or 0.96, respectively, which were statistically different from the control, P<0.01), and the difference was more significant in tetrandrine at 1 and 2 mg/L. The content of LN in supernatants was significantly decreased in tetrandrine groups to 58.5%, 69.1%, 65.8% or 60.0% that of the control respectively, and that of PCII] to 84.6%, 81.5%, 75.7% or 80.7% respectively (P<0.05 vs control), with no significant difference among tetrandrine groups. RTPCR showed that TGF-β1 mRNA expression was reduced by tetrandrine treatments from 56.56% to 87.90% in comparison with the control, while Smad 7 mRNA was increased 1.4-4.8 times. The TGF-β1 mRNA and Smad 7 mRNA expression was in a significant negative correlation (r = -0.755, P<0.01), and both were significantly correlated with α-SMA protein expression (r = -0.938, P<0.01; r = 0.938, P<0.01, respectively). The up-regulation of Smad 7 protein by tetrandrine (1 mg/L) was confirmed by Western blotting as well. CONCLUSION: Tetrandrine has a direct inhibiting effect on the activation of rat HSCs in culture. It up-regulates the expression of Smad 7 which in turn blocks TGF-β1 expression and signaling.     

PPARγ对肝纤维化中肝星状细胞相关信号通路的影响

肝星状细胞（HSC）是肝纤维化发展的核心,其激活和增殖受到多种细胞因子网络调控。过氧化物酶体增殖物激活受体（PPAR）是配体激活的核转录因子超家族成员。PPARγ是其一亚型,通过干扰调节HSC增殖与凋亡的信号途径参与肝纤维化的发生发展,可能为肝纤维化的治疗提供新方向。     

Hedgehog通路与肝纤维化及肝星状细胞活化的关系研究

目的:探讨Hedgehog通路与肝纤维化及肝星状细胞(HSC)活化的关系.方法:清洁级SD雄性大鼠20只,均分为模型组和对照组.模型组采用腹腔注射四氯化碳(CCl4)和高脂饮食诱导肝纤维化,对照组予以正常饮食.第8周末取模型组存活大鼠与对照组中大鼠各5只处死,取左叶肝脏组织.HE、Masson染色观察两组肝组织病理变化;逆转录-多聚酶链反应(RT-PCR)检测纤维化大鼠肝脏中表达Hedgehog通路成员超音速Hedgehog信号通路(Shh)、膜受体patched(Ptc)、smoothened(Smo)和核转录因子Gli表达;实时荧光定量PCR法检测Hedgehog通路成员及HSC活化标志基因α-平滑肌肌动蛋白(α-SMA)mRNA在两组大鼠肝脏中的表达差异.体外培养HSC-T6细胞,RT-PCR检测HSC-T6细胞株中Hedgehog通路成员的表达;四甲基偶氮唑盐比色分析(MTT)法检测不同浓度环耙明(Cyclopamine,Cyc)对HSC-T6增殖的影响;分别用0、100/μmol/L的Cye干预HSC-T6,实时荧光定量PCR法检测Shh、Smo、Ptc、Gli-1及αSMA mRNA表达差异.结果:模型组大鼠肝脏有大量脂质及胶原沉积,且肝脏组织中均有Shh、Smo、Ptc、Gli-1表达.荧光定量PCR结果示模型组大鼠Shh、Smo、Gli-1及αSMA mRNA表达均较对照大鼠升高(20.45±3.31、12.78±0.53、10.88±2.41、4.91±2.59比1;P值均<0.05).Cyc在体外对HSC-T6有明显的抑制作用,且抑制作用呈剂量依赖性(F=636.81,P<0.01).荧光定量PCR结果示,用Cyc 100 μmol/L干预的HSC-T6中,Ptc、Smo、Gli-1和αSMA表达量分别为0.20±0.11、0.21±0.08、0.28±0.05和0.27±0.10,与Cyc 0μmol/L干预比较差异均有统计学意义(P值均<0.01).结论:肝纤维化过程中Hedgehog通路成员表达增高,抑制Hedgehog通路可抑制HSC活化,推测Hedgehog通路通过活化HSC促进肝纤维化的发生.