肝巨噬细胞与肝星状细胞交互作用对肝纤维化发生与逆转的影响

肝纤维化是由各种病因所导致的肝脏病理性反应,是发展成肝硬化甚至肝癌的必经途径。以往研究发现,肝纤维化甚至是肝硬化早期都可以通过一定的干预治疗抑制与逆转病情,该过程有多种肝实质以及非实质细胞参与,肝星状细胞(hepatic stellate cell, HSC)与肝巨噬细胞是肝纤维化进程中关键的细胞类型。HSCs是肝纤维化的核心细胞,而肝巨噬细胞是肝纤维化进程中的主要调控细胞,HSCs与巨噬细胞间可通过分泌趋化因子、炎症因子以及凋亡因子诱导双方细胞的活化、分化、增殖和凋亡,并且能够调节细胞外基质(ECM)的生成与降解,进而影响肝纤维化的发生发展与抑制逆转。该文立足于HSCs与肝巨噬细胞的各自特征性功能,通过对它们之间的相互影响的阐述,探究两者在促进与逆转肝纤维化中的作用,以期探究肝纤维化复杂病理过程中的机制,为治疗逆转肝纤维化提供新的思路和有效靶点。     

肝星形细胞在肝纤维化发生及治疗中的作用

肝纤维化是指在修复肝脏损害和炎症的过程中,肝脏细胞外基质过度增多和过度沉积的病理过程。目前认为,肝星形细胞在肝纤维化形成的过程中起着非常重要的作用。细胞外基质主要来自肝星形细胞,肝实质中降解细胞外基质的基质金属蛋白酶也来自肝星形细胞。肝星形细胞已成为肝纤维化治疗的新靶点。     

肝窦内皮细胞在肝纤维化发生发展中的作用及其机制

肝窦内皮细胞(liver sinusoidal endothelial cells,LSECs)是肝脏抵御炎症和免疫反应的第一道防线,其独特的窗孔结构及功能特性决定它在肝纤维化发生发展中起重要作用。LSECs主要通过介导肝脏炎症反应、肝窦毛细血管化、活化肝星状细胞(hepatic stellate cells,HSC)、引发细胞外基质(extracellular matrix,ECM)的生成与降解失衡等途径参与肝纤维化的发生发展。靶向LSECs对治疗肝纤维化极具潜力,阐述清楚二者调控关系,将为抗肝纤维化治疗提供新的理论依据。     

细胞自噬与肝纤维化

自噬是细胞内一种进化保守的依赖溶酶体的蛋白降解途径,存在于大多数真核细胞中,细胞自噬可以为应激状态(如炎症、肿瘤、毒物等)的细胞提供能量。已有研究表明,肝纤维化的发病机制中涉及肝星状细胞的活化,伴随着细胞内脂质小滴的消耗,当肝细胞经历自噬,溶酶体的胞内基质将下降。研究还发现肝星状细胞的活化过程中伴随着其自噬水平的升高。本文将就自噬在肝纤维化中的作用做一综述。     

人肝星状细胞激活和抑制及其相关分子机制的研究进展

肝纤维化是肝脏对一系列慢性刺激的损伤修复反应,以细胞外基质的过度沉积为主要特征。许多研究证明人肝星状细胞(hepatic stellate cells,HSCs)的活化与增殖是肝纤维化形成的中心环节。因此,肝星状细胞激活机制及抑制活化途径的研究和发现成为防治肝纤维化的关键。目前,国际上肝纤维化药物研发的思路之一是从肝纤维化发生的机制,即肝星状细胞激活机制中寻找分子靶点。近年来,对各种使肝星状细胞活化的信号通路及相关抑制机制的研究取得了一些进展,但由于肝星状细胞活化是多条信号通路相互协调的结果,其复杂性、未知性造成了阻断方式的特异性、多样性,使该研究还仅限于实验室阶段,要想应用于临床还需要大量实验证明。该文就最新发现的肝星状细胞激活和抑制及相关分子机制作一综述。     

肝星状细胞中表皮形态发生素表达调控机制及其作用研究

肝星状细胞是肝脏中重要的间质细胞,是肝细胞外基质的主要来源.表皮形态发生素(epimorphin、EPM、syntaxin2)在肝脏发育、再生及癌变过程中发挥了重要的作用,目前其表达变化的调控机制及对肝星状细胞的作用还未有报道.通过对肝组织标本进行检测,发现肝纤维化过程中肝星状细胞表达EPM上调.从表观遗传学的角度对EPM表达变化调控机制进行研究,发现DNA去甲基化促进了EPM的表达.为了研究EPM对肝星状细胞的可能的调节作用,将EPM表达质粒转染肝星状细胞,之后检测了EPM对肝星状细胞增殖及迁移能力的变化.结果证明EPM能够促进肝星状细胞的增殖与迁移.本研究发现,激活的肝星状细胞高表达EPM可能是由于DNA去甲基化引起的,同时,高表达的EPM能够促进肝星状细胞的增殖与迁移,进而促进肝纤维化进展.     

siRNA对肝纤维化的抑制作用

目的:研究肝星状细胞(HSC)中smad2特异性小干扰RNA(siRNA)对Ⅰ型胶原表达的抑制作用,探讨抗肝纤维化的基因治疗新方法。方法:设计合成靶向Smad2基因的siRNA,将筛选成功的siRNA瞬时转染入体外培养的肝星状细胞(HSC),并给予转化生长因子β(TGF-β)刺激,应用RT-PCR和Western blot技术检测对照组与实验组Ⅰ型胶原mRNA水平和蛋白水平表达差异,研究siRNA对Ⅰ型胶原表达的抑制作用。结果:siRNA能明显降低肝星状细胞中Smad2的RNA和蛋白的表达水平,证实筛选的siRNA有效,能特异性抑制Smad2的基因表达;TGF-β刺激肝星状细胞后,与对照组比较,siRNA转染组细胞外基质(ECM)成分Ⅰ型胶原的表达水平明显降低(P<0.05)。结论:siRNA能够抑制TGFβ对肝星状细胞的激活,阻断TGFβ-Smads传导通路,使Ⅰ型胶原分泌下调,有效抑制TGFβ诱导的肝纤维化。     

siRNA对肝纤维化的抑制作用

目的：研究肝星状细胞（use）中smad2特异性小干扰RNA（siRNA）对I型胶原表达的抑制作用,探讨抗肝纤维化的基因治疗新方法。方法：设计合成靶向Smad2基因的siRNA,将筛选成功的siRNA瞬时转染入体外培养的肝星状细胞（HSC）,并给予转化生长因子p（TGF．B）刺激,应用RT—PCR和Westernblot技术检测对照组与实验组I型胶原mRNA水平和蛋白水平表达差异,研究siRNA对I型胶原表达的抑制作用。结果：siRNA能明显降低肝星状细胞中Smad2的RNA和蛋白的表达水平,证实筛选的siRNA有效,能特异性抑制Smad2的基因表达;TGF-β刺激肝星状细胞后,与对照组比较,siRNA转染组细胞外基质（ECM）成分I型胶原的表达水平明显降低（P〈0．05）。结论：siRNA能够抑制TGFβ对肝星状细胞的激活,阻断TGFB—Smads传导通路,使I型胶原分泌下调,有效抑制TGFB诱导的肝纤维化。     

中药乙肝散逆转肝纤维化过程中α-SMA阳性肝星状细胞变化的实验研究

目的探讨乙肝散逆转实验性肝纤维化过程中α-SMA阳性HSC变化.方法雄性Wistar大鼠尾静脉注射白蛋白建立免疫损伤性肝纤维化模型后,分组加用不同浓度的乙肝散颗粒饲料喂养12周,流式细胞仪定量分析大鼠肝组织α-SMA阳性肝星状细胞(HSC),观察大鼠肝组织病理、肝组织Lillie和苦味酸-天狼红染色、Ⅳ胶原免疫组化、α-SMA免疫组化、肝匀浆羟脯氨酸.结果乙肝散应用组肝组织α-SMA阳性HSC较模型对照组明显降低(P<0.01),同时肝组织Ⅰ胶原、Ⅲ胶原、Ⅳ胶原水平和Hyp含量明显降低(P<0.01或P<0.05).结论α-SMA阳性HSC减少是实验性肝纤维化逆转过程中重要的因素,乙肝散促进活化HSC减少.     

肝纤维化的表观遗传学研究进展

肝纤维化是常见的慢性进行性肝病,是慢性肝病发展到肝硬化阶段的必经阶段,却有逆转的可能性。肝纤维化的中心环节是肝星状细胞的激活。关于肝星状细胞的激活,除了经典的肝纤维化通路,不断有新的通路和机制出现,包括自噬、内质网应激、氧化应激、胆固醇代谢和表观遗传,这些都揭示了肝星状细胞的活化机制。表观遗传包括DNA甲基化、组蛋白修饰和调节性非编码RNAs,这些机制也参与调节肝星状细胞活化和肝纤维化发生,对表观遗传和肝纤维化治疗之间的关系研究具有重要意义。     

Silencing p53 inhibits interleukin 10-induced activated hepatic stellate cell senescence and fibrotic degradation in vivo

Activated hepatic stellate cells are reported to play a significant role in liver fibrogenesis. Beside the phenotype reversion and apoptosis of activated hepatic stellate cells, the senescence of activated hepatic stellate cells limits liver fibrosis. Our previous researches have demonstrated that interleukin-10 could promote hepatic stellate cells senescence via p53 signaling pathway in vitro. However, the relationship between expression of p53 and senescence of activated hepatic stellate cells induced by interleukin-10 in fibrotic liver is unclear. The purpose of present study was to explore whether p53 plays a crucial role in the senescence of activated hepatic stellate cells and degradation of collagen mediated by interleukin-10. Hepatic fibrosis animal model was induced by carbon tetrachloride through intraperitoneal injection and transfection of interleukin-10 gene to liver was performed by hydrodynamic-based transfer system. Depletions of p53 in vivo and in vitro were carried out by adenovirus-based short hairpin RNA against p53. Regression of fibrosis was assessed by liver biopsy and collagen staining. Cellular senescence in the liver was observed by senescence-associated beta-galactosidase (SA-β-Gal) staining. Immunohistochemistry, immunofluorescence double staining, and Western blot analysis were used to evaluate the senescent cell and senescence-related protein expression. Our data showed that interleukin-10 gene treatment could lighten hepatic fibrosis induced by carbon tetrachloride and induce the aging of activated hepatic stellate cells accompanied by up-regulating the expression of aging-related proteins. We further demonstrated that depletion of p53 could abrogate up-regulation of interleukin-10 on the expression of senescence-related protein in vivo and vitro. Moreover, p53 knockout in fibrotic mice could block not only the senescence of activated hepatic stellate cells, but also the degradation of fibrosis induced by interleukin-10 gene intervention. Taken together, our results suggested that interleukin-10 gene treatment could attenuate carbon tetrachloride-induced hepatic fibrosis by inducing senescence of activated hepatic stellate cells in vivo, and this induction was closely related to p53 signaling pathway.     

DHFR silence alleviated the development of liver fibrosis by affecting the crosstalk between hepatic stellate cells and macrophages

Liver fibrogenesis is a dynamic cellular and tissue process which has the potential to progress into cirrhosis of even liver cancer and liver failure. The activation of hepatic stellate cells (HSCs) is the central event underlying liver fibrosis. Besides, hepatic macrophages have been proposed as potential targets in combatting fibrosis. As for the relationship between HSCs and hepatic macrophages in liver fibrosis, it is generally considered that macrophages promoted liver fibrosis via activating HSCs. However, whether activated HSCs could in turn affect macrophage polarization has rarely been studied. In this study, mRNAs with significant differences were explored using exosomal RNA-sequencing of activated Lx-2 cells and normal RNA-sequencing of DHFR loss-of-function Lx-2 cell models. Cell functional experiments in both Lx-2 cells and macrophages animal model experiments were performed. The results basically confirmed exosomes secreted from activated HSCs could promote M1 polarization of macrophages further. Exosome harbouring DHFR played an important role in this process. DHFR silence in HSCs could decrease Lx-2 activation and M1 polarization of M0 macrophages and then alleviate the development of liver fibrosis both in vitro and vivo. Our work brought a new insight that exosomal DHFR derived from HSCs had a crucial role in crosstalk between HSCs activation and macrophage polarization, which may be a potential therapeutic target in liver fibrosis.     

Impaired proteolysis of collagen I inhibits proliferation of hepatic stellate cells: implications for regulation of liver fibrosis

Myofibroblastic-activated hepatic stellate cells are the major source of the collagen I-rich extracellular matrix in liver fibrosis but also produce matrix metalloproteinases, which remodel this protein. We have investigated the role of collagen I proteolysis in both regulating proliferation and maintaining the activated myofibroblastic phenotype of stellate cells in vitro. Compared with stellate cells plated on normal collagen I, those plated on a collagenase-resistant form of collagen I (r/r collagen) had reduced thymidine incorporation and proliferating cell nuclear antigen expression but increased p21 expression. Collagen I was shown to be rendered resistant to matrix metalloproteinases by artificial cross-linking in vitro using tissue transglutaminase exerted similar antiproliferative effects on stellate cells to r/r collagen. Of the stellate cell activation markers examined (tissue inhibitor of metalloproteinases-1, alpha-smooth muscle actin, matrix metalloproteinases-2 and -9, and procollagen I) only the last was decreased by culture on r/r collagen relative to normal collagen I. Antagonists of integrin alphavbeta3, an integrin reported to stimulate stellate cell proliferation, significantly inhibited adhesion, proliferation, and procollagen I synthesis of stellate cells plated on normal collagen I but had reduced effectiveness on these parameters in cells on r/r collagen. We conclude that proliferation of stellate cells is promoted by pericellular collagen I proteolysis acting via alphavbeta3 integrin. Cross-linking of collagen I by tissue transglutaminase, a process known to occur in chronic liver fibrosis, might not only increase its resistance to matrix metalloproteinases thereby inhibiting resolution of fibrosis but also functions to constrain the fibroproliferative process.     

Molecular pathogenesis of hepatic fibrosis and current therapeutic approaches

The pathogenesis of hepatic fibrosis involves significant deposition of fibrilar collagen and other extracellular matrix proteins. It is a rather dynamic process of wound healing in response to a variety of persistent liver injury caused by factors such as ethanol intake, viral infection, drugs, toxins, cholestasis, and metabolic disorders. Liver fibrosis distorts the hepatic architecture, decreases the number of endothelial cell fenestrations and causes portal hypertension. Key events are the activation and transformation of quiescent hepatic stellate cells into myofibroblast-like cells with the subsequent up-regulation of proteins such as α-smooth muscle actin, interstitial collagens, matrix metalloproteinases, tissue inhibitor of metalloproteinases, and proteoglycans. Oxidative stress is a major contributing factor to the onset of liver fibrosis and it is typically associated with a decrease in the antioxidant defense. Currently, there is no effective therapy for advanced liver fibrosis. In its early stages, liver fibrosis is reversible upon cessation of the causative agent. In this review, we discuss some aspects on the etiology of liver fibrosis, the cells involved, the molecular pathogenesis, and the current therapeutic approaches.     

Engagement of alphavbeta3 integrin regulates proliferation and apoptosis of hepatic stellate cells

Hepatic stellate cells are the major source of the extracellular matrix that accumulates in fibrotic liver. During progressive liver fibrosis, hepatic stellate cells proliferate, but during resolution of fibrosis there is extensive stellate cell apoptosis that coincides with degradation of the liver scar. We have examined the possibility that the fate of stellate cells is influenced by the extracellular matrix through the intermediary of alpha(v)beta(3) integrin. alpha(v)beta(3) integrin was expressed by activated, myofibroblastic rat and human stellate cells in culture. Antagonism of this integrin using neutralizing antibodies, echistatin, or small inhibitory RNA to silence alpha(v) subunit expression inhibited stellate cell proliferation and their expression of proliferating cell nuclear antigen and activated forms of p44 and p42 MAPK. These alpha(v)beta(3) antagonists also increased apoptosis of cultured stellate cells, and this was associated with an increase in the BAX/BCL-2 protein ratio, induction of nuclear DNA fragmentation, and activation of intracellular caspase-3. Expression of tissue inhibitor of metalloproteinases-1 by activated stellate cells was reduced by the alpha(v)beta(3) antagonists, while matrix metalloproteinase-9 synthesis was enhanced. Stellate cells incubated with active recombinant matrix metalloproteinase-9 showed enhanced apoptosis, while cells treated with a synthetic inhibitor of this protease showed increased survival. Our studies suggest that alpha(v)beta(3) integrin regulates the fate of hepatic stellate cells. Degradation of alpha(v)beta(3) ligands surrounding activated stellate cells during resolution of liver fibrosis might decrease alpha(v)beta(3) integrin ligation, suppressing stellate cell proliferation and inducing a fibrolytic, matrix metalloproteinase-secreting phenotype that may prime stellate cells for apoptosis.     

Selective suppression of M1 macrophages is involved in zinc inhibition of liver fibrosis in mice

Zinc deficiency is common in the liver of patients with chronic liver disease. Zinc supplementation suppresses the progression of liver fibrosis induced by bile duct ligation (BDL) in mice. The present study was undertaken to specifically investigate a possible mechanism by which zinc plays this role in the liver. Kunming mice were subjected to BDL for 4 weeks to induce liver fibrosis, and concomitantly treated with zinc sulfite or saline as control by gavage once a day. The results showed that zinc supplementation significantly suppressed liver fibrosis and inflammation along with inhibition of hepatic stellate cells activation induced by BDL. These inhibitory effects were accompanied by the reduction of collagen deposition and a significant reduction of macrophage infiltration affected livers. Importantly, zinc selectively inhibited M1 macrophage polarization and M1-related inflammatory cytokines. This inhibitory effect was further confirmed by the reduction of relevant biomarkers of M1 macrophages including inducible NO synthase (iNOS), monocyte chemotactic protein-1 (MCP-1/CCL2), and tumor necrosis factor-α in the zinc supplemented BDL livers. In addition, zinc inhibition of M1 macrophages was associated with a decrease of Notch1 expression. Taken together, these data indicated that zinc supplementation inhibited liver inflammation and fibrosis in BDL mice through selective suppression of M1 macrophages, which is associated with inhibition of Notch1 pathway in M1 macrophage polarization.     

Hepatitis C virus RNA replication in human stellate cells regulates gene expression of extracellular matrix-related molecules

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease, including chronic hepatitis, fibrosis, and cirrhosis. Fibrosis often develops in HCV-infected livers and ultimately leads to cirrhosis and carcinoma. During fibrosis, hepatic stellate cells (HSC) play important roles in the control of extracellular matrix synthesis and degradation in fibrotic livers. In this study, we established a subgenomic replicon (SGR) cell line with human hepatic stellate cells to investigate the effect of HCV RNA replication on HSC. Isolated SGR clones contained HCV RNA copy numbers ranging from 10⁴ to 10⁷ per μg total RNA, and long-term culture of low-copy number SGR clones resulted in markedly increased HCV RNA copy numbers. Furthermore, HCV RNA replication affected gene expression of extracellular matrix-related molecules in both hepatic stellate cells and hepatic cells, suggesting that HCV RNA replication and/or HCV proteins directly contribute to liver fibrosis. The HCV RNA-replicating hepatic stellate cell line isolated in this study will be useful for investigating hepatic stellate cell functions and HCV replication machinery.     

Basement membrane-like matrix inhibits proliferation and collagen synthesis by activated rat hepatic stellate cells: evidence for matrix-dependent deactivation of stellate cells.

During liver fibrosis hepatic stellate cells become activated, transforming into proliferative myofibroblastic cells expressing type I collagen and alpha-smooth muscle actin. They become the major producers of the fibrotic neomatrix in injured liver. This study examines if activated stellate cells are a committed phenotype, or whether they can become deactivated by extracellular matrix. Stellate cells isolated from normal rat liver proliferated and expressed mRNA for activation markers, alpha-smooth muscle actin, type I procollagen and tissue inhibitor of metalloproteinases-1 following 5-7 day culture on plastic, but culture on Matrigel suppressed proliferation and mRNA expression. Activated stellate cells were recovered from plastic by trypsinisation and replated onto plastic, type I collagen films or Matrigel. Cells replated on plastic and type I collagen films proliferated and remained morphologically myofibroblastic, expressing alpha-smooth muscle actin and type I procollagen. However, activated cells replated on Matrigel showed <30% of the proliferative rate of these cells, and this was associated with reduced cellular expression of proliferating cell nuclear antigen and phosphorylation of mitogen-activated protein kinase in response to serum. Activated HSC replated on Matrigel for 3-7 days progressively reduced their expression of mRNA for type I procollagen and alpha-smooth muscle actin and both became undetectable after 7 days. We conclude that basement membrane-like matrix induces deactivation of stellate cells. Deactivation represents an important potential mechanism mediating recovery from liver fibrosis in vivo where type I collagen is removed from the liver and stellate cells might re-acquire contact with their normal basement membrane-like pericellular matrix.     

Endoplasmic reticulum stress and autophagy are involved in adipocyte-induced fibrosis in hepatic stellate cells

Liver fibrosis, with the characterization of progressive accumulation of extracellular matrix (ECM), is the common pathologic feature in the process of chronic liver disease. Hepatic stellate cells (HSCs) which are activated and differentiate into proliferative and contractile myofibroblasts are recognized as the main drivers of fibrosis. Obesity-related adipocytokine dysregulation is known to accelerate liver fibrosis progression, but the direct fibrogenic effect of mature adipocytes on HSCs has been rarely reported. Therefore, the purpose of this study was to explore the fibrogenic effect of adipocyte 3T3-L1 cells on hepatic stellate LX-2 cells. The results showed that incubating LX-2 cells with the supernatant of 3T3-L1 adipocytes triggered the expression of ECM related proteins, such as α-smooth muscle actin (α-SMA), type I collagen (CO-I), and activated TGF β/Smad2/3 signaling pathway in LX-2 cells. In addition, 3T3-L1 cells inhibited insulin sensitivity, activated endoplasmic reticulum stress and autophagy to promote the development of fibrosis. These results supported the notion that mature adipocytes can directly activate hepatic stellate cells, and the establishment of an in vitro model of adipocytes on HSCs provides an insight into screening of drugs for liver diseases, such as nonalcoholic fatty liver disease.