Inhibitory effects of saikosaponin-d on CCl4-induced hepatic fibrogenesis in rats

AIM： To investigate the suppressive effect of saikosaponin-d （SSd） on hepatic fibrosis in rats induced by CCh injections in combination with alcohol and high fat, low protein feeding and its relationship with the expression of nuclear factor-κB （NF-κB）, tumor necrosis factor-alpha （TNF-α） and interleukins-6 （IL-6）. METHODS： Hepatic fibrosis models were induced by subcutaneous injection of CCh at a dosage of 3 mL/kg in rats. At the same time, rats in treatment groups were injected intraperitoneally with SSd at different doses （1.0, 1.5 and 2.0 mg/kg） once daily for 6 wk in combination with CCh, while the control group received olive oil instead of CCh. At the end of the experiment, rats were anesthetized and killed （except for 8 rats which died during the experiment; 2 from the model group, 3 in high-dose group, 1 in medium-dose group and 2 in lowdose group）. Hernatoxylin and eosin （HE） staining and Van Gieson staining were used to examine the changes in liver pathology. The levels of alanine aminotransferase （ALT）, triglyeride （TG）, albumin （ALB）, globulin （GLB）, hyaluronic acid （HA） and larninin （LN） in serum and the content of hydroxyproline （HYP） in liver were measured by biochemical examinations and radioimmuneoassay, respectively. In addition, the expression of TNF-α and IL-6 in liver homogenate was evaluated by enzymelinked immunosorbent assay （ELISA） and the levels of NF-κBp65 and I-κBa in liver tissue were analyzed by Western blotting. RESULTS： Both histological examination and Van Gieson staining demonstrated that SSd could attenuate the area and extent of necrosis and reduce the scores of liver fibrosis. Similarly, the levels of ALT, TG, GLB, HA, and LN in serum, and the contents of HYP, TNF-α and IL-6 in liver were all significantly increased in model group in comparison with those in control group. Whereas, the treatment with SScl markedly reduced all the above parameters compared with the model group, especially in the medium gr     

Acetaldehyde increases endogenous adiponectin and fibrogenesis in hepatic stellate cells but exogenous adiponectin inhibits fibrogenesis

Background: Adiponectin has antifibrogenic properties. Acetaldehyde, the principal metabolite of ethanol, is known to stimulate the expression of type I collagen genes and the production of type I collagen by wild‐type (wt) but not by obese gene (ob/ob) stellate cells. The aim of this study was to determine the expression of adiponectin in activated stellate cells obtained from wt and ob/ob mice and to determine the effects of acetaldehyde on adiponectin in relation to the expression of type I collagen. Methods: Stellate cells were isolated from wt and ob/ob mice by perfusion of the portal vein and cultured. Cell adiponectin was visualized by immunohistochemistry and confocal microscopy and determined by radioimmunoassay and by western blot. Adiponectin mRNA and α₁(I) collagen mRNA were determined by quantitative real time polymerase chain reaction. Results: Adiponectin levels were similar in wt and ob/ob stellate cells. Adiponectin receptor 2 mRNA (AdipoR2 mRNA) and AdipoR2 immunoprotein were higher in ob/ob than in wt stellate cells (p < 0.01). Acetaldehyde (200 μM) increased adiponectin both in wt and in ob/ob stellate cells (p < 0.05), but increased AdipoR2 immunoprotein only in ob/ob stellate cells (p < 0.01). However, in the presence of leptin, acetaldehyde decreased adiponectin in ob/ob stellate cells (p < 0.01). Acetaldehyde enhanced α₁(I) collagen mRNA in wt (p < 0.05), but decreased it in ob/ob stellate cells (p < 0.01). Leptin abrogated the effect of acetaldehyde in decreasing α₁(I) collagen mRNA in ob/ob stellate cells (p < 0.01). Adiponectin inhibited α₁(I) collagen mRNA in the basal state in wt stellate cells or when enhanced by acetaldehyde. Conclusions: Adiponectin and adiponectin receptor are present in activated stellate cells. Adiponectin has a negative regulatory role on the enhancing effect of acetaldehyde on fibrogenesis in alcoholic liver disease.     

褪黑素抑制大鼠肝脏星状细胞增殖机制

目的 观察褪黑素抑制大鼠肝脏星状细胞增殖的机制。方法 MTT法测定活细胞数量,流式细胞仪检测凋亡率和细胞周期, western blotting 检测NF-κB表达水平,放射免疫法检测细胞内cAMP浓度,免疫荧光法测定细胞内钙离子浓度。结果 Mel作用后HSCs增殖能力下降,亚G1凋亡峰增高,细胞内钙离子浓度、cAMP浓度和NF-κB表达水平下降。结论 Mel可能下调细胞内钙离子浓度、cAMP浓度及NF-κB表达水平而促进HSCs凋亡和G1期阻滞,从而抑制增殖。     

Inhibitory effects of Ligusticum chuanxiong on the proliferation of rat hepatic stellate cells

BACKGROUND AND AIMS: Platelet-derived growth factor (PDGF) is a very potent mitogen for hepatic stellate cells (HSC) in hepatic fibrogenesis. Ligusticum chuanxiong Hort. (LC), a traditional Chinese herb used for cerebrovascular diseases, has been shown to exert anti-inflammatory and free radical scavenging effects. The aims of the present study were to investigate the effects of LC extract on the proliferation-related biomarkers in a rat HSC cell line (HSC-T6) stimulated with PDGF. METHODS: DNA synthesis via bromodeoxyuridine (BrdU) incorporation, cell cycle related proteins and apoptosis markers were determined to evaluate the inhibitory effects of LC. RESULTS: The results revealed that LC extract (25-100 microg/mL) concentration-dependently decreased the PDGF-induced cell proliferation as well as alpha-smooth muscle actin expression in HSC. The inhibitory activity of LC on HSC was associated with: (i) inhibition of BrdU incorporation; (ii) induction of apoptosis with the activation of caspase-3, up-regulation of cell cycle inhibitory proteins p21 and p27, and down-regulation of cell cycle stimulatory proteins cyclins D1 and D2; and (iii) increased phosphorylation of mitogen-activated protein kinases (JNK). LC at the studied concentrations showed no direct cytotoxicity on primary hepatocytes. CONCLUSION: The results suggest that LC significantly inhibited PDGF-activated HSC proliferation, possibly through apoptotic mechanisms and the potential of LC as an antifibrotic agent warrants further investigation.     

Inhibitory effects of prostaglandin E_1 on activation of hepatic stellate cells in rabbits with schistosomiasis

BACKGROUND: Liver fibrosis is the result of an imbalance between synthesis and degradation of extracellular matrix proteins of the liver. At the cellular and molecular levels, this progressive process is mainly characterized by activation of hepatic stellate cells (HSCs). Schistosoma japonica is one of the most prevalent causes of liver fibrosis in China. It is characterized by hepatocyte damage, inflammation, and chronic parasite egg-induced granuloma formation leading to fibrosis. This study aimed to investigate the inhibitory effects of prostaglandin E1 (PGE1) on activation of HSCs and the alteration of type Ⅰ and Ⅲ collagen in rabbits with schistosomiasis. The study may promote the clinical application of praziquantel and PGE1 as a combined therapy to reverse hepatic fibrosis caused by schistosomiasis. METHODS: Rabbits were percutaneously infected with cercaria of S. japonicum. Seven rabbits were subjected to intravenous injections of PGE1 (2.5 μg/kg daily) from days 60 to 120 after infection. The ultrastructural changes in activated HSCs were observed under transmission electron microscopy. The expression of α-smooth muscle actin (α-SMA) was detected by immunohistochemistry. Fibril-forming collagens were detected by picrosirius staining. RESULTS: Activation of HSCs was a characteristic alteration in schistosome-induced hepatic fibrosis. The expression of contraction-related α-SMA and thecontent of collagens were increased. Exogenous PGE1 markedly inhibited the activation of HSCs and reduced the expression of α-SMA around the hepatic sinusoids (P<0.01). The contents of type Ⅰ and Ⅲ collagens were significantly attenuated. The ratio of staining area to the whole field (10×3.3) under a polarized light microscope in the untreated and treated groups was 37.25±9.71 vs. 13.38±4.24 (P<0.01) and 9.66±3.52 vs. 6.23±1.81 (P<0.05), respectively. CONCLUSIONS: Activation of HSCs may play a key role in the progress of schistosome-induced hepatic fibrosis. PGE1 effectively protects rabbit liver from fibrosis, at least in part by inhibiting the activation of HSCs.     

Suppression of macrophage infiltration inhibits activation of hepatic stellate cells and liver fibrogenesis in rats

BACKGROUND & AIMS: Monocytes/macrophages infiltrate into injured livers. We tried to clarify their roles in inflammation and subsequent fibrogenesis by inhibiting their infiltration with a mutated form (7ND; 7 amino acids at the N-terminal were deleted) of monocyte chemoattractant protein 1, which may function as a dominant-negative mutant. METHODS: Rats were injected via the tail vein with an adenovirus expressing either human 7ND (Ad7ND), a truncated type II transforming growth factor beta receptor (AdTbeta-TR), which works as a dominant-negative receptor, bacterial beta-galactosidase (AdLacZ), or saline. Seven days later, the rats were treated with dimethylnitrosamine for 1-21 days. RESULTS: Within 24 hours after a single dimethylnitrosamine injection, macrophages were observed in livers. With a 3-day dimethylnitrosamine treatment, activated hepatic stellate cells were detectable in livers in AdLacZ-, AdTbeta-TR-, and saline-injected rats. In contrast, in the Ad7ND-treated rats, infiltration of macrophages was markedly reduced, and activated hepatic stellate cells were not detectable. After a 3-week dimethylnitrosamine treatment, fibrogenesis was almost completely inhibited, and activated hepatic stellate cells were hardly seen in livers in both Ad7ND- and AdTbeta-TR-treated rats. CONCLUSIONS: Our results show that blockade of macrophage infiltration inhibits activation of hepatic stellate cells and leads to suppression of liver fibrogenesis. The presence of activated hepatic stellate cells in the initial phase after injury and its absence at a later phase in the AdTbeta-TR-treated livers indicate that transforming growth factor beta is not an activating factor for hepatic stellate cells, and this suggests that transforming growth factor beta is required for the survival of activated hepatic stellate cells. Our study suggests that infiltrated macrophages may themselves produce an activating factor for hepatic stellate cells.     

黄芩甙抗肝星状细胞增殖及胶原合成的作用

目的研究黄芩甙对大鼠肝星状细胞活化、增殖及细胞外基质合成的作用,探讨黄芩甙抗肝纤维化的机制。方法采用胶原酶循环灌流法和密度梯度离心法分离肝星状细胞。不同浓度黄芩甙作用后,通过观察细胞贴壁及细胞形态变化情况来检测细胞活化：采用MTT法检测细胞增殖;免疫细胞化学法检测Ⅰ、Ⅲ型胶原及层粘连蛋白的合成。结果黄芩甙（75～1200μg/mL）可抑制HSC活化,MTT法示75μg／mL、150μg／mL、300μmL、600μg／mL、1200μg／mL黄芩甙作用于HSC的A值分别为（0．060±6．53）×10^-2、（0．052±7．38）×10^9、（0．036±1．26）×10^-3、（0．023±1．72）×10^-3、（0．013±4．01）×10^-3,空白对照组A值为（0．065±1．32）×10^4,F值=1147．611,P〈0．05。黄芩甙可抑制活化的HSC增殖,减少HSCⅠ、Ⅲ型胶原及层粘连蛋白合成。结论黄芩甙抗肝纤维化作用主要机制可能为黄芩甙抑制肝星状细胞活化、增殖,抑制细胞胶原蛋白及糖蛋“等细胞外基质合成.     

Rosmarinic acid attenuates hepatic fibrogenesis via suppression of hepatic stellate cell activation/proliferation and induction of apoptosis

Objective:To investigate the antifibrotic role of rosmarinic acid(RA),a natural polyphenolic compound,on HSCs activation/proliferation and apoptosis in vitro and in vivo. Methods:The impact of RA on stellate cell line(HSC-T6) proliferation,activation and apoptosis was assessed along with its safety on primary hepatocytes. In vivo,rats were divided into:(i) normal;(ii) thioacetamide(TAA)-intoxicated rats for 12 weeks;(iii) TAA+silymarin or(iv) TAA+RA. At the end of experiment,liver functions,oxidative stress,inflammatory and profibrogenic markers,tissue inhibitor metalloproteinases type-1(TIMP-1) and hydroxyproline(HP) levels were evaluated. Additionally,liver histopathology and immunohistochemical examinations of alpha-smooth muscle actin(α-SMA),caspase-3 and proliferation cellular nuclear antigen(PCNA) were determined. Results:RA exhibited anti-proliferative effects on cultured HSCs in a time and concentration dependent manner showing an IC50 of 276 μg/mL and 171 μg/mL for 24 h and 48 h,respectively,with morphological reversion of activated stellate cell morphology to quiescent form. It significantly improved ALT,AST,oxidative stress markers and reduced TIMP-1,HP levels,inflammatory markers and fibrosis score(S1 vs S4). Furthermore,reduction in α-SMA plus elevation in caspase-3 expressions of HSCs in vitro and in vivo associated with an inhibition in proliferation of damaged hepatocytes were recorded. Conclusions:RA impeded the progression of liver fibrosis through inhibition of HSCs activation/proliferation and induction of apoptosis with preservation of hepatic architecture.     

Activation of hepatic stellate cells after phagocytosis of lymphocytes: A novel pathway of fibrogenesis

Increased CD8-T lymphocytes and reduced natural killer (NK) cells contribute to hepatic fibrosis. We have characterized pathways regulating the interactions of human hepatic stellate cells (HSCs) with specific lymphocyte subsets in vivo and in vitro. Fluorescence-activated cell sorting (FACS) was used to characterize human peripheral blood lymphocytes (PBLs) and intrahepatic lymphocytes (IHLs) obtained from healthy controls and from patients with either hepatitis B virus (HBV) or hepatitis C virus (HCV) with advanced fibrosis. Liver sections were analyzed by immunohistochemistry and confocal microscopy. To investigate in vitro interactions, PBLs from healthy controls or patients with HCV cirrhosis were co-cultured with an immortalized human HSC line (LX2 cells) or with primary HSCs. Significant alterations in lymphocyte distribution were identified in IHLs but not PBLs. The hepatic CD4/CD8 ratio and NK cells were significantly reduced in HBV/HCV patients. Expression of alpha-smooth muscle actin and infiltration of CD4, CD8, and NK cells were readily apparent in liver sections from patients with cirrhosis but not in healthy controls. Lymphocytes from each subset were in proximity to HSCs primarily within the periportal regions, and some were directly attached or engulfed. In culture, HSC activation was stimulated by HCV-derived CD8-subsets but attenuated by NK cells. Confocal microscopy identified lymphocyte phagocytosis within HSCs that was completely prevented by blocking intracellular adhesion molecule 1 (ICAM-1) and integrin molecules, or by irradiation of HSCs. LX2 knockdown of either Cdc42 or Rac1 [members of the Rho-guanosine triphosphatase (GTPase) family] prevented both phagocytosis and the activation of HSC by HCV-derived lymphocytes. CONCLUSION: The CD4/CD8 ratio and NK cells are significantly decreased in livers with advanced human fibrosis. Moreover, disease-associated but not healthy lymphocytes are engulfed by cultured HSCs, which is mediated by the Rac1 and Cdc42 pathways. Ingestion of lymphocytes by HSCs in hepatic fibrosis is a novel and potentially important pathway regulating the impact of lymphocytes on the course of hepatic fibrosis.     

Lipid peroxidation, stellate cell activation and hepatic fibrogenesis in a rat model of chronic steatohepatitis

BACKGROUND/AIMS: We explored the involvement of cell types, cytokines and lipid peroxidation in a rat dietary model of fibrosing steatohepatitis. METHODS: Male rats were fed a high fat diet deficient in methionine and choline (MCD) for up to 17 weeks. Whole liver, hepatocytes and non-parenchymal cells were analysed for reduced glutathione (GSH) levels, products of lipid peroxidation (thiobarbituric acid reactive substances, TBARS), liver injury, and fibrosis. RESULTS: MCD diet-fed rats developed hepatic steatosis at week 2 and focal necroinflammatory change by week 5, while pericellular fibrosis evolved and progressed between weeks 12 and 17. Collagen alpha(1)(1) gene expression was upregulated by week 5 and increased fivefold by week 17. Stellate cells were the unique source of collagen gene expression. TIMP-1 and -2 were increased at week 12. Livers of MCD diet-fed rats exhibited lowered levels of GSH and elevated TBARS. Hepatocytes were the source of lipid peroxidation, and mRNA levels for TGFbeta1 were increased only in this cell type. CONCLUSIONS: The MCD model of 'fibrosing steatohepatitis' replicates the histologic features of human steatohepatitis, and the sequence of steatosis, inflammatory cell injury and fibrogenesis. The temporal sequence is consistent with a concept for involvement of oxidative injury in inflammatory recruitment and pathogenesis of hepatic fibrogenesis.     

Mechanisms of hepatic fibrogenesis

Multiple etiologies of liver disease lead to liver fibrosis through integrated signaling networks that regulate the deposition of extracellular matrix. This cascade of responses drives the activation of hepatic stellate cells (HSCs) into a myofibroblast-like phenotype that is contractile, proliferative and fibrogenic. Collagen and other extracellular matrix (ECM) components are deposited as the liver generates a wound-healing response to encapsulate injury. Sustained fibrogenesis leads to cirrhosis, characterized by a distortion of the liver parenchyma and vascular architecture. Uncovering the intricate mechanisms that underlie liver fibrogenesis forms the basis for efforts to develop targeted therapies to reverse the fibrotic response and improve the outcomes of patients with chronic liver disease.     

细胞外信号调节激酶1与肝星状细胞增殖关系的体内实验研究

目的　探讨在纤维化发生中 ,细胞外信号调节激酶1(ERK1)与肝星状细胞 (HSCs)增殖的关系。方法　采用胆总管结扎 (BDL)方法建立大鼠肝纤维化模型 ,应用免疫组织化学及逆转录聚合酶链式反应 (RT PCR)技术研究ERK1及其mRNA在肝纤维化不同时期肝组织中的分布及含量的动态变化 ;采用免疫组织化学方法测定α SMA。结果　正常肝组织有少量α SMA、ERK1分布 ,随着肝纤维化发展 ,α SMA、ERK1阳性细胞明显增多 ;正常大鼠肝组织中有ERK1mRNA表达 ,分别于造模 2d开始上调 ,造模 4w表达最多。ERK1与α SMA呈显著正相关 (r =0 958,P <0 0 5)。结论　肝纤维化形成过程中ERK1及其mRNA表达明显增加 ,ERK1在HSCs增殖及肝纤维化形成过程中发挥重要作用     

Cyclin E1 controls proliferation of hepatic stellate cells and is essential for liver fibrogenesis in mice

Liver fibrogenesis is associated with the transition of quiescent hepatocytes and hepatic stellate cells (HSCs) into the cell cycle. Exit from quiescence is controlled by E-type cyclins (cyclin E1 [CcnE1] and cyclin E2 [CcnE2]). Thus, the aim of the current study was to investigate the contribution of E-type cyclins for liver fibrosis in man and mice. Expression of CcnE1, but not of its homolog, CcnE2, was induced in fibrotic and cirrhotic livers from human patients with different etiologies and in murine wild-type (WT) livers after periodical administration of the profibrotic toxin, CCl(4) . To further evaluate the potential function of E-type cyclins for liver fibrogenesis, we repetitively treated constitutive CcnE1(-/-) and CcnE2(-/-) knock-out mice with CCl(4) to induce liver fibrosis. Interestingly, CcnE1(-/-) mice were protected against CCl(4) -mediated liver fibrogenesis, as evidenced by reduced collagen type I α1 expression and the lack of septum formation. In contrast, CcnE2(-/-) mice showed accelerated fibrogenesis after CCl(4) treatment. We isolated primary HSCs from WT, CcnE1(-/-) , and CcnE2(-/-) mice and analyzed their activation, proliferation, and survival in vitro. CcnE1 expression in WT HSCs was maximal when they started to proliferate, but decreased after the cells transdifferentiated into myofibroblasts. CcnE1(-/-) HSCs showed dramatically impaired survival, cell-cycle arrest, and strongly reduced expression of alpha smooth muscle actin, indicating deficient HSC activation. In contrast, CcnE2-deficient HSCs expressed an elevated level of CcnE1 and showed enhanced cell-cycle activity and proliferation, compared to WT cells. Conclusions: CcnE1 and CcnE2 have antagonistic roles in liver fibrosis. CcnE1 is indispensable for the activation, proliferation, and survival of HSCs and thus promotes the synthesis of extracellular matrix and liver fibrogenesis. (HEPATOLOGY 2012;56:1140-1149).