Taurine attenuates activation of hepatic stellate cells by inhibiting autophagy and inducing ferroptosis

BACKGROUND Liver fibrosis is a compensatory response during the tissue repair process in chronic liver injury,and finally leads to liver cirrhosis or even hepatocellular carcinoma.The pathogenesis of hepatic fibrosis is associated with the progressive accumulation of activated hepatic stellate cells(HSCs),which can transdiffer-entiate into myofibroblasts to produce an excess of the extracellular matrix(ECM).Myofibroblasts are the main source of the excessive ECM responsible for hepatic fibrosis.Therefore,activated hepatic stellate cells(aHSCs),the principal ECM producing cells in the injured liver,are a promising therapeutic target for the treatment of hepatic fibrosis.AIM To explore the effect of taurine on aHSC proliferation and the mechanisms involved.METHODS Human HSCs(LX-2)were randomly divided into five groups:Normal control group,platelet-derived growth factor-BB(PDGF-BB)(20 ng/mL)treated group,mmol/L,respectively)with PDGF-BB(20 ng/mL)treated group.Cell Counting Kit-8 method was performed to evaluate the effect of taurine on the viability of aHSCs.Enzyme-linked immunosorbent assay was used to estimate the effect of taurine on the levels of reactive oxygen species(ROS),malondialdehyde,glutathione,and iron concen-tration.Transmission electron microscopy was applied to observe the effect of taurine on the autophagosomes and ferroptosis features in aHSCs.Quantitative real-time polymerase chain reaction and Western blot analysis were performed to detect the effect of taurine on the expression ofα-SMA,Collagen I,Fibronectin 1,LC3B,ATG5,Beclin 1,PTGS2,SLC7A11,and p62.RESULTS Taurine promoted the death of aHSCs and reduced the deposition of the ECM.Treatment with taurine could alleviate autophagy in HSCs to inhibit their activation,by decreasing autophagosome formation,downregulating LC3B and Beclin 1 protein expression,and upregulating p62 protein expression.Meanwhile,treatment with taurine triggered ferroptosis and ferritinophagy to eliminate aHSCs characterized by iron overload,lipid ROS accumu-lation,glutathione depletion,and lipid peroxidation.Furthermore,bioinformatics analysis demonstrated that taurine had a direct targeting effect on nuclear receptor coactivator 4,exhibiting the best average binding affinity of-20.99 kcal/mol.CONCLUSION Taurine exerts therapeutic effects on liver fibrosis via mechanisms that involve inhibition of autophagy and trigger of ferroptosis and ferritinophagy in HSCs to eliminate aHSCs.

HepG2.2.15-derived exosomes facilitate the activation and fibrosis of hepatic stellate cells

BACKGROUND The role of exosomes derived from HepG2.2.15 cells,which express hepatitis B virus(HBV)-related proteins,in triggering the activation of LX2 liver stellate cells and promoting liver fibrosis and cell proliferation remains elusive.The focus was on comprehending the relationship and influence of differentially expressed microRNAs(DE-miRNAs)within these exosomes.AIM To elucidate the effect of exosomes derived from HepG2.2.15 cells on the activation of hepatic stellate cell(HSC)LX2 and the progression of liver fibrosis.METHODS Exosomes from HepG2.2.15 cells,which express HBV-related proteins,were isolated from parental HepG2 and WRL68 cells.Western blotting was used to confirm the presence of the exosomal marker protein CD9.The activation of HSCs was assessed using oil red staining,whereas DiI staining facilitated the observation of exosomal uptake by LX2 cells.Additionally,we evaluated LX2 cell proliferation and fibrosis marker expression using 5-ethynyl-2′-deoxyuracil staining and western blotting,respectively.DE-miRNAs were analyzed using DESeq2.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathways were used to annotate the target genes of DE-miRNAs.RESULTS Exosomes from HepG2.2.15 cells were found to induced activation and enhanced proliferation and fibrosis in LX2 cells.A total of 27 miRNAs were differentially expressed in exosomes from HepG2.2.15 cells.GO analysis indicated that these DE-miRNA target genes were associated with cell differentiation,intracellular signal transduction,negative regulation of apoptosis,extracellular exosomes,and RNA binding.KEGG pathway analysis highlighted ubiquitin-mediated proteolysis,the MAPK signaling pathway,viral carcinogenesis,and the toll-like receptor signaling pathway,among others,as enriched in these targets.CONCLUSION These findings suggest that exosomes from HepG2.2.15 cells play a substantial role in the activation,proliferation,and fibrosis of LX2 cells and that DE-miRNAs within these exosomes contribute to the underlying mechanisms.

Linking fatty liver diseases to hepatocellular carcinoma by hepatic stellate cells

Hepatic stellate cells(HSCs),a distinct category of non-parenchymal cells in the liver,are critical for liver homeostasis.In healthy livers,HSCs remain non-proliferative and quiescent.However,under conditions of acute or chronic liver damage,HSCs are activated and participate in the progression and regulation of liver diseases such as liver fibrosis,cirrhosis,and liver cancer.Fatty liver diseases(FLD),including nonalcoholic(NAFLD)and alcoholrelated(ALD),are common chronic inflammatory conditions of the liver.These diseases,often resulting from multiple metabolic disorders,can progress through a sequence of inflammation,fibrosis,and ultimately,cancer.In this review,we focused on the activation and regulatory mechanism of HSCs in the context of FLD.We summarized the molecular pathways of activated HSCs(aHSCs)in mediating FLD and their role in promoting liver tumor development from the perspectives of cell proliferation,invasion,metastasis,angiogenesis,immunosuppression,and chemo-resistance.We aimed to offer an in-depth discussion on the reciprocal regulatory interactions between FLD and HSC activation,providing new insights for researchers in this field.

Ginsenoside Rb1 induces hepatic stellate cell ferroptosis to alleviate liver fibrosis via the BECN1/SLC7A11 axis

Liver fibrosis is primarily driven by the activation of hepatic stellate cells(HSCs),a process associated with ferroptosis.Ginsenoside Rb1(GRb1),a major active component extracted from Panax ginseng,inhibits HSC activation.However,the potential role of GRb1 in mediating HSC ferroptosis remains unclear.This study examined the effect of GRb1 on liver fibrosis both in vivo and in vitro,using CCl4-induced liver fibrosis mouse model and primary HSCs,LX-2 cells.The findings revealed that GRb1 effectively inactivated HSCs in vitro,reducing alpha-smooth muscle actin(a-SMA)and type I collagen(Col1A1)levels.Moreover,GRb1 significantly alleviated CCl4-induced liver fibrosis in vivo.From a mechanistic standpoint,the ferroptosis pathway appeared to be central to the antifibrotic effects of GRb1.Specifically,GRb1 promoted HSC ferroptosis both in vivo and in vitro,characterized by increased glutathione depletion,malondialdehyde production,iron overload,and accumulation of reactive oxygen species(ROS).Intriguingly,GRb1 increased Beclin 1(BECN1)levels and decreased the System Xc-key subunit SLC7A11.Further experiments showed that BECN1 silencing inhibited GRb1-induced effects on HSC ferroptosis and mitigated the reduction of SLC7A11 caused by GRb1.Moreover,BECN1 could directly interact with SLC7A11,initiating HSC ferroptosis.In conclusion,the suppression of BECN1 counteracted the effects of GRb1 on HSC inactivation both in vivo and in vitro.Overall,this study highlights the novel role of GRb1 in inducing HSC ferroptosis and promoting HSC inactivation,at least partly through its modulation of BECN1 and SLC7A11.

Perilipin 5 regulates hepatic stellate cell activation and high-fat diet-induced non-alcoholic fatty liver disease

Background:Nonalcoholic fatty liver disease(NAFLD)is one of the most common chronic liver diseases globally.Hepatic stellate cells(HSCs)are the major effector cells of liver fibrosis.HSCs contain abundant lipid droplets(LDs)in their cytoplasm during quiescence.Perilipin 5(PLIN 5)is a LD surface-associated protein that plays a crucial role in lipid homeostasis.However,little is known about the role of PLIN 5 in HSC activation.Methods:PLIN 5 was overexpressed in HSCs of Sprague–Dawley rats by lentivirus transfection.At the same time,PLIN 5 gene knockout mice were constructed and fed with a high-fat diet(HFD)for 20 weeks to study the role of PLIN 5 in NAFLD.The corresponding reagent kits were used to measure TG,GSH,Caspase 3 activity,ATP level,and mitochondrial DNA copy number.Metabolomic analysis of mice liver tissue metabolism was performed based on UPLC-MS/MS.AMPK,mitochondrial function,cell proliferation,and apoptosis-related genes and proteins were detected by western blotting and qPCR.Results:Overexpression of PLIN 5 in activated HSCs led to a decrease in ATP levels in mitochondria,inhibition of cell proliferation,and a significant increase in cell apoptosis through AMPK activation.In addition,compared with the HFD-fed C57BL/6J mice,PLIN 5 knockout mice fed with HFD showed reduced liver fat deposition,decreased LD abundance and size,and reduced liver fibrosis.Conclusion:These findings highlight the unique regulatory role of PLIN 5 in HSCs and the role of PLIN 5 in the fibrosis process of NAFLD.

KLF4 Inhibits the Activation of Human Hepatic Stellate Cell In Vitro

Objective Hepatic stellate cells(HSCs)play a crucial role in liver fibrosis.Early-stage liver fibrosis is reversible and intimately associated with the state of HSCs.Kruppel-like factor 4(KLF4)plays a pivotal role in a wide array of physiological and pathological processes.This study aimed to investigate the effect of KLF4 on the proliferation,apoptosis and phenotype of quiescent HSCs Methods We designed a KLF4 lentiviral vector and a KLF4 siRNA lentiviral vector,to upregulate and silence KLF4 expression in human HSC LX-2 cells via transfection.Cell proliferation was assessed using the CCK-8 assay.Flow cytometry was used to detect the cell cycle distribution and apoptosis rate.Western blotting was used to determine the levels of some quiescence and activation markers of HSCs Results Overexpression of KLF4 significantly increased the levels of E-cadherin and ZO-1,which are quiescent HSC markers,while significantly decreased the levels of N-cadherin and a-SMA,known activated HSC markers.In contrast,cell proliferation and apoptosis rates were elevated in LX-2 cells in which KLF4 expression was silenced Conclusion KLF4 inhibits the proliferation and activation of human LX-2 HSCs.It might be a key regulatory protein in the maintenance of HSC quiescence and may serve as a target for the inhibition of hepatic fibrosis.

Liver fibrosis and hepatic stellate cells: Etiology, pathological hallmarks and therapeutic targets

Liver fibrosis is a reversible wound-healing process aimed at maintaining organ integrity, and presents as the critical pre-stage of liver cirrhosis, which will eventually progress to hepatocellular carcinoma in the absence of liver transplantation. Fibrosis generally results from chronic hepatic injury caused by various factors, mainly viral infection, schistosomiasis, and alcoholism; however, the exact pathological mechanisms are still unknown. Although numerous drugs have been shown to have antifibrotic activity in vitro and in animal models, none of these drugs have been shown to be efficacious in the clinic. Importantly, hepatic stellate cells(HSCs) play a key role in the initiation, progression, and regression of liver fibrosis by secreting fibrogenic factors that encourage portal fibrocytes, fibroblasts, and bone marrow-derived myofibroblasts to produce collagen and thereby propagate fibrosis. These cells are subject to intricate cross-talk with adjacent cells, resulting in scarring and subsequent liver damage. Thus, an understanding of the molecular mechanisms of liver fibrosis and their relationships with HSCs is essential for the discovery of new therapeutic targets. This comprehensive review outlines the role of HSCs in liver fibrosis and details novel strategies to suppress HSC activity, thereby providing new insights into potential treatments for liver fibrosis.

Hes1,an important gene for activation of hepatic stellate cells,is regulated by Notch1 and TGF-β/BMP signaling

AIM:To determine the role of Notch1 and Hes1 in regulating the activation of hepatic stellate cells(HSCs) and whether Hes1 is regulated by transforming growth factor(TGF)/bone morphogenetic protein(BMP) signaling.METHODS:Immunofluorescence staining was used to detect the expression of desmin,glial fibrillary acidic protein and the myofibroblastic marker α-smooth muscle actin(α-SMA) after freshly isolated,normal rat HSCs had been activated in culture for different numbers of days(0,1,3,7 and 10 d).The expression of α-SMA,collagen1α2(COL1α2),Notch receptors(Notch1-4),and the Notch target genes Hes1 and Hey1 were analyzed by reverse transcriptase-polymerase chain reaction.Luciferase reporter assays and Western blot were used to study the regulation of α-SMA,COL1α1,COL1α2 and Hes1 by NICD1,Hes1,CA-ALK3,and CA-ALK5 in HSC-T6 cells.Moreover,the effects of inhibiting Hes1 function in HSC-T6 cells using a Hes1 decoy were also investigated.RESULTS:The expression of Notch1 and Hes1 m RNAs was significantly down-regulated during the culture of freshly isolated HSCs.In HSC-T6 cells,Notch1 inhibited the promoter activities of α-SMA,COL1α1 and COL1α2.On the other hand,Hes1 enhanced the promoter activities of α-SMA and COL1α2,and this effect could be blocked by inhibiting Hes1 function with a Hes1 decoy.Furthermore,co-transfection of pc DNA3-CAALK3(BMP signaling activin receptor-like kinase 3) and pc DNA3.1-NICD1 further increased the expression of Hes1 compared with transfection of either vector alone in HSC-T6 cells,while pc DNA3-CA-ALK5(TGF-β signaling activin receptor-like kinase 5) reduced the effect of NICD1 on Hes1 expression.CONCLUSION:Selective interruption of Hes1 or maintenance of Hes1 at a reasonable level decreases the promoter activities of α-SMA and COL1α2,and these conditions may provide an anti-fibrotic strategy against hepatic fibrosis.

Effects of retinoic acid on proliferation,phenotype and expression of cyclin-dependent kinase inhibitors in TGF-β1-stimulated rat hepatic stellate cells

AIM To study the molecular mechanisms ofretinoic acid(RA)on proliferation andexpression of cyclin-dependent kinase inhibitors(CKI),i.e.p16,p21 and p27 in cultured rathepatic stellate cells(HSC)stimulated withtransforming growth factor beta 1(TGF-β1).METHODS HSC were isolated from healthy ratlivers and cultured.After stimulated with1 mg/L TGF-β1,subcultured HSC were treatedwith or without 1 nmol/L RA.MTT assay,immunocytochemistry(ICC)for p16,p21,p27and α-smooth muscle actin(α-SMA)protein,insitu hybridization(ISH)for retinoic acidreceptor beta 2(RAR-β2)and p16,p21 and p27mRNA and quantitative image analysis(partially)were performed.RESULTS RA inhibited HSC proliferation(41.50%,P【0.05),decreased the protein levelof α-SMA(55.09%,P【0.05),and induced HSCto express RAR-β2 mRNA.In addition,RAincreased the protein level of p16(218.75%,P【0.05)and induced p21 protein expression;meanwhile,p27 was undetectable by ICC in bothcontrol and RA-treated HSC.However,RA hadno influence on the mRNA levels of p16,p21 orp27 as determined by ISH.CONCLISION Up-regulation of p16 and p21 on post-transcriptional level may contribule, in part to RA inhibition of TGF-β1-initiated rat HSC activation in vitro.

Effect of lipid on proliferation and activation of rat hepatic stellate cells(Ⅰ)

AIM To study the effect of lipid (triglyceride and very low density lipoprotein, VLDL) on proliferation and activation of rat hepatic stellate cells (HSC). METHODS HSC were isolated and cultured from liver of Wistar rats by in situ perfusion with pronase and collagenase and density gradient centrifugation with Nycodenz. HSC proliferation was examined with MTT colorimetric assay. RESULT Triglyceride of 12.5mg/L had a promoting effect on proliferation of HSC ( P <0 05), 25, 50, 100 and 200mg/L had no effects ( P >0 05), but 400mg/L had an inhibiting effect ( P <0 01). VLDL of 6 25 and 12 5mg/L had no effect on proliferation of HSC ( P >0 05), but increased concentration of VLDL could promote the HSC proliferation ( P <0 05). CONCLUSION Lipid had an effect on proliferation of HSC. Triglyceride and VLDL may promote HSC proliferation and may be associated with fatty liver and hepatic fibrogenesis.

Natural taurine promotes apoptosis of human hepatic stellate cells in proteomics analysis

AIM:To study the differential expression of proteins between natural taurine treated hepatic stellate cells and controls, and investigate the underlying regulatory mechanism of natural taurine in inhibiting hepatic fibrosis.METHODS: A proteomic strategy combining two-dimensional gel electrophoresis and ultraperform ance liquid chromatographyelectrospray ionizationtandem mass spectrometry (UPLCESIMS/MS) was used to study the differential expression of proteins and Western blotting was used to validate the results. Gene ontology (GO) method was utilized to analyze the functional enrichment of differentially expressed proteins. Flow cytometry was performed to compare the apoptosis rate between taurinetreated and untreated hepatic stellate cells (HSCs).RESULTS: Nineteen differentially expressed proteins (11 upregulated and 8 downregulated) were identifiedby 2D/MS, and the expression profiles of GLO1 and ANXA1 were validated by Western blotting. GO analysis found that these differentially expressed proteins were enriched within biological processes such as "cellular apoptosis", "oxidation reaction" and "metabolic process" in clusters. Flow cytometric analysis showed that taurinetreated HSCs had a significantly increased apoptosis rate when compared with the control group.CONCLUSION: Natural taurine can promote HSC apoptosis so as to inhibit hepatic fibrosis.

Effects of Fuzhenghuayu decoction on collagen synthesis of cultured hepatic stellate cells,hepatocytes and fibroblasts in rats

AIM To study the mechanism of Fuzhenghuayu (FZHY) decoction on anti liver fibrosis. METHODS FZHY 10% decoction sera was incubated with rat normal subcultured hepatic stellate cells (HSC) and fibrotic primarily cultured HSC, normal and fibrotic hepatocytes and subcultured skin fibroblasts separately. Cell intracellular and extracellular collagen synthesis rates were measured by the method of Proline impulse and collagenase digestion. RESULTS For primarily cultured HSC and hepatocytes, both of intracellular and extracellular collagen synthesis rates decreased in the drug sera group. For the normal subcultured HSC and primarily cultured hepatocytes, the extracellular collagen secretion was decreased obviously by the drug sera, and intracellular collagen synthesis rates were inhibited to some extents. For fibroblasts, both intracellular and extracellular collagen synthesis rates were inhibited some what, but no significant differences were found. CONCLUSION The mechanism of FZHY decoction on anti liver fibrosis may be associated with inhibition of liver collagen production.

Effect of Fuzhenghuayu decoction on vascular endothelial growth factor secretion in hepatic stellate cells

Objective: To investigate the effect of Fuzhenghuayu decoction on autocrine activation of hepatic stellate cell (HSC). Methods: The drug serum containing Fuzhenghuayu decoction was collected from normal rats, and cul- tured with activated HSC in vitro. The conditioned medium from the drug serum treated HSC was added to primary cultured quiescent HSC. Cell prolifera- tion was assayed by tetrazolium colorimetric test, and the contents of type Ⅰ collagen and vascular endo- thelial growth factor (VEGF) in the supernatant were measured with ELISA. Results: The conditioned medium from activated HSC could stimulate the quiescent HSC proliferation and type Ⅰ collagen secretion. The drug serum inhibi- ted this stimulating action and VEGF secretion from the activated HSC. Conclusion: Fuzhenghuayu decoction acts effectively against the autocrine activation pathway of HSC. The mechanism may be associated with the inhibition of the secretion of VEGF by activated HSC.

Metformin attenuates motility,contraction,and fibrogenic response of hepatic stellate cells in vivo and in vitro by activating AMP-activated protein kinase

AIM To investigate the effect of metformin on activated hepatic stellate cells(HSCs) and the possible signaling pathways involved. METHODS A fibrotic mouse model was generated by intraperitoneal injection of carbon tetrachloride(CCl_4) and subsequent treatment with or without metformin. The level of fibrosis was detected by hematoxylin-eosin staining, Sirius Red staining, and immunohistochemistry. The HSC cell line LX-2 was used for in vitro studies. The effect of metformin on cell proliferation(CCK8 assay),motility(scratch test and Transwell assay), contraction(collagen gel contraction assay), extracellular matrix(ECM) secretion(Western blot), and angiogenesis(ELISA and tube formation assay) was investigated. We also analyzed the possible signaling pathways involved by Western blot analysis.RESULTS Mice developed marked liver fibrosis after intraperitoneal injection with CCl_4 for 6 wk. Metformin decreased the activation of HSCs, reduced the deposition of ECM, and inhibited angiogenesis in CCl_4-treated mice. Platelet-derived growth factor(PDGF) promoted the fibrogenic response of HSCs in vitro, while metformin inhibited the activation, proliferation, migration, and contraction of HSCs, and reduced the secretion of ECM. Metformin decreased the expression of vascular endothelial growth factor(VEGF) in HSCs through inhibition of hypoxia inducible factor(HIF)-1α in both PDGF-BB treatment and hypoxic conditions, and it down-regulated VEGF secretion by HSCs and inhibited HSC-based angiogenesis in hypoxic conditions in vitro. The inhibitory effects of metformin on activated HSCs were mediated by inhibiting the Akt/mammalian target of rapamycin(m TOR) and extracellular signal-regulated kinase(ERK) pathways via the activation of adenosine monophosphate-activated protein kinase(AMPK).CONCLUSION Metformin attenuates the fibrogenic response of HSCs in vivo and in vitro, and may therefore be useful for the treatment of chronic liver diseases.

Anti-proliferative and pro-apoptotic effects of tectorigenin on hepatic stellate cells

AIM: To investigate the effect of tectorigenin on proliferation and apoptosis of hepatic stellate cells (HSC)-T6 cells. METHODS: HSC-T6 cells were incubated with tectorigenin at different concentrations, and their proliferation was assessed by bromodeoxyuridine incorporation assay. Apoptosis was detected by flow cytometry assay with Hoechst 33342 staining. Also, generation of reactive oxygen species (ROS), intracellular [Ca2+]i, potential of mitochondrial membrane, activities of cytochrome c and caspase-9 and-3 were investigated to explore a conceivable apoptotic pathway. RESULTS: Tectorigenin suppressed the proliferation of HSC-T6 cells and induced apoptosis of HSC-T6 cells in a time-and dose-dependent manner. Tectorigenin at the concentration of 100 μg/mL greatly inhibited the viability of HSC-T6 cells and induced the condensation of chromatin and fragmentation of nuclei. When treated for 48 h, the percentage of cell growth and apoptosis reached 46.3% ± 2.37% (P = 0.004) and 50.67% ± 3.24% (P = 0.003), respectively. Furthermore, tectorigenin-induced apoptosis of HSC-T6 cells was associated with the generation of ROS, increased intracellular [Ca2+]i, loss of mitochondrial membrane potential, translocation of cytochrome c, and activation of caspase-9 and -3. CONCLUSION: Tectorigenin inhibits proliferation of HSC-T6 cells and induces apoptosis of HSC-T6 cells.

Schistosoma japonicum egg antigen up-regulates fibrogenesis and inhibits proliferation in primary hepatic stellate cells in a concentration-dependent manner

AIM:To investigate the effects of different concentrations of Schistosoma japonicum(S.japonicum) egg antigen on fibrogenesis and apoptosis in primary hepatic stellate cells(HSCs).METHODS:A mouse model of schistosomiasis-associated liver fibrosis(SSLF) was established by infecting mice with schistosomal cercaria via the abdomen.HSCs were isolated from SSLF mice by discontinuous density gradient centrifugation,and their identity was confirmed by immunofluorescence double staining of α-smooth muscle actin(α-SMA) and desmin.The growth inhibitory effect and 50% inhibitory concentration(IC50) of S.japonicum egg antigen for primary HSCs(24 h) were determined using a cell counting kit-8(CCK-8) assay.The expression levels of α-SMA,matrix metalloproteinase-9(MMOL/LP-9) and tissue inhibitor of metalloproteinases-1(TIMP-1) in HSCs in response to different concentrations of S.japonicum egg antigen were detected by Western blotting and real-time reverse transcription-polymerase chain reaction.The levels of phospho-P38(P-P38),phospho-Jun N-terminal kinase(P-JNK) and phospho-Akt(P-AKT) in HSCs were detected by Western blotting.RESULTS:An SSLF mouse model was established,and primary HSCs were successfully isolated and cultured.S.japonicum egg antigen inhibited HSC proliferation in a concentration-dependent manner.The IC50 of the S.japonicum egg antigen was 244.53 ± 35.26 μg/mL.S.japonicum egg antigen enhanced α-SMA expression at both the mRNA and protein levels and enhanced TIMP-1 expression at the mRNA level in HSCs(P < 0.05),whereas the expression of MMOL/LP-9 was attenuated at both the mRNA and protein levels in a concentration-dependent manner(P < 0.05).A high concentration of S.japonicum egg antigen enhanced P-P38,P-JNK and P-AKT activation(P < 0.05).The changes in α-SMA and MMOL/LP-9 expression induced by S.japonicum egg antigen were closely correlated with P-P38 and P-JNK activation(P < 0.05).The attenuation of MMOL/LP-9 was also correlated with P-AKT activation(P < 0.05),but the increase in α-SMA expression was not.TIMP-1 expression was not correlated with P-P38,P-JNK or P-AKT activation.CONCLUSION:S.japonicum egg antigen promotes fibrogenesis,activates the P38/JNK mitogen-activated protein kinase and AKT/PI3K signaling pathways and inhibits proliferation in primary HSCs isolated from SSLF mice in a concentration-dependent manner.

Expression of matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-1 in hepatic stellate cells during rat hepatic fibrosis and its intervention by IL-10

AIM: To investigate the expression of matrix metallopr-oteinase-2 and tissue inhibitor of metalloproteinase-1 in hepatic fibrosis and the antifibrogenic role of exogenous interleukin-10 (IL-10). METHODS: Hepatic fibrosis was induced by CCI4 administration and 60 male Sprague-Dawley rats were randomly divided into normal control group (group N, 8 rats), CCI4-induced group (group C, 28 rats) and IL-10-treated group (group I, 24 rats). At the beginning of the 7th and 11th wk, rats in each group were routinely perfused with pronase E and type IV collagenase through portal vein catheter and the suspension was centrifuged by 11% Nycodenz density gradient to isolate hepatic stellate cells (HSCs). RT-PCR was used to analyze mRNA of MMP-2 and TIMP-1 from freshly isolated cells. Densitometric data were standardized with β-actin signals. Immunocytochemistry was performed to detect MMP-2 and TIMP-1 expression in HSC cultured for 72 h. RESULTS: Compared to group N in the 7th wk, MMP-2 and TIMP-1 mRNA increased in group C (P= 0.001/0.001) and group I (P= 0.001/0.009). The level of MMP-2 and TIMP-1 mRNA in group I was significantly lower than that in group C (P= 0.001/0.001). In the 11th wk, MMP-2 mRNA in group I was still lower than that in group C (P = 0.005), but both dropped compared with that in the 7th week (P = 0.001/0.004). TIMP-1 mRNA in group I was still lower than that in group C (P= 0.001), and increased in group C (P= 0.001) while decreased in group I (P = 0.042) compared with that in the 7th wk. Same results were found by immunocytochemistry. CONCLUSION: Expression of MMP-2 and TIMP-1 is increased in hepatic fibrosis. IL-10 exhibits an antifibrogenic effect by suppressing MMP-2 and TIMP-1 expression.

Roles of hepatic stellate cells in acute liver failure: From the perspective of inflammation and fibrosis

Acute liver failure(ALF)usually results in hepatocellular dysfunction and coagulopathy and carries a high mortality rate.Hepatic stellate cells(HSCs)are famous for their role in liver fibrosis.Although some recent studies revealed that HSCs might participate in the pathogenesis of ALF,the accurate mechanism is still not fully understood.This review focuses on the recent advances in understanding the functions of HSCs in ALF and revealed both protective and promotive roles during the pathogenesis of ALF:HSC activation participates in the maintenance of cell attachment and the architecture of liver tissue via extracellular matrix production and assists liver regeneration by producing growth factors;and HSC inflammation plays a role in relaying inflammation signaling from sinusoids to parenchyma via secretion of inflammatory cytokines.A better understanding of roles of HSCs in the pathogenesis of ALF may lead to improvements and novel strategies for treating ALF patients.

Role of free fatty acids in proliferation of rat hepatic stellate cells(Ⅱ)

AIM To study the role of free fatty acids (arachidonic acid and linoleic acid) in proliferation of rat hepatic stellate cells (HSC). METHODS HSC were isolated and cultured from liver of Wistar rats by in situ perfusion with pronase and collagenase, and density gradient centrifugation with Nycodenz. MTT colorimetric assay was detected for HSC proliferation. RESULTS Arachidonic acid and linoleic acid had an effect on proliferation of HSC. Arachidonic acid of 25mg/L promoted HSC proliferation ( P <0 01), but 50 and 100mg/L had an inhibitory effect on HSC ( P <0 01), and showed cytotoxity on HSC under inverted microscope; 6 25, 12 5 and 25mg/L of linoleic acid had no effect on HSC proliferation ( P >0 05), but with concentration increasing, 50 and 100mg/L of linoleic acid might promote HSC proliferation ( P <0 05 or 0 01). CONCLUSION Arachidonic acid and linoleic acid may promote HSC proliferation, but increased concentration can have cytotoxity on HSC. Arachidonic acid and linoleic acid might be associated with fatty liver and hepatic fibrogenesis by lipid peroxidation.

Hepatic immune tolerance induced by hepatic stellate cells

The liver, which is a metabolic organ, plays a pivotal role in tolerance induction. Hepatic stellate cells(Hp SCs), which are unique non-parenchymal cells, exert potent immunoregulatory activity during cotransplantation with allogeneic islets effectively protecting the islet allografts from rejection. Multiple mechanisms participate in the immune tolerance induced by Hp SCs, including the marked expansion of myeloid-derived suppressor cells(MDSCs), attenuation of effector T cell functions and augmentation of regulatory T cells. Hp SC conditioned MDSC-based immunotherapy has been conducted in mice with autoimmune disease and the results show that this technique may be promising. This article demonstrates how Hp SCs orchestrate both innate immunity and adaptive immunity to build a negative network that leads to immune tolerance.