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.

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.

Notch3 regulates the activation of hepatic stellate cells

AIM： To investigate whether Notch signaling is involved in liver fibrosis by regulating the activation of hepatic stellate cells （HSCs）.METHODS： Immunohistochemistry was used to detect the expression of Notch3 in fibrotic liver tissues of patients with chronic active hepatitis. The expression of Notch3 in HSC-T6 cells treated or not with transforming growth factor （TGF）-β1 was analyzed by iramunofluorescence staining, The expression of Notch3 and myofibroblastic marker （z-smooth muscle actin （（z-SMA） and collagen 1 in HSC-T6 cells transfected with pcDNA3. I-N3ICD or control vector were detected by Western blotting and immunofluorescence staining. Moreover, effects of Notch3 knockdown in HSC-T6 by Notch3 siRNA were investigated by Western blotting and immunofluorescence staining.RESULTS： The expression of Notch3 was significantly up-regulated in fibrotic liver tissues of patients withchronic active hepatitis, but not detected in normal liver tissues. Active Notch signaling was found in HSC-T6 cells. TGF-β1 treatment led to up-regulation of Notch3 expression in HSC-T6 cells, and over-expression of Notch3 increased the expression of α-SMA and collagen I in HSC-T6 without TGF-β1 treatment. Interestingly, transient knockdown of Notch3 decreased the expression of myofibroblastic marker and antagonized TGF-β1 induced expression of α-SMA and collagen I in HSC-T6.CONCLUSION： Notch3 may regulate the activation of HSCs, and the selective interruption of Notch3 may provide an anti -fibrotic strategy in hepatic fibrosis.

Lipid accumulation in hepatocytes induces fibrogenic activation of hepatic stellate cells

Despite the initial belief that non-alcoholic fatty liver disease is a benign disorder, it is now recognized that fibrosis progression occurs in a significant number of patients. Furthermore, hepatic steatosis has been identified as a risk factor for the progression of hepatic fibrosis in a wide range of other liver diseases. Here, we established an in vitro model to study the effect of hepatic lipid accumulation on hepatic stellate cells （HSCs）, the central mediators of liver fibrogenesis. Primary human hepatocytes were incubated with the saturated fatty acid palmitate to induce intracellular lipid accumulation. Subsequently, human HSCs were incubated with conditioned media （CM） from steatotic or control hepatocytes. Lipid accumulation in hepatocytes induced the release of factors that accelerated the activation and proliferation of HSC, and enhanced their resistance to apoptosis, largely mediated via activation of the PI-3-kinase pathway. Furthermore, CM from steatotic hepatocytes induced the expression of the profibrogenic genes TGF-β, tissue inhibitor of metallo-proteinase-1 （TIMP-1）, TIMP-2 and matrix-metallo-proteinase-2, as well as nuclear-factor κB-dependent MCP-1 expression in HSC. In summary, our in vitro data indicate a potential mechanism for the pathophysiological link between hepatic steatosis and fibrogenesis in vivo. Herewith, this study provides an attractive in vitro model to study the molecular mechanisms of steatosis-induced fibrogenesis, and to identify and test novel targets for antifibrotic therapies in fatty liver disease.

Insulin-like growth factor binding protein-7 induces activation and transdifferentiation of hepatic stellate cells in vitro

AIM:To investigate the role of insulin-like growth factor binding protein-7 (IGFBP-7) in the activation and transdifferentiation of hepatic stellate cells (HSC) in vitro.METHODS:Rat HSC-T6 cells were cultured in separate dishes and treated with various concentration of transforming growth factor (TGF)-β1,IGFBP-7 or antiIGFBP-7 antibody for 24 h.The supernatant or a cytoplasm suspension was obtained from cultured HSC,followed by transfer of cells to form cell-coated dishes.Immunocytochemistry and Western blotting were used to analyze the expression of IGFBP-7 induced by TGF-β1 and the level of fibronectin,collagen and α-smooth muscle actin (SMA).The pro-apoptotic effect of antiIGFBP-7 antibody was determined by flow cytometry.RESULTS:Immunocytochemistry and Western blotting revealed that the expression of IGFBP-7 in TGF-β1 treated HSC was significantly up-regulated compared to that in the control group.In addition,fibronectin,collagen and α-SMA also showed enhanced expression in accordance with the transdifferentiation process in a dose-dependent manner to some extent.Moreover,flow cytometry suggested that anti-IGFBP-7 antibody induced apoptosis of activated HSC,which is responsible for the development of liver fibrosis,and may represent a novel pathway and target for therapeutic intervention.CONCLUSION:IGFBP-7 showed increased expression in activated HSC and played an important role in the activation and transdifferentiation process of HSC.AntiIGFBP-7 antibody may ameliorate liver fibrogenesis.

Inhibition of high-mobility group box 1 expression by siRNA in rat hepatic stellate cells

AIM:To explore the role of high-mobility group box 1 (HMGB1) protein during liver fibrogenesis and investigate the functional effects of HMGB1 gene silencing in hepatic stellate cells (HSCs) using siRNA.METHODS:Hepatic fibrosis in rats was induced through serial subcutaneous injections of dimethylnitrosamine,and expression of HMGB1 was detected by immunohistochemistry.HMGB1 siRNAs were developed and transiently transfected into HSC-T6 cells using Lipofectamine 2000.HMGB1 expression was evaluated by real-time polymerase chain reaction (PCR) and Western blotting analysis.Expression of α-smooth muscle actin (α-SMA) and collagen typesⅠand Ⅲ was evaluated by real-time PCR.Cell proliferation and the cell cycle were determined using the methyl thiazolyl tetrazolium method.Finally,collagen content in HSC supernatant was evaluated by an enzyme-linked immunosorbent assay.RESULTS:The results showed that HMGB1 was upregulated during liver fibrosis and that its expression was closely correlated with the deposition of collagen.siRNA molecules were successfully transfected into HSCs and induced inhibition of HMGB1 expression in a time-dependent manner.Moreover,HMGB1 siRNA treatment inhibited synthesis of α-SMA and collagen types Ⅰ and Ⅲ in transfected HSCs.CONCLUSION:This study suggests a significant functional role for HMGB1 in the development of liver fibrosis.It also demonstrates that downregulation of HMGB1 expression might be a potential strategy to treat liver fibrosis.

miRNA studies in in vitro and in vivo activated hepatic stellate cells

AIM: To understand which and how different miRNAs are implicated in the process of hepatic stellate cell (HSC) activation. METHODS: We used microarrays to examine the differential expression of miRNAs during in vitro activation of primary HSCs (pHSCs). The transcriptome changes upon stable transfection of rno-miR-146a into an HSC cell line were studied using cDNA microarrays. Selected differentially regulated miRNAs were investigated by quantitative real-time polymerase chain reaction during in vivo HSC activation. The effect of miRNA mimics and inhibitor on the in vitro activation of pHSCs was also evaluated.RESULTS: We found that 16 miRNAs were upregulated and 26 were downregulated significantly in 10-d in vitro activated pHSCs in comparison to quiescent pHSCs. Overexpression of rno-miR-146a was characterized by marked upregulation of tissue inhibitor of metalloproteinase-3, which is implicated in the regulation of tumor necrosis factor-α activity. Differences in the regulation of selected miRNAs were observed comparing in vitro and in vivo HSC activation. Treatment with miR-26a and 29a mimics, and miR-214 inhibitor during in vitro activation of pHSCs induced significant downregulation of collagen type Ⅰ transcription. CONCLUSION: Our results emphasize the different regulation of miRNAs in in vitro and in vivo activated pHSCs. We also showed that miR-26a, 29a and 214 are involved in the regulation of collagen type I mRNA.

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.

Specific shRNA targeting of FAK influenced collagen metabolism in rat hepatic stellate cells

AIM:To investigate the effects and mechanism of disruption of focal adhesion kinase(FAK) expression on collagen metabolism in rat hepatic stellate cells(HSC).METHODS:The plasmids expressing FAK short hairpin RNA(shRNA) were transfected into HSC-T6 cells,and the level of FAK expression was determined by both real-time quantitative polymerase chain reaction(QPCR) and Western blotting analysis.The production of type collagen and type collagen in FAK-disrupted cells was analyzed by real-time Q-PCR.The level of collagen metabolism proteins,including matrix metalloproteinases-13(MMP-13) and tissue inhibitors of metalloproteinases-1(TIMP-1) was also determined by both real-time Q-PCR and Western blotting analysis.RESULTS:The transfection of FAK shRNA plasmids into HSC resulted in disrupted FAK expression.Compared with the HK group,the levels of type collagen and type collagen mRNA transcripts in FAK shRNA plas-mid group were signif icantly decreased(0.69 ± 0.03 vs 1.96 ± 0.15,P = 0.000;0.59 ± 0.07 vs 1.62 ± 0.12,P = 0.020).The production of TIMP-1 in this cell type was also signif icantly reduced at both mRNA and protein levels(0.49 ± 0.02 vs 1.72 ± 0.10,P = 0.005;0.76 ± 0.08 vs 2.31 ± 0.24,P = 0.000).However,the expression of MMP-13 mRNA could be significantly up-regulated by the transfection of FAK shRNA plasmids into HSC(1.74 ± 0.20 vs 1.09 ± 0.09,P = 0.000).CONCLUSION:These data support the hypothesis that shRNA-mediated disruption of FAK expression could attenuate extracellular matrix(ECM) synthesis and promote ECM degradation,making FAK a potential target for novel anti-f ibrosis therapies.

In vivo effects of Chinese herbal recipe, Danshaohuaxian, on apoptosis and proliferation of hepatic stellate cells in hepatic fibrotic rats

AIM: To investigate the effects of Danshaohuaxian (DSHX), a Chinese herbal recipe, on the apoptosis and cell cycles of hepatic stellate cells (HSCs) in rat hepatic fibrosis and its possible mechanisms. METHODS: Seventy-six male Wistar rats were randomly divided into normal control group, hepatic fibrosis group, non-DSHX-treated group and DSHX-treated group. Except for the normal control group, rat hepatic fibrotic models were induced by subcutaneous injection of carbon tetrachloride (CCl4), drinking alcohol, giving diet of hyperlipid and hypoprotein for 8 wk. When the hepatic fibrotic models were produced, 12 rats of hepatic fibrosis group (15 rats survived, others died during the 8 wk) were sacrificed to collect blood and livers. HSCs were isolated from the other 3 rats to detect the apoptotic index (AI) and cell cycles by flow cytometry. DSHX was then given to the DSHX-treated group (1.0 g/kg, PO, daily) for 8 wk. At the same time, normal control group and non-DSHX-treated group were given normal saline for 8 wk. At end of the experiment, some rats in these three groups were sacrificed to collect blood and livers, the other rats were used for HSC isolation to detect the apoptotic index (AI) and cell cycles. Then the liver index, serum hyaluronic acid (HA) and alanine aminotransferase (ALT), degree of hepatic fibrosis, urinary excretion of hydroxyproline (Hyp) and expression of collagen types Ⅰ and Ⅲ (COL Ⅰ and Ⅲ) in these four groups were detected respectively. RESULTS: Compared with the indexes of the hepatic fibrosis group and non-DSHX-treated group, the DSHX-treated group revealed a liver index of (0.0267±0.0017 vs 0.0423±0.0044, 0.0295±0.0019, P<0.05), levels of serum HA (200.78±31.71 vs 316.17±78.48, 300.86±72.73, P<0.05) and ALT(93.13±5.79 vs 174.5±6.02, 104.75±6.54, P<0.01), and stage of hepatic fibrosis (1.30 vs 4.25, 2.60, P<0.01) all reduced. The urinary excretion of Hyp increased (541.09±73.39 vs 62.00±6.40, 182.44±30.83, P<0.01), the COL Ⅰ and Ⅲ expression decreased (COL I: 1.07±0.96 vs 4.18±2.26, 3.22±1.44, P<0.01; COL Ⅲ: 1.09±0.58 vs 3.04±0.62, 2.23±0.58, P<0.01), the HSCs apoptotic index of HSCs (7.81±0.47 vs 1.63±0.25, 1.78±0.4, P<0.05) and the ratio of G0-G1 phase cells increased (94.30±1.33 vs 62.27±17.96, 50.53±2.25, P<0.05). The ratios of S-phase cells (3.11±1.27 vs 9.83±1.81, 11.87±1.9, P<0.05) and G2-M phase cells (2.58±0.73 vs 23.26±10.95, 13.60±1.15, P<0.01) declined. CONCLUSION: DSHX capsule shows certain therapeutic effects on hepatic fibrosis in rats and inhibits abnormal deposition of COL I and III in rat livers by promoting the apoptosis of HSCs and preventing their proliferation.

Pathophysiological mechanisms of hepatic stellate cells activation in liver fibrosis

Liver fibrosis is a complex pathological process controlled by a variety of cells,mediators and signaling pathways.Hepatic stellate cells play a central role in the development of liver fibrosis.In chronic liver disease,hepatic stellate cells undergo dramatic phenotypic activation and acquire fibrogenic properties.This review focuses on the pathophysiological mechanisms of hepatic stellate cells activation in liver fibrosis.They enter the cell cycle under the influence of various triggers.The“Initiation”phase of hepatic stellate cells activation overlaps and continues with the“Perpetuation”phase,which is characterized by a pronounced inflammatory and fibrogenic reaction.This is followed by a resolution phase if the injury subsides.Knowledge of these pathophysiological mechanisms paved the way for drugs aimed at preventing the development and progression of liver fibrosis.In this respect,impairments in intracellular signaling,epigenetic changes and cellular stress response can be the targets of therapy where the goal is to deactivate hepatic stellate cells.Potential antifibrotic therapy may focus on inducing hepatic stellate cells to return to an inactive state through cellular aging,apoptosis,and/or clearance by immune cells,and serve as potential antifibrotic therapy.It is especially important to prevent the formation of liver cirrhosis since the only radical approach to its treatment is liver transplantation which can be performed in only a limited number of countries.

Herbal compound 861 regulates mRNA expression of collagen synthesis- and degradation-related genes in human hepatic stellate cells

AIM： To identify the role of herbal compound 861 （Cpd 861） in the regulation of mRNA expression of collagen synthesis- and degradation-related genes in human hepatic stellate cells （HSCs）. METHODS： mRNA levels of collagen types I and III, matrix metalloproteinase 1 （MMP-1）, matrix metalloproteinase 2 （MMP-2）, membrane type-1 matrix metalloproteinase （MT1-MMP）, tissue inhibitor of metalloproteinase 1 （TIMP-1）, and transforming growth factor β1 （TGF-β1） in cultured-activated HSCs treated with Cpd 861 or interferon-γ, （IFN-γ,） were determined by real-time PCR. RESULTS： Both Cpd 861 and IFN-γ reduced the mRNA levels of collagen type Ⅲ, MMP-2 and TGF-β1. Moreover, Cpd 861 significantly enhanced the MMP-1 mRNA levels while down-regulated the TIMP-1 mRNA expression, increasing the ratio of MMP-1 to TIMP-1 to （6.3 ＋ 0.3）- fold compared to the control group. CONCLUSION： The anti-fibrosis function of Cpd 861 may be mediated by both decreased interstitial collagen sythesis by inhibiting the transcription of collagen type Ⅲ and TGF-β1 and increased degradation of these collagens by up-regulating MMP-1 and down-regulating TIMP-1 mRNA levels.

Mitochondria: A critical hub for hepatic stellate cells activation during chronic liver diseases

Background: Upon liver injury, quiescent hepatic stellate cells(q HSCs), reside in the perisinusoidal space, phenotypically transdifferentiate into myofibroblast-like cells(MFBs). The q HSCs in the normal liver are less fibrogenic, migratory, and also have less proliferative potential. However, activated HSCs(a HSCs) are more fibrogenic and have a high migratory and proliferative MFBs phenotype. HSCs activation is a highly energetic process that needs abundant intracellular energy in the form of adenosine triphosphate(ATP) for the synthesis of extracellular matrix(ECM) in the injured liver to substantiate the injury. Data sources: The articles were collected through Pub Med and EMBASE using search terms "mitochondria and hepatic stellate cells", "mitochondria and HSCs", "mitochondria and hepatic fibrosis", "mitochondria and liver diseases", and "mitochondria and chronic liver disease", and relevant publications published before September 31, 2020 were included in this review. Results: Mitochondria homeostasis is affected during HSCs activation. Mitochondria in a HSCs are highly energetic and are in a high metabolically active state exhibiting increased activity such as glycolysis and respiration. a HSCs have high glycolytic enzymes expression and glycolytic activity induced by Hedgehog(Hh) signaling from injured hepatocytes. Increased glycolysis and aerobic glycolysis(Warburg effect) endproducts in a HSCs consequently activate the ECM-related gene expressions. Increased Hh signaling from injured hepatocytes downregulates peroxisome proliferator-activated receptor-γ expression and decreases lipogenesis in a HSCs. Glutaminolysis and tricarboxylic acid cycle liberate ATPs that fuel HSCs to proliferate and produce ECM during their activation. Conclusions: Available studies suggest that mitochondria functions can increase in parallel with HSCs activation. Therefore, mitochondrial modulators should be tested in an elaborate manner to control or prevent the HSCs activation during liver injury to subsequently regress hepatic fibrosis.

Migration of hepatic stellate cells in fibrotic microenvironment of diseased liver model

BACKGROUND: In liver fibrosis, alterations within the space of Disse microenvironment facilitate the progression of chronic liver disease. The normal basement membranelike matrix in the space of Disse converts to a matrix rich in fibril-forming collagens during the fibrosis. This study aimed to investigate the impact of alterations in the space of Disse microenvironment on the migration of hepatic stellate cells (HSCs) in the process of liver fibrosis, and to explore the novel mechanism ofliver fibrosis from the viewpoint of cell migration. METHODS: A modified in vitro Boyden chamber system was employed to partially mimic the in vitro microenvironment of the Disse space in normal liver and in fibrosis. The effects of fibrogenetic growth factors on the migration of HSCs in simulated liver fibrosis were assessed by cell migration and cell proliferation experiments. RESULTS: Enhanced platelet-derived growth factor (PDGF)-BB, transforming growth factor-beta 1 (TGF-beta 1) and/or epithelial growth factor (EGF) in liver fibrosis resulted in an increase in migratory capacity of activated HSCs. The enhanced migration of HSCs, induced by PDGF-BB was proliferation -independent. The elevation of basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF) during liver fibrosis had no effect on the migration of HSCs. CONCLUSIONS: The study provides valuable insights into the role of the space of Disse microenvironment in regulating the migratory behavior of HSCs. TGF-beta 1, PDGF-BB and EGF, which increase in liver fibrosis, induce the migration of activated HSCs. However, bFGF and VEGF have no effect although they also increase during liver fibrosis.

Cross-talk between hepatic stellate cells and T lymphocytes in liver fibrosis

Background: Fibrosis results from inflammation and healing following injury. The imbalance between extracellular matrix(ECM) secretion and degradation leads to the ECM accumulation and liver fibrosis. This process is regulated by immune cells. T lymphocytes, including alpha beta( αβ) T cells, which have adaptive immune functions, and gamma delta( γδ) T cells, which have innate immune functions, are considered regulators of liver fibrosis. This review aimed to present the current understanding of the cross-talk between T lymphocytes and hepatic stellate cells(HSCs), which are the key cells in liver fibrosis. Data sources: The keywords "liver fibrosis", "immune", and "T cells" were used to retrieve articles published in Pub Med database before January 31, 2020. Results: The ratio of CD8 +(suppressor) T cells to CD4+(helper) T cells is significantly higher in the liver than in the peripheral blood. T cells secrete a series of cytokines and chemokines to regulate the inflammation in the liver and the activation of HSCs to influence the course of liver fibrosis. In addition, HSCs also regulate the differentiation and proliferation of T cells. Conclusions: The cross-talk between T cells and HSCs regulates liver fibrosis progression. The elucidation of this communication process will help us to understand the pathological process of liver fibrosis.

Metabolomic profiling for identification of metabolites and relevant pathways for taurine in hepatic stellate cells

AIM To develop a reliable and simple method to identify important biological metabolites and relevant pathways for taurine in hepatic stellate cells(HSCs), in order to provide more data for taurine therapy.METHODS All the biological samples were analyzed by using highperformance liquid chromatography-time electrospray ionization/quadrupole-time of flight mass spectrometry. Principal component analysis and partial least squares discriminant analysis were used to identify statistically different metabolites for taurine in HSCs, and metabolomic pathway analysis was used to do pathway analysis for taurine in HSCs. The chemical structure of the related metabolites and pathways was identified by comparing the m/z ratio and ion mode with the data obtained from free online databases.RESULTS A total of 32 significant differential endogenous metabolites were identified, which may be related to the mechanism of action of taurine in HSCs. Among the seven relevant pathways identified, sphingolipid metabolism pathway, glutathione metabolism pathway and thiamine metabolism pathway were found to be the most important metabolic pathways for taurine in HSCs.CONCLUSION This study showed that there were distinct changes in biological metabolites of taurine in HSCs and three differential metabolic pathways including sphingolipid pathway, glutathione pathway and thiamine metabolism pathway might be of key importance in mediating the mechanism of action of taurine in HSCs.

Angiotensin-converting enzyme 2 improves liver fibrosis in mice by regulating autophagy of hepatic stellate cells

BACKGROUND Liver fibrosis is the common pathological process associated with the occurrence and development of various chronic liver diseases.At present,there is still a lack of effective prevention and treatment methods in clinical practice.Hepatic stellate cell(HSC)plays a key role in liver fibrogenesis.In recent years,the study of liver fibrosis targeting HSC autophagy has become a hot spot in this research field.Angiotensin-converting enzyme 2(ACE2)is a key negative regulator of reninangiotensin system,and its specific molecular mechanism on autophagy and liver fibrosis needs to be further explored.AIM To investigate the effect of ACE2 on hepatic fibrosis in mice by regulating HSC autophagy through the Adenosine monophosphate activates protein kinases(AMPK)/mammalian target of rapamycin(mTOR)pathway.METHODS Overexpression of ACE2 in a mouse liver fibrosis model was induced by injection of liver-specific recombinant adeno-associated virus ACE2 vector(rAAV2/8-ACE2).The degree of liver fibrosis was assessed by histopathological staining and the biomarkers in mouse serum were measured by Luminex multifactor analysis.The number of apoptotic HSCs was assessed by terminal deoxynucleoitidyl transferase-mediated dUTP nick-end labeling(TUNEL)and immunofluorescence staining.Transmission electron microscopy was used to identify the changes in the number of HSC autophagosomes.The effect of ACE2 overexpression on Wu Y et al.ACE2 improves liver fibrosis through autophagy WJG https://www.wjgnet.com 4976 September 7,2023 Volume 29 Issue 33 autophagy-related proteins was evaluated by multicolor immunofluorescence staining.The expression of autophagy-related indicators and AMPK pathway-related proteins was measured by western blotting.RESULTS A mouse model of liver fibrosis was successfully established after 8 wk of intraperitoneal injection of carbon tetrachloride(CCl4).rAAV2/8-ACE2 administration reduced collagen deposition and alleviated the degree of liver fibrosis in mice.The serum levels of platelet-derived growth factor,angiopoietin-2,vascular endothelial growth factor and angiotensin II were decreased,while the levels of interleukin(IL)-10 and angiotensin-(1-7)were increased in the rAAV2/8-ACE2 group.In addition,the expression of alpha-smooth muscle actin,fibronectin,and CD31 was down-regulated in the rAAV2/8-ACE2 group.TUNEL and immunofluorescence staining showed that rAAV2/8-ACE2 injection increased HSC apoptosis.Moreover,rAAV2/8-ACE2 injection notably decreased the number of autophagosomes and the expression of autophagy-related proteins(LC3I,LC3II,Beclin-1),and affected the expression of AMPK pathway-related proteins(AMPK,p-AMPK,p-mTOR).CONCLUSION ACE2 overexpression can inhibit HSC activation and promote cell apoptosis by regulating HSC autophagy through the AMPK/mTOR pathway,thereby alleviating liver fibrosis and hepatic sinusoidal remodeling.

Interaction between insulin-like growth factor binding protein-related protein 1 and transforming 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 (TGF beta 1) is known as the strongest effector of liver fibrosis. Therefore, we aimed to investigate the detailed interaction between IGFBPrP1 and TGF beta 1 in primary hepatic stellate cells (HSCs). METHODS: We overexpressed TGF beta 1 or IGFBPrP1 and inhibited TGF beta 1 expression in primary HSCs for 6, 12, 24, 48, 72, and 96 hours to investigate their interaction and observe the accompanying expressions of a-smooth muscle actin (alpha-SMA), collagen I, fibronectin, and phosphorylated-mothers against decapentaplegic homolog 2/3 (p-Smad2/3). RESULTS: We found that the adenovirus vector encoding the TGF beta 1 gene (AdTGF beta 1) induced IGFBPrP1 expression while that of alpha-SMA, collagen I, fibronectin, and TGF beta 1 increased gradually. Concomitantly, AdIGFBPrP1 upregulated TGF beta 1, alpha-SMA, collagen I, fibronectin, and p-Smad2/3 in a time-dependent manner while IGFBPrP1 expression was decreased at 96 hours. Inhibition of TGF beta 1 expression reduced the IGFBPrP1-stimulated expression of alpha-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 TGF beta 1, which likely accelerates liver fibrosis progression. Furthermore, IGFBPrP1 likely participates in liver fibrosis in a TGF beta 1-depedent manner, and may act as an upstream regulatory factor of TGF beta 1 in the Smad pathway.