Hepatic Stellate Cell Fibrosis Research Articles

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

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. 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. 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. 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. 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.

Hypoxia-inducible factor 1α/IL-6 axis in activated hepatic stellate cells aggravates liver fibrosis

Hepatic stellate cells (HSCs) upregulate hypoxia inducible factor 1 alpha (HIF-1α) expression in response to fibrosis-induced hypoxia. The mechanism by which HIF-1α promotes liver fibrosis in HSCs is not fully understood. In this study, we found that increased expression of α-SMA, HIF-1α and IL-6, as well as colocalization of α-SMA and HIF-1α, and HIF-1α and IL-6, were observed in liver fibrotic tissues of patients and a mouse model. HIF-1α expression induced IL-6 secretion in activated HSCs and the increase could be abolished by HIF-1α suppression or HIF1A gene knockdown. HIF-1α directly bound to the hypoxia response element (HRE) region in HSC IL6/Il6 promoters. Additionally, culturing naïve CD4 T cells with supernatant from HSCs in which HIF-1α is highly expressed increased IL-17A expression, and the expression could be abolished by HIF1A knockdown in LX2. In turn, the IL-17A-enriched supernatant induced IL-6 secretion in HSCs. Together, these results show that HIF-1α upregulates IL-6 expression in HSCs and induces IL-17A secretion through directly binding to the HRE of IL6 promoter.

Umbilical Cord Wharton's Jelly-Derived Mesenchymal Stem Cells Inhibit the TGF-β1 Pathway in Hepatic Fibrosis Rats Through a Paracrine Regulation Process

The aim is to verify the therapeutic effect and possible mechanism of human umbilical cord Wharton's jelly-derived transplantation of mesenchymal stem cells (UMSCs) on CCl4-induced hepatic fibrosis rats through in vivo studies and to explore the regulatory mechanism of UMSCs on fibrosis of hepatic stellate cells (HSCs) through in vitro experiments. In vivo experiment: Rats were randomly divided into blank control group and hepatic fibrosis group. During the entire trial, the blank control group received subcutaneous injection of normal saline, while in the hepatic fibrosis group received injections of 50% CCl4-olive oil subcutaneously for 10 weeks to establish the rat model of liver fibrosis. Hepatic fibrosis rats were then randomly and evenly divided into umbilical cord mesenchymal stem cell (UMSC) group, bone marrow mesenchymal stem cell (BMSC) group, UMSC-culture medium (CM) group, and control group. Rats in each group were infused with the following substances through the caudal vein as follows: 1 mL UMSCs (2 × 106/mL) in UMSC group, 1 mL BMSCs (2 × 106/mL) in BMSC group, 1 mL UMSCs-CM in CM group, and 1 mL saline in control group. Rats of each group were closely observed (weight, hair condition, activity, appetite, diarrhea, etc.), venous blood samples were collected, the number of white blood cells and lymphocytes were measured, and liver function indicators (ALT, AST, TBIL, ALB) were determined. Three weeks later, rat liver specimens were taken, HE stained, pathological changes were examined and quantified. In vitro experiments: HSCs were seeded in 6-well plates at 1.0 × 105/mL, with a serum-free medium for 24 hours. Then, 2 mL of UMSCs-CM was added in the study group, while an equal amount of complete medium was added to the control group. RT-PCR was used to detect TGF-β1, Collagen-I, TIMP-2 mRNA expression in HSCs, and western blot was used to detect TGF-β1 protein expression in HSCs. In vivo experiment: Compared with the control group, after the transplantation, the activity status (weight, spirit, appetite, movement, hair, diarrhea, etc.) of rats in the UMSC group, BMSC group, and CM group were improved. The liver function indexes of these groups, such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin (TBIL) were significantly decreased (p < 0.05), while albumin (ALB) levels were mildly but not significantly increased (p > 0.05). The Knodell score (reflecting the degree of liver inflammation) and Chevallier score (reflecting the degree of liver fibrosis) of liver specimens in pathological examination were also significantly reduced, and the difference in the quantitative scores of those indexes was statistically significant (p < 0.05). There was no statistically significant difference in the number of venous white blood cells and lymphocytes, liver function indexes (ALT, AST, TBIL, ALB), Knodell score, and Chevallier score of liver samples among the UMSC group, BMSC group, and CM group. In vitro experiments: After treatment with UMSCs-CM, the expression of TGF-β1, Collagen-I, and TIMP-2 mRNA in HSCs was significantly down-regulated compared with that of the control group (treated with complete medium), and it gradually decreased with the extension of the treatment time. Compared with the control group, the expression of TGF-β1 protein in the HSCs of the experimental group was down-regulated, and this effect was time-dependent, specifically, the control group (2.49 ± 0.43) > the experimental group at 48 hours (1.98 ± 0.26) > the experimental group at 72 hours (1.62 ± 0.20) (F = 7.796, p < 0.05). In rats with liver fibrosis, transplantation of UMSCs can improve liver function and reduce the inflammatory activity and fibrosis of the liver, possibly through the paracrine mechanism. UMSCs inhibit HSCs fibrosis through a paracrine mechanism, which is time-dependent, possibly by targeting TGF-β1 and its downstream gene products.

CaM/CaMKII mediates activation and proliferation of hepatic stellate cells regulated by ASIC1a.

The activation of hepatic stellate cells (HSCs) is closely related to hepatic fibrosis and plays a key role in its occurrence and development. In the damaged liver, inhibition of the activation, proliferation, and clearance of HSCs is an important therapeutic strategy. However, the mechanism underlying the activation of HSCs is not completely clear. Acid-sensitive ion channel 1a (ASIC1a) is a cation channel activated by extracellular acid, which is responsible for the transport of Ca2+ and Na+ and participates in the activation of HSCs and the occurrence and development of many inflammatory diseases, suggesting that ASIC1a plays an important role in liver fibrosis. A previous study by the project team found that when the membrane channel protein ASIC1a was opened, intracellular Ca2+ levels increased, the expression of CaM/CaMKII in HSCs was high, and HSC was activated and proliferated. Therefore, we established an SD rat model of hepatic fibrosis and induced HSC-T6 activation by stimulating ASIC1a with acid in vitro. In vivo, CCl4 was used to induce liver fibrosis in rats, and different doses of KN93 (0.5, 1, and 2 mg/kg/d) and colchicine (0.1 mg/kg/d) were administered. Eight weeks later, the activities of ALT and AST in serum were measured and hematoxylin-eosin and Masson staining in liver tissue, and immunohistochemistry analysis were performed in SD rats. The expressions of ASIC1a, α-SMA, Collagen-1, CaM, and CaMKII were detected. In vitro, we activated HSC-T6 cells by stimulating ASIC1a with acid. The results showed that inhibition of ASIC1a could improve acid-induced HSCs activation. In addition, CaM/CaMKII was expressed in HSC of rats with hepatic fibrosis regulated by ASIC1a. After blocking or silencing the expression of CaMKII, the fibrosis marker protein can be down-regulated. KN93 also reduced inflammation and improved the activation, proliferation and fibrosis of HSC. In summary, we concluded that CaM/CaMKII participates in ASIC1a regulation of the proliferation and activation of HSC and promotes the occurrence of liver fibrosis.

TGF-\u03b2 activates NLRP3 inflammasome by an autocrine production of TGF-\u03b2 in LX-2 human hepatic stellate cells

Inflammation contributes to the pathogenesis of liver disease, and inflammasome activation has been identified as a major contributor to the amplification of liver inflammation. Transforming growth factor-beta (TGF-β) is a key regulator of liver physiology, contributing to all stages of liver disease. We investigated whether TGF-β is involved in inflammasome-mediated fibrosis in hepatic stellate cells. Treatment with TGF-β increased priming of NLRP3 inflammasome signaling by increasing NLRP3 levels and activating TAK1-NF-kB signaling. Moreover, TGF-β increased the expression of p-Smad2/3-NOX4 in LX-2 cells and consequently increased ROS content, which is a trigger for NLRP3 inflammasome activation. Elevated expression of NEK7 and active caspase-1 was also shown in TGF-β-induced LX-2 cells, and this level was reduced by (5Z)-oxozeaenol, a TAK inhibitor. Finally, TGF-β-treated cells significantly increased TGF-β secretion levels, and their production was inhibited by IL-1β receptor antagonist treatment. In conclusion, TGF-β may represent an endogenous danger signal to the active NLRP3 inflammasome, by which IL-1β mediates TGF-β expression in an autocrine manner. Therefore, targeting the NLRP3 inflammasome may be a promising approach for the development of therapies for TGF-β-induced liver fibrosis.

Involvement of TREK1 channels in the proliferation of human hepatic stellate LX-2 cells

Activation of hepatic stellate cells (HSCs) causes hepatic fibrosis and results in chronic liver diseases. Although activated HSC functions are facilitated by an increase in the cytosolic Ca2+ concentration ([Ca2+]cyt), the pathophysiological roles of ion channels are largely unknown. In the present study, functional analyses of the two-pore domain K+ (K2P) channels, which regulate the resting membrane potential and [Ca2+]cyt, were performed using the human HSC line, LX-2. Expression analyses revealed that TREK1 (also known as KCNK2 and K2P2.1) channels are expressed in LX-2 cells. Whole-cell K+ currents were activated by 10 μM arachidonic acid and the activation was abolished by 100 μM tetrapentylammonium, which are pharmacological characteristics of TREK1 channels. The siRNA knockdown of TREK1 channels caused membrane depolarization and reduced [Ca2+]cyt. In addition, TREK1 knockdown downregulated the gene expression of collage type I and platelet-derived growth factor. Furthermore, TREK1 knockdown inhibited the proliferation of LX-2 cells. In conclusion, the activity of TREK1 channels determines the resting membrane potential and [Ca2+]cyt, which play a role in extracellular matrix production and cell proliferation in HSCs. This study may help elucidate the molecular mechanism underlying hepatic fibrosis in HSCs and provide a potential therapeutic target for hepatic fibrosis.

MiR-34a promotes fibrosis of hepatic stellate cells via the TGF-β pathway.

BackgroundPrevious studies have confirmed that MicroRNA (miRNA) is a key regulator exhibiting different effects in human liver fibrosis. However, the function of miR-34a in liver fibrosis has not been reported. Hence, this study aimed to investigate the regulatory mechanism of miR-34a in liver fibrosis.MethodsThe expression of miR-34a was measured in fibrosis tissues via the quantitative real-time PCR (qRT-PCR) assay. Subsequently, 30 male C57BL/6J mice were divided into control and treatment groups and used to establish animal models of liver fibrosis to explore the expression level of miR-34a. Moreover, Cell Counting Kit 8 (CCK-8) and transwell assays were preformed to identify the regulatory mechanism of miR-34a in cells. The effect of miR-34a on the activity of transforming growth factor-β (TGF-β) pathway was observed by western blot.ResultsUp-regulation of miR-34a was detected in fibrosis cells. Moreover, the cellular phenotype was suppressed by miR-34a down-regulation in a primary culture of hepatic stellate cells (HSCs). Besides, it was found that increased miR-34a could significantly promote the invasion and migration of HSCs. Moreover, miR-34a activates HSCs through transforming TGF-β, α-smooth muscle actin (α-SMA), and Monocyte chemoattractant protein-1 (MCP-1), which further affects liver fibrosis.ConclusionsMiR-34a promotes the fibrosis of HSCs as well as cell proliferation, migration, and invasion.

IL-22 alleviates the fibrosis of hepatic stellate cells via the inactivation of NLRP3 inflammasome signaling.

Persistent and progressive liver injury causes liver fibrosis due to the inability of the liver to regenerate. Interleukin (IL)-22 serves an important role in liver fibrosis. However, the underlying mechanism by which IL-22 exerts its effects on liver fibrosis has not been fully elucidated. The aim of the present study was to investigate the underlying mechanism by which IL-22 affects the development of liver fibrosis. Following activation of the hepatic stellate cells (HSCs) using transforming growth factor β (TGF-β), HSC proliferation was measured using the Cell Counting Kit-8 assay. The indicators of oxidative stress were detected using specific kits. In addition, the mRNA and protein expression levels of fibrosis-associated genes were determined using reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. Subsequently, the protein expression levels of the NOD-like receptor protein 3 (NLRP3), caspase-1 and IL-1β were examined using western blotting. Following addition of Nigericin, a NLRP3 activator, the levels of oxidative stress and fibrosis were measured. IL-22 increased the viability of HSCs, which were activated by TGF-β. The malondialdehyde content was significantly decreased, whereas superoxide dismutase and glutathione levels were increased following IL-22 treatment. Moreover, IL-22 markedly downregulated the expression levels of fibrosis-associated genes, including α-smooth muscle actin, type I collagen and TIMP metallopeptidase inhibitor 1. Furthermore, the expression levels of NLRP3, caspase-1 and IL-1β were decreased in the IL-22-treated groups. However, the NLRP3 activator Nigericin reversed the inhibitory effects of IL-22 on the induction of oxidative stress and fibrosis of HSCs induced by TGF-β. In conclusion, the present study indicated that IL-22 alleviated the fibrosis of HSCs by inactivation of NLRP3 inflammasome signaling, which may provide further insight on the underlying mechanism by which IL-22 exerts protective effects on liver fibrosis.

LncRNA-HEIM Facilitated Liver Fibrosis by Up-Regulating TGF-β Expression in Long-Term Outcome of Chronic Hepatitis B.

BackgroundChronic liver fibrosis is an inevitable stage for the development of patients with chronic hepatitis B (CHB). However, anti-fibrotic therapies have been unsuccessful so far. The biological functions and molecular mechanisms of long non-coding RNAs (lncRNAs) in the host immune system during chronic hepatitis B virus (HBV) infection, especially in fibrosis, are still largely unknown.MethodThe total RNA of peripheral blood mononuclear cells (PBMCs) from asymptomatic carriers (ASCs) or CHB receiving at least 8 years of anti-viral treatments was analyzed using Arraystar microarray and validated via quantitative real-time PCR (qRT-PCR). Correlation analysis was conducted based on correlation coefficients, Clusterprofile, and RNA Interactome Database (RAID). The functions of lncRNA in monocytes were determined via loss-of-function RNAi or gain-of-function lentivirus assays. The expression levels of mRNAs or proteins were evaluated using qRT-PCR, western blotting assay, or enzyme linked immunosorbent assays (ELISA).ResultsA total of 1,042 mRNA transcripts (630 up-regulated and 412 down-regulated) were identified being differentially expressed between ASC and CHB patients. Through enrichment analysis we focused on the transforming growth factor beta (TGF-β) signaling pathway and validated their expression in a larger cohort. Moreover, we found that lncRNA ENST00000519726 (lncRNA-HEIM) was highly expressed in monocytes and further up-regulated upon HBV infection. LncRNA-HEIM played an important role in CHB patients with long-term antiviral treatments, and its elevated expression was remarkably correlated with the TGF-β signaling pathway, especially with the two members namely TGF-β and SMAD4. Furthermore, altering the endogenous lncRNA-HEIM level in monocytes significantly affected the production of TGF-β, as well as the fibrosis of hepatic stellate cells by affecting the expression of collagen I and α-smooth muscle actin (α-SMA).ConclusionThese findings not only added knowledge to the understanding of the roles of which lncRNA-HEIM played in the activation of HSCs in CHB patients with long-term medication, but also provided a promising therapeutic target in the future treatment for liver fibrosis.

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.

Specific Overexpression of Mitofusin-2 in Hepatic Stellate Cells Ameliorates Liver Fibrosis in Mice Model.

Liver fibrosis is a chronic liver disease that could further develop to cirrhosis and liver carcinoma. Hepatic stellate cells (HSCs) are primary effector cells to initiate liver fibrosis. We aimed to explore the function and underlying mechanisms of mitochondrial fusion protein Mitofusin-2 (MFN2) in liver fibrosis. First, we utilized an alpha-smooth muscle actin promoter to overexpress MFN2 specifically in HSCs using adeno-associated virus (AAV) vector (AAV-MFN2). Overexpression of MFN2 was specifically achieved in HSC-T6 cells, but not in murine bone marrow-derived macrophages or hepatocyte AML-12 cells. We found that high expression of MFN2 induced apoptosis of HSC-T6 cells. Mechanistically, we demonstrated that high level of MFN2 inhibited TGF-β1/Smad signaling pathway, triggered downregulation of type I, type III, and type IV collagen, and antagonized the formation of factors associated with liver fibrosis. Furthermore, we found that overexpression of MFN2 using AAV-MFN2 ameliorated CCl4-induced liver fibrosis in vivo with significantly decreased immune cell infiltration. Taken together, our findings indicate that MFN2 is critical in regulating apoptosis and liver fibrosis in HSCs, which might be a useful therapeutic target to treat liver fibrosis.

A Functional Role of GAS6/TAM in Nonalcoholic Steatohepatitis Progression Implicates AXL as Therapeutic Target

Background and AimsGAS6 signaling, through the TAM receptor tyrosine kinases AXL and MERTK, participates in chronic liver pathologies. Here, we addressed GAS6/TAM involvement in Non-Alcoholic SteatoHepatitis (NASH) development.MethodsGAS6/TAM signaling was analyzed in cultured primary hepatocytes, hepatic stellate cells (HSC) and Kupffer cells (KCs). Axl-/-, Mertk-/- and wild-type C57BL/6 mice were fed with Chow, High Fat Choline-Deficient Methionine-Restricted (HFD) or methionine-choline-deficient (MCD) diet. HSC activation, liver inflammation and cytokine/chemokine production were measured by qPCR, mRNA Array analysis, western blotting and ELISA. GAS6, soluble AXL (sAXL) and MERTK (sMERTK) levels were analyzed in control individuals, steatotic and NASH patients.ResultsIn primary mouse cultures, GAS6 or MERTK activation protected primary hepatocytes against lipid toxicity via AKT/STAT-3 signaling, while bemcentinib (small molecule AXL inhibitor BGB324) blocked AXL-induced fibrogenesis in primary HSCs and cytokine production in LPS-treated KCs. Accordingly; bemcentinib diminished liver inflammation and fibrosis in MCD- and HFD-fed mice. Upregulation of AXL and ADAM10/ADAM17 metalloproteinases increased sAXL in HFD-fed mice. Transcriptome profiling revealed major reduction in fibrotic- and inflammatory-related genes in HFD-fed mice after bemcentinib administration. HFD-fed Mertk-/- mice exhibited enhanced NASH, while Axl-/- mice were partially protected. In human serum, sAXL levels augmented even at initial stages, whereas GAS6 and sMERTK increased only in cirrhotic NASH patients. In agreement, sAXL increased in HFD-fed mice before fibrosis establishment, while bemcentinib prevented liver fibrosis/inflammation in early NASH.ConclusionAXL signaling, increased in NASH patients, promotes fibrosis in HSCs and inflammation in KCs, while GAS6 protects cultured hepatocytes against lipotoxicity via MERTK. Bemcentinib, by blocking AXL signaling and increasing GAS6 levels, reduces experimental NASH, revealing AXL as an effective therapeutic target for clinical practice.

Endostatin attenuates PDGF-BB- or TGF-β1-induced HSCs activation via suppressing RhoA/ROCK1 signal pathways.

AimTo testify the hypothesis that endostatin exerts antifibrotic effects in hepatic stellate cells (HSCs) by modulating RhoA (ras homolog gene family, member A)/ROCK 1 (Rho-associated protein kinase 1) signal pathways.Materials and methodsHSCs-T6 of passages 3–5 were cultured in DMEM and serum starved for 48 hours. HSCs were grouped as follows: control group, TGF-β1 (transforming growth factor β1) group, endostatin+TGF-β1 group, PDGF-BB (platelet-derived growth factor-BB) group, and endostatin+PDGF-BB group. In the PDGF-BB group, HSCs were treated with PDGF-BB (200 ng/mL) for 72 hours; in the TGF-β1 group, they were treated with TGF-β1 (10 ng/mL) for 72 hours. In the Endostatin+TGF-β1 group or Endostatin+PDGF-BB group, HSCs were treated with TGF-β1 (10 ng/mL) or PDGF-BB (200 ng/mL) for 72 hours after pretreatment with endostatin (5 µg/mL) for 1 hour. In the control group, HSCs were only treated with serum-free DMEM for 72 hours. Collagen I was analyzed with ELISA. F-actin was detected with immunofluorescent staining. The mRNAs and proteins of α-smooth muscle actin, RhoA, and ROCK1 were analyzed by using real-time PCR and Western blot, respectively.ResultsTGF-β1 and PDGF-BB promote the proliferation of HSCs significantly at 48 and 72 hours. Endostatin inhibits the proliferation effect induced by TGF-β1 or PDGF-BB significantly (P<0.01). The expression of collagen I and F-actin was significantly upregulated in both TGF-β1 and PDGF-BB groups than in the control group (P<0.01). Both the collagen I and F-actin expression were downregulated significantly in the endostatin-treated groups (P<0.05). Endostatin significantly inhibited the upregulated expression of α-smooth muscle actin, RhoA, and ROCK1 induced by TGF-β1 or PDGF-BB (P<0.01).ConclusionThese results suggested that endostatin inhibited TGF-β1- or PDGF-BB-induced fibrosis in HSCs by modulating RhoA/ROCK signal pathways.

In vitro study on the role and mechanism of interferon α-1b in regulating the inhibition of protein kinase Cε and Cα on fibrosis of hepatic stellate cells

Objective To investigate the regulation of interferon α-1b (IFNα-1b) on protein kinase Ce (PKCe) and protein kinase Cα (PKCα) which inhibit the fibrosis of hepatic stellate cells (HSC), and to explore its mechanism. Methods HSC-T6 cells were treated with different levels of IFNα-1b (100, 200, 400, 800 and 1 000 U/mL) and the proliferation of HSC-T6 cells was analyzed by methyl thiazol tetrazolium (MTT) assay. Changes of hydroxyproline level were analyzed. The expressions of PKCe and PKCα were detected by immunofluorescence staining. PKCe, PKCα, β-catenin and Survivin mRNA levels were detected by RT-PCR. PKCe, PKCα, β-catenin and Survivin protein levels were detected by Western blot. Variance analysis was conducted by using one-way ANOVA approach. Results The inhibition rates of 100, 200, 400, 800 and 1 000 U/mL IFNα-1b treatment after 24 hours of administration were (15.85±1.05)%, (36.59±1.03)%, (45.12±1.05)%, (50.00±1.01)% and (62.20±1.02)%, respectively, with statistically significant differences among groups (F=27.478, P<0.01). The 48h inhibition rates were (20.87±1.09)%, (43.96±1.08)%, (53.85±1.08)%, (64.84±1.06)% and (74.72±1.07)%, respectively, with statistically significant differences among groups (F=25.321, P<0.01). half maximal inhibitory concentration at 48 h was 343.47 U/mL. The levels of hydroxyproline in 100, 200 and 400 U/mL IFNα-1b groups were (7.48±0.28), (6.26±0.17) and (3.86±0.20) μg/mL, respectively, which were lower than that in control group (8.47±0.32) μg/mL. The differences were all statistically significant (t=4.033, 10.564 and 21.160, respective, all P<0.05). The fluorescence intensities of PKCe in 100, 200 and 400 U/mL IFNα-1b groups were all lower than that of control group. The differences were statistically significant (t=1.984, 2.457 and 7.771, respectively, all P<0.05). The fluorescence intensities of PKCα were also significantly lower than that of control group (t=9.232, 15.921 and 22.222, respectively, all P<0.01). With the increase of IFNα-1b level, the levels of HSC-T6 PKCe, PKCα, β-catenin and survivin were significantly lower than those of control group (t=7.020, 24.562, 45.701 and 14.241, respectively, all P<0.01). With the increase of IFNα-1b, the levels of HSC-T6 PKCe, PKCα, β-catenin and survivin were significantly lower than those of control group (t=9.564, 4.409, 10.036 and 6.794, respectively, all P<0.01). Conclusions IFNα-1b can down-regulate the expression of collagen in hepatic stellate cells in a dose-dependent manner, reduce the expressions of PKCe, PKCα, β-catenin and Survivin, and inhibit the proliferation of HSC-T6 hepatic stellate cells. Key words: IFNα-1b; hepatic stellate cells; proliferation; molecular mechanism

Lipopolysaccharide mediates hepatic stellate cell activation by regulating autophagy and retinoic acid signaling

ABSTRACTBacterial translocation and lipopolysaccharide (LPS) leakage occur at a very early stage of liver fibrosis in animal models. We studied the role of LPS in hepatic stellate cell (HSC) activation and the underlying mechanisms in vitro and in vivo. Herein, we demonstrated that LPS treatment led to a dramatic increase in autophagosome formation and autophagic flux in LX-2 cells and HSCs, which was mediated through the AKT-MTOR and AMPK-ULK1 pathway. LPS significantly decreased the lipid content, including the lipid droplet (LD) number and lipid staining area in HSCs; pretreatment with macroautophagy/autophagy inhibitors or silencing ATG5 attenuated this decrease. Furthermore, lipophagy was induced by LPS through the autophagy-lysosomal pathway in LX-2 cells and HSCs. Additionally, LPS-induced autophagy further reduced retinoic acid (RA) signaling, as demonstrated by a decrease in the intracellular RA level and Rar target genes, resulting in the downregulation of Bambi and promoting the sensitization of the HSC's fibrosis response to TGFB. Compared with CCl4 injection alone, CCl4 plus LPS injection exaggerated liver fibrosis in mice, as demonstrated by increased Col1a1 (collagen, type I, α 1), Acta2, Tgfb and Timp1 mRNA expression, ACTA2/α-SMA and COL1A1 protein expression, and Sirius Red staining area, which could be attenuated by injection of an autophagy inhibitor. LPS also reduced lipid content in HSCs in vivo, with this change being attenuated by chloroquine (CQ) administration. In conclusion, LPS-induced autophagy resulted in LD loss, RA signaling dysfunction, and downregulation of the TGFB pseudoreceptor Bambi, thus sensitizing HSCs to TGFB signaling.

Human bone marrow mesenchymal stem cells suppress the proliferation of hepatic stellate cells by inhibiting the ubiquitination of p27.

Bone marrow mesenchymal stem cells (BMSCs) have considerable therapeutic potential for the treatment of end-stage liver disease. Previous studies have demonstrated that BMSCs secrete growth factors and cytokines that inactivate hepatic stellate cells (HSCs), which inhibited the progression of hepatic fibrosis. The aim of this study was to determine the mechanism by which BMSCs suppress the function of HSCs in fibrosis. Our results showed that co-culture of BMSCs and HSCs induced cell cycle arrest at the G10/G1 phase and cell apoptosis of HSCs, which finally inhibited the cell proliferation of HSCs. Consistent with the cell cycle arrest, co-culture of BMSCs and HSCs increased the abundance of the cell cycle protein p27. Mechanistically, we further uncovered that following the co-culture with BMSCs, the expression level of the E3 ligase S-phase kinase-associated protein 2 (SKP2) that is responsible for the ubiquitination of p27 was decreased, which attenuated the ubiquitination of p27 and increased the stability of p27 in HSCs. Collectively, our results indicated the potential involvement of the SKP2-p27 axis for the inhibitory effect of BSMCs on the cell proliferation of HSCs.

Smad7-overexpressing rat BMSCs inhibit the fibrosis of hepatic stellate cells by regulating the TGF-β1/Smad signaling pathway.

Mesenchymal stem cells (MSCs) are able to differentiate into hepatocytes, promote the regeneration of hepatic cells and inhibit the progression of hepatic fibrosis. Transforming growth factor (TGF)-β1 is one of the key factors in the development of liver fibrosis, which also promotes extracellular matrix (ECM) formation. Drosophila mothers against decapentaplegic 7 (Smad7) is an essential negative regulator in the TGF-β1/Smad signaling pathway. In the present study, bone mesenchymal stem cells (BMSCs) were isolated from rat bone marrow and transfected with lentiviral vectors carrying the Smad7 gene. Smad7-enhanced green fluorescent protein (EGFP)-BMSCs stably expressing Smad7 were subsequently co-cultured with hepatic stellate cells (HSCs) for 48 h. Smad7 and TGF-β1 levels in the culture medium were detected using ELISA, and the levels of collagen (Col) I, Col III, laminin (LN) and hyaluronic acid (HA) were measured using immunoassays. The early apoptosis rates of HSCs were determined via flow cytometry. Reverse transcription-quantitative polymerase chain reaction and western blotting were performed to evaluate the mRNA and protein expression profiles, respectively. The results indicated that Smad7-EGFP-BMSCs stably expressing Smad7 were successfully constructed. Upon co-culturing with rat Smad7-EGFP-BMSCs, the early apoptotic rate of HSCs was significantly increased (P<0.05). Levels of Smad7 in the culture medium were also significantly increased (P<0.05), whereas the levels of TGF-β1, Col I, Col III, LN and HA were significantly decreased (P<0.05). Furthermore, the mRNA and protein levels of Smad7 and matrix metalloproteinase 1 were significantly increased (P<0.05), whereas those of TGF-β1, α-SMA, Smad2, smad3, TGF-β receptor I, Col I, tissue inhibitors of metalloproteinase-1 and Col III were significantly decreased. The results of the present study suggest that rat BMSCs overexpressing Smad7 may inhibit the fibrosis of HSCs by regulating the TGF-β1/Smad signaling pathway. This provides a novel insight into future treatments for liver fibrosis.

Scoparone attenuates hepatic stellate cell activation through inhibiting TGF-β/Smad signaling pathway

Activation of hepatic stellate cells (HSCs) plays a critical role in liver fibrosis. Scoparone, a major constituent isolated from Artemisia capillaris, was reported to possess hepatoprotective effect. However, the role of scoparone in liver fibrosis remains unknown. In the present study, we investigated the effects of scoparone on liver fibrosis in HSCs. Our results demonstrated that scoparoene inhibited the proliferation of HSCs exposed to transforming growth factor (TGF)-β1. In addition, scoparoene significantly suppressed TGF-β1-induced the expression of α-smooth muscle actin (α-SMA) and collagen I in HSC-T6 cells, as well as attenuated the expression of NADPH oxidase (NOX) isoforms expression and ROS production in TGF-β1-stimialted HSC-T6 cells. Mechanistically, scoparoene suppressed the phosphorylation level of Smad3 in TGF-β1-stimialted HSC-T6 cells. In conclusion, these findings showed that scoparone significantly inhibited the proliferation and activation of HSCs through the inactivation of TGF-β/Smad signaling pathway. These data provide that scoparone may have potential to treat liver fibrosis.

The Microbial Metabolite Sensor Pregnane X Receptor (PXR) Restrains Fibroblasts from Promoting Gastrointestinal Inflammation and Fibrosis in Mice

INTRODUCTIONFibrosis contributes to intestinal strictures and obstruction in 30–50% of Crohn's disease patients. The pathogenesis of intestinal fibrosis is incompletely understood and has virtually no effective treatments. The pregnane X receptor (PXR), a xenobiotic receptor involved in detoxification responses, has recently been identified as a modulator of fibrosis in hepatic stellate cells. Of the number of foreign ligands for the PXR, the tryptophan metabolite indole‐3‐propionic acid (IPA) produced by the intestinal commensal Clostridium sporogenes can bind to the PXR to mediate signalling events that protect intestinal barrier function. The role of the PXR in intestinal fibrosis and if microbial metabolite sensing can affect intestinal fibrotic responses is unknown.AIMSTo examine the role of the PXR and its ability to sense microbial metabolites in the modulation of intestinal fibrosis.METHODSIntestinal inflammation was induced using DSS (3.5%) for 5 days followed by healing for 25 days. Fibrosis was assessed using Masson‐trichrome and Serius Red staining of histological sections. Mouse primary fibroblasts were grown from wild type (WT) and PXR−/minus; mice. Fibroblasts were stimulated with cytomix (TNF‐α, IL‐1β, and IFN‐γ) for 24 hours after which cell supernatants were assessed for cytokine concentrations using Luminex. Fibroblasts were also stimulated with cytomix in the presence of the PXR agonists pregnenolone 16α‐carbonitrile (PCN) or IPA and assessed for gene expression using qPCR. NFκB activity was assessed via Western blot for the p65 subunit. To examine the microbiota's role in fibrosis, the microbiota was depleted after the 5‐day course of DSS for 25 days using an antibiotic cocktail of vancomycin, neomycin, metronidazole and ampicillin.FINDINGSFollowing a 25‐day recovery after DSS, WT mice showed clear evidence of fibrosis as indicated by Masson‐trichrome and Serius Red staining. When compared to WT mice, PXR−/− mice demonstrated significantly greater levels of fibrosis. Mouse primary fibroblasts were found to express the PXR, and PXR−/− fibroblasts displayed morphological differences compared to WT fibroblasts. Following stimulation with cytomix, PXR−/− fibroblasts produced dramatically higher levels of proinflammatory cytokines including eotaxin, CXCL2, GM‐CSF, G‐CSF and IL‐9. Importantly, PCN and the microbial metabolite IPA were able to block the expression of these cytokines. These effects in PXR−/− fibroblasts may be linked to the increased activity of NFκB that was observed both basally and after stimulation with cytomix compared to WT fibroblasts. Depletion of the microbiota led to exacerbated intestinal fibrosis.CONCLUSIONSPXR signaling in intestinal fibroblasts appears to be required to restrain inflammation and fibrosis. Luminal sensing of bacterial derived indoles (ie. IPA) via PXR may be involved in this process, highlighting a xenobiotic receptor‐microbiota axis that could be targeted to prevent the intestinal fibrosis observed in Crohn's disease.Support or Funding InformationCrohn's &amp; Colitis Canada, Dr. Keith Sharkey's CCFC Chair in IBD Research, The Dr. Lloyd Sutherland Investigator in IBD/GI Research, Canadian Foundation for Innovation, Canada Research Chairs Program, US Department of Defense, Beverley Philips Rising Star Fellowship.

Anti-fibrotic effects of &lt;i&gt;Rhus javanica&lt;/i&gt; Linn (Anacardiaceae) extract against Activated hepatic stellate cells via regulation of TGF-beta and smad signaling

Purpose: To evaluate the anti-fibrotic effects of ethanol extract of Rhus javanica Linn. (Anacardiaceae) (RJE) in activated hepatic stellate cells (HSCs) as well as explore the underlying mechanisms.Methods: The cytotoxic effect of RJE (100, 300 and 500 μg/mL) was analyzed using 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT) assay in Chang liver cells. The mRNA expression of collagen type I, alpha 2 (COL1A2), transforming growth factor-beta (TGF-β), α-smooth muscle actin (α-SMA) and platelet-derived growth factor (PDGF) were determined using reverse transcription-polymerase chain reaction (RT-PCR) in HSCs. Protein expression of collagen and Smad were measured by Western blot analysis.Results: Treatment with RJE extract at 100, 300 and 500 μg/mL did not show any signs of cytotoxicity to Chang liver cells. RJE at 500 μg/mL concentration influenced the morphology, reduced the stretched fiber and decreased the number of viable cells in activated HSCs. The increased expressional levels of fibrosis mediators such as COL1A2, TGF-β, α-SMA were decreased by RJE (500 μg/mL) pre-treatment. Quantification data showed that the increased band intensity of COL1A2 (1.41 ± 0.08), TGF-β (1.23 ± 0.13), α-SMA (1.71 ± 0.14) were significantly (p &lt; 0.05) reduced to 0.39 ± 0.12, 0.35 ± 0.11 and 0.04 ± 0.08, respectively upon RJE treatment. However, RJE did not suppress the expression of PDGF gene. Mechanistic study revealed that RJE prevented fibrosis in HSCs via regulation of TGF-β and Smad signaling pathways.Conclusion: The findings show that RJE inhibits fibrosis production in HSCs and can be developed as a novel therapy for hepatic fibrosis. This is the first report showing the beneficial effects of R. javanica as an anti-fibrotic agent.