Induction Of Hepatic Stellate Cells Apoptosis Research Articles

Network Pharmacology, Molecular Docking Analysis and Molecular Dynamics Simulation of Scutellaria baicalensis in the Treatment of Liver Fibrosis.

Traditional Chinese medicine Scutellaria Baicalensis (SB), one of the clinical firstline heat-clearing drugs, has obvious symptomatic advantages for hepatic fibrosis with dampness-heat stasis as its syndrome. We aim to predict and validate the potential mechanism of Scutellaria baicalensis active ingredients against liver fibrosis more scientifically and effectively. The underlying mechanism of Scutellaria baicalensis in inhibiting hepatic fibrosis was studied by applying network pharmacology, molecular docking and molecular dynamics simulation. Expression levels of markers in activated Hepatic Stellate Cells (HSC) after administration of three Scutellaria baicalensis extracts were determined by Western blot and Real-time PCR, respectively, in order to verify the anti-fibrosis effect of the active ingredients Results: There are 164 common targets of drugs and diseases screened and 115 signaling pathways obtained, which were mainly associated with protein phosphorylation, senescence and negative regulation of the apoptotic process. Western blot and Real-time PCR showed that Scutellaria baicalensis extracts could reduce the expression of HSC activation markers, and Oroxylin A had the strongest inhibitory effect on it. Molecular docking results showed that Oroxylin A had high binding activity to target proteins. Molecular dynamics simulation demonstrates promising stability of the Oroxylin A-AKT1 complex over the simulated MD time of 200 ns. Scutellaria baicalensis active ingredients may inhibit HSC proliferation, reduce the generation of pro-inflammatory factors and block the anti-inflammatory effect of inflammatory signal transduction by inducing HSC apoptosis and senescence, thus achieving the effect of anti-fibrosis.

Ginkgetin exhibits antifibrotic effects by inducing hepatic stellate cell apoptosis via STAT1 activation.

Liver fibrosis affects approximately 800 million patients worldwide, with over 2 million deaths each year. Nevertheless, there are no approved medications for treating liver fibrosis. In this study, we investigated the impacts of ginkgetin on liver fibrosis and the underlying mechanisms. The impacts of ginkgetin on liver fibrosis were assessed in mouse models induced by thioacetamide or bile duct ligation. Experiments on human LX-2 cells and primary mouse hepatic stellate cells (HSCs) were performed to explore the underlying mechanisms, which were also validated in the mouse models. Ginkgetin significantly decreased hepatic extracellular matrix deposition and HSC activation in the fibrotic models induced by thioacetamide (TAA) and bile duct ligation (BDL). Beneficial effects also existed in inhibiting hepatic inflammation and improving liver function. In vitro experiments showed that ginkgetin markedly inhibited HSC viability and induced HSC apoptosis dose-dependently. Mechanistic studies revealed that the antifibrotic effects of ginkgetin depend on STAT1 activation, as the effects were abolished in vitro after STAT1 silencing and in vivo after inhibiting STAT1 activation by fludarabine. Moreover, we observed a meaningful cross-talk between HSCs and hepatocytes, in which IL-6, released by ginkgetin-induced apoptotic HSCs, enhanced hepatocyte proliferation by activating STAT3 signaling. Ginkgetin exhibits antifibrotic effects by inducing HSC apoptosis via STAT1 activation and enhances hepatocyte proliferation secondary to HSC apoptosis via the IL-6/STAT3 pathway.

Berberine attenuates liver fibrosis by autophagy inhibition triggering apoptosis via the miR-30a-5p/ATG5 axis

Berberine (BBR) is an effective drug against liver fibrosis (LF). Autophagy is involved in the pathogenesis of LF; however, the mechanism linking BBR to autophagy in LF remains unresolved. To explore the underlying mechanism, we assessed the effects of BBR on autophagy and apoptosis of activated hepatic stellate cells (HSCs) in vitro and in a murine model of fibrosis. The decreased expression of the autophagy activation marker ATG5, autophagosome formation, and autophagy flux in the HSC model confirmed that BBR inhibited autophagy in activated HSCs and in mice with liver fibrosis. Moreover, ATG5 was necessary for inducing autophagy and HSC activation. BBR suppressed ATG5 expression by upregulating miR-30a-5p expression, which affected the stability of ATG5 mRNA by binding to its 3′-untranslated region, an effect that was attenuated by treatment with a miR-30a-5p inhibitor. BBR also markedly induced HSC apoptosis, as indicated by the upregulated expression of the pro-apoptosis markers p53, BAX, and cleaved PARP and the downregulated expression of the anti-apoptosis marker BCL-2, effects that were reversed by ATG5 overexpression. In vivo, BBR improved mouse LF by decreasing collagen deposition, inflammatory cell infiltration, and expression of fibrosis markers hydroxyproline, α-smooth muscle actin, and collagen type 1-A1 and the autophagy marker LC3. BBR had a protective effect on mouse fibrotic livers and reduced serum aspartate aminotransferase and alanine aminotransferase levels. Collectively, these results reveal a novel mechanism of BBR-induced autophagy inhibition triggering apoptosis in HSCs, providing a reliable experimental and theoretical basis for developing BBR-based candidate drugs for LF.

Inhibition of hepatic natural killer cell function via the TIGIT receptor in schistosomiasis-induced liver fibrosis.

Schistosomiasis is a zoonotic parasitic disease. Schistosoma japonicum eggs deposited in the liver tissue induce egg granuloma formation and liver fibrosis, seriously threatening human health. Natural killer (NK) cells kill activated hepatic stellate cells (HSCs) or induce HSC apoptosis and inhibit the progression of liver fibrosis. However, the function of NK cells in liver fibrosis caused by S. japonicum infection is significantly inhibited. The mechanism of this inhibition remains unclear. Twenty mice were percutaneously infected with S. japonicum cercariae. Before infection and 2, 4, 6, and 8 weeks after infection, five mice were euthanized and dissected at each time point. Hepatic NK cells were isolated and transcriptome sequenced. The sequencing results showed that Tigit expression was high at 4-6 weeks post infection. This phenomenon was verified by reverse transcription quantitative PCR (RT-qPCR) and flow cytometry. NK cells derived from Tigit-/- and wild-type (WT) mice were co-cultured with HSCs. It was found that Tigit-/- NK cells induced apoptosis in a higher proportion of HSCs than WT NK cells. Schistosomiasis infection models of Tigit-/- and WT mice were established. The proportion and killing activity of hepatic NK cells were significantly higher in Tigit-/- mice than in WT mice. The degree of liver fibrosis in Tigit-/- mice was significantly lower than that in WT mice. NK cells were isolated from Tigit-/- and WT mice and injected via the tail vein into WT mice infected with S. japonicum. The degree of liver fibrosis in mice that received NK cell infusion reduced significantly, but there was no significant difference between mice that received NK cells from Tigit-/- and WT mice, respectively. Our findings indicate that Tigit knockout enhanced the function of NK cells and reduced the degree of liver fibrosis in schistosomiasis, thus providing a novel strategy for treating hepatic fibrosis induced by schistosomiasis.

LncRNA CCAT2, involving miR-34a/TGF-β1/Smad4 signaling, regulate hepatic stellate cells proliferation.

miR-34a targeting on Smad4 plays important role in TGF-β1 pathway which is a dominant factor for balancing collagen production and degradation in hepatic stellate cells. TGF-β1/Smad4 regulated collagen deposition is a hallmark of hepatic fibrosis. The potential regulation on miR-34a by LncRNAs in hepatic stellate cells (HSCs) is still reserved to be revealed. In current study, it was hypothesized that a miR-34a interactor, lncRNA CCAT2 may regulate TGF-β1 pathway in liver fibrotic remodeling. The interaction between CCAT2 and miR-34a-5p was checked by dual luciferase assay. the effects of CCAT2 and miR-34a-5p on cell proliferation and apoptosis were verified by MTT assay, colony formation assay, and flow cytometry assay. Dual luciferase activity showed CCAT2 are targets of miR-34a-5p. Sh-CCAT2 transfection prohibit HSCs proliferation and induce HSCs apoptosis, also inhibited ECM protein synthesis in HSCs. Decreased miR-34a-5p enhanced HSCs proliferation, blocked HSCs apoptosis and promoted ECM protein production. miR-34a-5p inhibitor undo protective regulation of sh-CCAT2 in liver fibrosis. Furthermore, clinical investigation showed that CCAT2 and Smad4 expression level were significantly induced, while miR-34a-5p was significantly decreased in HBV related liver fibrosis serum. In conclusion, activated HSCs via TGF-β1/Smad4 signaling pathway was successfully alleviated by CCAT2 inhibition through miR-34a-5p elevation.

Germacrone Attenuates Hepatic Stellate Cells Activation and Liver Fibrosis via Regulating Multiple Signaling Pathways.

Liver fibrosis is an abnormal proliferation of connective tissue in the liver caused by various pathogenic factors. Chronic liver injury leads to release of inflammatory cytokines and reactive oxygen species (ROS) from damaged hepatocytes, which activates hepatic stellate cells (HSCs) to secrete extracellular matrix proteins, thereby leading to fibrosis. Thus, inhibition of hepatocyte injury and HSC activation, and promotion of apoptosis of activated HSCs are important strategies for prevention of liver fibrosis. In this study, we showed that the germacrone (GER), the main component in the volatile oil of zedoary turmeric, inhibited hepatic fibrosis by regulating multiple signaling pathways. First, GER improved the cell survival rate by inhibiting the production of ROS after hepatocyte injury caused by acetaminophen (APAP). In addition, GER inhibited the activation of HSCs and expression of collagen I by blocking TGF-β/Smad pathway in LX-2 cells. However, when the concentration of GER was higher than 60 μM, it specifically induced HSCs apoptosis by promoting the expression and activation of apoptosis-related proteins, but it had no effect on hepatocytes. Importantly, GER significantly attenuated the methionine- and choline-deficient (MCD) diet-induced liver fibrosis by inhibiting liver injury and the activation of HSCs in vivo. In summary, GER can not only protect hepatocytes by reducing ROS release to avoid the liver injury-induced HSC activation, but also directly inhibit the activation and survival of HSCs by regulating TGF-β/Smad and apoptosis pathways. These results demonstrate that GER can be used as a potential therapeutic drug for the treatment of liver fibrosis.

Tunicamycin Induces Hepatic Stellate Cell Apoptosis Through Calpain-2/Ca2 +-Dependent Endoplasmic Reticulum Stress Pathway.

It has been reported that calpain/caspase-mediated apoptosis induced by endoplasmic reticulum stress (ERS) in hepatic stellate cells (HSCs) by previous studies. At present, the activation of HSC is an important cause of liver fibrosis, and the induction of HSC apoptosis plays an irreplaceable role in reversing liver fibrosis. Therefore, it is of great significance to explore mechanisms of action that can induce HSC apoptosis for the reversal of hepatic fibrosis and the clinical prevention and treatment of hepatic-fibrosis-related diseases such as hepatitis, cirrhosis, and liver cancer. In the current study, we demonstrated that tunicamycin (a novel ERS inducer) can induce the apoptosis of HSCs and increase the concentration of intracellular Ca2+ and the expression of ERS protein GRP78, apoptosis protein caspase-12, and Bax, while it can decrease the antiapoptosis protein expression of Bcl-2. Our findings indicate that tunicamycin can induce HSCs apoptosis through calpain-2/Ca2+-dependent ERS pathway.

Therapeutic Effects of an Inhibitor of Thioredoxin Reductase on Liver Fibrosis by Inhibiting the Transforming Growth Factor-β1/Smads Pathway.

Liver fibrosis is an important stage in the progression of liver injury into cirrhosis or even liver cancer. Hepatic stellate cells (HSCs) are induced by transforming growth factor-β1 (TGF-β1) to produce α-smooth muscle actin (α-SMA) and collagens in liver fibrosis. Butaselen (BS), which was previously synthesized by our group, is an organic selenium compound that exerts antioxidant and tumor cell apoptosis–promoting effects by inhibiting the thioredoxin (Trx)/thioredoxin reductase (TrxR) system. The aim of this study was to investigate the potential effects of BS on liver fibrosis and explore the underlying molecular mechanisms of its action. Liver fibrosis models were established using male BALB/c mice through intraperitoneal injection of CCl4. BS was administered orally once daily at a dose of 36, 90, or 180 mg/kg. Silymarin (Si), which is a drug used for patients with nonalcoholic fatty liver disease and nonalcoholic steatohepatitis, was administered at a dose of 30 mg/kg per day as a control. The action mechanisms of BS against liver fibrosis progression were examined in HSCs. The study revealed that the activity and expression levels of TrxR were elevated in the mouse liver and serum after CCl4-induced liver fibrosis. Oral administration of BS relieved the pathological state of mice with liver fibrosis, showing significant therapeutic effects against liver fibrosis. Moreover, BS not only induced HSC apoptosis but also inhibited the production of α-SMA and collagens by HSCs by downregulating the TGF-β1 expression and blocking the TGF-β1/Smads pathway. The results of the study indicated that BS inhibited liver fibrosis by regulating the TGF-β1/Smads pathway.

CaMK II/Ca2+ dependent endoplasmic reticulum stress mediates apoptosis of hepatic stellate cells stimulated by transforming growth factor beta 1

Previous studies by our group have demonstrated that the calcium imbalance in rat hepatic stellate cells (HSCs) can induce endoplasmic reticulum stress (ERS) and promote cell apoptosis. KN-62, an inhibitor of Calmodulin kinase II (CaMK II), can decrease the expression of CaMK II that plays a major role in regulating the steady state of intracellular Ca2+. Uridine triphosphate (UTP) plays a biological role in increasing indirectly the level of intracellular Ca2+. In the experiment, we demonstrate that KN-62 and UTP can inhibit the proliferation and promote the apoptosis in HSCs, increase the level of intracellular Ca2+ and the expression of ERS protein GRP78, and increase the apoptosis protein Caspase-12 and Bax expression, while decrease the expression of Bcl-2 protein. Our findings indicate that the CaMK II/Ca2+ signaling pathway regulates the ERS apoptosis pathway and induces HSC apoptosis.

4-Methoxy Sulfonyl Paeonol Inhibits Hepatic Stellate Cell Activation and Liver Fibrosis by Blocking the TGF-β1/Smad, PDGF-BB/MAPK and Akt Signaling Pathways

Liver fibrosis initiates the progression of cirrhosis, and, finally, hepatocellular carcinoma (HCC). The increased proliferation and activation of hepatic stellate cells (HSCs) are crucial for hepatic fibrogenesis. Paeonol is the major vigorous component of Cortex Moutan, a traditional herbal medicine widely used for treating various diseases. Here, we identified a novel paeonol derivative (4-methoxy sulfonyl paeonol, 4-MSP) that inhibits TGF-β1-induced Smad2/3 phosphorylation and collagen expression in HSCs. 4-MSP pretreatment suppressed the PDGF-BB–induced phosphorylation of MAPK pathway members (MEK/ERK, p38, JNK), Akt/p70S6K, and HSC proliferation. However, 4-MSP treatment had no effect on the induction of apoptosis in HSCs. The microarray experiments showed that 4-MSP treatment affects the TGF-β signaling, MAPK cascade, and other pathways related to HSCs activation and proliferation. The administration of 4-MSP to a liver fibrosis mouse model induced by CCl4 significantly decreased the expression of hepatic fibrosis markers (α-SMA, col1A2, TGF-β, and MMP2), and attenuated hepatic collagen deposition and liver damage. In addition, no adverse effects were observed in 4-MSP exposed mice. In conclusion, this novel paeonol-phenylsulfonyl derivative prevents the progression of liver fibrosis through blocking TGF-β1/Smad, PDGF-BB/MAPK, and Akt signaling, which suggests its use as a novel therapeutic against liver fibrosis.

Malvidin induces hepatic stellate cell apoptosis via the endoplasmic reticulum stress pathway and mitochondrial pathway.

Blueberries have great beneficial effects due to high level of anthocyanins, especially malvidin. Hepatic stellate cells (HSCs) can be activated and increase excessive extracellular matrix (ECM) components, which play a central role in liver fibrogenesis. Therefore, activated HSC’s apoptosis can be induced to recover liver fibrosis. Malvidin's effects on apoptosis in rat activated hepatic stellate T6 cells (HSC‐T6) in vitro were investigated here. High concentration of malvidin was found to significantly induce apoptosis, activate caspase‐3, increase malondialdehyde, upregulate Bax, but downregulate Bcl‐2. Moreover, malvidin upregulated the protein levels of some endoplasmic reticulum stress (ERS)‐typical markers, including caspase‐12, glucose‐regulated protein 78 (GRP78), and CCAAT/enhancer‐binding protein (C/EBP) homologous protein (CHOP), suggesting that malvidin induced HSC apoptosis by the ERS apoptosis pathway as well as the mitochondrial‐dependent pathway. These findings indicated that blueberry anthocyanins, especially malvidin, could induce activated hepatic stellate cell apoptosis and might act as one kind of functional food ingredient or a novel nutraceutical beneficial for liver health.

Light-triggered NO-releasing nanoparticles for treating mice with liver fibrosis

Liver fibrosis, resulting from chronic liver damage and characterized by the accumulation of extracellular matrix (ECM) proteins, is a characteristic of most types of chronic liver diseases. The activation of hepatic stellate cells (HSC) is considered an essential pathological hallmark in liver fibrosis. Although nitric oxide (NO) can effectively induce HSC apoptosis, the systemic administration of NO is ineffective and may cause severe complications such as hypotension. To overcome this limitation, nanoparticles were designed to target HSCs and release NO locally under the exposure of near infrared light (NIR). To achieve this, upconversion nanoparticle (UCNP) cores were enveloped in mesoporous silica shells (UCNP@mSiO2), which were modified with hyaluronic acid (HA-UCNP@mSiO2) and Roussin’s black salt (RBS). HA molecules recognize and bind to CD44 proteins, which are overexpressed on activated HSCs. Under exposure to a 980-nm NIR laser, the UCNP cores convert the 980-nm wavelength into ultraviolet (UV) light, which then energizes the RBS (NO donors), resulting in an efficient release of NO inside of the HSCs. Once released, NO triggers HSC apoptosis and reverses the liver fibrosis. This targeted and controlled release method provides the theoretical and experimental basis for novel therapeutic approaches to treat hepatic fibrosis.

α-Lipoic acid modulates liver fibrosis: A cross talk between TGF-β1, autophagy, and apoptosis.

Autophagy and apoptosis are important players in the progression of hepatic fibrosis via activation of hepatic stellate cells (HSCs). Despite the recently depicted antifibrotic effects of alpha-lipoic acid (ALA), however, its modulatory effects on HSCs autophagy remain unverified. Our study aimed to elucidate the underlying antifibrotic mechanisms through which ALA mediates HSC autophagy and apoptosis. Liver fibrosis was induced via thioacetamide (TAA) intoxication in rats; TAA-intoxicated rats were treated with either silymarin or ALA. Effect of ALA on biochemical parameters and immunohistopathological examinations was measured and compared to silymarin. ALA restored normal hepatic architecture (S1 vs. S4), liver functions, hepatic glutathione, and transforming growth factor-β1 levels. ALA ameliorated hepatic levels of malondialdehyde, platelet-derived growth factor, tissue inhibitor metalloproteinases-1, hydroxyproline, and expression of alpha-smooth muscle actin. Moreover, ALA significantly reduced messenger RNA expression of LC3-II genes and triggered caspase-3 expression. Interestingly, ALA exhibited superior activities over silymarin regarding suppression of proliferation, activation and autophagy of HSCs, collagen deposition, and induction of HSCs apoptosis. In conclusion, treatment of TAA-intoxicated rats with ALA inhibited autophagy and induced apoptotic clearance of activated HSCs. Accordingly, this study provides mechanistic insights into the possible applicability of ALA in the treatment of hepatic fibrosis.

Oroxylin A induces apoptosis of activated hepatic stellate cells through endoplasmic reticulum stress.

Hepatic stellate cell (HSC) activation plays an indispensable role in hepatic fibrosis. Inducing apoptosis of activated HSCs can attenuate or reverse fibrogenesis. In this study, we initially found that oroxylin A (OA) protected CCl4-induced liver injury accompanied by endoplasmic reticulum stress (ERS) activation of HSCs in mice. In vitro, OA treatment markedly reduced fibrogenesis by modulating extracellular matrix synthesis and degradation. OA inhibited cell proliferation and induced cell cycle arrest of HSCs at S phase. Further, OA was observed to induce HSC apoptosis, as indicated by caspase activation. Using the eIF2α dephosphorylation inhibitor salubrinal, we found that ERS pathway activation was required for OA to induce HSC apoptosis. ERS-related proteins were significantly upregulated by OA treatment, and salubrinal abrogated the effects of OA on HSCs. Thus, we inferred that OA attenuated HSC activation by promoting ERS. In vivo, inhibition of ERS by salubrinal partly abrogated the hepatoprotective effect of OA in CCl4-treated mice. In conclusion, our findings suggest a role for ERS in the mechanism underlying amelioration of hepatic fibrosis by OA.

Bone marrow-derived mesenchymal stem cells utilize the notch signaling pathway to induce apoptosis of hepatic stellate cells via NF-κB sensor.

The present study aimed at evaluating the mechanism by which functionality of hepatic stellate cells (HSCs) is modulated by bone marrow stromal cells (BMSCs). Induction of apoptosis in HSCs was found to be caused by directly co-culturing HSCs with BMSCs, where the expression of α-smooth muscle actin (α-SMA) increased significantly in HSCs, along with an increase in their proliferation rate. Additionally, expression of Hes1 and Notch1 in HSCs co-cultured with BMSCs increased significantly at both protein and mRNA levels. Blocking of the notch signaling pathway (NSP) either by Notch1 siRNA or by DAPT treatment increased the proliferation rate while decreasing apoptosis and led to activation of the NF-κB signaling pathway in HSCs co-cultured with BMSCs. These effects were found to be reversed in HSCs overexpressing IκB S32/S36 mutants. The Notch signaling-mediated cell-cell contact was partially involved in the significant inhibition of proliferation of HSCs by BMSCs. Additionally, the NF-κB pathway was found to be responsible for NSP-mediated inhibition of growth of HSCs in the co-culture system. Thus, BMSCs might have a potential therapeutic significance in treating hepatic fibrosis.

Hepatic Stellate Cells Play a Functional Role in Exacerbating Ischemia-Reperfusion Injury in Rat Liver

Purpose: The involvement of hepatic stellate cells (HSCs) with ischemia-reperfusion (I/R) injury in rat liver was examined using gliotoxin, which is known to induce HSC apoptosis. Methods: Male Sprague-Dawley rats were used. HSC was represented by a glial fibrillary acidic protein (GFAP)-positive cell. Liver ischemia was produced by cross-clamping the hepatoduodenal ligament. The degree of I/R injury was evaluated by a release of aminotransferases. Sinusoidal diameter and sinusoidal perfusion rates were examined using intravital fluorescence microscopy. Results: Gliotoxin significantly decreased the number of GFAP-positive cells 48 h after dosing (2.50 ± 0.19% [mean ± SD] in the nontreated group vs. 1.91 ± 0.46% in the gliotoxin-treated group). Liver damage was significantly suppressed by the pretreatment with gliotoxin. Sinusoidal diameters in zone 3 were wider in the gliotoxin group (10.25 ± 0.35 µm) than in the nontreated group (8.21 ± 0.50 µm). The sinusoidal perfusion rate was maintained as well in the gliotoxin group as in normal livers, even after I/R. Conclusions: Pretreatment with gliotoxin significantly reduced the number of HSCs in the liver and further suppressed liver injury following I/R. It is strongly suggested that HSCs play a functional role in exacerbating the degree of I/R injury of the liver.

Heme oxygenase-1 exerts pro-apoptotic effects on hepatic stellate cells in vitro through regulation of nuclear factor-κB.

Heme oxygenase-1 (HO-1) is an antioxidant and cytoprotective protein, which has been proven to alleviate the proliferation of hepatic stellate cells (HSCs) and the development of liver fibrosis. However, the role of HO-1 in HSC apoptosis remains unclear. The aim of the present study was to investigate the effect of HO-1 on HSC apoptosis and its possible underlying mechanisms. HSCs-T6 were incubated with different concentrations of hemin (HO-1 chemical inducer) and Znpp-IX (HO-1 chemical inhibitor) for 12, 24 and 48 h. Cell viability was determined using an MTT assay. HSCs were classified into 4 groups as follows: Control, hemin, Znpp-IX and hemin+Znpp-IX co-treatment groups. Apoptosis was quantitatively measured by Annexin V/propidium iodide double staining and a terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. The mRNA and protein expression of HO-1, α-smooth muscle actin, B-cell lymphoma (Bcl)-2, caspase-3 and nuclear factor (NF)-κB p65 were measured using quantitative polymerase chain reaction and western blotting. The levels of tumor growth factor (TGF)-β and interleukin (IL)-6 in HSC supernatants were examined by ELISA. The results demonstrated that HO-1 exerted antiproliferative effects on HSCs in a time- and concentration-dependent manner. Increasing HO-1 expression induced HSC apoptosis in vitro as demonstrated by a significant decrease in Bcl-2 and an increase in caspase-3 expression. Additionally, the expression of NF-κB p65 and its downstream inflammatory factors TGF-β and IL-6 in the HO-1 overexpression group was significantly decreased compared with the control group. Therefore, the present study provided evidence that HO-1 serves an anti-fibrosis role in the liver by enhancing HSC apoptosis, which was partially associated with the regulation of NF-κB and its downstream effectors.

Oleanolic Acid-amino Acids Derivatives: Design, Synthesis, and Hepatoprotective Evaluation In Vitro and In Vivo.

Activated hepatic stellate cells (HSCs) are the main extracellular matrix (ECM)-producing cells in the injured liver and the key mediators of liver fibrosis; they also promote the progression of hepatocellular carcinoma (HCC). In the acidic extracellular microenvironment of HCC, HSCs are activated to promote the migration of HCC cells. It is worth attempting to alter the weak acidic microenvironment to promote activated HSC apoptosis to treat liver fibrosis and liver cancer. In the present study, a series of novel OA-amino acids analogues were designed and synthesized to introduce different amino acids in the 3-hydroxyl of OA using the ester condensation reaction to enhance hydrophilicity, alkalinity, and biological activity. We found that OA-lysine derivative (3g) could improve the hydrophilic of OA and induce HSCs apoptosis via inducing MMP depolarization and increasing intracellular Ca2+ levels. Additionally, 3g displayed a better hepatoprotective effect than OA (20 mg/kg, intragastric administration) against the acute liver injury induced by carbon tetrachloride (CCl4) in mice. The results suggested that basic amino acids (lysine) could effectively enhance OA’s hydrophilicity, alkalinity, and hepatoprotective activity in vitro and in vivo, which might be likely associated with increasing bioavailability and altering an extracellular weak acidic microenvironment with further verification. Therefore, the OA-lysine derivative (3g) has the potential to be developed as an agent with hepatoprotective activity.

MiR-185 Inhibits Fibrogenic Activation of Hepatic Stellate Cells and Prevents Liver Fibrosis.

Recent studies have shown the effect of microRNAs on HSC activation and transformation, which is essential for the pathogenesis of liver fibrosis. In our study, we explored the role of miR-185 in liver fibrosis. Plasma miR-185 was detected in hepatitis B virus-related liver fibrosis patients (S2/3, n = 10) by Illumina HiSeq sequencing, and healthy volunteers were selected (n = 8) as the control group. We found that the plasma miR-185 level in fibrosis patients was significantly downregulated. CCl4-induced fibrosis tissues in mouse livers and TGF-β1-activated HSCs also presented downregulated miR-185 concomitant with an increased expression of RHEB and RICTOR. To explore the correlations, LX-2 cells were transiently transfected with miR-185 mimics. The expression levels of α-SMA, collagen I, and collagen III were decreased as well as RHEB and RICTOR. Inhibition of endogenous miR-185 increased fibrogenic activity. Furthermore, dual-luciferase reporter assays indicated that miR-185 inhibited the expression of RHEB and RICTOR by directly targeting their 3′ UTRs. Moreover, silencing RHEB and RICTOR suppressed α-SMA and collagen expression levels. In conclusion, miR-185 prevents liver fibrogenesis by inhibiting HSC activation via inhibition of RHEB and RICTOR. These results provide new insights into the mechanisms behind the anti-fibrotic effect of miR-185.

Gypsophila elegans isoorientin-2″-O-α-l-arabinopyranosyl ameliorates porcine serum-induced immune liver fibrosis by inhibiting NF-κB signaling pathway and suppressing HSC activation

The present study was to investigate the inhibitory effect of Gypsophila elegans isoorientin-2″-O-α-l-arabinopyranosyl (GEI) on hepatic stellate cells (HSCs), to reveal the underlying mechanism of GEI against hepatic fibrosis. Our study showed that GEI significantly alleviated liver injury induced by porcine serum (PS) in rats; it notably alleviated collagen accumulation as evidenced by a significant decrease in the levels of collagen biomarkers including hyaluronic acid, laminin, hydroxyproline and procollagen III N-terminal peptide. Moreover, GEI treatment markedly decreased the secretion of inflammatory cytokines by inhibiting the NF-κB pathway and significantly inhibited the generation of excessive extracellular matrix (ECM) components by restoring the balance between matrix metalloproteinases (MMPs) and tissue inhibitor of matrix metalloproteinases (TIMPs). Additionally, the cell experiments in vitro showed that GEI strongly inhibited HSC proliferation, migration and clonogenicity and markedly induced HSC apoptosis. Moreover, GEI caused cell cycle arrest at G2 phase. In conclusion, our study demonstrates that GEI significantly alleviates PS-induced hepatic fibrosis by inhibiting the NF-κB pathway, restoring the balance between MMPs and TIMPs, and suppressing HSC activation.