Autophagy In Hepatic Stellate Cells Research Articles

Bone mesenchymal stem cells improve cholestatic liver fibrosis by targeting ULK1 to regulate autophagy through PI3K/AKT/mTOR pathway.

Cholestatic liver disease (CLD) is a severe disease, which can progress to liver cirrhosis, even liver cancer. Hepatic stellate cells (HSCs) activation plays a crucial role in CLD development. Bone mesenchymal stem cells (BMSCs) treatment was demonstrated to be beneficial in liver diseases. However, the therapeutic effect and mechanism of BMSCs on CLD are poorly known. In the present study, we investigated the therapeutic effects and underlying mechanisms of BMSCs transplantation in mouse models of bile duct ligation-induced cholestatic liver fibrosis (CLF). The results revealed that BMSCs significantly improved liver function and reduced the formation of fibrosis after portal vein transplantation. Mechanistically, after coculturing BMSCs and HSCs, we identified that BMSCs alleviated starvation-induced HSCs activation. Further, BMSCs inhibited HSCs activation by decreasing autophagy, and PI3K/AKT/mTOR pathway was involved in the regulation. More importantly, ULK1 is identified as the main autophagy-related gene regulated by BMSCs in HSCs autophagy. Overexpression of ULK1 reversed the suppression of HSCs autophagy by BMSCs. Collectively, our results provide a theoretical basis for BMSCs targeting ULK1 to attenuate HSCs autophagy and activation and suggest that BMSCs or ULK1 may be an alternative therapeutic approach/target for the treatment of CLF.

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

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 transdifferentiate 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. To explore the effect of taurine on aHSC proliferation and the mechanisms involved. 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, and low, medium, and high dosage of taurine (10 mmol/L, 50 mmol/L, and 100 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 concentration. 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 of α-SMA, Collagen I, Fibronectin 1, LC3B, ATG5, Beclin 1, PTGS2, SLC7A11, and p62. 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 accumulation, 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. 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.

Targeted inhibition of autophagy in hepatic stellate cells by hydroxychloroquine: An effective therapeutic approach for the treatment of liver fibrosis.

Liver fibrosis is a wound-healing reaction which is the main cause of chronic liver diseases worldwide. The activated hepatic stellate cell (aHSC) is the main driving factor in the development of liver fibrosis. Inhibiting autophagy of aHSC can prevent the progression of liver fibrosis, but inhibiting autophagy of other liver cells has opposite effects. Hence, targeted inhibition of autophagy in aHSC is quite necessary for the treatment of liver fibrosis, which prompts us to explore the targeted delivery system of small molecule autophagy inhibitor hydroxychloroquine (HCQ) that can target aHSC and alleviate the liver fibrosis. The delivery system of HCQ@retinol-liposome nanoparticles (HCQ@ROL-LNPs) targeting aHSC was constructed by the film dispersion and pH-gradient method. TGF-β-induced HSC activation and thioacetamide (TAA)-induced liver fibrosis mice model were established, and the targeting ability and therapeutic effect of HCQ@ROL-LNPs in liver fibrosis were studied subsequently invitro and invivo. HCQ@ROL-LNPs have good homogeneity and stability. They inhibited the autophagy of aHSC selectively by HCQ and reduced the deposition of extracellular matrix (ECM) and the damage to other liver cells. Compared with the free HCQ and HCQ@LNPs, HCQ@ROL-LNPs had good targeting ability, showing enhanced therapeutic effect and low toxicity to other organs. Construction of HCQ@ROL-LNPs delivery system lays a theoretical and experimental foundation for the treatment of liver fibrosis and promotes the development of clinical therapeutic drugs for liver diseases.

GATA3 promotes the autophagy and activation of hepatic stellate cell in hepatic fibrosis via regulating miR-370/HMGB1 pathway

BackgroundHepatic fibrosis (HF) is a common result of the repair process of various chronic liver diseases. Hepatic stellate cells (HSCs) activation is the central link in the occurrence of HF. MethodsELISA and histological analysis were performed to detect the pathological changes of liver tissues. In vitro, HSCs were treated with TGF-β1 as HF cell model. Combination of GATA-binding protein 3 (GATA3) and miR-370 gene promoter was ensured by ChIP and luciferase reporter assay. Autophagy was monitored by observing the GFP-LC3 puncta formation. The interaction between miR-370 and high mobility group box 1 protein (HMGB1) was verified by luciferase reporter assay. ResultsCCl4-induced HF mice exhibited an increase of ALT and AST, and severe damage and fibrosis of liver tissues. GATA3 and HMGB1 were up-regulated, and miR-370 was down-regulated in CCl4-induced HF mice and activated HSCs. GATA3 enhanced expression of the autophagy-related proteins and activation markers in the activated HSCs. Inhibition of autophagy partly reversed GATA3-induced activation of HSCs and the promotion of GATA3 to hepatic fibrosis. Moreover, GATA3 suppressed miR-370 expression via binding with its promotor, and enhanced HMGB1 expression in HSCs. Increasing of miR-370 inhibited HMGB1 expression by directly targeting its mRNA 3′-UTR. The promotion of GATA3 to TGF-β1-induced HSCs autophagy and activation was abrogated by miR-370 up-regulation or HMGB1 knockdown. ConclusionsThis work demonstrates that GATA3 promotes autophagy and activation of HSCs by regulating miR-370/HMGB1 signaling pathway, which contributes to accelerate HF. Thus, this work suggests that GATA3 may be a potential target for prevention and treatment of HF.

Angiotensin-converting enzyme 2 alleviates liver fibrosis through the renin-angiotensin system.

The present letter to the editor is related to the study titled 'Angiotensin-converting enzyme 2 improves liver fibrosis in mice by regulating autophagy of hepatic stellate cells'. Angiotensin-converting enzyme 2 can alleviate liver fibrosis by regulating autophagy of hepatic stellate cells and affecting the renin-angiotensin system.

Curcumin inhibits the activity and induces apoptosis of activated hepatic stellate cell by suppressing autophagy.

Curcumin, a kind of natural compound, has been previously proven to inhibit the autophagy in hepatic stellate cells (HSCs) and induce their apoptosis. However, it is not clear whether the enhanced apoptosis of activated HSCs (aHSCs) caused by curcumin depends on autophagy inhibition. We aim to verify this hypothesis and explore the potential mechanisms in this study. Immortalized human HSC line LX-2 was used as an experimental specimen and pretreated with transforming growth factor β1(TGF-β1) for 24 h to activate it before drug application. The levels of autophagy, apoptosis, cell activity, lipid metabolism, and the activity of the PI3K/Akt/mTOR signal pathway were evaluated by multiple methods, such as Western blotting, mcherry-EGFP-LC3B adenoviruses transfection, immunofluorescence, Nile Red staining, flow cytometry among others. Our results showed that rapamycin, an autophagy activator, could partly offset the effects of curcumin on autophagy and apoptosis of LX-2 cells, while 3-Methyladenine (3-MA), an autophagy inhibitor, could enhance these effects. Furthermore, curcumin could promote the activity of the PI3K/Akt/mTOR signal pathway in LX-2 cells, while PI3K inhibitor could partly offset this effect and increase the autophagy level. Overall, we demonstrated that curcumin could inhibit the activity and promote LX-2 cells apoptosis by suppressing autophagy by activating the PI3K/Akt/mTOR signal pathway. In addition, lipid recovery and energy deprivation due to autophagy inhibition may be the exact mechanism by which curcumin attenuates the pro-fibrotic activity of LX-2.

Vanillic acid alleviates liver fibrosis through inhibiting autophagy in hepatic stellate cells via the MIF/CD74 signaling pathway

MIF/CD74 signaling pathway and autophagy may be closely related to liver fibrosis. Vanillic acid (VA) is likely to have an anti-liver fibrosis effect, although related studies have not been reported. The aim of this study was to verify the role of hepatic stellate cells (HSCs) autophagy and the MIF/CD74 signaling pathway in the pathogenesis of liver fibrosis, and to investigate the effect of VA on liver fibrosis through in vivo and in vitro experiments. Our results showed that VA significantly attenuated CCl4-induced liver fibrosis. The alleviation of liver fibrosis with VA treatment was associated with a reduction of MIF, CD74, α-SMA, LC3B and Collagen 1. In addition, VA, MIF inhibitor (ISO-1) and autophagy inhibitor (3-MA) markedly inhibited the proliferation and migration of HSCs. This study indicates that VA could protect against HSCs activation, proliferation and migration by inhibiting the autophagy in HSCs via the MIF/CD74 signaling pathway so that alleviates liver fibrosis.

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

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 renin-angiotensin system, and its specific molecular mechanism on autophagy and liver fibrosis needs to be further explored. 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. 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 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. 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). 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.

Helicobacter pylori-derived outer membrane vesicles suppress liver autophagy: A novel mechanism for H. pylori-mediated hepatic disorder

BackgroundHelicobacter pylori outer membrane vesicles (OMVs) are nano-sized structures, which have been recently suggested to play a crucial role in H. pylori pathogenesis. There are growing evidence indicating the relationship of H. pylori infection with extra-gastroduodenal diseases, especially liver-related disorders. This study was aimed to investigate the effects of H. pylori-derived OMVs on autophagy in hepatic stellate cells (HSCs). Material and methodsA selection of five clinical strains of H. pylori with different virulence genotypes were included. The OMVs were isolated by ultracentrifugation and characterized by scanning electron microscopy (SEM) and dynamic light scattering (DLS). The protein concentration of OMVs was measured by BCA assay. MTT assay was used to determine the viability of LX-2 cells (human HSCs) treated with OMVs. The expression level of MTOR, AKT, PI3K, BECN1, ATG16 and LC3B genes was assessed in OMVs-treated LX-2 cells using quantitative real-time PCR. Moreover, immunocytochemistry was performed to evaluate the protein expression of MTOR and LC3B autophagy markers. ResultsH. pylori strains produced round shape nano-vesicles ranging from 50 to 500 nm. Treatment of HSCs with H. pylori-derived OMVs at concentration of 10 μg/mL for 24 h significantly elevated the expression of autophagy inhibitory markers (PI3K, AKT, and MTOR) and suppressed the mRNA expression level of autophagy core proteins (BECN1, ATG16 and LC3B). Immunocytochemistry also presented a substantial reduction in the concentration of LC3B autophagy core protein, and a marked elevation in the amount of MTOR autophagy inhibitory protein. ConclusionThis study revealed that H. pylori-derived OMVs could potentially suppress autophagy flux in HSCs as a novel mechanism for H. pylori-mediated liver autophagy impairment and liver disease development. Further studies are required to elucidate the exact role of OMV-carried contents in liver autophagy, and liver-associated disorders.

CD73 Blockade Alleviated Hepatic Fibrosis via Inhibiting Hepatic Stellate Cells Proliferation and Activation.

Liver fibrosis is associated with the activation of hepatic stellate cells (HSCs). Inhibition of HSCs activation is a strategy for alleviating hepatic fibrogenesis. CD73 is involved in liver disease development, while the mechanism remains unclear. This study aimed to investigate the effect of CD73 targeting inhibition on liver fibrosis. Intraperitoneal injection of CCl4 was used to induce liver fibrosis in mice models. Adenosine 5'-(α, β-methylene) diphosphate sodium salt (APCP) was used for CD73 blockade. The siRNA was used to induce CD73 knockdown in HSCs. LX2 and HSC-T6 were used to investigate the role of CD73 in HSCs activation in vitro. The results showed that APCP treatment could alleviate hepatic fibrosis. In fibrotic liver tissues, CD73 exhibited a positive correlation with markers of HSCs activation. Furthermore, APCP treatment and CD73 knockdown could inhibit HSCs (LX2 and HSC-T6) activation and proliferation. By using RNA sequencing of liver tissues from control, CCl4-mice, and APCP-treated mice, 851 genes that were significantly changed in CCl4 mice (vs. control) were reversed by APCP treatment. These genes were mainly enriched in cell division-associated biological processes. Moreover, we found that CD73 might be associated with autophagy in HSCs. In fibrotic liver tissues and HSCs, ATG5 and Beclin1 expression could be downregulated by CD73 knockdown and APCP treatment. This study demonstrated the effects and mechanism of CD73 in HSCs activation and proliferation, which presents the therapeutical potential of CD73 blockage for liver fibrosis.

GATA3 promotes the autophagy and activation of hepatic stellate cell in hepatic fibrosis via regulating miR-370/HMGB1 pathway

BackgroundHepatic fibrosis (HF) is a common result of the repair process of various chronic liver diseases. Hepatic stellate cells (HSCs) activation is the central link in the occurrence of HF. MethodsELISA and histological analysis were performed to detect the pathological changes of liver tissues. In vitro, HSCs were treated with TGF-β1 as HF cell model. Combination of GATA-binding protein 3 (GATA3) and miR-370 gene promoter was ensured by ChIP and luciferase reporter assay. Autophagy was monitored by observing the GFP-LC3 puncta formation. The interaction between miR-370 and high mobility group box 1 protein (HMGB1) was verified by luciferase reporter assay. ResultsCCl4-induced HF mice exhibited an increase of ALT and AST, and severe damage and fibrosis of liver tissues. GATA3 and HMGB1 were up-regulated, and miR-370 was down-regulated in CCl4-induced HF mice and activated HSCs. GATA3 enhanced expression of the autophagy-related proteins and activation markers in the activated HSCs. Inhibition of autophagy partly reversed GATA3-induced activation of HSCs and the promotion of GATA3 to hepatic fibrosis. Moreover, GATA3 suppressed miR-370 expression via binding with its promotor, and enhanced HMGB1 expression in HSCs. Increasing of miR-370 inhibited HMGB1 expression by directly targeting its mRNA 3′-UTR. The promotion of GATA3 to TGF-β1-induced HSCs autophagy and activation was abrogated by miR-370 up-regulation or HMGB1 knockdown. ConclusionsThis work demonstrates that GATA3 promotes autophagy and activation of HSCs by regulating miR-370/HMGB1 signaling pathway, which contributes to accelerate HF. Thus, this work suggests that GATA3 may be a potential target for prevention and treatment of HF.

Curcumol alleviates liver fibrosis through inducing autophagy and ferroptosis in hepatic stellate cells.

To explore the effect of curcumol on autophagy and ferroptosis of hepatic stellate cells, and to clarify the molecular mechanism of its anti-hepatic fibrosis. In the present study, we report that curcumol promotes the death of activated HSCs and reduces the deposition of extracellular matrix. Interestingly, curcumol treatment can trigger ferroptosis to eliminate activated HSCs characterized by iron overload, lipid ROS accumulation, glutathione depletion, and lipid peroxidation. Curcumol promotes HSC autophagy, which may be the key mechanism for its induction of ferroptosis. It is worth noting that the upregulation of nuclear receptor coactivator 4 (NCOA4) may play a key molecular mechanism. NCOA4 mediates the release of iron ions and induces the occurrence of ferroptosis. Overall, curcumol promotes autophagy in hepatic stellate cells, mediates the degradation of NCOA4 and FTH1 complexes, releases iron ions, leads to iron overload, and induces ferroptosis, which may be an important mechanism for its anti-hepatic fibrosis effect.

CD73 aggravates alcohol-related liver fibrosis by promoting autophagy mediated activation of hepatic stellate cells through AMPK/AKT/mTOR signaling pathway

CD73 is a membrane-bound glycoprotein that can dephosphorylate AMP to adenosine. Increasing evidence has shown that CD73 is involved in the occurrence and development of liver fibrosis. However, the potential mechanism by which CD73 affects the progression of alcohol-related liver fibrosis (ALF) remains unknown. This study aimed to examine the role and mechanism of CD73 in autophagy in HSC-T6 cells and its role in ALF in mice that treated with alcohol plus CCl4. We found that CD73 knockout reduced serum alanine aminotransferase and aspartate aminotransferase levels and decreased liver injury and collagen deposition. Furthermore, autophagy-related indicators were downregulated in the liver fibrosis tissues of CD73-/- (EtOH + CCl4) mice. In vitro, the expression of CD73 and autophagy increased in activated HSC-T6 cells. Autophagy inhibitor, 3-methyladenine, reduced autophagy and activation of acetaldehyde-induced HSC-T6 cells. When using CD73-siRNA, autophagy in HSC-T6 cells was found to be downregulated. However, the CD73 plasmid increased the activation and autophagy of hepatic stellate cells (HSCs). In addition, CD73 induced autophagy through the AMPK/AKT/mTOR pathway, which is characterized by an increase in the ratio of P-AMPKα/AMPKα and a decrease in the ratio of P-AKT/AKT and P-mTOR/mTOR. Our study found that CD73 promotes HSCs activation by regulating autophagy through the AMPK/AKT/mTOR signaling pathway.

Chrysophanol-8-O-glucoside protects mice against acute liver injury by inhibiting autophagy in hepatic stellate cells and inflammatory response in liver-resident macrophages.

Acute liver failure (ALF) is an unfavorable condition characterized by the rapid loss of liver function and high mortality. Chrysophanol-8-O-glucoside (CPOG) is an anthraquinone derivative isolated from rhubarb. This study aims to evaluate the protective effect of CPOG on lipopolysaccharide (LPS)/D-GalN-induced ALF and its underlying mechanisms. LPS/D-GalN-induced mice ALF model and LPS treatment model in RAW 264.7 and LX2 cells were established. It was found that CPOG ameliorated LPS/D-GalN-induced liver injury and improved mortality as indicated by Hematoxylin-eosin (H&E) staining. Molecularly, qPCR and ELISA results showed that CPOG alleviated LPS/D-GalN-induced release of alanine aminotransferase and aspartate transaminase and the secretion of TNF-α and IL-1β in vivo. LPS/D-GalN-induced intracellular ROS production was also attenuated by CPOG in liver tissue. Further, CPOG attenuated ROS generation and inhibited the expression of p-IκB and p-p65 as well as the expression of TNF-α and IL-1β stimulated by LPS in RAW 264.7 cells. In addition, CPOG alleviated LPS-induced up-regulation of LC3B, p62, ATG5 and Beclin1 by attenuating ROS production and inhibiting MAPK signaling in LX2 cells. Taken together, our data indicated that the CPOG protected against LPS/D-GalN-induced ALF by inhibiting oxidative stress, inflammation response and autophagy. These findings suggest that CPOG could be potential drug for the treatment of ALF in clinic.

Salvianolic acid B inhibits autophagy and activation of hepatic stellate cells induced by TGF-β1 by downregulating the MAPK pathway.

In liver fibrosis, transforming growth factor-β1 (TGF-β1) can stimulate autophagy and activation of hepatic stellate cells (HSCs). Autophagy, playing a crucial role in HSCs activation, is related to liver fibrosis. Increasing evidence have suggested that antifibrosis effects of salvianolic acid B (Sal B) and their mechanisms of action, however, remain unclear. The aim of the article is to understand the role of Sal B in HSCs autophagy in liver fibrosis. Herein, we demonstrated that inducing TGF-β1 led to dramatic increase in autophagosome formation and autophagic flux in JS1 and LX2, which was mediated through the ERK, JNK, and p38 MAPK cascades. TGF-β1 significantly increased the protein of autophagy and liver fibrosis, including LC3BⅡ, ATG5, α-SMA, and Col.I; Sal B inhibits JS1 autophagy and activation by inhibiting the formation of autophagosomes and autophagic flux. Sal B significantly decreased the LC3BⅡ, ATG5, α-SMA, and Col.I protein expressions; pretreatment with autophagy inhibitors, chloroquine (CQ) and 3-methyladenine (3-MA) or silencing ATG7 further increase these reductions. However, pretreatment with autophagy agonist, rapamycin (Rapa), or overexpressed ATG5 attenuated this decrease. To further assess the importance of this mechanism, the antibody chip was used to detect the change of phosphorylation protein expression of the MAPK signaling pathway after treating JS1 with Sal B. Eleven differentially expressed proteins were verified. Sal B inhibits activation and autophagy of JS1 induced by TGF-β1 through downregulating the ERK, p38, and JNK signaling pathways, as demonstrated by downregulating p-ERK, p-JNK, and p-p38 MAPK protein expressions. In conclusion, Sal B inhibits autophagy and activation induced by TGF-β1 of HSCs possibly by downregulating the MAPK pathway.

Macrophages evoke autophagy of hepatic stellate cells to promote liver fibrosis in NAFLD mice via the PGE2/EP4 pathway

The pathogenesis of liver fibrosis in nonalcoholic fatty liver disease (NAFLD) remains unclear and the effective treatments have not been explored yet. The activation of hepatic stellate cells (HSCs) is considered as the most critical factor in the progression of liver fibrosis and cirrhosis. Autophagy has recently been identified as a new mechanism to regulate HSC activation. Here, we found that liver macrophages were polarized toward type 2 (M2) during the progression of nonalcoholic steatohepatitis (NASH) and liver fibrosis in both patients and NAFLD mice. Using the methionine-choline-deficient (MCD) diet NAFLD murine model and the in vitro cell culture system, we identified that the M2 macrophages promoted HSC autophagy by secreting prostaglandin E2 (PGE2) and binding its receptor EP4 on the surface of HSCs, which consequently enhanced HSC activation, extracellular matrix deposition, and liver fibrosis. Mechanistically, PGE2/EP4 signals enhanced HSC autophagy through the Erk pathway. A specific PGE2/EP4 antagonist E7046 significantly inhibited M2 macrophage-mediated HSC autophagy and improved liver fibrosis and histopathology in NAFLD mice. Our study provides novel mechanistic insights into the regulation of HSC activation and liver fibrosis. Our findings suggest that the PGE2/EP4 pathway is a promising therapeutic target to prevent NASH progression into cirrhosis.

Modification of lysine deacetylation regulates curcumol-induced necroptosis through autophagy in hepatic stellate cells.

The excessive deposition of extracellular matrix (ECM) is the main characteristic of liver fibrosis, and hepatic stellate cells (HSCs) are the main source of ECM. The removal of activated HSCs has a reversal effect on liver fibrosis. Western blot and MTT analysis indicated that curcumol could relieve hepatic fibrosis by promoting HSCs receptor-interacting protein kinase 1/3 (RIP1/RIP3)-dependent necroptosis. Importantly, autophagy flow was monitored by constructing the mRFP-GFP-LC3 plasmid, and it was found that curcumol cleared activated HSCs in a necroptosis manner that was dependent on autophagy. Our study suggested that the activation of necrosome formed by RIP1 and RIP3 depended on Atg5, and that autophagosomes were also necessary for curcumol-induced necroptosis. Furthermore, microscale thermophoresis and co-immunoprecipitation assay results proved that curcumol could target Sirt1 to regulate autophagy by reducing the acetylation level of Atg5. The HSCs-specific silencing of Sirt1 exacerbated CCl4 -induced liver fibrosis in mice. The deacetylation of Atg5 not only accelerated the accumulation of autophagosomes but also enhanced the interaction between Atg5 and RIP1/RIP3 to induce necroptosis. Overall, our study indicated that curcumol could activate Sirt1 to promote Atg5 deacetylation and enhanced its protein-protein interaction function, thereby inducing autophagy and promoting the necroptosis of HSCs to reduce liver fibrosis.

M6A methylation is required for dihydroartemisinin to alleviate liver fibrosis by inducing ferroptosis in hepatic stellate cells

Activation of hepatic stellate cells (HSCs) is a central event in the development of liver fibrosis, and the elimination of activated HSCs is considered to be an effective anti-fibrotic strategy. Here, we report that dihydroartemisinin (DHA) prevented the activation of HSCs via ferroptosis pathway. Importantly, DHA treatment increased the level of autophagy in HSCs. The inhibition of autophagy by 3-MA dramatically abolished the DHA-induced ferroptosis in HSCs. Mechanistically, the up-regulated m6A modification is essential for the activation of autophagy by DHA through the reduction of fat mass and obesity-associated gene (FTO). Down-regulation of m6A modification by FTO overexpression could impair autophagy and the classical ferroptotic events. Interestingly, the m6A modification of BECN1 mRNA was evidently up-regulated compared with other autophagy-related genes. More importantly, YTHDF1 was identified as a key m6A reader protein for BECN1 mRNA stability, and knockdown of YTHDF1 could prevent DHA-induced HSC ferroptosis. Noteworthy, YTH domain was essential for YTHDF1 to prolong the half-life of BECN1 mRNA in DHA-induced HSC ferroptosis. In mice, DHA treatment alleviated liver fibrosis by triggering HSC ferroptosis. HSC-specific inhibition of m6A modification and autophagy could impair DHA-induced HSC ferroptosis in murine liver fibrosis. Overall, these results provided novel implications to reveal the molecular mechanism of DHA-induced ferroptosis, by which pointed to m6A modification-dependent ferroptosis as a potential target for the treatment of liver fibrosis.

Interleukin-10 regulates starvation-induced autophagy through the STAT3-mTOR-p70s6k axis in hepatic stellate cells.

The degree of activation of hepatic stellate cells (HSCs) is closely related to the level of autophagy in HSCs. We previously showed that interleukin-10 (IL-10) strongly inhibits HSC activation in rat fibrotic liver. However, little is known about the effect of IL-10 on HSC autophagy. For investigation of the effect of IL-10 on starvation-induced autophagy in immortal rat hepatic stellate cells (HSC-T6) and the molecular mechanism, HSC-T6 cells were incubated with serum-free DMEM for different periods and treated with IL-10 at different concentrations. Transmission electron microscopy (TEM), analysis of autophagic flux and Western blotting (WB) assays were used to observe changes in autophagosome morphology and number and autophagy-related protein expression in HSC-T6 cells and to evaluate the regulatory effect of IL-10 on starvation-induced autophagy. Cryptotanshinone (CPT) and rapamycin (Rapa) were used to block activation of the signal transducer and activator of transcription 3 (STAT3) and mTOR signaling pathways, respectively. STAT3-mTOR-p70s6k signaling pathway proteins were analyzed by WB to assess the signaling pathway by which IL-10 regulates autophagy. WB showed an increased LC3II/I ratio, increased Beclin1 expression, and decreased p62 expression in HSC-T6 cells starved for 3 h (p < 0.05). IL-10 inhibited the increases in the LC3II/I ratio and Beclin1 expression and upregulated p62 expression (p < 0.05), and the optimal IL-10 concentration was 20 ng/mL. TEM and double-labeled immunofluorescence analysis showed that IL-10 inhibited autophagosome formation and autophagic flux, as indicated by the decreased numbers of double-membrane autophagosomes and yellow autophagic puncta. Further examination of signaling pathway molecules showed that phosphorylation of the mTOR, STAT3, and p70s6k proteins was significantly decreased during starvation-induced autophagy, but IL-10 could increase mTOR, STAT3, and p70s6k protein phosphorylation (p < 0.05). Blocking either the mTOR or STAT3 pathway reversed the inhibitory effect of IL-10 on starvation-induced autophagy in HSC-T6 cells (p < 0.05). IL-10 suppresses starvation-induced autophagosome formation through activation of the STAT3-mTOR-p70s6k axis in HSC-T6 cells.

Probiotics counteract the expression of hepatic profibrotic genes via the attenuation of TGF-β/SMAD signaling and autophagy in hepatic stellate cells.

Hepatic fibrosis is caused by the increased accumulation and improper degradation of extracellular matrix (ECM) proteins in the liver. Hepatic stellate cells (HSCs) activation is a key process in initiating hepatic fibrosis and can be ameliorated by the administration of probiotic strains. This study hypothesized that LAB strains (Lactiplantibacillus plantarum, Lactobacillus brevis, and Weissella cibaria) might attenuate pro-fibrogenic cytokine TGF-β mediated HSCs activation and induce collagen deposition, expression of other fibrogenic/inflammatory markers, autophagy, and apoptotic processes in vitro. Few studies have evaluated the probiotic effects against fibrogenesis in vitro. In this study, TGF-β exposure increased collagen deposition in LX-2 cells, but this increase was diminished when the cells were pretreated with LAB strains before TGF-β stimulation. TGF-β not only increased collagen deposition, but it also significantly upregulated the mRNA levels of Col1A1, alpha-smooth muscle actin (α-SMA), matrix metalloproteinases-2 (MMP-2), IL-6, CXCL-8, CCL2, and IL-1β in LX-2 cells. Pretreatment of the cells with LAB strains counteracted the TGF-β-induced pro-fibrogenic and inflammatory markers by modulating SMAD-dependent and SMAD-independent TGF-β signaling. In addition, LX-2 cells exposed to TGF-β induced the autophagic and apoptotic associated proteins that were also positively regulated by the LAB strains. These findings suggest that LAB can attenuate TGF-β signaling that is associated with liver fibrogenesis.