MiR-4465-modified mesenchymal stem cell-derived small extracellular vesicles inhibit liver fibrosis development via targeting LOXL2 expression. / MiR-4465修饰的间质干细胞来源的小细胞外囊泡通过靶向LOXL2表达抑制肝纤维化的进展.

Liver fibrosis is a significant health burden, marked by the consistent deposition of collagen. Unfortunately, the currently available treatment approaches for this condition are far from optimal. Lysyl oxidase-like protein 2 (LOXL2) secreted by hepatic stellate cells (HSCs) is a crucial player in the cross-linking of matrix collagen and is a significant target for treating liver fibrosis. Mesenchymal stem cell-derived small extracellular vesicles (MSC-sEVs) have been proposed as a potential treatment option for chronic liver disorders. Previous studies have found that MSC-sEV can be used for microRNA delivery into target cells or tissues. It is currently unclear whether microRNA-4465 (miR-4465) can target LOXL2 and inhibit HSC activation. Additionally, it is uncertain whether MSC-sEV can be utilized as a gene therapy vector to carry miR-4465 and effectively inhibit the progression of liver fibrosis. This study explored the effect of miR-4465-modified MSC-sEV (MSC-sEVmiR-4465) on LOXL2 expression and liver fibrosis development. The results showed that miR-4465 can bind specifically to the promoter of the LOXL2 gene in HSC. Moreover, MSC-sEVmiR-4465 inhibited HSC activation and collagen expression by downregulating LOXL2 expression in vitro. MSC-sEVmiR-4465 injection could reduce HSC activation and collagen deposition in the CCl4-induced mouse model. MSC-sEVmiR-4465 mediating via LOXL2 also hindered the migration and invasion of HepG2 cells. In conclusion, we found that MSC-sEV can deliver miR-4465 into HSC to alleviate liver fibrosis via altering LOXL2, which might provide a promising therapeutic strategy for liver diseases.

Single-cell RNA sequencing reveals reduced intercellular adhesion molecule crosstalk between activated hepatic stellate cells and neutrophils alleviating liver fibrosis in hepatitis B virus transgenic mice post menstrual blood-derived mesenchymal stem cell transplantation.

Liver fibrosis can cause hepatitis B virus (HBV)-associated hepatocellular carcinoma. Menstrual blood-derived mesenchymal stem cells (MenSCs) can ameliorate liver fibrosis through paracrine. Single-cell RNA sequencing (scRNA-seq) may be used to explore the roadmap of activated hepatic stellate cell (aHSC) inactivation to target liver fibrosis. This study established HBV transgenic (HBV-Tg) mouse model of carbon tetrachloride (CCl4)-induced liver fibrosis and demonstrated that MenSCs migrated to the injured liver to improve serological indices and reduce fibrotic accumulation. RNA-bulk analysis revealed that MenSCs mediated extracellular matrix accumulation and cell adhesion. Liver parenchymal cells and nonparenchymal cells were identified by scRNA-seq in the control, CCl4, and MenSC groups, revealing the heterogeneity of fibroblasts/HSCs. A CellChat analysis revealed that diminished intercellular adhesion molecule (ICAM) signaling is vital for MenSC therapy. Specifically, Icam1 in aHSCs acted on Itgal/Itgb2 and Itgam/Itgb2 in neutrophils, causing decreased adhesion. The expression of Itgal, Itgam, and Itgb2 was higher in CCl4 group than in the control group and decreased after MenSC therapy in neutrophil clusters. The Lcn2, Pglyrp1, Wfdc21, and Mmp8 had high expression and may be potential targets in neutrophils. This study highlights interacting cells, corresponding molecules, and underlying targets for MenSCs in treating HBV-associated liver fibrosis.

Arctigenin induces activated HSCs quiescence via AMPK-PPARγ pathway to ameliorate liver fibrosis in mice.

Arctigenin (ATG), a traditional Chinese herbal medicine, is a natural lignan compound extracted from the seeds of burdock (Arctium lappa L, Asteraceae). As a natural product with multiple biological activities, the effect and mechanism of ATG against liver fibrosis are not fully elucidated yet. In current work, we first discovered that ATG could improve CCl4-induced liver injury reflected by lower plasma ALT and AST levels, liver coefficient and pathological scoring of ballooning. Furthermore, it also could reduce the positive areas of Masson, Sirius red and α-SMA staining, inhibit the expression of fibrosis-related genes (Col1a1, Col3a1, Acta2), and decrease the content of hydroxyproline, indicated ATG treatment had benefits in alleviating CCl4-induced liver fibrosis. In vitro, we observed that ATG can inhibit collagen production stimulated by TGF-ß1 in LX2 cells. By analysis of the information obtained from SymMap and GeneCards databases and in vitro validation experiments, ATG was proven to be an indirect PPARγ agonist and its effect on collagen production was dependent on PPARγ. Subsequently, we confirmed that ATG activating AMPK was the contributor of its effect on PPARγ and collagen production. Finally, the transformation of activated hepatic stellate cells was determined after treated with ATG, in which ATG treatment could return activated LX2 cells to quiescence because of the elevated quiescent markers and lipid droplets. Our work has highlighted the potential of ATG in the treatment of liver fibrosis and clarified that ATG can activate AMPK/PPARγ pathway to restore the activated hepatic stellate cell to quiescence thereby improving liver fibrosis.

Promotion of liver fibrosis by Y-box binding protein 1 via the attenuation of transforming growth factor-beta 3 transcription.

Background: Spurred by the seriousness of liver fibrosis, we evaluated the correlation between Y-box binding protein 1 (YB-1) and transforming growth factor-beta 3 (TGF-ß3) expression levels in the signaling pathways of the disease. Methods: Based on a mouse model of carbon tetrachloride-induced liver fibrosis, YB-1 overexpression lentivirus was used to explore the effect of YB-1 on liver fibrosis in vivo. In addition, a hepatic stellate cell (HSC) activation model in the HSC line LX-2 was developed using TGF-ß1. Western blot assays were used to investigate the effects of YB-1 overexpression and knockdown on liver fibrosis. Finally, chromatin immunoprecipitation and luciferase reporter assays were used to elucidate the relationship between YB-1 and its downstream signaling pathways. Results: YB-1 was overexpressed in fibrotic liver tissue, which enhanced both fibrosis and the relative protein expressions of the TGF-ß pathway. Moreover, YB-1 overexpression promoted HSC activation in response to TGF-ß1 stimulation, but its knockdown inhibited liver fibrosis in vitro. Both in vitro and in vivo experiments indicated the expression of TGF-ß3 in the YB-1 overexpression group to be suppressed, and liver fibrosis was more obvious in the YB-1-overexpression group than in the YB-1-inhibition group. YB-1 attenuated TGF-ß3 transcription by binding to its promoter, which is involved in the effect of YB-1 on liver fibrosis. Conclusions: YB-1 overexpression in HSCs promoted liver fibrosis by attenuating TGF-ß3 transcription.

Vitamin D receptor (VDR) mediates the quiescence of activated hepatic stellate cells (aHSCs) by regulating M2 macrophage exosomal smooth muscle cell-associated protein 5 (SMAP-5). / ç»´ç”Ÿç´ D受体（VDR）通过调节M2巨噬细胞外泌体SMAP-5介导肝星状细胞的静息.

An effective therapeutic regimen for hepatic fibrosis requires a deep understanding of the pathogenesis mechanism. Hepatic fibrosis is characterized by activated hepatic stellate cells (aHSCs) with an excessive production of extracellular matrix. Although promoted activation of HSCs by M2 macrophages has been demonstrated, the molecular mechanism involved remains ambiguous. Herein, we propose that the vitamin D receptor (VDR) involved in macrophage polarization may regulate the communication between macrophages and HSCs by changing the functions of exosomes. We confirm that activating the VDR can inhibit the effect of M2 macrophages on HSC activation. The exosomes derived from M2 macrophages can promote HSC activation, while stimulating VDR alters the protein profiles and reverses their roles in M2 macrophage exosomes. Smooth muscle cell-associated protein 5 (SMAP-5) was found to be the key effector protein in promoting HSC activation by regulating autophagy flux. Building on these results, we show that a combined treatment of a VDR agonist and a macrophage-targeted exosomal secretion inhibitor achieves an excellent anti-hepatic fibrosis effect. In this study, we aim to elucidate the association between VDR and macrophages in HSC activation. The results contribute to our understanding of the pathogenesis mechanism of hepatic fibrosis, and provide potential therapeutic targets for its treatment.

The Ultrastructure of Hepatic Stellate Cell-Macrophage Intercellular Crosstalk as a New Morphological Insight into Phenomenon of Fibrogenesis in Pediatric Autoimmune Hepatitis.

The aim of the study was the pioneering retrospective ultrastructural evaluation of respective forms of hepatic stellate cells (HSCs) and analysis of their crosstalk with other adjacent nonparenchymal cells (NPCs), especially Kupffer cells/macrophages (KCs/MPs), in pediatric autoimmune hepatitis (AIH). METHODS: Ultrastructural assessment of the HSC population and NPCs was performed in transmission electron microscopy (TEM) using pretreatment liver biopsies from 25 children (8 boys and 17 girls) aged 4-17 with clinic-pathologically diagnosed untreated AIH. RESULTS: Submicroscopic evaluation allowed easy identification of numerous HSCs in the form of transitory cells, i.e., T-HSCs, accompanied by signs of fibrosis. T-HSCs included cells with features of activation initiation (iHSCs) and activation perpetuation (pHSCs), indicating high HSC activation plasticity. The pHSCs were markedly elongated and mainly showed a distinct loss of lipid cytoplasmic material, expanded and dilated channels of granular endoplasmic reticulum, and linear bundles of microfilaments beneath the cell membrane. They were surrounded by usually mature collagen fibers. Frequently activated KCs/MPs adhered directly to T-HSCs. Between them, tight intercellular junctions were formed by means of point desmosomes. CONCLUSIONS: Our qualitative TEM observations indicate a key role of T-HSCs in liver fibrogenesis in pediatric AIH, with the essential involvement of activated KCs/MPs that directly adhere to them. Tight intercellular junctions, being the ultrastructural exponent of the specific cellular mechanisms of the crosstalk between NPCs, can play a vital role in hepatic collagen fibroplasia. A better understanding of HSC population morphology at the ultrastructural level in AIH seems important not only to improve the disease morphological diagnostics but to also provide new insights into therapeutic interventions for the phenomenon of liver fibrogenesis.

The well-developed actin cytoskeleton and Cthrc1 expression by actin-binding protein drebrin in myofibroblasts promote cardiac and hepatic fibrosis.

Fibrosis is mainly triggered by inflammation in various tissues, such as heart and liver tissues, and eventually leads to their subsequent dysfunction. Fibrosis is characterized by the excessive accumulation of extracellular matrix proteins (e.g., collagens) produced by myofibroblasts. The well-developed actin cytoskeleton of myofibroblasts, one of the main features differentiating them from resident fibroblasts in tissues under inflammatory conditions, contributes to maintaining their ability to produce excessive extracellular matrix proteins. However, the molecular mechanisms via which the actin cytoskeleton promotes the production of fibrosis-related genes in myofibroblasts remain unclear. In this study, we found, via single-cell analysis, that developmentally regulated brain protein (drebrin), an actin-binding protein, was specifically expressed in cardiac myofibroblasts with a well-developed actin cytoskeleton in fibrotic hearts. Moreover, our immunocytochemistry analysis revealed that drebrin promoted actin cytoskeleton formation and myocardin-related transcription factor-serum response factor signaling. Comprehensive single-cell analysis and RNA-Seq revealed that the expression of collagen triple helix repeat containing 1 (Cthrc1), a fibrosis-promoting secreted protein, was regulated by drebrin in cardiac myofibroblasts via myocardin-related transcription factor-serum response factor signaling. Furthermore, we observed the profibrotic effects of drebrin exerted via actin cytoskeleton formation and the Cthrc1 expression regulation by drebrin in liver myofibroblasts (hepatic stellate cells). Importantly, RNA-Seq demonstrated that drebrin expression levels increased in human fibrotic heart and liver tissues. In summary, our results indicated that the well-developed actin cytoskeleton and Cthrc1 expression due to drebrin in myofibroblasts promoted cardiac and hepatic fibrosis, suggesting that drebrin is a therapeutic target molecule for fibrosis.

LPS-induced macrophage exosomes promote the activation of hepatic stellate cells and the intervention study of total astragalus saponins combined with glycyrrhizic acid.

Huangqi decoction, also known as Huangqi Liuyi decoction, was first recorded in the prescriptions of the Bureau of Taiping People's Welfare Pharmacy. It comprises astragalus and licorice, which is a commonly used prescription in traditional Chinese medicine for the clinical treatment of chronic liver disease, especially liver cirrhosis. Total astragalus saponins (AST) is the main component of astragalus, and glycyrrhizic acid (GA) is the main component of licorice. In this study, normal macrophage exosomes were extracted, and the exosomes incubated with lipopolysaccharides (LPS) and those incubated with LPS + AST + GA were co-cultured with JS1 cells (hepatic stellate cell line). The survival rate and the activation of key signaling pathways of JS1 cells in each group were detected and compared. We found that the co-culture of LPS-induced macrophage exosomes with JS1 cells could significantly increase the expression levels of Collagen-1 (Col-1) and Alpha smooth muscle actin (α-SMA)in JS1 cells. However, a significant reversal effect was observed after pretreatment with AST combined with GA. Further evaluation found that the expression levels of phospho (p)-Smad2 and p-Smad3 in the JS1 cells were significantly increased after macrophages were induced with LPS, whereas pretreatment with AST + GA could significantly decrease the expression levels of p-Smad2 and p-Smad3. Preliminary results of this study indicated that LPS-induced macrophage exosomes can promote the activation of hepatic stellate cells, and the pretreatment of AST combined with GA can exert a significant intervention effect. In this study, the new mechanism of anti-hepatic fibrosis effect of traditional Chinese medicine components of Huangqi Decoction was analyzed from the perspective of exosomes.

Vitamin D receptor (VDR) mediates the quiescence of activated hepatic stellate cells (aHSCs) by regulating M2 macrophage exosomal smooth muscle cell-associated protein 5 (SMAP-5) / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

An effective therapeutic regimen for hepatic fibrosis requires a deep understanding of the pathogenesis mechanism. Hepatic fibrosis is characterized by activated hepatic stellate cells (aHSCs) with an excessive production of extracellular matrix. Although promoted activation of HSCs by M2 macrophages has been demonstrated, the molecular mechanism involved remains ambiguous. Herein, we propose that the vitamin D receptor (VDR) involved in macrophage polarization may regulate the communication between macrophages and HSCs by changing the functions of exosomes. We confirm that activating the VDR can inhibit the effect of M2 macrophages on HSC activation. The exosomes derived from M2 macrophages can promote HSC activation, while stimulating VDR alters the protein profiles and reverses their roles in M2 macrophage exosomes. Smooth muscle cell-associated protein 5 (SMAP-5) was found to be the key effector protein in promoting HSC activation by regulating autophagy flux. Building on these results, we show that a combined treatment of a VDR agonist and a macrophage-targeted exosomal secretion inhibitor achieves an excellent anti-hepatic fibrosis effect. In this study, we aim to elucidate the association between VDR and macrophages in HSC activation. The results contribute to our understanding of the pathogenesis mechanism of hepatic fibrosis, and provide potential therapeutic targets for its treatment.

Inorganic arsenic exposure-induced premature senescence and senescence-associated secretory phenotype (SASP) in human hepatic stellate cells.

Exposure to inorganic arsenic has been known to induce cancers in various organs, however, the underlying mechanisms remain unclear. Premature senescence refers to the irreversible growth arrest induced by stress stimuli. The senescence-associated secretory phenotype (SASP), particularly in fibroblasts, has been shown to promote cancer development. In this study, we examined whether arsenite exposure causes premature senescence and induction of SASP in liver fibroblasts using the human hepatic stellate cell line, LX-2. Exposure of LX-2 cells to 5 or 7.5 µM of sodium arsenite for 144 h induced the features of senescence in the cells, including morphological changes, growth inhibition, increased senescence-associated ß-galactosidase activity, increased P21 gene expression, and decreased LAMINB1 gene expression. The mRNA expressions of SASP factors, such as MMP1, MMP3, IL-8, IL-1ß, and CXCL1, were also highly upregulated. The wound healing assay revealed that the conditioned medium from LX-2 cells with arsenite-induced senescence increased the migration activity of cells of the human hepatoma cell line, Huh-7. Gene expression data of liver cancer samples from the Human Protein Atlas showed that high expression levels of the SASP factors that were upregulated in the cells with arsenite-induced senescence were strongly associated with a poor prognosis. In addition, the cellular levels of γ-H2AX, a DNA double-strand break marker, were increased by arsenite exposure, suggesting that DNA damage could contribute to premature senescence induction. These results show that arsenite exposure induces premature senescence in hepatic stellate cells and suggest that the SASP factors from the senescent cells promote hepatic carcinogenesis.

Ethanol extract of Pharbitis nil ameliorates liver fibrosis through regulation of the TGFß1-SMAD2/3 pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Pharbitis nil (L.) Choisy is a medicinal herb, and herbal remedies based on its seeds have been used to treat of obesity and liver diseases, including fatty liver and liver cirrhosis in East Asia. AIM OF THE STUDY: Liver fibrosis is a major cause of morbidity and mortality in patients with chronic liver inflammation such as that caused by non-alcoholic steatohepatitis. However, no effective pharmaceutical treatment for liver fibrosis has been approved. In this study, we aimed to investigate that ethanol extract of pharbitis nil (PNE) alleviates the liver fibrosis. MATERIALS AND METHODS: We studied the effects of PNE on two preclinical models. Six-week-old male C57BL/6 mice were intraperitoneally injected with CCl4 twice weekly for 6 weeks and then treated with 5 or 10 mg/kg PNE daily from week 3 for weeks. Secondly, mice were fed HFD for 41 weeks and at 35 weeks treated with 5 mg/kg PNE daily for the remaining 6 weeks. In addition, we examined the antifibrotic effects of PNE in primary mouse hepatic stellate cells and LX-2 cells. RESULTS: PNE treatment ameliorated hepatocyte necrosis, inflammation, and liver fibrosis in CCl4-treated mice and inhibited the progression of liver fibrosis in mice with HFD-induced fibrosis. PNE reduced the expressions of fibrosis markers and SMAD2/3 activations in mouse livers and in TGFß1-treated primary mouse hepatic stellate and LX-2 cells CONCLUSIONS: This study demonstrates that PNE attenuates liver fibrosis by downregulating TGFß1-induced SMAD2/3 activation.

NAMPT-mediated NAD+ biosynthesis suppresses activation of hepatic stellate cells and protects against CCl4-induced liver fibrosis in mice.

Background: Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the rate-limiting step in the salvage pathway of mammalian nicotinamide adenine dinucleotide (NAD+) biosynthesis. Through its NAD+-biosynthetic activity, NAMPT is able to regulate the development of hepatic steatosis and inflammation induced by diet or alcohol. However, the roles NAMPT plays in the development of liver fibrosis remain obscure. Purpose: To investigate the roles of NAMPT-mediated NAD+ biosynthesis in hepatic stellate cell (HSC) activation and liver fibrosis. Research Design: Realtime RT-PCR and western blot analyses were performed to analyze the expression of profibrogenic genes. Sirius red staining was conducted to examine the fibrosis in liver. Mouse liver fibrosis was induced by intraperitoneal injection of carbon tetrachloride (CCl4) 2 times a week for 6 weeks. Adenovirus-mediated NAMPT overexpression or nicotinamide mononucleotide (NMN) administration was carried out to study the effects of elevation of NAD+ levels on protecting CCl4-induced liver fibrosis in mice. LX2 cells or primary HSCs were used to study the role of NAMPT overexpression or NMN treatment in reducing profibrogenic gene expression in vitro. ResultsCCl4 administration suppresses NAMPT expression in liver and reduces hepatic NAD+ content. Tgfß1 treatment decreases intracellular NAD+ levels and NAMPT expression in LX2 cells. Adenovirus-mediated NAMPT overexpression augments liver NAD+ levels, inhibits HSC activation and alleviates CCl4-induced liver fibrosis in mice. Administration of NMN also suppresses HSC activation and protects against CCl4-induced liver fibrosis in mice. Conclusions: NAMPT-mediated NAD+ biosynthesis inhibits HSC activation and protects against CCl4-induced liver fibrosis.

Integrin αVß1 regulates procollagen I production through a non-canonical transforming growth factor ß signaling pathway in human hepatic stellate cells.

Hepatic stellate cells (HSCs) are thought to play key roles in the development of liver fibrosis. Extensive evidence has established the concept that αV integrins are involved in the activation of latent transforming growth factor ß (TGF-ß), a master regulator of the fibrotic signaling cascade. Based on mRNA and protein expression profiling data, we found that αVß1 integrin is the most abundant member of the αV integrin family in either quiescent or TGF-ß1-activated primary human HSCs. Unexpectedly, either a selective αVß1 inhibitor, Compound 8 (C8), or a pan-αV integrin inhibitor, GSK3008348, decreased TGF-ß1-activated procollagen I production in primary human HSCs, in which the role of ß1 integrin was confirmed by ITGB1 siRNA. In contrast with an Activin receptor-like kinase 5 (Alk5) inhibitor, C8 and GSK3008348 failed to inhibit TGF-ß1 induced SMAD3 and SMAD2 phosphorylation, but inhibited TGF-ß-induced phosphorylation of ERK1/2 and STAT3, suggesting that αVß1 integrin is involved in non-canonical TGF-ß signaling pathways. Consistently, ITGB1 siRNA significantly decreased phosphorylation of ERK1/2. Furthermore, a selective inhibitor of MEK1/2 blocked TGF-ß1 induced phosphorylation of ERK1/2 and decreased TGF-ß1 induced procollagen I production, while a specific inhibitor of STAT3 had no effect on TGF-ß1 induced procollagen I production. Taken together, current data indicate that αVß1 integrin can regulate TGF-ß signaling independent of its reported role in activating latent TGF-ß. Our data further support that αVß1 inhibition is a promising therapeutic target for the treatment of liver fibrosis.

Mechanotransduction-Targeting Drugs Attenuate Stiffness-Induced Hepatic Stellate Cell Activation in Vitro.

In hepatitis, activated hepatic stellate cells (HSCs) produce collagens, causing liver fibrosis. Microenvironmental stiffness is a known trigger of HSC activation and is communicated through mechanotransduction. Cell proliferation, alpha smooth muscle actin (α-SMA) and collagen type Iα (Col1α) are indicative of activated HSCs. We hypothesized that certain compounds could interfere with the HSC's recognition of microenvironmental stiffness by blocking cell adhesion signaling. To verify the potential of mechanotransduction, and in particular of focal adhesion proteins, as liver fibrosis drug targets, we evaluated existing drugs. We examined the effects of the integrin antagonist, BS-1417; the focal adhesion kinase (FAK) inhibitor, defactinib; the cyclin-dependent kinase (CDK) inhibitor, roscovitine; and two microtubule modulators, paclitaxel and colchicine, on stiffness-induced HSC activation. To determine the extent of transforming growth factor ß (TGF-ß) participation in mechanotransduction, we measured gene expression levels of α-SMA and Col1α. We also measured ATP levels to determine cell number. Results revealed that interestingly, although TGF-ß did not show additional HSC activation after stiffness stimulation, the TGF-ß receptor inhibitor, SB525334, markedly suppressed stiffness-induced α-SMA and Col1α mRNA expression. BS-1417, roscovitine, defactinib and colchicine suppressed α-SMA and Col1α mRNA expression as well as the number of HSCs. Paclitaxel also suppressed stiffness-induced α-SMA mRNA expression and the number of HSCs, but mildly reduced that of Col1α mRNA. Together, these results show that an integrin antagonist and mechanotransduction-targeting drugs reduced stiffness-induced HSC activation in a dose-dependent fashion. The targeting of focal adhesion proteins involved in mechanotransduction is promising in liver fibrosis drug development.

Role of B Cell Lymphoma 2 in the Regulation of Liver Fibrosis in miR-122 Knockout Mice.

MicroRNA-122 (miR-122) has been identified as a marker of various liver injuries, including hepatitis- virus-infection-, alcoholic-, and non-alcoholic steatohepatitis (NASH)-induced liver fibrosis. Here, we report that the extracellular miR-122 from hepatic cells can be delivered to hepatic stellate cells (HSCs) to modulate their proliferation and gene expression. Our published Argonaute crosslinking immunoprecipitation (Ago-CLIP) data identified several pro-fibrotic genes, including Ctgf, as miR-122 targets in mice livers. However, treating Ctgf as a therapeutic target failed to rescue the fibrosis developed in the miR-122 knockout livers. Alternatively, we compared the published datasets of human cirrhotic livers and miR-122 KO livers, which revealed upregulation of BCL2, suggesting its potential role in regulating fibrosis. Notably, ectopic miR-122 expression inhibited BCL2 expression in human HSC (LX-2) cells). Publicly available ChIP-seq data in human hepatocellular cancer (HepG2) cells and mice livers suggested miR-122 could regulate BCL2 expression indirectly through c-MYC, which was confirmed by siRNA-mediated depletion of c-MYC in Hepatocellular Carcinoma (HCC) cell lines. Importantly, Venetoclax, a potent BCL2 inhibitor approved for the treatment of leukemia, showed promising anti-fibrotic effects in miR-122 knockout mice. Collectively, our data demonstrate that miR-122 suppresses liver fibrosis and implicates anti-fibrotic potential of Venetoclax.

Palmitic acid stimulates NLRP3 inflammasome activation through TLR4-NF-κB signal pathway in hepatic stellate cells.

BACKGROUND: The NLRP3 inflammasome activation plays an important role in the development of NASH and fibrogenesis. However, the mechanisms involved in NLRP3 activation in hepatic stellate cells (HSCs) have been unclear. The aim of this study was to investigate the mechanism of NLRP3 activation in HSCs and the role of NLPR3 inflammasome activation in HSCs on the development of nonalcoholic steatohepatitis (NASH) to fibrosis. METHODS: Primary HSCs isolated from SD rats were incubated with palmitic acid and/or LPS, respectively. For in vivo animal experiment, 4-week-old SD rats were fed with high fat diet (HF-diet) for 12 weeks, SD rats were sacrificed at 0, 4, 8 and 12 w. In another group of animal experiment, 4-week-old SD rats were fed with HF-diet and a NLRP3 inhibitor (intraperitoneal injection of NLRP3 inhibitor glybenclamide 5 mg/kg, injected every 3 days) for 12 weeks. Liver tissue and serum were harvested. RT-PCR, WB, ELISA, immunofluorescence and immunohistochemistry were performed to assess the NLRP3 inflammasome activation and signal molecules. RESULTS: Palmitic acid stimulated NLPR3 inflammasome activation and fibrotic phenotype change in primary HSCs, LPS sensitizes the response of HSCs to palmitic acid. TLR4-NF-κB signal pathway was involved in NLRP3 inflammasome activation in palmitic acid-exposed HSCs and HF diet-induced NASH. It is evident that administration of NLRP3 inhibitor reduced the development of NASH to liver fibrosis in the NASH rat model. CONCLUSIONS: Palmitic acid stimulates NLRP3 inflammasome activation through the TLR4-NF-κB signal pathway in HSCs. NLRP3 inflammasome activation in HSCs exacerbates the development of NASH to liver fibrosis.

LRP6-CRISPR prevents activation of hepatic stellate cells and liver fibrogenesis in rats.

This study elaborated on the function of Low-density lipoprotein receptor-related protein 6 (LRP6), a critical component of Wnt signaling, in liver fibrosis. This study enrolled sixty-eight patients with liver fibrosis, with ten healthy liver tissue samples, served as the controls. A lentiviral vector expressing LRP6-CRISPR was constructed. Immortalized HSC-T6 cells were transfected with LRP6-CRISPR. A rat model of CCl4-induced liver fibrosis was established, and rats were injected with lentiviral vectors expressing LRP6-CRISPR. LRP6 expression and fibrosis biomarkers were examined by PCR, Western blot, and immunofluorescence assay, respectively. HSC growth and its ability of migration and invasion were evaluated by MTT and Transwell assay, separately. Wnt signaling activity was examined by Luciferase reporter assay. LRP6 was overexpressed in human fibrotic-liver tissues, and the expression of LRP6 was correlated with liver fibrosis stages. LRP6 knockout with CRISPR suppressed the Wnt signaling activities and consequently repressed HSC activation and relived liver injury in fibrotic-liver model rats. Our data revealed that the knockout of LRP6 weakens the binding of Wnt ligand with its cell surface receptors, the first step of Wnt transduction cascade, and consequently repressed HSC activation.

Hemistepsin A alleviates liver fibrosis by inducing apoptosis of activated hepatic stellate cells via inhibition of nuclear factor-κB and Akt.

Hemistepsin A (HsA), isolated from Hemistepta lyrata (Bunge) Bunge, has the ability to ameliorate hepatitis in mice. However, the effects of H. lyrata and HsA on other types of liver disease have not been explored. In this report, we investigated the effects of H. lyrata and HsA on liver fibrosis and the underlying molecular mechanisms in activated hepatic stellate cells (HSCs). Based on cell viability-guided isolation, we found HsA was the major natural product responsible for H. lyrata-mediated cytotoxicity in LX-2 cells. HsA significantly decreased the viability of LX-2 cells and primary activated HSCs, increased the binding of Annexin V, and altered the expression of apoptosis-related proteins, suggesting that HsA induces apoptosis in activated HSCs. HsA reduced the phosphorylation of IKKε and the transactivation of nuclear factor-κB (NF-κB). Moreover, HsA decreased the phosphorylation of Akt and its downstream signaling molecules. Transfection experiments suggested that inhibition of NF-κB or Akt is essential for HsA-induced apoptosis of HSCs. In a CCl4-induced liver fibrosis model, HsA administration significantly decreased ALT and AST activities. Furthermore, HsA attenuated CCl4-mediated collagen deposits and profibrogenic genes expression in hepatic tissue. Thus, HsA may serve as a natural product for managing liver fibrosis through inhibition of NF-κB/Akt-dependent signaling.

Hormone-sensitive lipase is a retinyl ester hydrolase in human and rat quiescent hepatic stellate cells.

Hepatic stellate cells (HSC) store vitamin A as retinyl esters and control circulating retinol levels. Upon liver injury, quiescent (q)HSC lose their vitamin A and transdifferentiate to myofibroblasts, e.g. activated (a)HSC, which promote fibrosis by producing excessive extracellular matrix. Adipose triglyceride lipase/patatin-like phospholipase domain-containing protein 2 (ATGL/PNPLA2) and adiponutrin (ADPN/PNPLA3) have so far been shown to mobilize retinol from retinyl esters in HSC. Here, we studied the putative role of hormone-sensitive lipase (HSL/LIPE) in HSC, as it is the major retinyl ester hydrolase (REH) in adipose tissue. Lipe/HSL expression was analyzed in rat liver and primary human and rat qHSC and culture-activated aHSC. Retinyl hydrolysis was analyzed after Isoproterenol-mediated phosphorylation/activation of HSL. Primary human HSC contain 2.5-fold higher LIPE mRNA levels compared to hepatocytes. Healthy rat liver contains significant mRNA and protein levels of HSL/Lipe, which predominates in qHSC and cells of the portal tree. Q-PCR comparison indicates that Lipe mRNA levels in qHSC are dominant over Pnpla2 and Pnpla3. HSL is mostly phosphorylated/activated in qHSC and partly colocalizes with vitamin A-containing lipid droplets. Lipe/HSL and Pnpla3 expression is rapidly lost during HSC culture-activation, while Pnpla2 expression is maintained. HSL super-activation by isoproterenol accelerates loss of lipid droplets and retinyl palmitate from HSC, which coincided with a small, but significant reduction in HSC proliferation and suppression of Collagen1A1 mRNA and protein levels. In conclusion, HSL participates in vitamin A metabolism in qHSC. Equivalent activities of ATGL and ADPN provide the healthy liver with multiple routes to control circulating retinol levels.

rSjp40 inhibits activated hepatic stellate cells by promoting nuclear translocation of YB1 and inducing BMP-7/Smad1/5/8 pathway.

BACKGROUND: Activation of hepatic stellate cells is the dominant pathogenic event during the process of liver fibrosis. Bone morphogenic protein (BMP)-7 has recently been identified as an anti-fibrotic factor and leads to phosphorylation of Smad1/5/8 in activated hepatic stellate cells. Its expression can be upregulated by the transcriptional activator, Y-Box protein-1 (YB1). Previous studies have found that the recombinant Schistosoma japonicum protein p40 (rSjp40) can inhibit the activation of hepatic stellate cells, and based on this evidence we attempted to investigate whether or not BMP-7 is involved in rSjp40's inhibition. METHODS: A human hepatic stellate cell line, the LX-2 cell line, was cultured and treated with rSjp40. The role of BMP-7 was analyzed by Western blot. RESULTS: Our findings testified that knockdown of BMP-7 impaired rSjp40-induced downregulation of α-SMA and phosphorylation of Smad1/5/8 in LX-2 cells. Furthermore, rSjp40 upregulated expression of BMP-7 at both mRNA and protein levels depending on YB1. Interestingly, YB1 was translocated from the cytoplasm to the nucleus upon treatment of rSjp40. CONCLUSIONS: These results suggest that rSjp40 inhibits the activation of hepatic stellate cells by promoting nuclear translocation of YB1 and inducing BMP-7/Smad1/5/8 pathway, which provide a new clue to guide ongoing research into the anti-fibrosis of rSjp40.