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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.
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Proteínas Quinases Ativadas por AMP , Furanos , Células Estreladas do Fígado , Lignanas , Cirrose Hepática , PPAR gama , Transdução de Sinais , Lignanas/farmacologia , Lignanas/uso terapêutico , Animais , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/patologia , Cirrose Hepática/metabolismo , Furanos/farmacologia , Furanos/uso terapêutico , Camundongos , Proteínas Quinases Ativadas por AMP/metabolismo , Masculino , PPAR gama/metabolismo , Transdução de Sinais/efeitos dos fármacos , Linhagem Celular , Tetracloreto de Carbono , Humanos , Camundongos Endogâmicos C57BL , Fígado/efeitos dos fármacos , Fígado/patologia , Fígado/metabolismo , Colágeno/metabolismoRESUMO
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.
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Aminoácido Oxirredutases , Vesículas Extracelulares , Células Estreladas do Fígado , Cirrose Hepática , Células-Tronco Mesenquimais , MicroRNAs , Animais , Humanos , Masculino , Camundongos , Aminoácido Oxirredutases/genética , Aminoácido Oxirredutases/metabolismo , Vesículas Extracelulares/metabolismo , Células Estreladas do Fígado/metabolismo , Cirrose Hepática/terapia , Cirrose Hepática/metabolismo , Cirrose Hepática/genética , Células-Tronco Mesenquimais/metabolismo , Camundongos Endogâmicos C57BL , MicroRNAs/genética , MicroRNAs/metabolismoRESUMO
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.
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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.
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Humanos , Células Estreladas do Fígado/patologia , Receptores de Calcitriol , Cirrose Hepática/patologia , Macrófagos/metabolismoRESUMO
Objective To investigate the effects of all-trans retinioc acid (ATRA)on proliferation of rat hepatic stellate cells (HSC-T6)and expressions of collagen Ⅰ,matrix metalloproteinase-2 (MMP-2),tissue inhibitor of metalloproteinases-1 (TIMP-1 )and signal protein Smad2/3 in TGF-β1-simulated HSC-T6 so as to explore the impact and molecular mechanisms of ATRA on liver fibrosis in vitro .Methods Cultured HSC-T6s were treated with different concentrations of ATRA (0.1,1,10 μmol/L)for fixed time (12,24,48 hours).After intervention time,cell proliferation was evaluated by MTT.Meanwhile,HSC-T6s stimulated by TGF-β1 (5 ng/mL)were treated with different concentrations of ATRA for 24 h.The mRNA expressions of COL1α2,MMP-2 and TIMP-1 were quantified by RT-PCR;the expression of Smad 2/3 protein was determined by cell immunochemistry.Results The proliferation of hepatic stellate cells was inhibited by ATRA in a dose-dependent manner (P < 0.05 ).After induced by TGF-β1,the mRNA expressions of COL1α2,MMP-2 and TIMP-1 and the expression of Smad 2/3 protein were increased significantly compared with control group (P <0.05).However,ATRA could obviously reduce themRNA expressions of COL1α2,MMP-2 and TIMP-1 and the expression of Smad 2/3 protein in HSC-T6 induced by TGF-β1 (P < 0.05 ).Conclusion ATRA can inhibit the proliferation of HSC-T6s and reduce the mRNA expressions of COL1α2,MMP-2 and TIMP-1 in HSC-T6 which were induced by TGF-β1.The anti-hepatic fibrosis function of ATRA may be related to its inhibition on the expression of Smad 2/3 protein in HSC-T6 to influence TGF-β1/Smad signaling pathway.
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Objective To investigate the effects of all-trans retinioc acid (ATRA)on proliferation of rat hepatic stellate cells (HSC-T6)and expressions of collagen Ⅰ,matrix metalloproteinase-2 (MMP-2),tissue inhibitor of metalloproteinases-1 (TIMP-1 )and signal protein Smad2/3 in TGF-β1-simulated HSC-T6 so as to explore the impact and molecular mechanisms of ATRA on liver fibrosis in vitro .Methods Cultured HSC-T6s were treated with different concentrations of ATRA (0.1,1,10 μmol/L)for fixed time (12,24,48 hours).After intervention time,cell proliferation was evaluated by MTT.Meanwhile,HSC-T6s stimulated by TGF-β1 (5 ng/mL)were treated with different concentrations of ATRA for 24 h.The mRNA expressions of COL1α2,MMP-2 and TIMP-1 were quantified by RT-PCR;the expression of Smad 2/3 protein was determined by cell immunochemistry.Results The proliferation of hepatic stellate cells was inhibited by ATRA in a dose-dependent manner (P < 0.05 ).After induced by TGF-β1,the mRNA expressions of COL1α2,MMP-2 and TIMP-1 and the expression of Smad 2/3 protein were increased significantly compared with control group (P <0.05).However,ATRA could obviously reduce themRNA expressions of COL1α2,MMP-2 and TIMP-1 and the expression of Smad 2/3 protein in HSC-T6 induced by TGF-β1 (P < 0.05 ).Conclusion ATRA can inhibit the proliferation of HSC-T6s and reduce the mRNA expressions of COL1α2,MMP-2 and TIMP-1 in HSC-T6 which were induced by TGF-β1.The anti-hepatic fibrosis function of ATRA may be related to its inhibition on the expression of Smad 2/3 protein in HSC-T6 to influence TGF-β1/Smad signaling pathway.
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BACKGROUND: Hepatic stellate cell (HSC) has been suggested to play a role in fibrogenesis in alcoholic liver disease. We evaluate the correlation with fibrogenesis and ultrastructure of hepatic stellate cells in alcoholic fatty liver. METHODS: We studied 6 patients with alcoholic fatty liver and 5 non-alcoholic fatty liver. The numbers of fat droplets in hepatic stellate cell was determined by electron microscopy. We also studied the grading of deposition of collagen fibers in the space of Disse. We were to evaluate the structure of hepatic stellate cells in the space of Disse by light and electron microscopy. RESULTS: Wider distribution of fat droplets in hepatic stellate cells in alcoholic fatty liver than in normal liver. The hypertrophied endoplasmic reticulum in hepatic stellate cells is a prominent findings in alcoholic fatty liver. We observed basement membrane-like materials in patients with alcoholic fatty liver with hepatic fibrosis. CONCLUSION: The results demonstrate that, in patients with alcoholic fatty liver by alcoholic liver injury, the hepatic stellate cells may play an important role in the fibrogenesis of perisinusoidal spaces in the liver.
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Adulto , Feminino , Humanos , Masculino , Membrana Basal/ultraestrutura , Biópsia por Agulha , Colágeno/ultraestrutura , Estudo Comparativo , Fígado Gorduroso Alcoólico/patologia , Hepatócitos/ultraestrutura , Lipídeos/análise , Microscopia Eletrônica , Pessoa de Meia-Idade , Probabilidade , Estudos Prospectivos , Valores de Referência , Estatísticas não Paramétricas , Técnicas de CulturaRESUMO
Objective To investigate the effects and mechanism of Safflower Injection on the proliferation,apoptosis,and expression of bcl-2/bax gene in hepatic stellate cell(HSC) in vitro.Methods HSC Line HSC-T6 was incubated with Safflower Injection at different concertration,cell proliferation was assessed by MTT colorimetric assay.After incubated with Safflower Injection 5,10,and 20 mg/mL,flow cytometry(FCM) was used to detect the content of DNA in HSC-T6.Morphological change of HSC-T6 was observed under microscope and agarose gel electrophoresis for DNA Ladder was used to detect apoptosis.Besides,the early stage of apoptosis was detected with Annexin V-FITC/PI double labbled assay.And real time RT-PCR was used to detect the expression of bcl-2/bax gene.Results The significant inhibition of Safflower Injection on HSC proliferation was observed in a dose-and time-dependent manner.Observed by FCM,the cell ratio in G0/G1 phase with Safflower Injection treatment(10 and 20 mg/mL) for 24 h was increased,which showed the significant difference compared with the control group(P
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Aim To investigate the effects of leptin on ?1(Ⅰ) collagen mRNA expression and protein production, and the roles of Janus kinase/signal transducers and activators transcription(JAK/STAT) signaling transduction pathway in increased ?1(Ⅰ) collagen mRNA expression stimulated by leptin in activated hepatic stellate cells(HSCs).Methods Firstly, ?1(Ⅰ) collagen mRNA expression and protein production as well as JAK1 and STAT3 phosphorylation induced by leptin at different doses in a human HSC cell line, LX-2 were determined by RT-PCR, ELISA, and Western-blot.Secondly, the effects of JAK1 inhibitor AG490 on JAK1 phosphorylation and ?1(Ⅰ) collagen mRNA expression stimulated by leptin were detected by Western blot and RT-PCR. Thirdly, the roles of AG490 and transfection with STAT3 antisense oligonucleotide(STAT3-ASON) in STAT3 phosphorylation after leptin were detected by Western blot. Finally, the effect of transfection with STAT3-ASON on ?1(Ⅰ) collagen mRNA expression after leptin was measured by RT-PCR.Results Leptin increased ?1(Ⅰ) collagen mRNA expression and protein production in a dose-dependent manner in LX-2, reaching a maximal level at 80 ?g?L-1 leptin. In addition, phosphorylation of JAK1 and STAT3 after leptin exhibited a time-dependent effect. Besides, JAK1 inhibitor AG490 completely blocked JAK1 and STAT3 phosphorylation and increased in ?1(Ⅰ) collagen gene expression after leptin in LX-2. Transfection with STAT3-ASON blocked STAT3 phosphorylation and increased ?1(Ⅰ) collagen mRNA by leptin in LX-2.Conclusion Leptin had a direct action on liver fibrogenesis by stimulating ?1(Ⅰ) collagen mRNA expression and protein production in activated HSC, and JAK/STAT signaling transduction pathway was involved in the process. JAK1 inhibitor AG490 and transfection with STAT3-ASON blocked the transduction pathway effectively in LX-2. Leptin may be an important factor in the development of hepatic fibrosis. Activated JAK1 and STAT3 signaling in human HSC line provided a novel molecular target for therapeutic intervention of liver fibrosis.
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Aim To explore the therapeutic effect of total flavones of Bidens Bipinnata L (TFB) on liver fibrosis in rats and its mechanism. Methods The model of rat liver fibrosis was adopted which was induced by CCl4 injection. The effects of TFB were observed on the levels of serum HA,PCⅢ,CIV and Hyp in rats liver fibrosis,and on liver histopathological changes as well as collagen hyperplasia formation in liver tissue. The apoptosis of HSC were detected by double-staining of ?-smooth muscle actin (?-SMA) and TUNEL. The study in vitro was carried out on the culture of isolated hepatic stellate cells. Cell proliferation was detected with MTT assay. Cell apoptosis was detected by electron microscopy and flow cytometry. Results TFB can significantly reduce serum HA,PCⅢ,CⅣ and Hyp contents in liver fibrosis of rats,improve the liver pathologic injury,reduce collagen hyperplasia in liver of liver fibrosis rats,inhibit the activation and proliferation of HSC,and promote the apoptosis of HSC. In addition TFB could significantly inhibit the proliferation and increased the apoptosis of isolated and cultured HSC compared with the control group. Conclusions TFB has a significant therapeutic effect on the liver fibrosis rats,probably its inhibition of proliferation and stimulation of activated HSC apoptosis may be an important mechanism of its therapeutical effect against liver fibrosis.
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Objective To investigate the effects of high glucose on the proliferation of hepatic stellate cell-T6(HSC-T6) and the expressions of transformation growth factor-?1(TGF-?1),platelet-derived growth factor(PDGF) and precollagen Ⅲ(PCⅢ).Methods Based on glucose groups of different concentration,we observed the proliferation of HSC in 30min-16h time period,and used the methods of immunohistochemistry and radioimmunoassay to measure the expressions of TGF-?1,PDGF and PCⅢ in the supernatant at 48h and 72h.Results In 2-16h time period,the proliferation of HSC was increased stepwise over time in 1500-6000mg/L group,and was more visible in 4500-6000mg/L group.The expressions of TGF-?1 and PDGF increased at 48h,and the expression of PCⅢ in the supernatant increased at 72h in 4500-6000mg/L group compared with that in glucose control and hypertonic control groups.Conclusion High glucose can promote hepatic fibrosis by stimulating the expressions of TGF-?1,PDGF and PCⅢ in HSC-T6.
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Objective To investigate the preventive effect of spironolactone on hepatic fibrosis. Methods Ninety SD rats were randomly divided into three groups. Control group consisted of 8 rats that , fed by normal food, were injected with peanut oil subcutaneously. Model group consisted of 42 rats whose liver fibrosis was induced by compound factors. Spiro nolactone-prevention group consisted of 40 rats that were given 100 mg?kg -1 spironolactone per day, by the same methods of making models as those of the model group. At the end of weeks 2, 4, 6 and 8, 8 rats were randomly taken out of model group and spironol actone group and then were sacrificed. The expressions of TGF- ? 1 , PDGF- BB and ? -SMA in hepatic tissues were detected with immunohistochemical met hods. Results The expressions of TGF- ? 1 , PDGF-BB a nd ? -SMA in spironolactone group decreased greatly than those in model gro up ( P