RESUMO
Liver fibrosis is a progressive pathological process that accompanies wound healing; however, therapeutics for reversing hepatic fibrosis are unavailable. Activation of hepatic stellate cells (HSCs) play a critical role in liver fibrosis. Recent reports showed that succinate and its receptor, G-protein coupled receptor 91 (GPR91), act as signaling molecules during the activation of HSCs. However, the role of succinate in proliferation, apoptosis, and migration of HSCs has not been studied. In this study, we determined whether succinate regulates proliferation, apoptosis, and migration of HSCs and induces liver fibrosis in a mouse model. Succinate treatment not only induced activation of HSCs, but also increased the proliferation and migration of LX-2 HSCs and inhibited apoptosis. To investigate whether succinate causes hepatic fibrosis, 100â¯mg/kg succinate or control PBS was administered by intraperitoneal injection to mice once a day for four weeks. There were significant molecular changes such as increased α-SMA and collagen type 1 production and increased production of inflammatory cytokines such as IL-6 and TNF-α, but not TGF-ß, in the succinate-treated group compared to the control group. However, no morphological changes were observed in Masson's trichrome staining. In conclusion, the present study demonstrated that succinate induces activation, proliferation, and migration of HSCs and attenuates apoptosis in LX-2 HSCs. Therefore, inhibition of succinate accumulation may be an effective method for reversing liver fibrosis by controlling HSC survival and growth.
Assuntos
Células Estreladas do Fígado/patologia , Cirrose Hepática/metabolismo , Fígado/patologia , Ácido Succínico/metabolismo , Actinas/análise , Actinas/metabolismo , Animais , Apoptose , Linhagem Celular , Movimento Celular , Proliferação de Células , Células Estreladas do Fígado/citologia , Células Estreladas do Fígado/metabolismo , Humanos , Fígado/citologia , Fígado/metabolismo , Cirrose Hepática/patologia , Masculino , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: Chronic liver disease is becoming a major cause of morbidity and mortality worldwide. During liver injury, hepatic stellate cells (HSCs) trans-differentiate into activated myofibroblasts, which produce extracellular matrix. Succinate and succinate receptor (G-protein coupled receptor91, GPR91) signaling pathway has now emerged as a regulator of metabolic signaling. A previous study showed that succinate and its specific receptor, GPR91, are involved in the activation of HSCs and the overexpression of α-smooth muscle actin (α-SMA). Metformin, a well-known anti-diabetic drug, inhibits hepatic gluconeogenesis in the liver. Many studies have shown that metformin not only prevented, but also reversed, steatosis and inflammation in a nonalcoholic steatohepatitis (NASH) animal model. However, the role of metformin in HSC activation and succinate-GPR91 signaling has not been clarified. METHODS: The immortalized human HSCs, LX-2â¯cells, were used for the in vitro study. For the in vivo study, male C57BL/J6 mice were randomly divided into 3 groups and were fed with a methionine-choline-deficient diet (MCD diet group) as a nonalcoholic steatohepatitis (NASH) mouse model with or without 0.1% metformin for 12 weeks, or were fed a control methionine-choline-sufficient diet (MCS diet group). RESULTS: In our study, metformin and 5-aminoimidazole-4-carboxamide 1-ß-d-ribofuranoside (AICAR), which is an analog of adenosine monophosphate, were shown to suppress α-SMA expression via enhanced phosphorylation of AMP-activated protein kinase (AMPK) and inhibition of succinate-GPR91 signaling in activated LX-2â¯cells induced by palmitate- or succinate. Metformin and AICAR also reduced succinate concentration in the cell lysates when LX-2â¯cells were treated with palmitate. Moreover, metformin and AICAR reduced interleukin-6 and, transforming growth factor-ß1 production in succinate-treated LX-2â¯cells. Both metformin and AICAR inhibited succinate-stimulated HSC proliferation and cell migration. Mice fed a MCD diet demonstrated increased steatohepatitis and liver fibrosis compared to that of mice fed control diet. Metformin ameliorated steatohepatitis, liver fibrosis, inflammatory cytokine production and decreased α -SMA and GPR91expression in the livers of the MCD diet-fed mice. CONCLUSION: This study shows that metformin can attenuate activation of HSCs by activating the AMPK pathway and inhibiting the succinate-GPR91 pathway. Metformin has therapeutic potential for treating steatohepatitis and liver fibrosis.
Assuntos
Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/metabolismo , Metformina/administração & dosagem , Receptores Acoplados a Proteínas G/metabolismo , Ácido Succínico/metabolismo , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Células Estreladas do Fígado/patologia , Humanos , Hipoglicemiantes/administração & dosagem , Cirrose Hepática/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Resultado do TratamentoRESUMO
BACKGRUOUND: Hepatic stellate cells (HSCs) are the major cells which play a pivotal role in liver fibrosis. During injury, extracellular stimulators can induce HSCs transdifferentiated into active form. Phloretin showed its ability to protect the liver from injury, so in this research we would like to investigate the effect of phloretin on succinate-induced HSCs activation in vitro and liver fibrosis in vivo study. METHODS: In in vitro, succinate was used to induce HSCs activation, and then the effect of phloretin on activated HSCs was examined. In in vivo, succinate was used to generated liver fibrosis in mouse and phloretin co-treated to check its protection on the liver. RESULTS: Phloretin can reduce the increase of fibrogenic markers and inhibits the proliferation, migration, and contraction caused by succinate in in vitro experiments. Moreover, an upregulation of proteins associated with aerobic glycolysis occurred during the activation of HSCs, which was attenuated by phloretin treatment. In in vivo experiments, intraperitoneal injection of phloretin decreased expression of fibrotic and glycolytic markers in the livers of mice with sodium succinate diet-induced liver fibrosis. These results suggest that aerobic glycolysis plays critical role in activation of HSCs and succinate can induce liver fibrosis in mice, whereas phloretin has therapeutic potential for treating hepatic fibrosis. CONCLUSION: Intraperitoneal injection of phloretin attenuated succinate-induced hepatic fibrosis and alleviates the succinate-induced HSCs activation.
Assuntos
Floretina , Ácido Succínico , Camundongos , Animais , Ácido Succínico/metabolismo , Ácido Succínico/farmacologia , Ácido Succínico/uso terapêutico , Floretina/farmacologia , Floretina/metabolismo , Floretina/uso terapêutico , Células Estreladas do Fígado , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/prevenção & controleRESUMO
BACKGROUND: Hepatic stellate cells (HSCs) are known to play a fundamental role in the progression of liver fibrosis. Once HSCs are activated, they are involved in proliferation, migration, and contractility which are characteristics of liver fibrogenesis. Recent studies have shown that irisin, a myokine secreted during physical exercise, has a protective effect in various metabolic diseases, especially in renal fibrosis. However, whether irisin is involved in HSC activation and other processes associated with liver fibrosis has not yet been investigated. In this study, we reveal the role of irisin in HSC activation as well as in proliferation, migration, and contractile properties of HSCs in vitro. METHODS: LX-2 cells, immortalized human HSCs, were treated with transforming growth factor beta 1 (TGF-ß1), a core regulator of HSC fibrosis, with or without irisin, and markers of the aforementioned processes were analyzed. Further, an inflammatory response was stimulated with TGF-ß1 and lipopolysaccharide (LPS) in combination with irisin and the expression of cytokines was measured. RESULTS: Recombinant irisin significantly suppressed the expression of TGF-ß1-stimulated fibrosis markers including alpha-smooth muscle actin and collagen type 1 alpha 1 and prevented the TGF-ß1-induced proliferation, migration, and contractility of LX-2 cells. Additionally, irisin ameliorated the production of interleukin-6 (IL-6) and IL-1ß induced by TGF-ß1 and LPS treatments. CONCLUSION: These findings suggested that irisin potently improved the progression of hepatic fibrosis by regulating HSC activation, proliferation, migration, contractility, and HSC-mediated production of inflammatory cytokine.
Assuntos
Fibronectinas/farmacologia , Células Estreladas do Fígado/metabolismo , Cirrose Hepática/patologia , Fator de Crescimento Transformador beta1/farmacologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Células Estreladas do Fígado/citologia , Células Estreladas do Fígado/efeitos dos fármacos , Humanos , Proteínas Recombinantes/farmacologiaRESUMO
Fibroblast growth factor 21 (FGF21) is an important metabolic regulator expressed predominantly in the liver. In this study, we evaluated the role of LY2405319, an analogue of FGF21, in hepatic stellate cell (HSC) activation and in a methionine and choline-deficient (MCD)-diet induced mouse model of liver fibrosis. During liver injury, HSCs trans-differentiate into activated myofibroblasts which produce alpha-smooth muscle actin (α-SMA) and become a major cell type in hepatic fibrogenesis. Succinate and succinate receptor (GPR91) signaling has emerged as a regulator to promote α-SMA production in MCD diet- induced mice. Treatment with palmitate or MCD medium on LX-2 cells (HSCs) increased succinate concentration in the conditioned medium and cell lysate of LX-2 cells and increased production of GPR91 and α-SMA. However, LY2405319 administration ameliorates palmitate or MCD media-induced succinate production and decreases over-expression of GPR91 and α-SMA in LX2-cells. In an in vivo study, the MCD diet treatment caused increased steatohepatitis and liver fibrosis compared with the control diet in mice. Administration of LY2405319 improved steatohepatitis ameliorated GPR91 and α -SMA production in the liver, decreased succinate concentration in both liver and serum of MCD diet -induced mice. These results suggest that FGF21 reduces production of α-SMA by inhibiting the succinate-GPR91 pathway. We conclude that FGF21 acts as an inhibitor of the succinate-GPR91 pathway to control liver fibrosis. This suggests that FGF21 has therapeutic potential for treating liver fibrogenesis.