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Abstract：
BACKGROUND: Enhanced glycolysis is a crucial metabolic event that drives the development of liver fibrosis, but the molecular mechanisms have not been fully understood. Lactate is the endproduct of glycolysis, which has recently been identified as a bioactive metabolite binding to G-protein-coupled receptor 81 (GPR81). We then questioned whether GPR81 is implicated in the development of liver fibrosis.
METHODS: The level of GPR81 was determined in mice with carbon tetrachloride (CCl4)-induced liver fibrosis and in transforming growth factor beta 1 (TGF-β1)-activated hepatic stellate cells (HSCs) LX-2. To investigate the significance of GPR81 in liver fibrosis, wild-type (WT) and GPR81 knockout (KO) mice were exposed to CCl4, and then the degree of liver fibrosis was determined. In addition, the GPR81 agonist 3,5-dihydroxybenzoic acid (DHBA) was supplemented in CCl4-challenged mice and TGF-β1-activated LX-2 cells to further investigate the pathological roles of GPR81 on HSCs activation.
RESULTS: CCl4 exposure or TGF-β1 stimulation significantly upregulated the expression of GPR81, while deletion of GPR81 alleviated CCl4-induced elevation of aminotransferase, production of pro-inflammatory cytokines, and deposition of collagen. Consistently, the production of TGF-β1, the expression of alpha-smooth muscle actin (α-SMA) and collagen I (COL1A1), as well as the elevation of hydroxyproline were suppressed in GPR81 deficient mice. Supplementation with DHBA enhanced CCl4-induced liver fibrogenesis in WT mice but not in GPR81 KO mice. DHBA also promoted TGF-β1-induced LX-2 activation. Mechanistically, GPR81 suppressed cAMP/CREB and then inhibited the expression of Smad7, a negative regulator of Smad3, which resulted in increased phosphorylation of Smad3 and enhanced activation of HSCs.
CONCLUSIONS: GPR81 might be a detrimental factor that promotes the development of liver fibrosis by regulating CREB/Smad7 pathway.
METHODS: The level of GPR81 was determined in mice with carbon tetrachloride (CCl4)-induced liver fibrosis and in transforming growth factor beta 1 (TGF-β1)-activated hepatic stellate cells (HSCs) LX-2. To investigate the significance of GPR81 in liver fibrosis, wild-type (WT) and GPR81 knockout (KO) mice were exposed to CCl4, and then the degree of liver fibrosis was determined. In addition, the GPR81 agonist 3,5-dihydroxybenzoic acid (DHBA) was supplemented in CCl4-challenged mice and TGF-β1-activated LX-2 cells to further investigate the pathological roles of GPR81 on HSCs activation.
RESULTS: CCl4 exposure or TGF-β1 stimulation significantly upregulated the expression of GPR81, while deletion of GPR81 alleviated CCl4-induced elevation of aminotransferase, production of pro-inflammatory cytokines, and deposition of collagen. Consistently, the production of TGF-β1, the expression of alpha-smooth muscle actin (α-SMA) and collagen I (COL1A1), as well as the elevation of hydroxyproline were suppressed in GPR81 deficient mice. Supplementation with DHBA enhanced CCl4-induced liver fibrogenesis in WT mice but not in GPR81 KO mice. DHBA also promoted TGF-β1-induced LX-2 activation. Mechanistically, GPR81 suppressed cAMP/CREB and then inhibited the expression of Smad7, a negative regulator of Smad3, which resulted in increased phosphorylation of Smad3 and enhanced activation of HSCs.
CONCLUSIONS: GPR81 might be a detrimental factor that promotes the development of liver fibrosis by regulating CREB/Smad7 pathway.
摘要：
背景：糖酵解增强是驱动肝纤维化发展的关键代谢事件，但是分子机制还没有被完全理解。乳酸是糖酵解的最终产物,最近被鉴定为与G蛋白偶联受体81（GPR81）结合的生物活性代谢物。然后，我们质疑GPR81是否与肝纤维化的发展有关。
方法：在四氯化碳（CCl4）诱导的肝纤维化小鼠和转化生长因子β1（TGF-β1）激活的肝星状细胞（HSCs）LX-2中测定GPR81的水平。探讨GPR81在肝纤维化中的意义,野生型(WT)和GPR81敲除(KO)小鼠暴露于CCl4,然后测定肝纤维化程度。此外,在CCl4攻击的小鼠和TGF-β1激活的LX-2细胞中补充了GPR81激动剂3,5-二羟基苯甲酸（DHBA），以进一步研究GPR81对HSC活化的病理作用。
结果：CCl4暴露或TGF-β1刺激显着上调GPR81的表达，而GPR81的缺失减轻了CCl4诱导的转氨酶升高，促炎细胞因子的产生，和胶原蛋白的沉积。始终如一，TGF-β1的产生，α-平滑肌肌动蛋白（α-SMA）和I型胶原（COL1A1）的表达，在GPR81缺陷小鼠中，羟脯氨酸的升高也受到抑制。在WT小鼠中补充DHBA可增强CCl4诱导的肝纤维化发生，但在GPR81KO小鼠中则没有。DHBA还促进TGF-β1诱导的LX-2活化。机械上,GPR81抑制cAMP/CREB，然后抑制Smad3的负调节因子Smad7的表达，这导致Smad3的磷酸化增加并增强HSC的活化。
结论：GPR81可能是通过调节CREB/Smad7通路促进肝纤维化发展的有害因素。
方法：在四氯化碳（CCl4）诱导的肝纤维化小鼠和转化生长因子β1（TGF-β1）激活的肝星状细胞（HSCs）LX-2中测定GPR81的水平。探讨GPR81在肝纤维化中的意义,野生型(WT)和GPR81敲除(KO)小鼠暴露于CCl4,然后测定肝纤维化程度。此外,在CCl4攻击的小鼠和TGF-β1激活的LX-2细胞中补充了GPR81激动剂3,5-二羟基苯甲酸（DHBA），以进一步研究GPR81对HSC活化的病理作用。
结果：CCl4暴露或TGF-β1刺激显着上调GPR81的表达，而GPR81的缺失减轻了CCl4诱导的转氨酶升高，促炎细胞因子的产生，和胶原蛋白的沉积。始终如一，TGF-β1的产生，α-平滑肌肌动蛋白（α-SMA）和I型胶原（COL1A1）的表达，在GPR81缺陷小鼠中，羟脯氨酸的升高也受到抑制。在WT小鼠中补充DHBA可增强CCl4诱导的肝纤维化发生，但在GPR81KO小鼠中则没有。DHBA还促进TGF-β1诱导的LX-2活化。机械上,GPR81抑制cAMP/CREB，然后抑制Smad3的负调节因子Smad7的表达，这导致Smad3的磷酸化增加并增强HSC的活化。
结论：GPR81可能是通过调节CREB/Smad7通路促进肝纤维化发展的有害因素。
