Abstract：
BACKGROUND: Berberine (BBR) is the main active component from Coptidis rhizome, a well-known Chinese herbal medicine used for metabolic diseases, especially diabetes for thousands of years. BBR has been reported to cure various metabolic disorders, such as nonalcoholic fatty liver disease (NAFLD). However, the direct proteomic targets and underlying molecular mechanism of BBR against NAFLD remain less understood.
OBJECTIVE: To investigate the direct target and corresponding molecular mechanism of BBR on NAFLD is the aim of the current study.
METHODS: High-fat diet (HFD)-fed mice and oleic acid (OA) stimulated HepG2 cells were utilized to verify the beneficial impacts of BBR on glycolipid metabolism profiles. The click chemistry in proteomics, DARTS, CETSA, SPR and fluorescence co-localization analysis were conducted to identify the targets of BBR for NAFLD. RNA-seq and shRNA/siRNA were used to investigate the downstream pathways of the target.
RESULTS: BBR improved hepatic steatosis, ameliorated insulin resistance, and reduced TG levels in the NAFLD models. Importantly, Aldo-keto reductase 1B10 (AKR1B10) was first proved as the target of BBR for NAFLD. The gene expression of AKR1B10 increased significantly in the NAFLD patients\' liver tissue. We further demonstrated that HFD and OA increased AKR1B10 expression in the C57BL/6 mice\'s liver and HepG2 cells, respectively, whereas BBR decreased the expression and activities of AKR1B10. Moreover, the knockdown of AKR1B10 by applying shRNA/siRNA profoundly impacted the beneficial effects on the pathogenesis of NAFLD by BBR. Meanwhile, the changes in various proteins (ACC1, CPT-1, GLUT2, etc.) are responsible for hepatic lipogenesis, fatty acid oxidation, glucose uptake, etc. by BBR were reversed by the knockdown of AKR1B10. Additionally, RNA-seq was used to identify the downstream pathway of AKR1B10 by examining the gene expression of liver tissues from HFD-fed mice. Our findings revealed that BBR markedly increased the protein levels of PPARα while downregulating the expression of PPARγ. However, various proteins of PPAR signaling pathways remained unaffected post the knockdown of AKR1B10.
CONCLUSIONS: BBR alleviated NAFLD via mediating PPAR signaling pathways through targeting AKR1B10. This study proved that AKR1B10 is a novel target of BBR for NAFLD treatment and helps to find new targets for the treatment of NAFLD by using active natural compounds isolated from traditional herbal medicines as the probe.
OBJECTIVE: To investigate the direct target and corresponding molecular mechanism of BBR on NAFLD is the aim of the current study.
METHODS: High-fat diet (HFD)-fed mice and oleic acid (OA) stimulated HepG2 cells were utilized to verify the beneficial impacts of BBR on glycolipid metabolism profiles. The click chemistry in proteomics, DARTS, CETSA, SPR and fluorescence co-localization analysis were conducted to identify the targets of BBR for NAFLD. RNA-seq and shRNA/siRNA were used to investigate the downstream pathways of the target.
RESULTS: BBR improved hepatic steatosis, ameliorated insulin resistance, and reduced TG levels in the NAFLD models. Importantly, Aldo-keto reductase 1B10 (AKR1B10) was first proved as the target of BBR for NAFLD. The gene expression of AKR1B10 increased significantly in the NAFLD patients\' liver tissue. We further demonstrated that HFD and OA increased AKR1B10 expression in the C57BL/6 mice\'s liver and HepG2 cells, respectively, whereas BBR decreased the expression and activities of AKR1B10. Moreover, the knockdown of AKR1B10 by applying shRNA/siRNA profoundly impacted the beneficial effects on the pathogenesis of NAFLD by BBR. Meanwhile, the changes in various proteins (ACC1, CPT-1, GLUT2, etc.) are responsible for hepatic lipogenesis, fatty acid oxidation, glucose uptake, etc. by BBR were reversed by the knockdown of AKR1B10. Additionally, RNA-seq was used to identify the downstream pathway of AKR1B10 by examining the gene expression of liver tissues from HFD-fed mice. Our findings revealed that BBR markedly increased the protein levels of PPARα while downregulating the expression of PPARγ. However, various proteins of PPAR signaling pathways remained unaffected post the knockdown of AKR1B10.
CONCLUSIONS: BBR alleviated NAFLD via mediating PPAR signaling pathways through targeting AKR1B10. This study proved that AKR1B10 is a novel target of BBR for NAFLD treatment and helps to find new targets for the treatment of NAFLD by using active natural compounds isolated from traditional herbal medicines as the probe.
摘要：
背景：小檗碱（BBR）是黄连根茎的主要活性成分，一种用于代谢疾病的著名中草药，尤其是数千年来的糖尿病。据报道，BBR可以治疗各种代谢紊乱，如非酒精性脂肪性肝病（NAFLD）。然而,BBR抗NAFLD的直接蛋白质组学靶标和潜在分子机制尚不清楚。
目的：探讨BBR对NAFLD的直接作用靶点及相应的分子机制是本研究的目的。
方法:利用高脂肪饮食(HFD)喂养的小鼠和油酸(OA)刺激的HepG2细胞来验证BBR对糖脂代谢谱的有益影响。蛋白质组学中的点击化学，达人,CETSA,进行SPR和荧光共定位分析以鉴定BBR用于NAFLD的靶标。RNA-seq和shRNA/siRNA用于研究靶标的下游途径。
结果：BBR改善了肝脏脂肪变性，改善胰岛素抵抗，并降低NAFLD模型中的TG水平。重要的是,Aldo-keto还原酶1B10（AKR1B10）首次被证明是NAFLDBBR的靶标。NAFLD患者肝组织中AKR1B10的基因表达明显增加。我们进一步证明了HFD和OA增加了AKR1B10在C57BL/6小鼠肝脏和HepG2细胞中的表达，分别,而BBR降低了AKR1B10的表达和活性。此外，通过应用shRNA/siRNA敲低AKR1B10对BBR对NAFLD发病机制的有益影响。同时,各种蛋白质(ACC1、CPT-1、GLUT2等)的变化。)负责肝脏脂肪生成，脂肪酸氧化，葡萄糖摄取，等。BBR被AKR1B10的敲低逆转。此外,RNA-seq用于通过检查来自HFD饲喂的小鼠的肝组织的基因表达来鉴定AKR1B10的下游途径。我们的发现表明，BBR显着增加PPARα的蛋白水平，同时下调PPARγ的表达。然而,在AKR1B10敲低后,PPAR信号通路的各种蛋白仍不受影响.
结论：BBR通过靶向AKR1B10介导PPAR信号通路减轻NAFLD。这项研究证明，AKR1B10是BBR治疗NAFLD的新靶点，并以从传统草药中分离出的活性天然化合物为探针，有助于寻找治疗NAFLD的新靶点。
目的：探讨BBR对NAFLD的直接作用靶点及相应的分子机制是本研究的目的。
方法:利用高脂肪饮食(HFD)喂养的小鼠和油酸(OA)刺激的HepG2细胞来验证BBR对糖脂代谢谱的有益影响。蛋白质组学中的点击化学，达人,CETSA,进行SPR和荧光共定位分析以鉴定BBR用于NAFLD的靶标。RNA-seq和shRNA/siRNA用于研究靶标的下游途径。
结果：BBR改善了肝脏脂肪变性，改善胰岛素抵抗，并降低NAFLD模型中的TG水平。重要的是,Aldo-keto还原酶1B10（AKR1B10）首次被证明是NAFLDBBR的靶标。NAFLD患者肝组织中AKR1B10的基因表达明显增加。我们进一步证明了HFD和OA增加了AKR1B10在C57BL/6小鼠肝脏和HepG2细胞中的表达，分别,而BBR降低了AKR1B10的表达和活性。此外，通过应用shRNA/siRNA敲低AKR1B10对BBR对NAFLD发病机制的有益影响。同时,各种蛋白质(ACC1、CPT-1、GLUT2等)的变化。)负责肝脏脂肪生成，脂肪酸氧化，葡萄糖摄取，等。BBR被AKR1B10的敲低逆转。此外,RNA-seq用于通过检查来自HFD饲喂的小鼠的肝组织的基因表达来鉴定AKR1B10的下游途径。我们的发现表明，BBR显着增加PPARα的蛋白水平，同时下调PPARγ的表达。然而,在AKR1B10敲低后,PPAR信号通路的各种蛋白仍不受影响.
结论：BBR通过靶向AKR1B10介导PPAR信号通路减轻NAFLD。这项研究证明，AKR1B10是BBR治疗NAFLD的新靶点，并以从传统草药中分离出的活性天然化合物为探针，有助于寻找治疗NAFLD的新靶点。
