Abstract：
BACKGROUND: The occurrence of hyperlipidemia is significantly influenced by lipid synthesis, which is regulated by sterol regulatory element binding proteins (SREBPs), thus the development of drugs that inhibit lipid synthesis has become a popular treatment strategy for hyperlipidemia. Alisol B (ALB), a triterpenoid compound extracted from Alisma, has been reported to ameliorate no-nalcoholic steatohepatitis (NASH) and slow obesity. However, the effect of ALB on hyperlipidemia and mechanism are unclear.
OBJECTIVE: To examine the therapeutic impact of ALB on hyperlipidemia whether it inhibits SREBPs to reduce lipid synthesis.
METHODS: HepG2, HL7702 cells, and C57BL/6J mice were used to explore the effect of ALB on hyperlipidemia and the molecular mechanism in vivo and in vitro.
METHODS: Hyperlipidemia models were established using western diet (WD)-fed mice in vivo and oleic acid (OA)-induced hepatocytes in vitro. Western blot, real-time PCR and other biological methods verified that ALB regulated AMPK/mTOR/SREBPs to inhibit lipid synthesis. Cellular thermal shift assay (CETSA), molecular dynamics (MD), and ultrafiltration-LC/MS analysis were used to evaluate the binding of ALB to voltage-dependent anion channel protein-1 (VDAC1).
RESULTS: ALB decreased TC, TG, LDL-c, and increased HDL-c in blood, thereby ameliorating liver damage. Gene set enrichment analysis (GSEA) indicated that ALB inhibited the biosynthesis of cholesterol and fatty acids. Consistently, ALB inhibited the protein expression of n-SREBPs and downstream genes. Mechanistically, the impact of ALB on SREBPs was dependent on the regulation of AMPK/mTOR, thereby impeding the transportation of SREBPs from endoplasmic reticulum (ER) to golgi apparatus (GA). Further investigations indicated that the activation of AMPK by ALB was independent on classical upstream CAMKK2 and LKB1. Instead, ALB resulted in a decrease in ATP levels and an increase in the ratios of ADP/ATP and AMP/ATP. CETSA, MD, and ultrafiltration-LC/MS analysis indicated that ALB interacted with VDAC1. Molecular docking revealed that ALB directly bound to VDAC1 by forming hydrogen bonds at the amino acid sites S196 and H184 in the ATP-binding region. Importantly, the thermal stabilization of ALB on VDAC1 was compromised when VDAC1 was mutated at S196 and H184, suggesting that these amino acids played a crucial role in the interaction.
CONCLUSIONS: Our findings reveal that VDAC1 serves as the target of ALB, leading to the inhibition of lipid synthesis, presents potential target and candidate drugs for hyperlipidemia.
OBJECTIVE: To examine the therapeutic impact of ALB on hyperlipidemia whether it inhibits SREBPs to reduce lipid synthesis.
METHODS: HepG2, HL7702 cells, and C57BL/6J mice were used to explore the effect of ALB on hyperlipidemia and the molecular mechanism in vivo and in vitro.
METHODS: Hyperlipidemia models were established using western diet (WD)-fed mice in vivo and oleic acid (OA)-induced hepatocytes in vitro. Western blot, real-time PCR and other biological methods verified that ALB regulated AMPK/mTOR/SREBPs to inhibit lipid synthesis. Cellular thermal shift assay (CETSA), molecular dynamics (MD), and ultrafiltration-LC/MS analysis were used to evaluate the binding of ALB to voltage-dependent anion channel protein-1 (VDAC1).
RESULTS: ALB decreased TC, TG, LDL-c, and increased HDL-c in blood, thereby ameliorating liver damage. Gene set enrichment analysis (GSEA) indicated that ALB inhibited the biosynthesis of cholesterol and fatty acids. Consistently, ALB inhibited the protein expression of n-SREBPs and downstream genes. Mechanistically, the impact of ALB on SREBPs was dependent on the regulation of AMPK/mTOR, thereby impeding the transportation of SREBPs from endoplasmic reticulum (ER) to golgi apparatus (GA). Further investigations indicated that the activation of AMPK by ALB was independent on classical upstream CAMKK2 and LKB1. Instead, ALB resulted in a decrease in ATP levels and an increase in the ratios of ADP/ATP and AMP/ATP. CETSA, MD, and ultrafiltration-LC/MS analysis indicated that ALB interacted with VDAC1. Molecular docking revealed that ALB directly bound to VDAC1 by forming hydrogen bonds at the amino acid sites S196 and H184 in the ATP-binding region. Importantly, the thermal stabilization of ALB on VDAC1 was compromised when VDAC1 was mutated at S196 and H184, suggesting that these amino acids played a crucial role in the interaction.
CONCLUSIONS: Our findings reveal that VDAC1 serves as the target of ALB, leading to the inhibition of lipid synthesis, presents potential target and candidate drugs for hyperlipidemia.
摘要：
背景：高脂血症的发生受到脂质合成的显著影响，它由固醇调节元件结合蛋白（SREBPs）调节，因此，开发抑制脂质合成的药物已成为高脂血症的流行治疗策略。AlisolB(ALB),从泽泻中提取的三萜类化合物，据报道可以改善非酒精性脂肪性肝炎（NASH）和缓慢肥胖。然而,ALB对高脂血症的作用及机制尚不清楚。
目的：观察ALB是否抑制SREBPs降低脂质合成对高脂血症的治疗作用。
方法：HepG2,HL7702细胞,以C57BL/6J小鼠为研究对象,探讨ALB对高脂血症的影响及其体内外分子机制。
方法：用西方饮食（WD）喂养的小鼠体内和油酸（OA）诱导的肝细胞体外建立高脂血症模型。蛋白质印迹,实时PCR和其他生物学方法证实ALB调节AMPK/mTOR/SREBPs抑制脂质合成。细胞热转移测定（CETSA），分子动力学（MD），和超滤-LC/MS分析用于评估ALB与电压依赖性阴离子通道蛋白1（VDAC1）的结合。
结果：ALB降低TC，TG,LDL-c,血液中的HDL-c升高,从而改善肝脏损伤。基因集富集分析（GSEA）表明ALB抑制胆固醇和脂肪酸的生物合成。始终如一，ALB抑制n-SREBPs和下游基因的蛋白表达。机械上,ALB对SREBP的影响依赖于AMPK/mTOR的调节，从而阻碍SREBP从内质网（ER）运输到高尔基体（GA）。进一步的研究表明，ALB对AMPK的激活与经典的上游CAMKK2和LKB1无关。相反，ALB导致ATP水平降低和ADP/ATP和AMP/ATP的比率增加。CETSA,MD,超滤-LC/MS分析表明ALB与VDAC1相互作用。分子对接显示，ALB通过在ATP结合区的氨基酸位点S196和H184处形成氢键直接与VDAC1结合。重要的是,当VDAC1在S196和H184突变时，ALB在VDAC1上的热稳定性受到损害，这表明这些氨基酸在相互作用中起着至关重要的作用。
结论：我们的研究结果表明，VDAC1作为ALB的靶标，导致脂质合成的抑制，提出了高脂血症的潜在靶点和候选药物。
目的：观察ALB是否抑制SREBPs降低脂质合成对高脂血症的治疗作用。
方法：HepG2,HL7702细胞,以C57BL/6J小鼠为研究对象,探讨ALB对高脂血症的影响及其体内外分子机制。
方法：用西方饮食（WD）喂养的小鼠体内和油酸（OA）诱导的肝细胞体外建立高脂血症模型。蛋白质印迹,实时PCR和其他生物学方法证实ALB调节AMPK/mTOR/SREBPs抑制脂质合成。细胞热转移测定（CETSA），分子动力学（MD），和超滤-LC/MS分析用于评估ALB与电压依赖性阴离子通道蛋白1（VDAC1）的结合。
结果：ALB降低TC，TG,LDL-c,血液中的HDL-c升高,从而改善肝脏损伤。基因集富集分析（GSEA）表明ALB抑制胆固醇和脂肪酸的生物合成。始终如一，ALB抑制n-SREBPs和下游基因的蛋白表达。机械上,ALB对SREBP的影响依赖于AMPK/mTOR的调节，从而阻碍SREBP从内质网（ER）运输到高尔基体（GA）。进一步的研究表明，ALB对AMPK的激活与经典的上游CAMKK2和LKB1无关。相反，ALB导致ATP水平降低和ADP/ATP和AMP/ATP的比率增加。CETSA,MD,超滤-LC/MS分析表明ALB与VDAC1相互作用。分子对接显示，ALB通过在ATP结合区的氨基酸位点S196和H184处形成氢键直接与VDAC1结合。重要的是,当VDAC1在S196和H184突变时，ALB在VDAC1上的热稳定性受到损害，这表明这些氨基酸在相互作用中起着至关重要的作用。
结论：我们的研究结果表明，VDAC1作为ALB的靶标，导致脂质合成的抑制，提出了高脂血症的潜在靶点和候选药物。
