二苯乙烯类化合物通过NLRP3炎症小体改善脂肪细胞胰岛素抵抗的作用机制

Mechanism of stilbene compounds on the improvement of insulin resistance of adipocytes by NLRP3 inflammasome

关键词:  二苯乙烯类化合物  胰岛素抵抗  NLRP3炎症小体  炎症  网络药理学

Key words:  stilbene compounds  insulin resistance  NLRP3 inflammasome  inflammation  network pharmacology

摘要:

[目的] 探讨源自三种中药的二苯乙烯类化合物白藜芦醇（Re）、桑皮苷A（SP）、何首乌苷（HSW）改善胰岛素抵抗（IR）的作用机制。[方法] 网络药理学预测三种二苯乙烯类化合物的潜在靶点；利用肿瘤坏死因子-α（TNF-α）诱导3T3-L1脂肪细胞建立胰岛素抵抗细胞模型（IR-3T3-L1）；使用NOD样受体热蛋白结构域相关蛋白3（NLRP3）的抑制剂MCC950预处理细胞，分别采用噻唑蓝（MTT）法和葡萄糖氧化酶法检测Re、SP、HSW对IR-3T3-L1细胞生存率和葡萄糖消耗的影响；采用分子对接技术和细胞热转移实验（CETSA）考察Re、SP、HSW与NLRP3的结合情况；采用蛋白免疫印迹（Western blot）法检测NLRP3相关蛋白表达情况；采用逆转录实时定量聚合酶链式反应（RT-qPCR）法检测相关炎症因子基因表达情况。[结果] 网络药理学分析表明，NOD样受体信号通路在Re、SP、HSW改善炎症与治疗糖尿病方面有着重要影响；与Re、SP、HSW单独给药组相比，同时使用NLRP3的抑制剂MCC950进一步促进葡萄糖消耗；CETSA和分子对接结果显示，SP、HSW能够与NLRP3结合；与空白对照组相比，模型组的NLRP3、剪切型含半胱氨酸的天冬氨酸蛋白水解酶1（cle-caspase 1）蛋白表达明显增多，caspase 1前体（pro-caspase 1）蛋白表达明显减少，模型组的白细胞介素-6（IL-6）、白细胞介素-18（IL-18）、白细胞介素-1β（IL-1β）的mRNA表达明显增加，脂联素（Adipoq）的mRNA的表达明显下降；与模型组相比，Re、SP、HSW组明显下调NLRP3的蛋白表达，SP、HSW组明显增加pro-caspase 1的蛋白表达，HSW组明显下调cle-caspase 1的蛋白表达。Re显著降低IL-6、IL-18、IL-1β的mRNA表达，SP显著降低IL-6、IL-1β的mRNA表达，HSW显著降低IL-1β的mRNA表达。[结论] Re、SP、HSW可能通过抑制NLRP3炎性小体改善脂肪细胞炎症反应和IR，从而发挥降糖活性。

Abstract:

[Objective] To investigate the mechanism of stilbene compounds，including resveratrol（Re），mulberroside A（SP），and polygonum multiflorum glycosides（HSW），from three traditional Chinese medicines on the improvement of insulin resistance（IR） of adipocytes. [Methods] Network pharmacology was used to predict the potential therapeutic targets of the tested compounds；insulin resistant 3T3-L1 adipocytes model（IR-3T3-L1） was established by the stimulation of tumor necrosis factor α（TNF-α）；after pretreated with NLRP3（NOD-like receptor pyrin domain containing 3） inhibitor MCC950，the growth inhibitory effect and glucose consumption of the tested compound on the IR-3T3-L1 cells were determined by thiazole blue（MTT） method and Glucose oxidase method，respectively；molecular docking and cell thermal shift assay（CETSA） were used to investigate the binding of NLRP3 with Re，SP and HSW；Western Blot was used to test the expression of NLRP3 inflammasome-related protein；reverse transcription-quantitative polymerase chain reaction（RT-qPCR） was performed to investigate the mRNA expression of inflammatory factors. [Results] The results of network pharmacology analysis indicated that NOD-like receptor signaling pathway might play an important role in the beneficial effects of Re，SP，and HSW on inflammation and diabetes mellitus；the addition of MCC950 further increased the glucose uptake compared with Re，SP，HSW alone treatment；the results of molecular docking and CETSA showed that SP，HSW could bind to NLRP3；compared with the control group，the protein expression of NLRP3 and cleaved caspase-1（cle-caspase 1） in the model group was upregulated，and the pro-caspase 1 protein was downregulated. Meanwhile TNF-α significantly upregulated the mRNA expression of interleukin-1β（IL-1β），interleukin-6（IL-6） and interleukin-18（IL-18），and downregulated the mRNA expression of adiponectin（Adipoq）；but compared with the model group，Re，SP and HSW could significantly decrease the protein expressions of NLRP3，while SP and HSW significantly increased the protein expression of pro-caspase 1，and HSW significantly reduced the protein expression of cle-caspase 1. In addition，compared with the model group，Re significantly inhibited the mRNA expression of IL-6，IL-18 and IL-1β，and SP decreased the mRNA expression of IL-6 and IL-18，and HSW significantly decreased the mRNA expression of IL-1β. [Conclusion] Re，SP and HSW exhibited anti-diabetic effects through suppressing inflammatory responses and improving IR of adipocytes by inhibition of NLRP3 inflammasome.