姜黄素激活Nrf2系统缓解线粒体损伤和胰岛素抵抗作用机制研究

发布时间：2019-07-09 06:22

【摘要】：目的氧化应激导致的线粒体功能损害参与胰岛素抵抗(Insulin resistance,IR)。姜黄素(Curcumin,Cur)可激活Nrf2系统对抗线粒体氧化应激及缓解胰岛素抵抗。但其作用机制尚不明确。本研究围绕着姜黄素是否以及如何通过调控Nrf2功能缓解线粒体损伤与IR发病这一核心问题,进一步深度解析Nrf2增敏胰岛素的作用途径、作用靶点以及相关信号机理。研究将阐明姜黄素转化应用的药理基础从而为应用于糖尿病的防治奠定理论基础。方法首先选用C57BL/6J小鼠进行高脂饲料干预诱导IR模型,再给予Cur干预,观察姜黄素的作用,探讨Cur保护线粒体和逆转IR作用靶点。为深入了解Nrf2抗氧化系统在其中所起作用,利用棕榈酸诱导HepG2肝细胞IR模型给予Nrf2抑制剂维甲酸RA处理,观察对Cur作用影响。由于线粒体形态调控与其功能关系密切。随后,在此模型上进行促线粒体分裂抑制剂Mdivi-1处理,Cur是否通过影响线粒体形态起到调控线粒体功能以及与激活Nrf2系统和增强胰岛素信号关系。由于线粒体氧化还原平衡与钙稳态关系密切,最后,我们观察了 Cur以及Nrf2系统对线粒体钙含量影响,并利用线粒体钙通道抑制剂,进一步探讨其对胰岛素信号的调控作用。具体实验如下:动物IR模型采用60%的高脂饲料饲喂雄性C57BL/6J小鼠并根据葡萄糖耐量实验(IPGTT)结果判定模型是否成功。细胞IR模型采用人肝癌细胞HepG2并给予60μM饱和脂肪酸棕榈酸(Palmitate,PA)处理细胞诱导IR模型。动物IR模型成功后,连续2周进行Cur灌胃干预,Cur干预剂量为50mg/kg/d。细胞用棕榈酸IR造模时,给予不同的工具药处理,包括1μM Nrf2抑制剂维甲酸(Retinoic acid,RA)、50μM促线粒体分裂抑制剂 Mdivi-1 处理、30μM钌红(Ruthenium red,RuR)和10μM Cur干预。随后进行细胞线粒体膜电位、ROS、钙离子含量测定以及WB蛋白含量检测。蛋白免疫印迹法(Western blot，，WB)检测线粒体促分裂和融合蛋白(Drp1、Mfr2及OPA1)及相关调控因子(NRF1、mtTFA、TFB1M及TFB2M等)在C57BL/6J小鼠组织的变化。磷钼酸比色法测定小鼠肝脏组织内ATP含量及ATPase(Na+ K+-ATPase,Mg2+-ATPase,Ca2+-ATPase,Ca2+ Mg2+-ATPase)的活力,评价 Cur 对 IR 模型小鼠肝脏线粒体能量代谢的影响。细胞线粒体氧化还原状态检测采用TAB结合法测定线粒体中脂质过氧化指标MDA(malondialdehyde)含量;利用JC-1试剂盒测定细胞线粒体膜电位(Mitochondrial membrane potential,MMP)的改变;MitoSOXTM 试剂盒对 HepG2 肝癌细胞染色检测细胞线粒体内ROS的含量进行测定;同时,在RA、Mdivi-1和RuR处理细胞以后,利用WB方法检测胰岛素刺激的PKBSer473磷酸化、GRP78、IκBα及相关线粒体蛋白及调控因子的表达。结果无论在高脂饲料诱导的动物IR模型中还是在PA诱导的细胞IR模型中,线粒体融合蛋白均受到抑制(Mfn2、OPA1融合蛋白减少)、促分裂调控蛋白表达增多(Drp1促分裂蛋白增加)、转录调控蛋白下调(Nrf1、mtTFA、TFB1M及TFB2M表达降低),Cur均可以明显减轻甚至逆转高脂所造成的这些影响。此外,Cur干预可明显纠正IR模型小鼠肝脏组织 ATP 含量降低并增强 Na+ K+-ATPase,Mg2+-ATPase,Ca2+-ATPase,Ca2+ Mg2+-ATPase的酶活性活性。另外,Cur可部分对抗PA所致的线粒体活性氧ROS含量升高以及线粒体功能损害,即线粒体膜电位降低;反之,对细胞进行Nrf2抑制剂处理后,Cur的上述改善作用受到明显抑制,说明Cur通过激活Nrf2实现其对线粒体功能以及胰岛素信号的调控作用。进一步的研究表明,无论线粒体分裂抑制剂Mdivi-1以及Cur处理处理PAIR细胞,均能上调线粒体合成及转录调控蛋白,增强线粒体融合蛋白含量,同时抑制线粒体促分裂调控蛋白表达。最后,由于姜黄素具有减轻IR细胞线粒体钙超载作用。为了进一步评价姜黄素这一作用的药效意义,我们利用线粒体钙通道抑制剂处理IR细胞发现:IR细胞的线粒体钙超载受到抑制后,可以起到抑制炎症、内质网应激,同时激活Nrf2系统并增强胰岛素信号传导。结论Cur无论在高脂饲喂动物以及棕榈酸诱导的IR细胞均能上调促进线粒体合成及转录调控蛋白,增强线粒体融合蛋白并抑制促分裂调控蛋白表达,而且这些对线粒体的调控作用与姜黄素激活Nrf2抗氧化功能以及胰岛素信号的调控作用有关。因此,姜黄素通过激活Nrf2系统改善线粒体氧化应激并对线粒体形态和功能进行调控,这些作用是其改善胰岛素抵抗药效作用的重要机理,而姜黄素通过激活Nrf2系统对抗线粒体氧化应激并抑制钙超载诱发的炎症激活、内质网应激以及Nrf2系统的功能抑制,并依此改善胰岛素敏感性。
[Abstract]:Objective To investigate the role of mitochondrial function induced by oxidative stress in insulin resistance (IR). Curcumin (Curcuin, Cur) can activate the Nrf2 system to counter mitochondrial oxidative stress and to relieve insulin resistance. However, its mechanism of action is not clear. This study focused on whether curcumin and how to alleviate the core problem of mitochondrial injury and IR by regulating the function of Nrf2, and to further analyze the role of Nrf2 to sensitize the insulin, the target of action and the related signal mechanism. The study will clarify the pharmacological basis of curcumin conversion and lay a theoretical foundation for the prevention and treatment of diabetes. Methods C57BL/ 6J mice were first selected for high-fat feed intervention to induce IR, and then Cur intervention was given to observe the effect of curcumin on the protection of mitochondria and reverse the IR target. In order to understand the role of Nrf2 anti-oxidation system, the effect of Nrf2 inhibitor-retinoic acid RA on the effect of Nrf2 on the effect of Nrf2 on the effect of Nrf2 on the effect of Nrf2 on the effect of the anti-oxidation system was observed. The regulation of mitochondrial morphology is closely related to its function. Subsequently, the Mdivi-1 treatment of the mitogen-stimulating inhibitor Mdivi-1 was carried out on this model, and whether the Cur had the function of regulating the mitochondrial function by affecting the mitochondrial morphology and the relationship with the activation of the Nrf2 system and the enhanced insulin signal. In the end, the effects of Cur and Nrf2 on the content of calcium in mitochondria were observed, and the regulation of the insulin signal was further discussed by using the mitochondrial calcium channel inhibitor. The specific experiment is as follows: The animal IR model uses 60% high-fat feed to feed the male C57BL/ 6J mice and determines whether the model is successful according to the results of the glucose tolerance test (IPGTT). The cellular IR model used human liver cancer cell HepG2 and given a 60 & mu; M saturated fatty acid palmitate (Palmitate, PA) to process the cell-induced IR model. After the animal IR model was successful, Cur was given intragastric intervention for 2 weeks, and the intervention dose of Cur was 50 mg/ kg/ day. When the cells were made with palmitic acid IR, different administration treatments were administered, including 1. m u.M Nrf2 inhibitor Retinoic acid (RA),50. m u.M mitogen-mitogen inhibitor Mdivi-1 treatment,30. m u.M of red (RuR) and 10. m u.M Cur intervention. Then the cell mitochondrial membrane potential, the ROS, the calcium ion content determination, and the WB protein content detection were performed. The changes of mitochondrial mitogen and fusion protein (Dr1, Mfr2 and OPPA1) and related regulatory factors (NF1, mtTFA, TFB1M, and TFB2M, etc.) in C57BL/ 6J mice were detected by Western blot and WB. The activity of ATP content and ATPase (Na + K +-ATPase, Mg 2 +-ATPase, Ca 2 +-ATPase, Ca 2 + Mg 2 +-ATPase) in the liver of mice was determined by the P-P-acid colorimetric method, and the effect of Cur on the energy metabolism of liver mitochondria in the mouse liver was evaluated. The content of MDA (malonic dehyde) in the mitochondria was determined by a TAB-binding method, and the changes of the mitochondrial membrane potential (MMP) were determined by the JC-1 kit. The content of ROS in the mitochondria of HepG2 liver cancer cells was determined by the MiteSXTM kit. After the cells were treated with RA, Mdivi-1 and RuR, the phosphorylation of PKBSer473, the expression of GRP78, I-B and the related mitochondrial proteins and regulatory factors were detected by WB method. Results In the animal IR model induced by high-fat diet or in the PA-induced cellular IR model, the mitochondrial fusion protein was inhibited (Mfn2, OPA1 fusion protein decreased), the expression of the mitogen-controlled protein was increased (the DDr1 mitogen increased), and the transcription regulatory protein was down-regulated (Nrf1, Both mtTFA, TFB1M, and TFB2M expression decreased, and Cur could significantly reduce or even reverse the effects of high fat. In addition, the activity of the activity of Na + K +-ATPase, Mg2 +-ATPase, Ca2 +-ATPase, Ca2 + Mg2 +-ATPase can be significantly corrected by Cur intervention. in addition, that Cur can partially antagonize the increase in the ROS content of the mitochondria and the damage of the function of the mitochondria, that is, the mitochondrial membrane potential is reduced; on the contrary, after the Nrf2 inhibitor treatment is carried out on the cell, the above-mentioned improvement effect of the Cur is obviously inhibited, The effect of Cur on the function of mitochondria and the regulation of the insulin signal is explained by the activation of Nrf2. The further study shows that the mitochondrial synthesis and transcription regulation protein can be up-regulated, the content of the mitochondrial fusion protein can be enhanced, and the expression of the mitochondrial mitogen-promoting regulatory protein can be inhibited, regardless of the mitochondrial division inhibitor Mdivi-1 and the treatment of PAIR cells by the Cur treatment. And finally, the curcumin has the function of reducing the mitochondrial calcium overload of the IR cells. In order to further evaluate the effect of curcumin, we use the mitochondrial calcium channel inhibitor to treat IR cells, which can inhibit the inflammation, endoplasmic reticulum stress, and activate Nrf2 system and enhance the conduction of insulin signal. Conclusion Cur can upregulate the mitochondrial synthesis and transcriptional regulation protein, enhance the mitochondrial fusion protein and inhibit the expression of mitogen-regulated protein, whether in high-fat feeding animals and palmitic acid-induced IR cells. And the regulatory effects of these on the mitochondria are related to the activation of the anti-oxidation function of the curcumin and the regulation of the insulin signal. Therefore, the curcumin can improve the mitochondrial oxidative stress by activating the Nrf2 system and regulate the shape and function of the mitochondria, which is an important mechanism for improving the effect of insulin resistance, Curcumin inhibits the mitochondrial oxidative stress by activating the Nrf2 system and inhibits the inflammatory activation induced by calcium overload, endoplasmic reticulum stress, and the functional inhibition of the Nrf2 system, and accordingly improves insulin sensitivity.
【学位授予单位】：福建中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285.5

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