【摘要】：目的:作為一種由多種原因引起的以慢性高血糖為特征的終身代謝性疾病,糖尿病已經(jīng)成為21世紀(jì)人類(lèi)所面臨的最具挑戰(zhàn)性的疾病之一。長(zhǎng)期持續(xù)的血糖增高會(huì)導(dǎo)致大血管、微血管損害而導(dǎo)致心、腦、腎、周?chē)窠?jīng)、眼睛、足等發(fā)生病變。據(jù)估計(jì),糖尿病患者中75%的死亡率都是由于心血管疾病所引起的。越來(lái)越多的研究表明炎癥和氧化應(yīng)激在糖尿病血管系統(tǒng)并發(fā)癥的形成和發(fā)展的起著重要作用。α-硫辛酸(LA),是一種線粒體中的輔酶,因有很強(qiáng)的還原性,被稱(chēng)為“萬(wàn)能抗氧化劑”。本課題通過(guò)高濃度葡萄糖誘導(dǎo)人主動(dòng)脈內(nèi)皮細(xì)胞損傷,建立糖尿病細(xì)胞模型,用不同劑量的α-硫辛酸進(jìn)行干預(yù)處理,探討α-硫辛酸對(duì)高糖誘導(dǎo)的人主動(dòng)脈內(nèi)皮細(xì)胞炎癥反應(yīng)和氧化應(yīng)激作用的影響并揭示其相關(guān)機(jī)制。方法:體外建立高糖(30mM)誘導(dǎo)的人主動(dòng)脈細(xì)胞(HAECs)損傷模型,分別加入不同濃度硫辛酸(50,100,200μM)。用還原性谷胱甘肽(GSH)測(cè)試盒檢測(cè)細(xì)胞中GSH的含量。采用流式細(xì)胞技術(shù)檢測(cè)硫辛酸對(duì)高糖誘導(dǎo)的HAECs內(nèi)活性氧(ROS)產(chǎn)生量的影響。利用蛋白質(zhì)印跡法(Western Blot)方法檢測(cè)HAECs中NADPH氧化酶-4(Nox4)、p22phox、Caspase-3、Bcl-2蛋白的表達(dá)水平,細(xì)胞色素C的線粒體釋放以及Nuclear Factor-κB(NF-κB)信號(hào)通路的激活情況。采用實(shí)時(shí)熒光測(cè)定PCR(Real-time RT-PCR)檢測(cè)Nox4、p22phox mRNA的表達(dá)水平。結(jié)果:1.與空白組相比較,高糖誘導(dǎo)后HAECs中GSH含量明顯降低(p0.01),硫辛酸給予后可以劑量依賴(lài)性的增加人細(xì)胞中GSH的表達(dá)量(p0.01);2.與空白組相比較,高糖增加HAECs中ROS的過(guò)量產(chǎn)生(p0.01)、增加Nox4和p22phox mRNA和蛋白的過(guò)量表達(dá)(p0.01,p0.01),加入不同濃度的硫辛酸后高糖誘導(dǎo)人主動(dòng)脈內(nèi)皮細(xì)胞中活性氧ROS的產(chǎn)生量明顯減少(p0.05),且硫辛酸抑制高糖誘導(dǎo)的Nox4和p22phox mRNA和蛋白的過(guò)量表達(dá)(p0.01,p0.01);3.與空白組相比較,高糖顯著增加I-κB的降解,促進(jìn)P65的核轉(zhuǎn)位,從而增加NF-κB的轉(zhuǎn)錄活性,而硫辛酸可以抑制高糖誘導(dǎo)的細(xì)胞中I-κB的降解,以及P65的核轉(zhuǎn)位,進(jìn)而抑制NF-κB信號(hào)通路的激活(p0.05);4.高糖誘導(dǎo)后,凋亡相關(guān)蛋白Caspcase-3表達(dá)上調(diào)而抗凋亡蛋白Bcl-2表達(dá)明顯下降(p0.01),硫辛酸干預(yù)后使抗凋亡蛋白Bcl-2的表達(dá)呈濃度依賴(lài)性增加(p0.01),同時(shí)硫辛酸還能下調(diào)凋亡蛋白Caspcase-3的表達(dá)(p0.01);5.與空白組相比較,高糖使人主動(dòng)脈內(nèi)皮細(xì)胞中細(xì)胞色素C的線粒體釋放量增加,而加入硫辛酸后減少高糖誘導(dǎo)的細(xì)胞色素C的線粒體釋放(p0.05)。結(jié)論:硫辛酸可以抑制高糖誘導(dǎo)的HAECs中ROS過(guò)量產(chǎn)生,抑制還原型煙酰胺腺嘌呤二核苷酸磷酸(NADPH)家族中Nox4、p22phox亞基的mRNA和蛋白的過(guò)量表達(dá);硫辛酸能夠顯著降低高糖誘導(dǎo)的Caspase-3的表達(dá),抑制由高糖導(dǎo)致的Bcl-2蛋白表達(dá)量的下降。硫辛酸抑制NF-κB信號(hào)通路的轉(zhuǎn)錄活性,同時(shí)還可以抑制HAECs中細(xì)胞色素C的線粒體釋放,由此來(lái)抑制高糖誘導(dǎo)的人主動(dòng)脈內(nèi)皮細(xì)胞的氧化應(yīng)激和炎癥反應(yīng)以及抑制細(xì)胞凋亡。我們推測(cè)硫辛酸可能是通過(guò)阻斷Nox4的表達(dá)來(lái)抑制細(xì)胞中ROS的過(guò)量產(chǎn)生,并通過(guò)影響NF-κB信號(hào)通路的轉(zhuǎn)錄活性從而減弱高糖誘導(dǎo)的HAECs中的氧化應(yīng)激和炎癥反應(yīng)。
[Abstract]:Objective: as a life-long metabolic disease characterized by chronic hyperglycemia, diabetes has become one of the most challenging diseases in twenty-first Century. Prolonged continuous hyperglycemia can lead to large blood vessels and microvascular damage and cause lesions in the heart, brain, kidney, peripheral nerves, eyes, and feet. It is estimated that 75% of the mortality rates in diabetic patients are caused by cardiovascular disease. More and more studies have shown that inflammation and oxidative stress play an important role in the formation and development of diabetic vascular system complications. Alpha lipoic acid (LA), a cofactor in the mitochondria, is called "universal anti oxygen" because of its strong reducibility. Using high concentration glucose to induce the injury of human aortic endothelial cells, the diabetic cell model was established. The effects of alpha lipoic acid on the inflammatory response and oxidative stress induced by high glucose induced human aortic endothelial cells were investigated by interfering with different doses of alpha lipoic acid, and the mechanism was revealed. A model of human aortic cell (HAECs) injury induced by high glucose (30mM) was established with different concentrations of lipoic acid (50100200 u M). The content of GSH in cells was detected by a reduced glutathione (GSH) test box. The effect of thioctanoic acid on the production of ROS in high glucose induced HAECs was detected by flow cytometry. Western Blot method was used to detect the expression level of NADPH oxidase -4 (Nox4), p22phox, Caspase-3, Bcl-2 protein, mitochondrial release of cytochrome C, and activation of Nuclear Factor- kappa signaling pathway. Results: 1. and blank Compared with the group, the content of GSH in HAECs decreased significantly (P0.01) after high glucose induction. The dose dependent increase of GSH expression in human cells (P0.01) after the administration of lipoic acid was increased. 2. compared with the blank group, high glucose increased the excessive production of ROS in HAECs (P0.01), increased Nox4 and p22phox mRNA and protein overexpression (P0.01,), and added different concentrations. High glucose induced the production of reactive oxygen ROS in human aortic endothelial cells (P0.05), and lipoic acid inhibited the excessive expression of Nox4 and p22phox mRNA and protein (P0.01, P0.01) induced by high glucose (P0.01, P0.01). 3. compared with the blank group, high glucose significantly increased the degradation of I- kappa B and promoted the nuclear transposition of P65, thus increasing the transcriptional activity of NF- kappa B. Lipoic acid can inhibit the degradation of I- kappa B in high glucose induced cells, and the nuclear transposition of P65, and then inhibit the activation of NF- kappa B signaling pathway (P0.05). After the induction of 4. high glucose, the expression of apoptosis related protein Caspcase-3 is up and the expression of anti apoptotic protein Bcl-2 is obviously decreased (P0.01), and the expression of anti apoptotic protein Bcl-2 is thicker after the intervention of lipoic acid. Degree dependence increased (P0.01), while lipoic acid also lowered the expression of apoptotic protein Caspcase-3 (P0.01); 5. compared with the blank group, high glucose increased the mitochondrial release of cytochrome C in human aortic endothelial cells, and after adding lipoic acid, it reduced the mitochondrial release of hyperpigmented C induced by high glucose (P0.05). Conclusion: lipoic acid can inhibit the mitochondrial release of cytochrome C (P0.05). The excessive production of ROS induced by high glucose induced HAECs inhibited the overexpression of mRNA and protein in the Nox4, p22phox subunit of the nicotinamide adenine dinucleotide phosphate (NADPH) family; thioctanoic acid could significantly reduce the expression of high glucose induced Caspase-3 and inhibit the decrease of Bcl-2 protein expression caused by high sugar. Thioctanoic acid inhibited NF- kappa B The transcriptional activity of the signal pathway also inhibits the mitochondrial release of cytochrome C in HAECs, which inhibits oxidative stress and inflammatory response and inhibits apoptosis of human aortic endothelial cells induced by high glucose. We speculate that lipoic acid may inhibit the excessive production of ROS in cells by blocking the expression of Nox4 and through shadow The transcriptional activity of the NF- - kappa B signaling pathway weakens the oxidative stress and inflammatory response in HAECs induced by high glucose.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R587.2

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