过氧化氢诱导的L6细胞胰岛素抵抗及EGB对胰岛素抵抗的改善作用

发布时间：2018-04-30 06:24

本文选题：氧化应激 + 胰岛素抵抗　；参考：《吉林大学》2017年硕士论文

【摘要】：研究背景:目前糖尿病是全世界严重的公共卫生问题,患病率呈现世界性上升趋势。胰岛素抵抗是2型糖尿病的重要发病机制,氧化应激是骨骼肌发生胰岛素抵抗的一个重要病因,ROS会激活细胞应激敏感性通路炎症信号通路NF-κB,进而影响胰岛素信号通路的传导,引起胰岛素抵抗。NF-κB是氧化应激重要的一个靶点,了解及明确NF-κB在氧化应激引起的胰岛素抵抗过程中的具体机制尤其重要。氧化应激可被内在和外在的因素可控制,通过降低氧化应激预防和治疗胰岛素抵抗是目前的一个新的研究方向。本研究通过一定浓度过氧化氢刺激大鼠骨骼肌细胞(L6细胞)建立氧化应激模型,检测葡萄糖摄取能力及胰岛素抵抗情况,并应用NF-κB抑制剂PDTC预处理,观察胰岛素抵抗水平情况,探讨其具体机制;在了解过氧化氢诱导大鼠骨骼肌胰岛素抵抗的具体机制之后,我们欲探讨天然中药银杏叶提取物能否改善L6细胞的胰岛素抵抗状态,并探讨具体机制。本研究分为以下两个部分。第一部分:过氧化氢诱导的胰岛素抵抗及机制研究目的:研究过氧化氢对L6细胞胰岛素抵抗的影响,并探讨具体机制。方法:首先体外培养L6细胞并诱导分化为成熟的骨骼肌细胞,MTT法测定细胞存活率,确定H_2O_2和PDTC的作用浓度和作用时间。将细胞分为4组:正常组、H_2O_2组、H_2O_2+PDTC组、PDTC组。应用DCFH-DA探针检测细胞内ROS的生成,DAPI观察细胞凋亡情况,2-NBDG法检测细胞摄取葡萄糖能力,ELISA法检测细胞上清中炎性因子TNF-α、IL-1β、IL-6的分泌水平,Western Blot检测细胞中Bax、Bcl2、Caspase-8、IκB-a、P-IκB-a、P38-MAPK、P-P38-MAPK、NF-κB、P-NF-κB、IRS1、P-IRS1 Tyr612、PI3K、GLUT4等蛋白的表达。结果:1.L6细胞的诱导分化:诱导分化后第7d细胞融合成明显的肌管。2.氧化应激模型建立:H_2O_2浓度1.0mmol/L时,细胞存活率70%左右,确定H_2O_2的刺激浓度为1.0mmol/L。当PDTC浓度为100μmol/L时,细胞存活率为80%左右,高于其他实验浓度组。孵育4h时,PDTC对细胞存活率抑制作用相对较小,确定PDTC的孵育浓度和时间为100μmol/L,4h。DCFH-DA结果显示,1.0mmol/LH_2O_2处理L6细胞4h后,细胞内ROS生成显著升高,确定氧化应激模型建立。3.细胞凋亡情况:正常组细胞核完整,边缘清晰,染色质均一;H_2O_2组细胞出现典型凋亡特征,如细胞核皱缩,细胞核破碎,凋亡小体形成;而H_2O_2+PDTC组除个别细胞出现凋亡,大多数均正常。与正常组相比较,H_2O_2组Bax相对表达量、Bax/Bcl2比值、Caspase-8相对表达量显著升高,Bcl2相对表达量显著降低,差异均有统计学意义(P0.05)。与H_2O_2组相比较,H_2O_2+PDTC组Bax相对表达量无变化,差异无统计学意义(P0.05);Bcl2相对表达量显著升高,Bax/Bcl2比值、Caspase-8相对表达量均显著降低,差异均有统计学意义(P0.05)。4.NF-κB活化及胰岛素信号传导改变:2-NBDG摄取试验结果显示,与正常组相比较,H_2O_2组2-NBDG摄取能力显著下降,差异有统计学意义(P0.05);与H_2O_2组相比较,H_2O_2+PDTC组2-NBDG摄取能力显著升高,差异有统计学意义(P0.05)。ELISA结果显示,与正常组相比较,H_2O_2组炎症因子TNF-α、IL-1β、IL-6产生显著增加,差异均有统计学意义(P0.05);与H_2O_2组相比较,H_2O_2+PDTC组TNF-α、IL-1β、IL-6产生显著降低,差异均有统计学意义(P0.05)。与正常组相比较,H_2O_2组IκB-a、P38-MAPK、NF-κB相对表达量均显著升高,P-IκB-a/IκB-a、P-P38/P38、P-NF-κB/NF-κB相对水平均显著升高,IRS1、PI3K、GLUT4相对表达量及P-IRS1 Tyr612/IRS1相对水平均显著降低,差异均有统计学意义(P0.05);与H_2O_2组相比较,H_2O_2+PDTC组IκB-a、P38-MAPK、NF-κB相对表达量均显著降低,P-IκB-a/IκB-a、P-P38/P38、P-NF-κB/NF-κB相对水平均显著降低,H_2O_2+PDTC组IRS1、PI3K、GLUT4相对表达量,P-IRS1 Tyr612/IRS1相对水平均显著升高,差异均有统计学意义(P0.05)。结论:1.H_2O_2能够导致L6细胞出现氧化应激和凋亡。2.H_2O_2能够导致细胞炎性因子TNF-α、IL-1β、IL-6产生增加。3.H_2O_2能够导致细胞摄取葡萄糖能力下降,引起胰岛素抵抗,具体机制与NF-κB通路激活有关。4.应用PDTC可以缓解细胞凋亡、减少ROS和炎性因子的产生、提高摄取葡萄糖能力、抑制NF-κB通路的激活,进而改善胰岛素抵抗。第二部分银杏叶提取物对过氧化氢诱导的胰岛素抵抗的改善作用目的:研究EGB对过氧化氢诱导的胰岛素抵抗的改善作用及具体机制。方法:首先体外培养L6细胞并诱导分化为成熟的骨骼肌细胞,MTT法测定细胞存活率,确定EGB的作用浓度。将细胞分为7组:正常组、H_2O_2组、H_2O_2+EGB组、H_2O_2+PDTC组、H_2O_2+EGB+PDTC组、PDTC组、EGB组。应用DCFH-DA探针检测细胞内ROS的生成,2-NBDG摄取试验检测细胞摄取葡萄糖能力,ELISA法检测细胞上清中炎性因子TNF-α、IL-1β、IL-6的分泌水平,Western Blot检测L6细胞中IκB-a、P-IκB-a、NF-κB、P-NF-κB、IRS1、P-IRS1 Tyr612、GLUT4等蛋白的表达。结果:1.EGB对氧化应激的改善:MTT结果显示,随着EGB浓度的增加,细胞的存活率呈先升高后稳定,反而稍微下降的趋势。在EGB浓度为200μg/m L时,细胞存活率达到最大值108%,确定EGB的预处理浓度为200μg/m L。DCFH-DA流式结果显示,与正常组相比较,H_2O_2组细胞内ROS显著升高,差异有统计学意义(P0.05),确定氧化应激模型建立;与H_2O_2组相比较,H_2O_2+EGB、H_2O_2+PDTC和H_2O_2+EGB+PDTC组细胞内ROS显著降低,差异均有统计学意义(P0.05)。且H_2O_2+EGB+PDTC组细胞内ROS均低于H_2O_2+EGB组和H_2O_2+PDTC组,差异均有统计学意义(P0.05)。2.EGB对NF-κB活化及胰岛素信号传导的影响:2-NBDG摄取试验结果显示,与正常组相比较,H_2O_2组2-NBDG摄取能力显著下降,差异有统计学意义(P0.05);与H_2O_2组相比较,H_2O_2+EGB、H_2O_2+PDTC和H_2O_2+EGB+PDTC组2-NBDG摄取能力均显著升高,差异均具有统计学意义(P0.05)。并且H_2O_2+EGB+PDTC组细胞2-NBDG摄取能力显著高于H_2O_2+PDTC组,差异有统计学意义(P0.05)。ELISA结果显示,与正常组相比较,H_2O_2组炎症因子TNF-α、IL-1β、IL-6产生显著增加,差异均有统计学意义(P0.05);与H_2O_2组相比较,H_2O_2+EGB、H_2O_2+PDTC和H_2O_2+EGB+PDTC组TNF-α、IL-1β、IL-6产生均显著降低,差异均有统计学意义(P0.05);且H_2O_2+EGB+PDTC组TNF-α产生低于H_2O_2+PDTC组,差异有统计学意义(P0.05);Western blot结果显示,与正常组相比较,H_2O_2组IκB-a、NF-κB、P-IκB-a/IκB-a、P-NF-κB/NF-κB相对表达量均显著升高,IRS1、GLUT4、P-IRS1 Tyr612/IRS1相对表达量均显著降低,差异均有统计学意义(P0.05);与H_2O_2组相比较,H_2O_2+EGB、H_2O_2+PDTC和H_2O_2+EGB+PDTC组IκB-a、NF-κB、P-IκB-a/IκB-a、P-NF-κB/NF-κB相对表达量均显著降低,IRS1、GLUT4、P-IRS1 Tyr612/IRS1相对表达量均显著升高,差异均有统计学意义(P0.05)。结论:1.EGB能够提高L6细胞存活率,降低氧化应激程度,且联合应用EGB和PDTC在降低细胞氧化应激程度上优于单独应用EGB和单独应用PDTC。2.EGB能够减少L6细胞炎性因子TNF-α、IL-1β、IL-6产生,且联合应用EGB和PDTC在减少TNF-α产生上优于单独应用PDTC。3.EGB能够提高细胞摄取葡萄糖能力,改善胰岛素抵抗,具体机制与抑制NF-κB通路的激活有关,且联合应用EGB和PDTC在提高细胞摄取葡萄糖能力上优于单独应用PDTC。
[Abstract]:Background: at present, diabetes is a serious public health problem in the world. The incidence of disease is rising worldwide. Insulin resistance is an important pathogenesis of type 2 diabetes. Oxidative stress is an important cause of insulin resistance in skeletal muscle. ROS activates the inflammatory signaling pathway NF- kappa B of the cell stress sensitivity pathway and further shadows The transmission of the insulin signaling pathway causes insulin resistance to.NF- kappa B as an important target for oxidative stress. It is particularly important to understand and clarify the specific mechanism of NF- kappa B in the process of insulin resistance induced by oxidative stress. Oxidative stress can be controlled by internal and external factors, and the prevention and treatment of insulin against oxidative stress can be used to prevent and treat insulin. Resistance is a new research direction at present. In this study, the oxidative stress model of rat skeletal muscle cells (L6 cells) was stimulated by a certain concentration of hydrogen peroxide, and the ability of glucose uptake and insulin resistance was detected. The condition of islet resistance was observed by NF- kappa B inhibitor PDTC, and the specific mechanism was discussed. After the specific mechanism of insulin resistance induced by hydrogen peroxide in rat skeletal muscle, we would like to explore whether the extract of Ginkgo biloba leaves can improve the insulin resistance of L6 cells and discuss the specific mechanism. This study is divided into two parts. The first part: the purpose of the study of hydrogen peroxide induced insulin resistance and its mechanism: the study of peroxidation The effect of hydrogen on insulin resistance in L6 cells was discussed. Methods: first, L6 cells were cultured in vitro and induced into mature skeletal muscle cells. The survival rate of cells was determined by MTT method. The action concentration and time of H_2O_2 and PDTC were determined. The cells were divided into 4 groups: normal group, H_2O_2 group, H_2O_2+PDTC group, PDTC group. Detection of ROS production in cells, DAPI observation of cell apoptosis, 2-NBDG method to detect cell uptake of glucose, ELISA method to detect the secretion of TNF- alpha, IL-1 beta, IL-6 in cell supernatant, Western Blot detection cells Bax, Bcl2, Caspase-8. The expression of UT4 and other proteins. Results: induced differentiation of 1.L6 cells: the differentiation of 7D cells after induction of differentiation into a distinct.2. oxidative stress model of myotubes: the survival rate of cells was about 70% when H_2O_2 concentration was 1.0mmol/L, and the survival rate of H_2O_2 was about 80% when PDTC concentration was 100 micron mol/L, which was higher than that of other experiments. Concentration group. When incubated for 4h, the inhibitory effect of PDTC on cell survival was relatively small, and the incubation concentration and time of PDTC were 100 u mol/L. The results of 4h.DCFH-DA showed that after 1.0mmol/LH_2O_2 treated L6 cell 4h, the formation of ROS in the cells increased significantly, and the oxidative stress model established.3. fine cell apoptosis: normal group nuclei were complete, edge clear, dyed. The cells of group H_2O_2 showed typical characteristics of apoptosis, such as nuclear crinkle, nucleus fragmentation, and apoptotic body formation, while group H_2O_2+PDTC except some cells appeared apoptosis, most of them were normal. Compared with normal group, the relative expression of Bax in group H_2O_2, Bax/Bcl2 ratio, relative expression of Caspase-8 increased significantly, and the relative expression of Bcl2 was significant. The difference was statistically significant (P0.05). Compared with the H_2O_2 group, the relative expression of Bax in group H_2O_2+PDTC had no significant difference (P0.05), the relative expression of Bcl2 increased significantly, the ratio of Bax/Bcl2 and the relative expression of Caspase-8 decreased significantly, and the difference was statistically significant (P0.05).4.NF- kappa B activation and insulin signal transduction Changes: 2-NBDG uptake test results showed that compared with the normal group, the 2-NBDG uptake ability of H_2O_2 group decreased significantly (P0.05). Compared with the H_2O_2 group, the 2-NBDG uptake ability of H_2O_2+PDTC group increased significantly, and the difference was statistically significant (P0.05).ELISA results, compared with the normal group, the H_2O_2 group inflammatory factor TNF- alpha, IL-1 beta, IL-6 produced a significant increase, the difference was statistically significant (P0.05). Compared with the H_2O_2 group, TNF- alpha, IL-1 beta and IL-6 produced a significant decrease in H_2O_2+PDTC group, and the difference was statistically significant (P0.05). The relative levels of IRS1, PI3K, GLUT4 and the relative level of P-IRS1 Tyr612/IRS1 were significantly decreased, and the difference was statistically significant (P0.05). Compared with the H_2O_2 group, the relative expression of I kappa B-a, P38-MAPK, NF- kappa, H_2O_2+PDTC group was significantly reduced, and the relative level of kappa kappa was significantly reduced. The relative expression of IRS1, PI3K, GLUT4 in group H_2O_2+PDTC, P-IRS1 Tyr612/IRS1 relative levels were significantly increased, and the difference was statistically significant (P0.05). Conclusion: 1.H_2O_2 can lead to oxidative stress and apoptosis of L6 cells, and.2.H_2O_2 can lead to the inflammatory factor TNF- alpha, IL-1 beta, which can lead to the uptake of grapes. The decrease of sugar capacity causes insulin resistance. The specific mechanism related to the activation of NF- kappa B pathway related to the activation of.4. application PDTC can alleviate apoptosis, reduce the production of ROS and inflammatory factors, increase the uptake of glucose, inhibit the activation of NF- kappa B pathway, and then improve insulin resistance. Second parts of Ginkgo biloba extract against hydrogen peroxide induced insulin against the insulin resistance. Objective: To study the effect of EGB on the improvement of insulin resistance induced by hydrogen peroxide and the specific mechanism. Methods: first, L6 cells were cultured in vitro and induced into mature skeletal muscle cells. MTT method was used to determine the cell survival rate and determine the concentration of EGB. The cells were divided into 7 groups: normal group, H_2O_2 group, H_2O_2+EGB group, H_2O_2+PDTC Group H_2O_2+EGB+PDTC, group PDTC, group EGB. Using DCFH-DA probe to detect the formation of ROS in cells, 2-NBDG uptake test to detect the ability of cell uptake of glucose, ELISA method to detect the inflammatory factor TNF- a, IL-1 beta, IL-6 secreted in cell supernatant. Results: the results of the expression of equal protein. Results: the improvement of oxidative stress by 1.EGB: MTT results showed that with the increase of EGB concentration, the survival rate of cells increased first and then decreased slightly. The cell survival rate reached the maximum of 108% when the concentration of EGB was 200 mu g/m L, and the preconditioning concentration of EGB was shown to be 200 u g/m L.DCFH-DA flow results. Compared with the normal group, the intracellular ROS in the H_2O_2 group increased significantly, and the difference was statistically significant (P0.05), and the oxidative stress model was established. Compared with the H_2O_2 group, the intracellular ROS in H_2O_2+EGB, H_2O_2+PDTC and H_2O_2+EGB+PDTC groups decreased significantly (P0.05), and the ROS in the H_2O_2+EGB+PDTC group was lower than that in the H_2O_2+EGB group and in the H_2O_2+EGB+PDTC group. H_2O_2+PDTC group, the difference was statistically significant (P0.05) the effect of.2.EGB on the activation of NF- kappa B and insulin signal transduction. 2-NBDG uptake test results showed that the 2-NBDG uptake ability of H_2O_2 group decreased significantly compared with the normal group, and the difference was statistically significant (P0.05); H_2O_2+EGB, H_2O_2+PDTC, and groups were compared with the H_2O_2 group. The ability of G uptake increased significantly (P0.05), and the ability of 2-NBDG uptake in H_2O_2+EGB+PDTC group was significantly higher than that in H_2O_2+PDTC group, and the difference was statistically significant (P0.05).ELISA results showed that the inflammatory factors TNF- alpha, IL-1 beta and IL-6 were significantly increased in H_2O_2 group, and the difference was statistically significant. (P0.05); compared with group H_2O_2, the production of TNF- alpha, IL-1 beta and IL-6 in H_2O_2+EGB, H_2O_2+PDTC and H_2O_2+EGB+PDTC groups decreased significantly, and the difference was statistically significant (P0.05), and TNF- alpha production in H_2O_2+EGB+PDTC group was lower than that of H_2O_2+PDTC group. - kappa B, P-I kappa B-a/I kappa B-a, P-NF- kappa B/NF- kappa B significantly increased, IRS1, GLUT4, P-IRS1 Tyr612/IRS1 relative expression decreased significantly, the difference was statistically significant (P0.05). Low, IRS1, GLUT4, P-IRS1 Tyr612/IRS1 relative expression increased significantly, the difference was statistically significant (P0.05). Conclusion: 1.EGB can improve the survival rate of L6 cells and reduce oxidative stress, and the combination of EGB and PDTC in reducing cell oxidative stress is better than single application EGB and single application PDTC.2.EGB can reduce L6 cell inflammation Factor TNF- alpha, IL-1 beta, IL-6 production, and combined use of EGB and PDTC in reducing TNF- alpha production is superior to single application PDTC.3.EGB can improve cell uptake of glucose ability and improve insulin resistance. The specific mechanism is related to the inhibition of NF- kappa B pathway activation, and the combined application of EGB and PDTC is superior to the ability to increase cell uptake of glucose. Using PDTC.

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.1

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