槲皮素改善炎症微环境逆转多囊卵巢综合征胰岛素抵抗的机制研究
发布时间：2018-09-16 21:30
【摘要】：目的观察槲皮素对多囊卵巢综合征胰岛素抵抗的治疗作用，并探讨其对多囊卵巢综合征胰岛素抵抗微环境的重塑作用及相关机制。 方法动物实验：选择21日龄Wistar雌性大鼠132只，按随机数目表法随机分为病理模型组（120只）和正常对照组（12只），病理模型组用脱氢表雄酮诱导多囊卵巢综合征胰岛素抵抗大鼠动物模型。造模成功的36只随机分为模型对照组、槲皮素组、二甲双胍组。正常对照组及模型对照组给予生理盐水灌胃处理，槲皮素组用槲皮素灌胃干预，二甲双胍组用二甲双胍灌胃干预，持续灌胃28天。灌胃结束后观察大鼠的性周期情况、大鼠体重、卵巢系数、大鼠卵巢形态学变化；并用氧化酶-过氧化物酶方法测定空腹血糖；用酶联免疫方法检测血睾酮、胰岛素水平；用免疫组织化学方法检测卵巢组织白介素-6、白介素-1β、肿瘤坏死因子、胰岛素受体底物-1酪氨酸磷酸化、胰岛素受体底物-2的表达；采用免疫组织荧光方法，测大鼠卵巢组织核转录因子核转位情况；采用反转录聚合酶链式反应和免疫印迹的方法检测大鼠卵巢组织的还原型烟酰胺腺嘌呤二核苷酸磷酸氧化酶p22phox亚基、氧化型低密度脂蛋白、Toll样受体-4的基因和蛋白表达。体外实验：选择小鼠前脂肪细胞株3T3-L1，将其诱导分化为脂肪细胞，设正常对照组和病理模型组。病理模型组采用高葡萄糖、高胰岛素方法将细胞诱导为胰岛素抵抗细胞。造模成功的细胞设模型对照组、槲皮素组、二甲双胍组。正常对照组及模型对照组给予普通培养基处理，槲皮素组用槲皮素干预，二甲双胍组用二甲双胍干预。作用24小时后，采用氧化酶-过氧化物酶法测细胞葡萄糖消耗量；采用葡萄糖摄取法测细胞葡萄糖摄取量；用酶联免疫方法检测细胞白介素-6、白介素-1β、肿瘤坏死因子的表达；采用细胞免疫荧光方法，测细胞核转录因子核转位情况；采用反转录聚合酶链式反应和免疫印迹的方法检测细胞的还原型烟酰胺腺嘌呤二核苷酸磷酸氧化酶p22phox亚基、氧化型低密度脂蛋白、Toll样受体-4、胰岛素受体底物-1酪氨酸磷酸化的基因和蛋白表达。单独采用Toll样受体-4抑制剂TAK-242、NF-κB抑制剂PDTC及同时联合TAK-242和PDTC作用模型组细胞，，作用24小时后，采用氧化酶-过氧化物酶法测细胞葡萄糖消耗量；采用葡萄糖摄取法测细胞葡萄糖摄取量；用酶联免疫方法检测细胞白介素-6、白介素-1β、肿瘤坏死因子表达；采用细胞免疫荧光方法，测细胞核转录因子核转位情况；采用反转录聚合酶链式反应和免疫印迹的方法检测胰岛素受体底物-1酪氨酸磷酸化的基因和蛋白表达。 结果动物实验结果表明槲皮素不仅可以降低PCOS胰岛素抵抗模型组大鼠的血胰岛素，还能减少核转录因子核转位，降低白介素-6、白介素-1β、肿瘤坏死因子炎性因子水平，对还原型烟酰胺腺嘌呤二核苷酸磷酸氧化酶p22phox亚基、氧化型低密度脂蛋白、Toll样受体-4的基因和蛋白表达有抑制作用；可以升高胰岛素受体底物-1酪氨酸磷酸化、胰岛素受体底物-2的基因和蛋白表达。大鼠性周期恢复率58.33％。体外实验结果表明槲皮素可以减少胰岛素抵抗细胞核转录因子核转位，降低炎性因子白介素-6、白介素-1β、肿瘤坏死因子水平，改善细胞胰岛素抵抗；同时还能降低还原型烟酰胺腺嘌呤二核苷酸磷酸氧化酶p22phox亚基、氧化型低密度脂蛋白、Toll样受体-4的基因和蛋白表达；能升高胰岛素受体底物-1酪氨酸磷酸化的基因和蛋白表达。TLR/NF-κB信号通路被抑制后，胰岛素抵抗细胞的转录因子核转位、白介素-6、白介素-1β、肿瘤坏死因子炎性因子水平均下降，胰岛素受体底物-1酪氨酸磷酸化的基因和蛋白表达上升，细胞的胰岛素抵抗得以逆转。TLR/NF-κB信号通路被抑制后，槲皮素对胰岛素抵抗的治疗作用被阻断。 结论TLR/NF-κB信号通路参与多囊卵巢综合征胰岛素抵抗的形成；槲皮素治疗多囊卵巢综合征胰岛素抵抗有较好的疗效；其机制可能是通过抑制TLR/NF-κB信号通路，改善多囊卵巢胰岛素抵抗微环境炎症，从而逆转胰岛素抵抗；槲皮素是一个潜在的，对胰岛素抵抗有较好治疗效果的药物。
[Abstract]:Objective To observe the therapeutic effect of quercetin on insulin resistance in polycystic ovary syndrome (PCOS), and to explore the remodeling effect of quercetin on insulin resistance microenvironment in PCOS.
Methods Animal experiment: 132 Wistar female rats aged 21 days were randomly divided into pathological model group (120 rats) and normal control group (12 rats) according to the random number table method. Metformin group: normal control group and model control group were treated with normal saline, quercetin group was treated with quercetin, metformin group was treated with metformin for 28 days. The levels of serum testosterone and insulin were measured by enzyme linked immunosorbent assay (ELISA), interleukin-6 (IL-6), interleukin-1 beta (IL-1 beta), tumor necrosis factor (TNF), tyrosine phosphorylation of insulin receptor substrate-1 (IR-1) and expression of IR-2 (IR-2) in ovarian tissues were detected by immunohistochemistry. The expression of reduced nicotinamide adenine dinucleotide phosphate oxidase p22phox subunit, oxidized low density lipoprotein and Toll-like receptor-4 in rat ovary was detected by reverse transcription polymerase chain reaction and Western blot. Preadipocyte line 3T3-L1 was induced to differentiate into adipocytes. Normal control group and pathological model group were set up. Pathological model group was induced to insulin-resistant cells by high glucose and high insulin. Successful cells were set up as model control group, quercetin group, metformin group. Normal control group and model control group were given general administration. After 24 hours, cell glucose consumption was measured by oxidase-peroxidase method, cell glucose uptake was measured by glucose uptake method, cell interleukin-6, interleukin-1 beta and tumor necrosis factor were detected by enzyme-linked immunosorbent assay. The expression of p22phox subunit, oxidized low density lipoprotein, Toll-like receptor-4, insulin receptor substrate-1 were detected by reverse transcription polymerase chain reaction and immunoblotting. Tyrosine phosphorylation gene and protein expression were measured by oxidase-peroxidase assay, glucose uptake was measured by glucose uptake, glucose uptake was measured by enzyme assay, and glucose uptake was measured by enzyme assay. Interleukin-6, interleukin-1 beta and tumor necrosis factor (TNF) expression were detected by immunofluorescence assay, and tyrosine phosphorylation gene and protein expression of insulin receptor substrate-1 were detected by reverse transcription polymerase chain reaction and immunoblotting.
Results The results of animal experiment showed that quercetin could not only reduce the insulin level in plasma, but also reduce the nuclear translocation of nuclear transcription factor, interleukin-6, interleukin-1 beta, tumor necrosis factor inflammatory factor, nicotinamide adenine dinucleotide phosphate oxidase p22phox subunit and oxidative hypodense in PCOS insulin resistance model rats. The gene and protein expression of DOL and Toll-like receptor-4 were inhibited, and the tyrosine phosphorylation of insulin receptor substrate-1 and the gene and protein expression of insulin receptor substrate-2 were increased. The recovery rate of sexual cycle in rats was 58.33%. The results of in vitro experiments showed that quercetin could reduce the nuclear transcription factor translocation of insulin resistance cells and decrease the expression of nuclear transcription factor. Low inflammatory factors IL-6, IL-1beta, tumor necrosis factor levels, improve cell insulin resistance; also reduce reduced nicotinamide adenine dinucleotide phosphate oxidase p22phox subunit, oxidized low density lipoprotein, Toll-like receptor-4 gene and protein expression; can increase insulin receptor substrate-1 tyrosine phosphate After the TLR/NF-kappa B signaling pathway was inhibited, the levels of transcription factor nuclear translocation, interleukin-6, interleukin-1 beta, tumor necrosis factor inflammatory factor, tyrosine phosphorylation gene and protein expression of insulin receptor substrate-1 increased, and insulin resistance was reversed. After the inhibition of B signaling pathway, the therapeutic effect of quercetin on insulin resistance was blocked.
Conclusion TLR/NF-kappa B signaling pathway is involved in the formation of insulin resistance in polycystic ovary syndrome and quercetin is effective in the treatment of insulin resistance in polycystic ovary syndrome. A potential drug that has a good therapeutic effect on insulin resistance.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R711.75
本文编号：2244862
[Abstract]:Objective To observe the therapeutic effect of quercetin on insulin resistance in polycystic ovary syndrome (PCOS), and to explore the remodeling effect of quercetin on insulin resistance microenvironment in PCOS.
Methods Animal experiment: 132 Wistar female rats aged 21 days were randomly divided into pathological model group (120 rats) and normal control group (12 rats) according to the random number table method. Metformin group: normal control group and model control group were treated with normal saline, quercetin group was treated with quercetin, metformin group was treated with metformin for 28 days. The levels of serum testosterone and insulin were measured by enzyme linked immunosorbent assay (ELISA), interleukin-6 (IL-6), interleukin-1 beta (IL-1 beta), tumor necrosis factor (TNF), tyrosine phosphorylation of insulin receptor substrate-1 (IR-1) and expression of IR-2 (IR-2) in ovarian tissues were detected by immunohistochemistry. The expression of reduced nicotinamide adenine dinucleotide phosphate oxidase p22phox subunit, oxidized low density lipoprotein and Toll-like receptor-4 in rat ovary was detected by reverse transcription polymerase chain reaction and Western blot. Preadipocyte line 3T3-L1 was induced to differentiate into adipocytes. Normal control group and pathological model group were set up. Pathological model group was induced to insulin-resistant cells by high glucose and high insulin. Successful cells were set up as model control group, quercetin group, metformin group. Normal control group and model control group were given general administration. After 24 hours, cell glucose consumption was measured by oxidase-peroxidase method, cell glucose uptake was measured by glucose uptake method, cell interleukin-6, interleukin-1 beta and tumor necrosis factor were detected by enzyme-linked immunosorbent assay. The expression of p22phox subunit, oxidized low density lipoprotein, Toll-like receptor-4, insulin receptor substrate-1 were detected by reverse transcription polymerase chain reaction and immunoblotting. Tyrosine phosphorylation gene and protein expression were measured by oxidase-peroxidase assay, glucose uptake was measured by glucose uptake, glucose uptake was measured by enzyme assay, and glucose uptake was measured by enzyme assay. Interleukin-6, interleukin-1 beta and tumor necrosis factor (TNF) expression were detected by immunofluorescence assay, and tyrosine phosphorylation gene and protein expression of insulin receptor substrate-1 were detected by reverse transcription polymerase chain reaction and immunoblotting.
Results The results of animal experiment showed that quercetin could not only reduce the insulin level in plasma, but also reduce the nuclear translocation of nuclear transcription factor, interleukin-6, interleukin-1 beta, tumor necrosis factor inflammatory factor, nicotinamide adenine dinucleotide phosphate oxidase p22phox subunit and oxidative hypodense in PCOS insulin resistance model rats. The gene and protein expression of DOL and Toll-like receptor-4 were inhibited, and the tyrosine phosphorylation of insulin receptor substrate-1 and the gene and protein expression of insulin receptor substrate-2 were increased. The recovery rate of sexual cycle in rats was 58.33%. The results of in vitro experiments showed that quercetin could reduce the nuclear transcription factor translocation of insulin resistance cells and decrease the expression of nuclear transcription factor. Low inflammatory factors IL-6, IL-1beta, tumor necrosis factor levels, improve cell insulin resistance; also reduce reduced nicotinamide adenine dinucleotide phosphate oxidase p22phox subunit, oxidized low density lipoprotein, Toll-like receptor-4 gene and protein expression; can increase insulin receptor substrate-1 tyrosine phosphate After the TLR/NF-kappa B signaling pathway was inhibited, the levels of transcription factor nuclear translocation, interleukin-6, interleukin-1 beta, tumor necrosis factor inflammatory factor, tyrosine phosphorylation gene and protein expression of insulin receptor substrate-1 increased, and insulin resistance was reversed. After the inhibition of B signaling pathway, the therapeutic effect of quercetin on insulin resistance was blocked.
Conclusion TLR/NF-kappa B signaling pathway is involved in the formation of insulin resistance in polycystic ovary syndrome and quercetin is effective in the treatment of insulin resistance in polycystic ovary syndrome. A potential drug that has a good therapeutic effect on insulin resistance.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R711.75
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