【摘要】：目的觀察槲皮素對(duì)多囊卵巢綜合征胰島素抵抗的治療作用，并探討其對(duì)多囊卵巢綜合征胰島素抵抗微環(huán)境的重塑作用及相關(guān)機(jī)制。 方法動(dòng)物實(shí)驗(yàn)：選擇21日齡Wistar雌性大鼠132只，按隨機(jī)數(shù)目表法隨機(jī)分為病理模型組（120只）和正常對(duì)照組（12只），病理模型組用脫氫表雄酮誘導(dǎo)多囊卵巢綜合征胰島素抵抗大鼠動(dòng)物模型。造模成功的36只隨機(jī)分為模型對(duì)照組、槲皮素組、二甲雙胍組。正常對(duì)照組及模型對(duì)照組給予生理鹽水灌胃處理，槲皮素組用槲皮素灌胃干預(yù)，二甲雙胍組用二甲雙胍灌胃干預(yù)，持續(xù)灌胃28天。灌胃結(jié)束后觀察大鼠的性周期情況、大鼠體重、卵巢系數(shù)、大鼠卵巢形態(tài)學(xué)變化；并用氧化酶-過氧化物酶方法測定空腹血糖；用酶聯(lián)免疫方法檢測血睪酮、胰島素水平；用免疫組織化學(xué)方法檢測卵巢組織白介素-6、白介素-1β、腫瘤壞死因子、胰島素受體底物-1酪氨酸磷酸化、胰島素受體底物-2的表達(dá)；采用免疫組織熒光方法，測大鼠卵巢組織核轉(zhuǎn)錄因子核轉(zhuǎn)位情況；采用反轉(zhuǎn)錄聚合酶鏈?zhǔn)椒磻?yīng)和免疫印跡的方法檢測大鼠卵巢組織的還原型煙酰胺腺嘌呤二核苷酸磷酸氧化酶p22phox亞基、氧化型低密度脂蛋白、Toll樣受體-4的基因和蛋白表達(dá)。體外實(shí)驗(yàn)：選擇小鼠前脂肪細(xì)胞株3T3-L1，將其誘導(dǎo)分化為脂肪細(xì)胞，設(shè)正常對(duì)照組和病理模型組。病理模型組采用高葡萄糖、高胰島素方法將細(xì)胞誘導(dǎo)為胰島素抵抗細(xì)胞。造模成功的細(xì)胞設(shè)模型對(duì)照組、槲皮素組、二甲雙胍組。正常對(duì)照組及模型對(duì)照組給予普通培養(yǎng)基處理，槲皮素組用槲皮素干預(yù)，二甲雙胍組用二甲雙胍干預(yù)。作用24小時(shí)后，采用氧化酶-過氧化物酶法測細(xì)胞葡萄糖消耗量；采用葡萄糖攝取法測細(xì)胞葡萄糖攝取量；用酶聯(lián)免疫方法檢測細(xì)胞白介素-6、白介素-1β、腫瘤壞死因子的表達(dá)；采用細(xì)胞免疫熒光方法，測細(xì)胞核轉(zhuǎn)錄因子核轉(zhuǎn)位情況；采用反轉(zhuǎn)錄聚合酶鏈?zhǔn)椒磻?yīng)和免疫印跡的方法檢測細(xì)胞的還原型煙酰胺腺嘌呤二核苷酸磷酸氧化酶p22phox亞基、氧化型低密度脂蛋白、Toll樣受體-4、胰島素受體底物-1酪氨酸磷酸化的基因和蛋白表達(dá)。單獨(dú)采用Toll樣受體-4抑制劑TAK-242、NF-κB抑制劑PDTC及同時(shí)聯(lián)合TAK-242和PDTC作用模型組細(xì)胞，，作用24小時(shí)后，采用氧化酶-過氧化物酶法測細(xì)胞葡萄糖消耗量；采用葡萄糖攝取法測細(xì)胞葡萄糖攝取量；用酶聯(lián)免疫方法檢測細(xì)胞白介素-6、白介素-1β、腫瘤壞死因子表達(dá)；采用細(xì)胞免疫熒光方法，測細(xì)胞核轉(zhuǎn)錄因子核轉(zhuǎn)位情況；采用反轉(zhuǎn)錄聚合酶鏈?zhǔn)椒磻?yīng)和免疫印跡的方法檢測胰島素受體底物-1酪氨酸磷酸化的基因和蛋白表達(dá)。 結(jié)果動(dòng)物實(shí)驗(yàn)結(jié)果表明槲皮素不僅可以降低PCOS胰島素抵抗模型組大鼠的血胰島素，還能減少核轉(zhuǎn)錄因子核轉(zhuǎn)位，降低白介素-6、白介素-1β、腫瘤壞死因子炎性因子水平，對(duì)還原型煙酰胺腺嘌呤二核苷酸磷酸氧化酶p22phox亞基、氧化型低密度脂蛋白、Toll樣受體-4的基因和蛋白表達(dá)有抑制作用；可以升高胰島素受體底物-1酪氨酸磷酸化、胰島素受體底物-2的基因和蛋白表達(dá)。大鼠性周期恢復(fù)率58.33％。體外實(shí)驗(yàn)結(jié)果表明槲皮素可以減少胰島素抵抗細(xì)胞核轉(zhuǎn)錄因子核轉(zhuǎn)位，降低炎性因子白介素-6、白介素-1β、腫瘤壞死因子水平，改善細(xì)胞胰島素抵抗；同時(shí)還能降低還原型煙酰胺腺嘌呤二核苷酸磷酸氧化酶p22phox亞基、氧化型低密度脂蛋白、Toll樣受體-4的基因和蛋白表達(dá)；能升高胰島素受體底物-1酪氨酸磷酸化的基因和蛋白表達(dá)。TLR/NF-κB信號(hào)通路被抑制后，胰島素抵抗細(xì)胞的轉(zhuǎn)錄因子核轉(zhuǎn)位、白介素-6、白介素-1β、腫瘤壞死因子炎性因子水平均下降，胰島素受體底物-1酪氨酸磷酸化的基因和蛋白表達(dá)上升，細(xì)胞的胰島素抵抗得以逆轉(zhuǎn)。TLR/NF-κB信號(hào)通路被抑制后，槲皮素對(duì)胰島素抵抗的治療作用被阻斷。 結(jié)論TLR/NF-κB信號(hào)通路參與多囊卵巢綜合征胰島素抵抗的形成；槲皮素治療多囊卵巢綜合征胰島素抵抗有較好的療效；其機(jī)制可能是通過抑制TLR/NF-κB信號(hào)通路，改善多囊卵巢胰島素抵抗微環(huán)境炎癥，從而逆轉(zhuǎn)胰島素抵抗；槲皮素是一個(gè)潛在的，對(duì)胰島素抵抗有較好治療效果的藥物。
[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.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R711.75

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 周美霞;陳建林;;多囊卵巢綜合征胰島素抵抗的信號(hào)傳導(dǎo)[J];醫(yī)藥論壇雜志;2011年14期

2 吳小燕;梁瑞寧;;淺析多囊卵巢綜合征中醫(yī)病因病機(jī)[J];江西中醫(yī)藥;2012年02期

3 孫涓;余世春;;槲皮素的研究進(jìn)展[J];現(xiàn)代中藥研究與實(shí)踐;2011年03期

4 王莉;;兩種方案治療合并胰島素抵抗的多囊卵巢綜合征不孕患者臨床療效評(píng)價(jià)[J];吉林醫(yī)學(xué);2013年08期

5 梁靜;孫維峰;周建龍;;復(fù)方多囊調(diào)經(jīng)湯治療多囊卵巢綜合征的機(jī)制研究[J];華南國防醫(yī)學(xué)雜志;2013年06期

6 王針織;俞超芹;;補(bǔ)腎活血化痰法治療多囊卵巢綜合征的臨床觀察[J];實(shí)用中西醫(yī)結(jié)合臨床;2010年01期

7 路敏;呂亞麗;劉麗宏;;胰島素抵抗細(xì)胞模型概況[J];世界中西醫(yī)結(jié)合雜志;2012年10期

8 蘇健;李亞敏;田李軍;邵建英;;補(bǔ)腎化痰通絡(luò)法加針刺治療肥胖型多囊卵巢綜合征的臨床研究[J];天津中醫(yī)藥;2013年05期

9 霍文蘭;范小芹;趙天聰;;超聲法提取山楂果中熊果酸和齊墩果酸的工藝研究[J];應(yīng)用化工;2013年10期

10 崔山風(fēng);;槲皮素的研究進(jìn)展[J];西北藥學(xué)雜志;2006年06期

本文編號(hào)：2244862