本文關(guān)鍵詞： 2型糖尿病 降糖消渴顆粒 內(nèi)質(zhì)網(wǎng)應(yīng)激 糖脂代謝 糖尿病肝損傷 氧化應(yīng)激　出處：《北京中醫(yī)藥大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：目的本課題組采用高脂飼料誘導(dǎo)的自發(fā)性2型糖尿病KK-Ay小鼠模型,通過對KK-Ay小鼠血及肝臟中糖脂代謝相關(guān)指標(biāo)、胰島素信號通路相關(guān)指標(biāo)等進行檢測,觀察降糖消渴顆粒對糖尿病胰島素抵抗合并肝臟糖脂代謝異常的影響；通過對KK-Ay小鼠肝臟中氧化應(yīng)激、炎癥細(xì)胞因子、內(nèi)質(zhì)網(wǎng)應(yīng)激等相關(guān)指標(biāo)進行檢測,進一步研究探討降糖消渴顆粒治療2型糖尿病、改善糖尿病肝損傷的可能作用機制。方法雄性8周齡KK-Ay小鼠,高脂飼料喂養(yǎng)4周,將空腹血糖≥13.9 mmol/L的小鼠45只按照體重,血糖隨機分為模型組、降糖消渴顆粒高、中、低劑量組和吡格列酮組,9只／組,共給藥10周。10只C57BL/6J小鼠作為正常對照,基礎(chǔ)飼料喂養(yǎng),每日給予等比例純凈水灌胃。每周同一時間段測量小鼠空腹血糖、體重、攝食量,在第4、10周給予葡萄糖溶液灌胃檢測小鼠糖耐量。實驗結(jié)束后取材,稱量肝臟重量,計算肝體重比；腹主動脈取血,檢測血中TG、TC、HDL、LDL、FFA、FINS及HbAlc水平,計算ISI；檢測血清中肝功能相關(guān)指標(biāo)ALT,AST, γ-GT及氧化應(yīng)激相關(guān)指標(biāo)MDA,SOD的變化；肝組織勻漿后,相應(yīng)試劑盒檢測勻漿上清液中TG、TC、HDL、LDL含量及MDA、 SOD、GSH活性；HE染色觀察肝細(xì)胞中脂質(zhì)堆積情況及組織形態(tài)學(xué)改變；過碘酸雪夫染色觀察肝臟中糖原儲備情況；RT-PCR法檢測肝組織中胰島素信號通路相關(guān)基因IRS-1、IRS-2、Akt、PKCε mRNA的表達,糖脂代謝相關(guān)基因AMPKα、PPARα、GSK-3α、 SREBP-1c、SREBP-2和FAS的mRNA表達及炎癥因子NF-κB、TNF-α、IL-1β和內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)指標(biāo)eIF2α、ATF4、CHOP、IRE1α, XBP1和GRP78mRNA表達量變化；免疫組化檢測肝組織石蠟切片中p-eIF2α、GRP78和CHOP的蛋白表達量；Western Blot法檢測肝臟中p-eIF2α和GRP78的蛋白表達量。結(jié)果攝食量、體重和肝重：所有小鼠體重持續(xù)增長,各劑量降糖消渴顆粒對小鼠體重和攝食量都沒有明顯影響；高、中劑量降糖消渴顆粒顯著降低了小鼠的肝體重比(P0.01)。肝臟病理改變：中劑量降糖消渴顆�？娠@著改善肝細(xì)胞脂肪變性,減少肝臟中炎性細(xì)胞浸潤,維持肝組織正常結(jié)構(gòu)。糖代謝指標(biāo)：降糖消渴顆粒在10周的治療期內(nèi)有效降低了各組小鼠的空腹血糖(P0.01),但給藥4周時對糖耐量沒有改善作用(P0.05)；給藥10周時高劑量降糖消渴顆�？梢悦黠@改善小鼠口服糖耐量(P0.01)；高、中劑量降糖消渴顆�？梢燥@著降低血清FINS、HbAlc并提高小鼠ISI(P0.01),低劑量降糖消渴顆粒也可減少血清FINS (P0.05),提高ISI (P0.01),但對HbA1c沒有明顯改善效果；肝糖原PAS染色結(jié)果顯示,中劑量降糖消渴顆�？梢杂行岣吒闻K中糖原儲備,且高、中劑量降糖消渴顆粒都可以顯著下調(diào)肝臟中GSK-3α的mRNA表達量。脂代謝指標(biāo)：在血清脂質(zhì)含量的改變方面,高劑量降糖消渴顆�？擅黠@降低血清TG、LDL含量(P0.01),對血清TC、HDL、FFA無明顯改善作用(P0.05)；中劑量降糖消渴顆粒對TC、TG、HDL、LDL均有良好的改善作用(P0.01),且可以降低FFA含量(P0.05)；經(jīng)低劑量降糖消渴顆粒治療后,血清中TC、HDL、LDL、FFA含量顯著降低(P0.01),而血清TG并無改善(P0.05)。在肝臟脂質(zhì)含量的改變方面,低劑量降糖消渴顆粒可降低肝臟中TG(P0.05)并顯著提升HDL含量(P0.01),但對TC、LDL無明顯作用(P0.05)；中、高劑量降糖消渴顆粒均可顯著降低肝臟中TC、TC、LDL并提高HDL含量(P0.05),而中劑量降糖消渴顆粒降低肝臟中TG、 TC含量作用較高劑量更佳。在脂質(zhì)代謝相關(guān)基因的表達量方面,高劑量降糖消渴顆�？梢陨险{(diào)肝臟中PPARα并減少SREBP1c的mRNA表達量(P0.01),對AMPKα、 Insig-1、SREBP2、FAS的mRNA表達量無影響(P0.05)；中劑量降糖消渴顆�？梢陨险{(diào)肝臟中AMPKα(P0.01)和Insig-1的1nRNA表達量(P0.05),并下調(diào)SREBP1c. SREBP2的mRNA表達量(P0.01)和FAS的mRNA表達量(P0.05),對PPARα的mRNA表達量無影響(P0.05)；低劑量降糖消渴顆�？梢陨险{(diào)肝臟中AMPKα的mRNA表達量(P0.01),下調(diào)FAS的mRNA表達量(P0.05),顯著下調(diào)SREBP1c SREBP2的mRNA表達量(P0.01),對Insig-1和PPARα的mRNA表達量無影響(P0.05)。胰島素信號通路：高、中、低劑量降糖消渴顆粒都可以提高IRS-2的mRNA表達量(P0.01,P0.01和P0.05)；中劑量降糖消渴顆粒還可以增加Akt的mRNA表達(P0.01),各劑量降糖消渴顆粒對IRS-1和PKCε的mRNA表達量都沒有明顯改善作用(P0.05)。肝功能：各劑量降糖消渴顆粒均可顯著降低肝臟ALT、γ-GT含量(P0.01),高劑量降糖消渴顆�？捎行Ы档脱錋ST含量(P0.01),低、中劑量降糖消渴顆粒對小鼠血清AST無明顯改善作用(P0.05)。氧化應(yīng)激：在血清SOD、MDA含量方面,各劑量降糖消渴顆粒均明顯改善了血清MDA、SOD (P0.01),以中劑量降糖消渴顆粒效果最佳；在肝組織氧化應(yīng)激相關(guān)指標(biāo)的活性方面,中、高劑量降糖消渴顆�？娠@著降低肝臟中MDA含量(P0.01)且提高SOD活性(P0.01), GSH活性也較模型組小鼠有所提高(P0.05),高劑量效果稍優(yōu)于中劑量(P0.05),低劑量降糖消渴顆粒對肝臟氧化應(yīng)激各指標(biāo)都無改善作用(P0.05)。炎癥因子：中、低劑量降糖消渴顆粒均明顯下調(diào)了糖尿病小鼠肝贓中NF-κB的mRNA表達量(P0.01),經(jīng)中、低劑量降糖消渴顆粒治療后,肝臟中TNF-α的mRNA表達量也有所降低(P0.05),與模型組相比,各治療組小鼠肝臟中IL-1β的mRNA表達量雖有所下調(diào),但無統(tǒng)計學(xué)意義(P0.05)。內(nèi)質(zhì)網(wǎng)應(yīng)激通路：高劑量降糖消渴顆�？梢燥@著下調(diào)肝臟中eIF2α、ATF4的]mRNA表達量(P0.01),對CHOP、IRE1α、XBP1和GRP78的mRNA表達量無影響(P0.05)；中劑量降糖消渴顆�？梢燥@著下調(diào)肝臟中eIF2α、ATF4和GRP78的mRNA表達量(P0.01),下調(diào)CHOP、IRE1α的mRNA表達量(P0.05),對XBP1的mRNA表達量沒有明顯影響(P0.05)；低劑量降糖消渴顆�？梢燥@著下調(diào)肝臟中ATF4的mRNA表達量(P0.01),下調(diào)eIF2α的nRNA表達量(P0.05),對CHOP、IRE1αXBP1和GRP78的mRNA表達量無影響(P0.05)。而在蛋白表達方面,各劑量降糖消渴顆粒均可顯著降低eIF2α的磷酸化水平(P0.01),降低GRP78和CHOP的蛋白表達。結(jié)論(1)降糖消渴顆�？山档透咧嬍痴T導(dǎo)的2型糖尿病KK-Ay小鼠的空腹血糖,增強糖耐量,增加胰島素敏感性,從而改善胰島素抵抗,以高、中劑量效果為佳；(2)降糖消渴顆粒對實驗小鼠糖尿病肝損傷具有保護作用,此外,還可降低血清和肝臟中脂質(zhì)含量。高、中劑量效果較好；(3)降糖消渴顆�？梢酝ㄟ^上調(diào)實驗小鼠肝臟胰島素信號通路中IRS-2的表達來促進肝臟對葡萄糖的攝取和利用,合成肝糖原并抑制肝臟葡萄糖的輸出,從而起到調(diào)節(jié)糖代謝,改善糖代謝紊亂的作用；(4)降糖消渴顆粒可以通過提高實驗小鼠肝臟AMPKα的mRNA表達,調(diào)節(jié)其下游與脂質(zhì)代謝相關(guān)的PPARα、SREBPs、FAS及Insig-1的mRNA表達,減少糖尿病胰島素抵抗?fàn)顟B(tài)下脂質(zhì)在肝臟的異位沉積,從而起到調(diào)節(jié)脂代謝,改善脂代謝紊亂的作用；(5)降糖消渴顆粒對實驗小鼠全身性的氧化應(yīng)激狀態(tài)起到整體的調(diào)節(jié)作用,提高機體抗氧化能力,保證胰島素信號的正常傳遞。高、中劑量效果最佳；(6)降糖消渴顆粒具有良好的整體調(diào)節(jié)效果及抗炎作用,可抑制NF-κB炎癥信號通路的激活,減少炎性因子分泌,從而增加IRS酪氨酸磷酸化,提高胰島素敏感性,減輕全身氧化應(yīng)激。中、低劑量效果較好；(7)降糖消渴顆�？删徑鈨�(nèi)質(zhì)網(wǎng)應(yīng)激反應(yīng),保證胰島素信號在胞內(nèi)的正常傳遞,從而改善胰島素的生理功能,減輕2型糖尿病胰島素抵抗。中劑量效果最佳。本研究采用高脂飼料誘導(dǎo)自發(fā)性2型糖尿病KK-Ay小鼠模型,模擬了人類的發(fā)病原因。首次將中醫(yī)臟腑相關(guān)理論與炎癥反應(yīng)、內(nèi)質(zhì)網(wǎng)應(yīng)激信號通路通路聯(lián)系起來,為“肝脾腎三臟同調(diào),辨證治療2型糖尿病”的理論提供新的實驗數(shù)據(jù)支持。以2型糖尿病小鼠和肝臟糖脂代謝為切入點,探索了降糖消渴顆粒對糖尿病肝損傷狀態(tài)下糖脂代謝紊亂的改善作用；從IR、肝臟氧化應(yīng)激、炎癥反應(yīng)以及內(nèi)質(zhì)網(wǎng)應(yīng)激四者之間的相互關(guān)系出發(fā),探討了降糖消渴顆粒改善糖尿病肝損傷的作用機制。
[Abstract]:The research group with spontaneous type 2 diabetic KK-Ay mice model induced by high fat diet, the relevant indicators of the glycolipid metabolism in liver and serum KK-Ay, insulin signaling pathway related indexes were detected, to observe the effect of Jiangtang Xiaoke granules resistance and liver abnormal glucose and lipid metabolism in diabetic insulin; oxidative stress on mouse liver by KK-Ay in inflammatory cytokines, endoplasmic reticulum stress related indexes were detected, further study of Jiangtang Xiaoke granules in treatment of type 2 diabetes mellitus, diabetic liver injury mechanism. Methods 8 week old male KK-Ay mice fed high fat diet for 4 weeks, the fasting blood glucose greater than 13.9 mmol/L 45 mice according to body weight, blood glucose were randomly divided into model group, Jiangtang Xiaoke granules high, low dose group and pioglitazone group, 9 rats in each group were administered for 10 weeks,.10 C57BL/6J mice were selected as normal Control, basic diet, daily for the proportion of pure water gavage. Every week the same time measured fasting blood glucose, body weight, food intake, glucose solution given intragastric glucose tolerance in mice were detected in 4,10 weeks. After the end of the experiment were weighing the liver weight, liver body weight ratio calculation; abdominal aortic blood the detection of serum TG, TC, HDL, LDL, FFA, ISI and FINS calculate the HbAlc level in serum; liver function indexes of ALT, AST, -GT and gamma related indicators of oxidative stress MDA, SOD changes; liver homogenate, the corresponding detection kit in TG homogenate supernatant, TC, HDL the content of LDL, and MDA, SOD, GSH activity in liver cells; to observe the morphological and histological changes of lipid accumulation in HE staining; periodic acid Schiff staining of glycogen reserves in the liver; insulin signaling pathway related genes IRS-1, RT-PCR method to detect the liver tissue IRS-2, Akt, PKC e mRNA table Da, glucose and lipid metabolism related genes of AMPK alpha, alpha PPAR, alpha GSK-3, SREBP-1c, SREBP-2 and FAS expression of mRNA and inflammatory cytokines NF- kappa B, TNF- alpha, IL-1 beta and endoplasmic reticulum stress related indicators of eIF2 alpha, ATF4, CHOP, IRE1 alpha, the amount of expression of XBP1 and GRP78mRNA; immunohistochemical detection of alpha p-eIF2 liver tissue, expression of GRP78 and CHOP protein; expression of p-eIF2 and GRP78 Western Blot method in the detection of liver protein. The food intake, body weight and liver weight of all mice continued to grow, the dose of Jiangtang Xiaoke Granule on mice body weight and food intake were not significantly affected; high dose Jiangtang Xiaoke granules significantly decreased the liver weight ratio (P0.01). The pathological changes of liver: the dose of Jiangtang Xiaoke granules can significantly improve the fatty degeneration of liver cells, reduce the infiltration of inflammatory cells in the liver, maintain the normal structure of liver tissue. Glucose metabolism index: hypoglycemic The treatment period of Xiaoke granule in 10 weeks reduced mice fasting blood glucose (P0.01), but after 4 weeks of administration had no effect on glucose tolerance (P0.05); after 10 weeks of administration of high dose of Jiangtang Xiaoke granule can improve oral glucose tolerance in mice (P0.01); high dose Jiangtang Xiaoke granules can significantly reduce serum FINS, HbAlc and ISI were increased (P0.01), low dose of Jiangtang Xiaoke granules can also reduce serum FINS (P0.05), ISI (P0.01), but the increase of HbA1c has no obvious improvement effect; glycogen PAS staining showed that the dose of Jiangtang Xiaoke granules can effectively improve the glycogen reserves the liver and high expression in dose of Jiangtang Xiaoke granules can significantly reduce GSK-3 alpha mRNA in liver. Lipid metabolism in serum lipid content changes, high-dose Jiangtang Xiaoke granule can significantly reduce serum TG, the content of LDL (P0.01), the Serum TC, HDL, FFA had no significant effect (P0.05); middle dose of Jiangtang Xiaoke Granule on TC, TG, HDL, LDL had a good effect (P0.01), and can reduce the content of FFA (P0.05); the low dose of Jiangtang Xiaoke granules after treatment, serum TC, HDL, LDL, FFA content significantly decreased (P0.01), serum TG (P0.05). There has been no improvement in the liver lipid content changes, low dose of Jiangtang Xiaoke granule can reduce liver TG (P0.05) and significantly improve the content of HDL (P0.01), but had no obvious effect on TC, LDL (P0.05); high dose in Jiangtang Xiaoke granules could significantly decrease the liver TC, TC, LDL and increase the content of HDL (P0.05), and the dose of Jiangtang Xiaoke granules can decrease TG, TC content of high dose better. Expression of genes related to lipid metabolism, high dose of Jiangtang Xiaoke granules can increase liver PPAR alpha and decrease the SREBP1c expression of mRNA (P0 .01, AMPK) of Insig-1, SREBP2, FAS alpha, mRNA expression had no effect (P0.05); middle dose Jiangtang Xiaoke granules can be AMPK upregulated in the liver (P0.01) and the expression level of Insig-1 1nRNA (P0.05), the expression and down-regulation of SREBP1c. SREBP2 mRNA (P0.01) expression and FAS (mRNA P0.05, PPAR) on the expression of mRNA had no effect on alpha (P0.05); low dose of Jiangtang Xiaoke granules can express AMPK upregulation of mRNA in liver (P0.01), down-regulation of FAS mRNA (P0.05), the expression of SREBP2 mRNA significantly reduced SREBP1c (P0.01), Insig-1 and PPAR alpha mRNA there was no effect (P0.05). The insulin signaling pathway: high, low dose of Jiangtang Xiaoke granules can increase the IRS-2 expression of mRNA (P0.01, P0.01 and P0.05); middle dose Jiangtang Xiaoke granules can also increase the expression of Akt mRNA (P0.01), different doses of mRNA on IRS-1 and PKC Jiangtang Xiaoke granules. The expression of no There is obvious improvement (P0.05). The liver function: each dose of Jiangtang Xiaoke granules could significantly decrease the content of liver ALT, -GT gamma (P0.01), high dose of Jiangtang Xiaoke granules can effectively reduce the content of serum AST (P0.01), low dose of Jiangtang Xiaoke granules had no obvious effect on serum AST (P0.05). Oxidative stress: in serum SOD, MDA levels, the dose of Jiangtang Xiaoke granules significantly improved serum MDA, SOD (P0.01), the middle dose of Jiangtang Xiaoke granules is best; in the relevant indicators of oxidative stress in liver tissue activity, and high dose of Jiangtang Xiaoke granules could significantly reduce the content of MDA in liver (P0.01) and increase the activity of SOD (P0.01), the activity of GSH is increased in the model group (P0.05), high dose effect is slightly better than that in (P0.05), low dose of Jiangtang Xiaoke granules have no effect on the index of oxidative stress in the liver (P0.05). Because of inflammation Son: in the low dose of Jiangtang Xiaoke granules were significantly decreased in the diabetic mice liver in NF- kappa B mRNA expression (P0.01), the low dose of Jiangtang Xiaoke granules after treatment with TNF- alpha in liver mRNA expression also decreased (P0.05), compared with the model group, the treatment group of mice the liver IL-1 beta mRNA expression is decreased, but there was no statistical significance (P0.05). The endoplasmic reticulum stress pathway: high dose of Jiangtang Xiaoke granules can significantly downregulate eIF2 alpha in liver, the expression of ATF4]mRNA (P0.01), CHOP, IRE1 XBP1 and GRP78 alpha, mRNA expression had no effect (P0.05); middle dose Jiangtang Xiaoke granules can significantly downregulate eIF2 alpha in liver, expression of ATF4 and GRP78 mRNA (P0.01), down CHOP, the expression of IRE1 alpha mRNA (P0.05), the XBP1 mRNA expression had no significant effect (P0.05); low dose of Jiangtang Xiaoke granules could reduce liver ATF4 mRN The expression of A (P0.01), expression of eIF2 alpha nRNA (P0.05), CHOP, IRE1 XBP1 and GRP78 alpha mRNA expression had no effect (P0.05). And the expression in protein, the dose of Jiangtang Xiaoke granules could significantly decrease the phosphorylation level of eIF2 alpha (P0.01), and the decreased expression of GRP78 CHOP (1) protein. Conclusion Jiangtang Xiaoke granules can reduce type 2 diabetic KK-Ay mice induced by high fat diet fasting blood glucose, enhanced glucose tolerance, increased insulin sensitivity, improve insulin resistance, with high, middle dose effect is better; (2) granule on experimental Jiangtang Xiaoke diabetic mice with liver injury in addition, the protective effect, but also can reduce the content of lipid in serum and liver. High dose, good in effect; (3) Jiangtang Xiaoke granules can increase the expression of IRS-2 in mice liver through the insulin signaling pathway to promote the uptake and utilization of glucose in the liver, liver synthesis Glycogen and inhibit hepatic glucose output, so as to regulate glucose metabolism, improve glucose metabolism disorder; (4) Jiangtang Xiaoke granules can improve the expression of AMPK in mouse liver alpha mRNA, regulate its downstream associated with lipid metabolism of PPAR alpha, SREBPs, expression of FAS and Insig-1 mRNA, to reduce insulin resistance in diabetes mellitus under the condition of lipid in ectopic deposition of liver, so as to regulate lipid metabolism and improve lipid metabolism function; (5) the oxidative stress state of Jiangtang Xiaoke Granule on experimental murine systemic regulation to the whole, improve the antioxidant capacity, to ensure the normal transmission of the insulin signal. High dose effect is best; (6) Jiangtang Xiaoke granules has a good overall effect and anti-inflammatory effect, can inhibit the activation of NF- kappa B signaling pathway, reduce the secretion of inflammatory factors, so as to increase the tyrosine phosphorylation of IRS, To improve insulin sensitivity, reduce systemic oxidative stress. In low dose, good effect; (7) Jiangtang Xiaoke granules can alleviate the endoplasmic reticulum stress, to ensure the normal insulin signal in intracellular delivery, so as to improve the physiological function of insulin, reduce insulin resistance in type 2 diabetes mellitus. Dose effect is the best. This study used high fat diet to induce spontaneous type 2 diabetic KK-Ay mice model, simulated the cause of mankind. For the first time the TCM theory and the inflammatory reaction, endoplasmic reticulum stress signaling pathways linked to liver and spleen kidney three dirty homology, syndrome differentiation and treatment of type 2 diabetes "theory provides new experimental data to support and type 2 diabetic mice. The liver lipid metabolism as a starting point, explore the effect of Jiangtang Xiaoke granules on diabetic liver injury under the condition of glucose and lipid metabolic disorder; from IR, liver oxidative stress, anti inflammation According to the relationship between four patients with endoplasmic reticulum stress, the mechanism of hypoglycemic and Xiaoke granule to improve the liver injury of diabetes was discussed.

【學(xué)位授予單位】：北京中醫(yī)藥大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R259
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本文編號：1454068