降糖消渴颗粒对T2DM小鼠肝脏糖脂代谢和内质网应激的影响

发布时间：2018-01-22 07:22

  本文关键词： 2型糖尿病 降糖消渴颗粒 内质网应激 糖脂代谢 糖尿病肝损伤 氧化应激　出处：《北京中医药大学》2016年博士论文　论文类型：学位论文

【摘要】：目的本课题组采用高脂饲料诱导的自发性2型糖尿病KK-Ay小鼠模型,通过对KK-Ay小鼠血及肝脏中糖脂代谢相关指标、胰岛素信号通路相关指标等进行检测,观察降糖消渴颗粒对糖尿病胰岛素抵抗合并肝脏糖脂代谢异常的影响；通过对KK-Ay小鼠肝脏中氧化应激、炎症细胞因子、内质网应激等相关指标进行检测,进一步研究探讨降糖消渴颗粒治疗2型糖尿病、改善糖尿病肝损伤的可能作用机制。方法雄性8周龄KK-Ay小鼠,高脂饲料喂养4周,将空腹血糖≥13.9 mmol/L的小鼠45只按照体重,血糖随机分为模型组、降糖消渴颗粒高、中、低剂量组和吡格列酮组,9只／组,共给药10周。10只C57BL/6J小鼠作为正常对照,基础饲料喂养,每日给予等比例纯净水灌胃。每周同一时间段测量小鼠空腹血糖、体重、摄食量,在第4、10周给予葡萄糖溶液灌胃检测小鼠糖耐量。实验结束后取材,称量肝脏重量,计算肝体重比；腹主动脉取血,检测血中TG、TC、HDL、LDL、FFA、FINS及HbAlc水平,计算ISI；检测血清中肝功能相关指标ALT,AST, γ-GT及氧化应激相关指标MDA,SOD的变化；肝组织匀浆后,相应试剂盒检测匀浆上清液中TG、TC、HDL、LDL含量及MDA、 SOD、GSH活性；HE染色观察肝细胞中脂质堆积情况及组织形态学改变；过碘酸雪夫染色观察肝脏中糖原储备情况；RT-PCR法检测肝组织中胰岛素信号通路相关基因IRS-1、IRS-2、Akt、PKCε mRNA的表达,糖脂代谢相关基因AMPKα、PPARα、GSK-3α、 SREBP-1c、SREBP-2和FAS的mRNA表达及炎症因子NF-κB、TNF-α、IL-1β和内质网应激相关指标eIF2α、ATF4、CHOP、IRE1α, XBP1和GRP78mRNA表达量变化；免疫组化检测肝组织石蜡切片中p-eIF2α、GRP78和CHOP的蛋白表达量；Western Blot法检测肝脏中p-eIF2α和GRP78的蛋白表达量。结果摄食量、体重和肝重：所有小鼠体重持续增长,各剂量降糖消渴颗粒对小鼠体重和摄食量都没有明显影响；高、中剂量降糖消渴颗粒显著降低了小鼠的肝体重比(P0.01)。肝脏病理改变：中剂量降糖消渴颗粒可显著改善肝细胞脂肪变性,减少肝脏中炎性细胞浸润,维持肝组织正常结构。糖代谢指标：降糖消渴颗粒在10周的治疗期内有效降低了各组小鼠的空腹血糖(P0.01),但给药4周时对糖耐量没有改善作用(P0.05)；给药10周时高剂量降糖消渴颗粒可以明显改善小鼠口服糖耐量(P0.01)；高、中剂量降糖消渴颗粒可以显著降低血清FINS、HbAlc并提高小鼠ISI(P0.01),低剂量降糖消渴颗粒也可减少血清FINS (P0.05),提高ISI (P0.01),但对HbA1c没有明显改善效果；肝糖原PAS染色结果显示,中剂量降糖消渴颗粒可以有效提高肝脏中糖原储备,且高、中剂量降糖消渴颗粒都可以显著下调肝脏中GSK-3α的mRNA表达量。脂代谢指标：在血清脂质含量的改变方面,高剂量降糖消渴颗粒可明显降低血清TG、LDL含量(P0.01),对血清TC、HDL、FFA无明显改善作用(P0.05)；中剂量降糖消渴颗粒对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)；经低剂量降糖消渴颗粒治疗后,血清中TC、HDL、LDL、FFA含量显著降低(P0.01),而血清TG并无改善(P0.05)。在肝脏脂质含量的改变方面,低剂量降糖消渴颗粒可降低肝脏中TG(P0.05)并显著提升HDL含量(P0.01),但对TC、LDL无明显作用(P0.05)；中、高剂量降糖消渴颗粒均可显著降低肝脏中TC、TC、LDL并提高HDL含量(P0.05),而中剂量降糖消渴颗粒降低肝脏中TG、 TC含量作用较高剂量更佳。在脂质代谢相关基因的表达量方面,高剂量降糖消渴颗粒可以上调肝脏中PPARα并减少SREBP1c的mRNA表达量(P0.01),对AMPKα、 Insig-1、SREBP2、FAS的mRNA表达量无影响(P0.05)；中剂量降糖消渴颗粒可以上调肝脏中AMPKα(P0.01)和Insig-1的1nRNA表达量(P0.05),并下调SREBP1c. SREBP2的mRNA表达量(P0.01)和FAS的mRNA表达量(P0.05),对PPARα的mRNA表达量无影响(P0.05)；低剂量降糖消渴颗粒可以上调肝脏中AMPKα的mRNA表达量(P0.01),下调FAS的mRNA表达量(P0.05),显著下调SREBP1c SREBP2的mRNA表达量(P0.01),对Insig-1和PPARα的mRNA表达量无影响(P0.05)。胰岛素信号通路：高、中、低剂量降糖消渴颗粒都可以提高IRS-2的mRNA表达量(P0.01,P0.01和P0.05)；中剂量降糖消渴颗粒还可以增加Akt的mRNA表达(P0.01),各剂量降糖消渴颗粒对IRS-1和PKCε的mRNA表达量都没有明显改善作用(P0.05)。肝功能：各剂量降糖消渴颗粒均可显著降低肝脏ALT、γ-GT含量(P0.01),高剂量降糖消渴颗粒可有效降低血清AST含量(P0.01),低、中剂量降糖消渴颗粒对小鼠血清AST无明显改善作用(P0.05)。氧化应激：在血清SOD、MDA含量方面,各剂量降糖消渴颗粒均明显改善了血清MDA、SOD (P0.01),以中剂量降糖消渴颗粒效果最佳；在肝组织氧化应激相关指标的活性方面,中、高剂量降糖消渴颗粒可显著降低肝脏中MDA含量(P0.01)且提高SOD活性(P0.01), GSH活性也较模型组小鼠有所提高(P0.05),高剂量效果稍优于中剂量(P0.05),低剂量降糖消渴颗粒对肝脏氧化应激各指标都无改善作用(P0.05)。炎症因子：中、低剂量降糖消渴颗粒均明显下调了糖尿病小鼠肝赃中NF-κB的mRNA表达量(P0.01),经中、低剂量降糖消渴颗粒治疗后,肝脏中TNF-α的mRNA表达量也有所降低(P0.05),与模型组相比,各治疗组小鼠肝脏中IL-1β的mRNA表达量虽有所下调,但无统计学意义(P0.05)。内质网应激通路：高剂量降糖消渴颗粒可以显著下调肝脏中eIF2α、ATF4的]mRNA表达量(P0.01),对CHOP、IRE1α、XBP1和GRP78的mRNA表达量无影响(P0.05)；中剂量降糖消渴颗粒可以显著下调肝脏中eIF2α、ATF4和GRP78的mRNA表达量(P0.01),下调CHOP、IRE1α的mRNA表达量(P0.05),对XBP1的mRNA表达量没有明显影响(P0.05)；低剂量降糖消渴颗粒可以显著下调肝脏中ATF4的mRNA表达量(P0.01),下调eIF2α的nRNA表达量(P0.05),对CHOP、IRE1αXBP1和GRP78的mRNA表达量无影响(P0.05)。而在蛋白表达方面,各剂量降糖消渴颗粒均可显著降低eIF2α的磷酸化水平(P0.01),降低GRP78和CHOP的蛋白表达。结论(1)降糖消渴颗粒可降低高脂饮食诱导的2型糖尿病KK-Ay小鼠的空腹血糖,增强糖耐量,增加胰岛素敏感性,从而改善胰岛素抵抗,以高、中剂量效果为佳；(2)降糖消渴颗粒对实验小鼠糖尿病肝损伤具有保护作用,此外,还可降低血清和肝脏中脂质含量。高、中剂量效果较好；(3)降糖消渴颗粒可以通过上调实验小鼠肝脏胰岛素信号通路中IRS-2的表达来促进肝脏对葡萄糖的摄取和利用,合成肝糖原并抑制肝脏葡萄糖的输出,从而起到调节糖代谢,改善糖代谢紊乱的作用；(4)降糖消渴颗粒可以通过提高实验小鼠肝脏AMPKα的mRNA表达,调节其下游与脂质代谢相关的PPARα、SREBPs、FAS及Insig-1的mRNA表达,减少糖尿病胰岛素抵抗状态下脂质在肝脏的异位沉积,从而起到调节脂代谢,改善脂代谢紊乱的作用；(5)降糖消渴颗粒对实验小鼠全身性的氧化应激状态起到整体的调节作用,提高机体抗氧化能力,保证胰岛素信号的正常传递。高、中剂量效果最佳；(6)降糖消渴颗粒具有良好的整体调节效果及抗炎作用,可抑制NF-κB炎症信号通路的激活,减少炎性因子分泌,从而增加IRS酪氨酸磷酸化,提高胰岛素敏感性,减轻全身氧化应激。中、低剂量效果较好；(7)降糖消渴颗粒可缓解内质网应激反应,保证胰岛素信号在胞内的正常传递,从而改善胰岛素的生理功能,减轻2型糖尿病胰岛素抵抗。中剂量效果最佳。本研究采用高脂饲料诱导自发性2型糖尿病KK-Ay小鼠模型,模拟了人类的发病原因。首次将中医脏腑相关理论与炎症反应、内质网应激信号通路通路联系起来,为“肝脾肾三脏同调,辨证治疗2型糖尿病”的理论提供新的实验数据支持。以2型糖尿病小鼠和肝脏糖脂代谢为切入点,探索了降糖消渴颗粒对糖尿病肝损伤状态下糖脂代谢紊乱的改善作用；从IR、肝脏氧化应激、炎症反应以及内质网应激四者之间的相互关系出发,探讨了降糖消渴颗粒改善糖尿病肝损伤的作用机制。
[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.

【学位授予单位】：北京中医药大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R259
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本文编号：1454068