吡格列酮对2型糖尿病大鼠肾脏雷帕霉素靶蛋白表达的影响
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摘要
目的：
1观察糖尿病组及吡格列酮干预组大鼠空腹血糖、胰岛素、甘油三酯、胆固醇、尿微量白蛋白、尿素氮、肌酐的变化。
2探讨PPAR-γ激动剂吡格列酮对糖尿病大鼠肾脏mTOR表达的影响。
方法：
体质量为(200±20)g的8周龄健康清洁级SD雄性大鼠33只,随机分成2组,正常对照组(A组)11只,糖尿病模型组22只。A组给予正常普通饮食,糖尿病模型组给予高糖高脂饮食造模,喂养4周,按照HOMA胰岛素抵抗指数[HOMA= (FPG×FNS)/22.5]判断动物出现胰岛素抵抗,本实验HOMA统计结果为(16.3±2.74)mmol.mU/L2,将链脲佐菌素(STZ)按30mg/kg体质量一次性腹腔注射,正常对照组腹腔内注射等体积柠檬酸缓冲液。1周后,尾静脉采血,血糖仪测定空腹血糖(采血前大鼠禁食12h),以空腹血糖≥13.9mmol/L作为糖尿病造模成功标准。将造模成功的糖尿病大鼠随机分成2组,即糖尿病模型组B组、吡咯列酮干预组C组,A、B组给予生理盐水灌胃(2ml/kg.d), C组给予吡格列酮(10mg/kg.d)灌胃给药,饲养6周。大鼠自实验结束前一日收集24小时尿标本,用于测定尿微量白蛋白；最后处死大鼠采血,用于测定血糖、血胰岛素、胆固醇、甘油三酯、尿素氮、肌酐；摘取肾脏,一个肾用4%中性多聚甲醛固定,行HE染色行形态学检查及免疫组化观察mTOR蛋白水平的变化；另一个肾组织保存于-80℃冰箱,用于测定mTOR mRNA的表达。
结果：
1各组大鼠实验室指标：与A组比较,B、C组大鼠FBG、FNS、HOMA-IR、TG、TC水平均明显升高(P<0.05),与B组比较,C组大鼠FBG.FNS.HOMA-IR、TG、TC水平均明显降低(P<0.05)。
2肾功能：各组大鼠BUN、Cr、UAlb比较：与A组比较,B、C组大鼠BUN、UAlb升高(P<0.01),B组Cr升高(P<0.05),A、C组Cr水平无明显差异,与B组比较,C组大鼠BUN、Cr、UAlb降低(P<0.05)。
3 HE染色：A组大鼠肾组织病理结构基本正常,B组大鼠肾小管上皮细胞可见玻璃样变和无结构红染物质,局灶区域可见肾小管液化坏死,肾小球囊内可见少量红细胞渗出,肾小球基底膜增厚,细胞外基质增多,C组较B组病变减轻。
4免疫组织化学染色结果：各组大鼠在肾小管、肾小球中均可见mTOR表达呈棕色颗粒样物,与A组比较,B、C组大鼠肾组织mTOR表达明显升高(P<0.01)；与B组比较,C组大鼠肾组织mTOR表达明显降低(P<0.05)。
5基因表达：与A组比较,B、C组大鼠肾组织mTOR mRNA表达强度增强(P<0.05)；与B组比较,C组大鼠肾组织mTOR mRNA表达强度减弱(P<0.05)。
6尿微量白蛋白与mTOR的表达密切相关,r=0.71(P<0.01)。结论：
1糖尿病组大鼠空腹血糖、胰岛素、甘油三酯、胆固醇、尿微量白蛋白、尿素氮、肌酐水平增加,吡格列酮干预后能降低各项生化指标(肌酐除外)。
2糖尿病组大鼠肾脏组织mTOR表达比正常组表达增强,吡格列酮干预后,肾脏组织mTOR的表达减弱。
3吡格列酮能改善糖尿病大鼠糖脂代谢和胰岛素抵抗,通过调控mTOR的表达,起到延缓糖尿病肾病进展的作用。
Objective:
(1)To observe FBG、FNS、TG、TC、Alb、BUN and Cr of the diabetic and Pioglitazone group rats.
(2)To investigat the effect of Pioglitazone on the expression of mTOR in renal tissue of type 2 diabetic rats.
Methods:
Eight weeks thirty-Three healthy、sanitary Sprague-Dawley maleness rats whose body weight was (200±20) g included in this experiment were randomly divided into two groups.①Control group (group A,n=11);②Diabetic model group (n=22); Rats in group B and C were made type 2 diabetic model by feeding with high glucose high fat diet for four weeks,judging the rats appear insulin resistance status by HOMA insulin resistance index[HOMA=(FPG×FNS)/22.5], The insulin resistance index was (16.3±2.74),and then STZ(30mg·kg-1) with intraperitoneally injection once, rats in group A recived common diet for four weeks and a same volume of vehicle citrate buffer by intraperitoneally injection. After one week of STZ injection(all the rats were fasted for 12 hours),the fasting blood glucose was carried out by getting blood from tail vein,the fasting blood glucose for successful standard of diabetic model is 13.9. The rats which were diabetic model successfully were randomly divided into two groups, Diabetic model group (group B,n=11);Pioglitazone group (group C,n=11).Rats in group A and B recived a volume of 0.9% saline (2ml·kg-1·d-1)by intragastric administration, rats in group C recived Pioglitazone (10mg·kg-1·d-1) by intragastric administration. After six weeks, we collected 24h urine before the day we accomplish the experiment to detect urine albumin, and gathered blood of rats when we killed the rats in the end to detect blood glucose、blood insulin、TG、TC、Cr and BuN; kidneys of rats were removed,one renal was put into 4% formalin to detect the expression of mTOR by immunohistochemical staining, histopathological changes were done by HE staining; another was put into freezer for subzero eighty centi-degree to detected the expression of mTORmRNA by PCR.
Results:
(1) Biochemistry:Compared with group A,FBG、FNS、HOMA-IR、TG and TC of group B and C were enhanced (P<0.05); compared with group B, FBG、HOMA -IR、TG and TC of group C were decreased(P<0.05).
(2) Renal function:Compared with group A,BUNand UAlb of group B and C were enhanced (P<0.05); compared with group B, BUN、Cr and UAlb of group C were decreased(P<0.05). Cr was no significant difference between group A and C.
(3) Histopathological characteristics(HE staining):In group A the form of kidney cytoplasm were normal, and in group B, the kidney tubules epithelium appeared hyalinization and structurelessness dye red substance,part of the kidney tubules appeared liquefative necrosis and the structure of kidney tubules was indistinct,the bowman's capsule in kidney glomerulus exudated a little red blood cell, glomerular basement membrane was thicken, extracellular matrixc increased,the histopathological changes of group C were wosern than group B.
(4) Immunohistochemical staining:The rats of every group express mTOR which appearansed brown granulation in kidney glomerulus and kidney glomerulus, compared with group A, the expression of mTOR in group B and C were enhanced (P<0.01), the expression of mTOR in group C was fewer than group B(P<0.05).
(5) Related genes:Compared with the group A, the expression of mTORmRNA in group B and C were enhanced(P<0.05), The expression of mTORmRNA in group C was significantly fewer than group B(P<0.05).
(6) The Correlation between UAlb and the expression of mTOR is positive Correlation,r=0.71(P<0.01).
Conclusion:
(1)The FBG、FNS、TG、TC and UAlb were significantly increased in diabetic rats, the drugs of Pioglitazone could reduce FBG、FNS、TG、TC and UAlb. (except Cr)
(2) The expression of mTOR increased in diabetic rats, the drugs of Pioglitazone could inhibit the expression of mTOR.
(3)It suggests that the drugs of Pioglitazone could improve glucolipid metabolism and insulin resistance in diabetic rats,thus they through regulating the expression of mTOR,which may put off diabetic nephropathy.
1观察糖尿病组及吡格列酮干预组大鼠空腹血糖、胰岛素、甘油三酯、胆固醇、尿微量白蛋白、尿素氮、肌酐的变化。
2探讨PPAR-γ激动剂吡格列酮对糖尿病大鼠肾脏mTOR表达的影响。
方法：
体质量为(200±20)g的8周龄健康清洁级SD雄性大鼠33只,随机分成2组,正常对照组(A组)11只,糖尿病模型组22只。A组给予正常普通饮食,糖尿病模型组给予高糖高脂饮食造模,喂养4周,按照HOMA胰岛素抵抗指数[HOMA= (FPG×FNS)/22.5]判断动物出现胰岛素抵抗,本实验HOMA统计结果为(16.3±2.74)mmol.mU/L2,将链脲佐菌素(STZ)按30mg/kg体质量一次性腹腔注射,正常对照组腹腔内注射等体积柠檬酸缓冲液。1周后,尾静脉采血,血糖仪测定空腹血糖(采血前大鼠禁食12h),以空腹血糖≥13.9mmol/L作为糖尿病造模成功标准。将造模成功的糖尿病大鼠随机分成2组,即糖尿病模型组B组、吡咯列酮干预组C组,A、B组给予生理盐水灌胃(2ml/kg.d), C组给予吡格列酮(10mg/kg.d)灌胃给药,饲养6周。大鼠自实验结束前一日收集24小时尿标本,用于测定尿微量白蛋白；最后处死大鼠采血,用于测定血糖、血胰岛素、胆固醇、甘油三酯、尿素氮、肌酐；摘取肾脏,一个肾用4%中性多聚甲醛固定,行HE染色行形态学检查及免疫组化观察mTOR蛋白水平的变化；另一个肾组织保存于-80℃冰箱,用于测定mTOR mRNA的表达。
结果：
1各组大鼠实验室指标：与A组比较,B、C组大鼠FBG、FNS、HOMA-IR、TG、TC水平均明显升高(P<0.05),与B组比较,C组大鼠FBG.FNS.HOMA-IR、TG、TC水平均明显降低(P<0.05)。
2肾功能：各组大鼠BUN、Cr、UAlb比较：与A组比较,B、C组大鼠BUN、UAlb升高(P<0.01),B组Cr升高(P<0.05),A、C组Cr水平无明显差异,与B组比较,C组大鼠BUN、Cr、UAlb降低(P<0.05)。
3 HE染色：A组大鼠肾组织病理结构基本正常,B组大鼠肾小管上皮细胞可见玻璃样变和无结构红染物质,局灶区域可见肾小管液化坏死,肾小球囊内可见少量红细胞渗出,肾小球基底膜增厚,细胞外基质增多,C组较B组病变减轻。
4免疫组织化学染色结果：各组大鼠在肾小管、肾小球中均可见mTOR表达呈棕色颗粒样物,与A组比较,B、C组大鼠肾组织mTOR表达明显升高(P<0.01)；与B组比较,C组大鼠肾组织mTOR表达明显降低(P<0.05)。
5基因表达：与A组比较,B、C组大鼠肾组织mTOR mRNA表达强度增强(P<0.05)；与B组比较,C组大鼠肾组织mTOR mRNA表达强度减弱(P<0.05)。
6尿微量白蛋白与mTOR的表达密切相关,r=0.71(P<0.01)。结论：
1糖尿病组大鼠空腹血糖、胰岛素、甘油三酯、胆固醇、尿微量白蛋白、尿素氮、肌酐水平增加,吡格列酮干预后能降低各项生化指标(肌酐除外)。
2糖尿病组大鼠肾脏组织mTOR表达比正常组表达增强,吡格列酮干预后,肾脏组织mTOR的表达减弱。
3吡格列酮能改善糖尿病大鼠糖脂代谢和胰岛素抵抗,通过调控mTOR的表达,起到延缓糖尿病肾病进展的作用。
Objective:
(1)To observe FBG、FNS、TG、TC、Alb、BUN and Cr of the diabetic and Pioglitazone group rats.
(2)To investigat the effect of Pioglitazone on the expression of mTOR in renal tissue of type 2 diabetic rats.
Methods:
Eight weeks thirty-Three healthy、sanitary Sprague-Dawley maleness rats whose body weight was (200±20) g included in this experiment were randomly divided into two groups.①Control group (group A,n=11);②Diabetic model group (n=22); Rats in group B and C were made type 2 diabetic model by feeding with high glucose high fat diet for four weeks,judging the rats appear insulin resistance status by HOMA insulin resistance index[HOMA=(FPG×FNS)/22.5], The insulin resistance index was (16.3±2.74),and then STZ(30mg·kg-1) with intraperitoneally injection once, rats in group A recived common diet for four weeks and a same volume of vehicle citrate buffer by intraperitoneally injection. After one week of STZ injection(all the rats were fasted for 12 hours),the fasting blood glucose was carried out by getting blood from tail vein,the fasting blood glucose for successful standard of diabetic model is 13.9. The rats which were diabetic model successfully were randomly divided into two groups, Diabetic model group (group B,n=11);Pioglitazone group (group C,n=11).Rats in group A and B recived a volume of 0.9% saline (2ml·kg-1·d-1)by intragastric administration, rats in group C recived Pioglitazone (10mg·kg-1·d-1) by intragastric administration. After six weeks, we collected 24h urine before the day we accomplish the experiment to detect urine albumin, and gathered blood of rats when we killed the rats in the end to detect blood glucose、blood insulin、TG、TC、Cr and BuN; kidneys of rats were removed,one renal was put into 4% formalin to detect the expression of mTOR by immunohistochemical staining, histopathological changes were done by HE staining; another was put into freezer for subzero eighty centi-degree to detected the expression of mTORmRNA by PCR.
Results:
(1) Biochemistry:Compared with group A,FBG、FNS、HOMA-IR、TG and TC of group B and C were enhanced (P<0.05); compared with group B, FBG、HOMA -IR、TG and TC of group C were decreased(P<0.05).
(2) Renal function:Compared with group A,BUNand UAlb of group B and C were enhanced (P<0.05); compared with group B, BUN、Cr and UAlb of group C were decreased(P<0.05). Cr was no significant difference between group A and C.
(3) Histopathological characteristics(HE staining):In group A the form of kidney cytoplasm were normal, and in group B, the kidney tubules epithelium appeared hyalinization and structurelessness dye red substance,part of the kidney tubules appeared liquefative necrosis and the structure of kidney tubules was indistinct,the bowman's capsule in kidney glomerulus exudated a little red blood cell, glomerular basement membrane was thicken, extracellular matrixc increased,the histopathological changes of group C were wosern than group B.
(4) Immunohistochemical staining:The rats of every group express mTOR which appearansed brown granulation in kidney glomerulus and kidney glomerulus, compared with group A, the expression of mTOR in group B and C were enhanced (P<0.01), the expression of mTOR in group C was fewer than group B(P<0.05).
(5) Related genes:Compared with the group A, the expression of mTORmRNA in group B and C were enhanced(P<0.05), The expression of mTORmRNA in group C was significantly fewer than group B(P<0.05).
(6) The Correlation between UAlb and the expression of mTOR is positive Correlation,r=0.71(P<0.01).
Conclusion:
(1)The FBG、FNS、TG、TC and UAlb were significantly increased in diabetic rats, the drugs of Pioglitazone could reduce FBG、FNS、TG、TC and UAlb. (except Cr)
(2) The expression of mTOR increased in diabetic rats, the drugs of Pioglitazone could inhibit the expression of mTOR.
(3)It suggests that the drugs of Pioglitazone could improve glucolipid metabolism and insulin resistance in diabetic rats,thus they through regulating the expression of mTOR,which may put off diabetic nephropathy.
引文
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[37]Goossens GH.The role of adipose tissue dysfunction in the pathogenesis of obesity-related insulin resistance[J].Physiol Behav,2008,94(2):206-218.
[38]苑红,牛燕媚,刘彦辉mTOR/S6K1信号通路与有氧运动改善小鼠高脂饮食诱导胰岛素抵抗问的关系[J].中国康复医学杂志,2009,24(4)：297-302.
[39]Niu YM, Yuan H,Zhang N,et al.The relationship between mTor/S6K1 signaling pathway and insulin resistance and the study of aerobic exercise on this pathway[J]. Zhongguo Ying Yong Sheng Li Xue Za Zhi,2010,26(4):399-403.
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[6]Mordier S,Iynedjian PB.Activation of mammalian target of rapamycin complex 1 and insulin resistance induced by palmitate in hepatocytes[J].Biochem Biophys Res Commun, 2007,362(1):206-211.
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[9]张君,褚志华.肥胖/糖尿病过程中大鼠脂肪细胞因子水平变化的比较[J].农垦医学,2009,31(1)：7-12.
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[11]郭颖,高毅.雷帕霉素逆转胰岛素胰岛素抵抗的动物试验[J].广东医学,2008,29(3)：387-389.
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[15]Mehta NN,Anderson PD,Hinkle CC,et al. Experimental endotoxemia induces adipose inflammation and insulin resistance in humans[J].Diabetes,2010,59(1):172-181.
[16]Liao Q,Shi DH,Zheng W,et al.Antiproliferation of cardamonin is involved in mTOR on aortic smooth muscle cells in high fructose-induced insulin resistance rats[J].Eur J Pharmacol,2010,641 (2-3):179-186.
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[1]金惠铭.加强微血.管病发病机制的研究[J].中国微循环杂志,2006,10(4)：77-79.
[2]Khamaisi M,Schrijvers BF,et al.The emerging role of VEGF in diabetic kidney disease[J].Nephrol Dial Transplant.2003,18(8):1427-1430.
[3]Suzuki M,Kanazawa A,Shiba M,et al.Insulin resistance in diabetic microangiopathies[J]. Diabetes Complication.2000,14(1):40-45.
[4]Murakami M,Ichisaka T,Maeda M,et al. mTOR is essential for growth and proliferation in early mouse embryos and embryonic stem cells[J].Mol Cel Bio,2004,24(15):6710-6718.
[5]Hu LY,Sun ZG,Wen YM,et al.ATP-mediated protein kinase B Akt/mammalian target of rapamycin mTOR/p70 ribosomal S6 protein p70s6k kinase signaling pathway activation promotes improvement of locomotor function after spinal cord injury in rats[J]. Neuroscience,2010,169(3):1046-1062.
[6]Mordier S,Iynedjian PB.Activation of mammalian target of rapamycin complex 1 and insulin resistance induced by palmitate in hepatocytes[J].Biochem Biophys Res Commun, 2007,362(1):206-211.
[7]Liu H, Remedi MS. Pappan KL,et al. Glycogen Synthase Kinase-3 and Mammalian Target of Rapamycin Pathways Contribute to DNA Synthesis, Cell Cycle Progression,and Proliferation in Human Islets[J].Diabetes,2009,58(3):663-672.
[8]Goossens GH.The role of adipose tissue dysfunction in thepathogenesis of obesity-related insulin resistance[J].Physiol Behav,2008,23(94):206-218.
[9]张君,褚志华.肥胖/糖尿病过程中大鼠脂肪细胞因子水平变化的比较[J].农垦医学,2009,31(1)：7-12.
[10]Um SH,Frigerio F,Watanabe M,el al. Absence of S6K1 protects against age-and diet-induced obesity while enhancing insulin sensitivity[J].Nature,2004,431(7005):200-205.
[11]郭颖,高毅.雷帕霉素逆转胰岛素胰岛素抵抗的动物试验[J].广东医学,2008,29(3)：387-389.
[12]Chen JK,Chen J,Thomas G,et al.S6 Kinase 1 Knockout Inhibits Uninephreetomy-or Diabetes-induced Renal Hypertrophy[J].Am J Physiol Renal Physiol,2009,297(3):585-593.
[13]Drummond MJ,Bell JA,Fujita S,el al.Amino acids are necessary for the insulin-induced activation of mTOR/S6Kl signaling and protein synthesis in healthy and insulin resistant human skeletal muscle [J].Clin Nutr,2008,27(3):447-456.
[14]Torres-Leal FL,Fonseca-Alaniz MH,Rogero MM,et al.The role of inflamed adipose tissue in the insulin resistance[J].cell biochem funct,2010,28(8):623-631.
[15]Mehta NN,Anderson PD,Hinkle CC,et al. Experimental endotoxemia induces adipose inflammation and insulin resistance in humans[J].Diabetes,2010,59(1):172-181.
[16]Liao Q,Shi DH,Zheng W,et al.Antiproliferation of cardamonin is involved in mTOR on aortic smooth muscle cells in high fructose-induced insulin resistance rats[J].Eur J Pharmacol,2010,641 (2-3):179-186.
[17]White ES, Sagana RL, Booth AJ,et al.Control of fibroblast fibronectin expression and alternative splicing via the P13K/Akt/mTOR pathway[J].Exp Cell Res,2010,316(16):2644-2653.
[18]校丽芳.雷帕霉素对糖尿病大鼠肾脏病变和VEGF及其受体表达的影响[J].上海交通大学学报(医学版),2009,29(9)：1040-1045.
[19]Fraenkel M,Ketzinel-Gilad M,Ariav Y,et al.mTOR Inhibition by Rapamycin Prevents β-Cell Adaptation to Hyperglycemia and Exacerbates the Metabolic State in Type 2 Diabetes[J].Diabetes,2008,57(4):945-957.
[20]Danielpour D. Functions and regulation of transforming growth Factor-beta(TGF-β)in the prostate[J].Eur J Cancer,2005,41 (6):846-857.