【作者】 李新；

【导师】 袁莉；

【作者基本信息】 华中科技大学 ， 内科学， 2009， 博士

【摘要】 目的近年来肾素血管紧张素系统（RAS）与2型糖尿病的关系日益得到关注，RAS在胰岛β细胞功能障碍发生中的作用及其干预价值成为2型糖尿病防治研究的热点之一。本课题通过长期高脂高热量饮食构建胰岛素抵抗大鼠模型，以高脂饮食加小剂量链尿佐菌素（STZ）腹腔注射构建糖尿病大鼠模型，观察在糖尿病发病的不同阶段阻断RAS对胰岛β细胞功能的保护效应及机制，及其对STZ致糖尿病发生率的影响，并通过体外对β细胞系INS-1细胞的培养及处理，探讨RAS活化参与β细胞功能损害的分子机制。方法（1）阻断RAS对胰岛素抵抗大鼠胰岛β细胞功能的效应及机制，及其对STZ致糖尿病发生率的影响：90只8周龄雄性Wistar大鼠，随机分为正常对照组（n=15）和胰岛素抵抗造模组（n=75）。胰岛素抵抗造模组给予高脂高热量饮食喂养16周，随机分为高脂对照组（n=30），培哚普利干预组（n=15），替米沙坦干预组（n=30），共干预8周。为了观察在胰岛素抵抗阶段阻断RAS对STZ致糖尿病发生率的影响，分别从高脂对照组及替米沙坦干预组中随机选择15只大鼠作为高脂+小剂量STZ腹腔注射组（n=15）及替米沙坦干预+小剂量STZ腹腔注射组（n=15），禁食10-12h，以20mg／kg的剂量腹腔注射STZ，1周后以非同日2次随机血糖≥16.7mmol／1者为糖尿病大鼠，继续喂养1周。（2）阻断RAS对糖尿病大鼠胰岛β细胞功能的效应及机制研究：50只8周龄雄性Wistar大鼠随机分为正常对照组（n=10）和糖尿病造模组（n=40）。糖尿病组以高脂高热量饮食喂养8周后予小剂量STZ（30mg／kg）腹腔注射诱导糖尿病，纳入标准同上，随机分为糖尿病组（n=8）、ACEI干预组（n=10）、ARB干预组（n=10），共干预8周。（3） RAS参与β细胞功能损害的相关分子机制研究：体外培养β细胞系INS-1细胞，分别用不同浓度葡萄糖处理不同时间，观察其对INS-1细胞血管紧张素Ⅱ（AngⅡ）受体1（AT1R）表达的影响；用不同浓度的AngⅡ处理24h，观察其对INS-1细胞凋亡、功能以及胰岛素信号分子的影响。动物研究中胰岛结构、β细胞内胰岛素含量、胰岛细胞凋亡、炎症、氧化应激、胰岛微血管密度及RAS表达采用免疫组化或反转录聚合酶链反应（RT-PCR）法检测，胰岛功能采用静脉葡萄糖耐量试验（IVGTT）与静脉胰岛素释放试验（IVIRT）检测，胰岛素敏感性采用高胰岛素正葡萄糖钳夹试验检测；体外研究中INS-1细胞AT1R及胰岛素基因表达采用RT-PCR检测，细胞活性、凋亡及胰岛素信号分子分别采用MTT法、免疫荧光与流式细胞仪及western-blot法检测。结果1．胰岛素抵抗阶段阻断RAS对胰岛β细胞功能的效应及机制，及其对STZ致糖尿病发生率的影响1．1长期高脂高热量饮食喂养大鼠胰岛素敏感性及胰岛β细胞功能与正常对照组（NC，n=15）相比，高脂对照组（FC，n=15）稳态葡萄糖输注率（GIR）显著降低[（7.80±0.51）mg·kg^-1·min^-1 vs（5.32±0.90）mg·kg^-1·min^-1，P＜0.01]，说明FC组出现了明显的胰岛素抵抗；FC组胰岛增生肥大，胰岛内胰岛素相对含量（IRC）较NC组显著降低（P＜0.01），提示FC组大鼠β细胞内胰岛素储备不足；胰岛素阳性细胞核密度（ICD）也有减少趋势，但是差异无统计学意义；葡萄糖负荷后第一时相胰岛素分泌高峰延迟，0-10min胰岛素曲线下面积（AUCI）低于NC组（P＜0.01），但是10-60minAUCI超过NC组68.8％（P＜0.01），提示FC组大鼠胰岛素第一时相分泌不足，而出现第二时相异常高分泌。1．2胰岛素抵抗大鼠胰岛内RAS表达、炎症反应及细胞凋亡与NC组比较，FC组AT1R mRNA相对表达量增加了1.16倍（P＜0.01），白细胞介素1β（IL-1β）相对表达量增加了1.95倍（P＜0.01），核因子κB（NF-KB）相对浓度增加了20.5％（P＜0.01），胰岛内凋亡信号分子Caspase-3相对表达量增加了19.1％（P＜0.01），单位胰岛面积TUNEL阳性细胞数增加了2.43倍（P＜0.01）。1．3阻断RAS对胰岛素抵抗大鼠胰岛β细胞功能的影响药物干预后，培哚普利干预组（FP，n=15）、替米沙坦干预组（FT，n=15）IRC较FC组明显增加，分别为（-4.99±0.12）（P＜0.05）与（-4.87±0.09）（P＜0.01）；ICD也有所增加，但是差异无统计学意义；AUCI（10-60）分别下降了15.6％、17.3％（均P＜0.01），AUCI（0-10）及AUCI（0-60）也有所下降，但差异无显著性。说明阻断RAS具有增加胰岛素抵抗大鼠β细胞内胰岛素含量及部分改善胰岛素分泌模型的的效应。FP组、FT组之间差异均无显著性。1．4阻断RAS对胰岛素抵抗大鼠胰岛内炎症及细胞凋亡的效应与FC组相比，FP组、FT组胰岛内AT1R mRNA相对表达量明显降低（均P＜0.01），IL-1β相对表达量降低了22.4％（P＜0.05）、28.5％（P＜0.01）；NF-KB相对含量分别下降了20.1％、22.8％（均P＜0.01）；Caspase-3相对表达量分别降低了15.5％、17.7％（均P＜0.01）；单位胰岛面积TUNEL阳性细胞数分别下降了60.0％及67.4％（均P＜0.01）。说明阻断RAS可以显著降低胰岛素抵抗大鼠胰岛内炎症反应及细胞凋亡水平。FP组、FT组之间差异均无显著性。1．5在胰岛素抵抗阶段阻断RAS对STZ致糖尿病发生率的影响与高脂+STZ腹腔注射组（FS，n=15）比较，替米沙坦干预+STZ腹腔注射组（TS，n=15）糖尿病的发生率显著降低，分别为80.0％（12／15）、33.3％（5／15）（P＜0.05）。两组平均空腹血糖也有显著差异[（17.5±5.1）mmol／l vs（13.2±4.7）mmol／l，P＜0.05]。2．糖尿病阶段阻断RAS对胰岛β细胞功能的效应及机制2．1高脂饮食加小剂量STZ腹腔注射构建的糖尿病大鼠模型胰岛结构与功能与正常对照组（NC，n=10）相比，糖尿病组（DM，n=8）大鼠胰岛形态不规则，边界模糊，结构紊乱，IRC显著下降（P＜0.01）；胰岛素第一时相分泌高峰延迟、峰值降低，AUCI（0-10）下降了67.0％（P＜0.01），早期胰岛素分泌指数（EISI）降低了81.1％（P＜0.01）。2．2糖尿病大鼠胰岛内RAS表达、氧化应激、微血管密度及细胞凋亡与NC组比较，DM组胰岛内血管紧张素原（AGT）mRNA表达显著增加（P＜0.01）；诱导型一氧化氮合酶（iNOS）相对浓度增加了10.3％（P＜0.01），提示糖尿病状态下胰岛局部氧化应激反应增强；胰岛微血管密度（MVD）降低了71.4％（P＜0.01），低氧诱导因子1α（HIF-1α）mRNA相对表达量增加了1.19倍（P＜0.01），提示糖尿病大鼠胰岛处于相对或绝对乏氧状态；单位面积胰岛细胞凋亡数增加了2.14倍（P＜0.01）。2．3阻断RAS对糖尿病大鼠胰岛β细胞功能的影响药物干预后，ACEI组（AE，n=10）、ARB组（AR，n=10）胰岛结构有所改善，IRC较DM组明显增加（均P＜0.01），AUCI（0-10）分别增加了41.2％和33.1％（均P＜0.01），EISI分别增加了1.84倍和1.74倍（均P＜0.01），说明阻断RAS后糖尿病大鼠胰岛素第一时相分泌有所恢复。AE组、AR组之间差异均无显著性。2．4阻断RAS对糖尿病大鼠胰岛氧化应激、微血管密度及细胞凋亡的影响AE组和AR组胰岛内iNOS含量较DM组分别下降了16.5％、18.2％（均P＜0.01）；MVD分别增加了62.5％（P＜0.05）与75.0％（P＜0.01），HIF-1α基因表达分别下降了27.2％与29.0％（均P＜0.01）；单位面积胰岛凋亡细胞数分别下降了29.0％、36.2％（均P＜0.01）。AE组、AR组之间差异均无显著性。3．RAS参与β细胞功能损害的相关分子机制3．1不同糖浓度处理不同时间INS-1细胞AT1R基因表达变化以5.6 mmol／l的葡萄糖（简称为5.6mG，下同）培养24h作为基础对照组，5.6mG培养48h AT1R mRNA表达无明显改变；16.7mG培养24h后AT1R mRNA表达增多，但差异无统计学意义，培养48h后AT1R mRNA表达较对照组增加了0.72倍（P＜0.05）；33.3mG培养24h，AT1R表达水平较对照组增加了1.33倍（P＜0.01），培养48h后，AT1R mRNA表达水平进一步增加，为对照组的2.6倍（P＜0.01）。3．2不同浓度AngⅡ处理24h对INS-1细胞胰岛素分泌功能的影响以5.6mG培养24h作为对照组，不同浓度（0.1、1、10、100nmol／1）AngⅡ处理组基础状态下的胰岛素分泌无明显改变，但葡萄糖刺激后胰岛素分泌（GSIS）分别较对照组下降了7.9％（P＜0.05）、21.1％（P＜0.01）、26.3％（P＜0.01）和34.2％（P＜0.01）。3．3不同浓度AngⅡ处理24h对INS-1细胞凋亡的影响分组同上，观察INS-1细胞的凋亡率变化。5.6mG培养48h，细胞凋亡率为5.7％，0.1 nmol／l AngⅡ培养24h组细胞凋亡率为10.3％，较对照组增加，但是差异无统计学意义，其余各组（1，10，100 nmol／l）随AngⅡ浓度增加，细胞凋亡率均显著增加（均P＜0.01）。3．4不同浓度AngⅡ处理24h对INS-1细胞胰岛素信号分子的影响以5.6mG培养24h为对照组，未检测到磷酸化的胰岛素受体底物丝氨酸270位点（IRS-ser270）的表达，而加入不同浓度（0.1、100nmol／l）AngⅡ培养24h后，可检测到磷酸化IRS-ser270蛋白表达，其相对表达量分别为0.31，0.72（均P＜0.01）；0.1nmol／l AngⅡ处理24h后，蛋白激酶B丝氨酸473位点（PKB-ser473）磷酸化水平较对照组下降了20.1％（P＜0.01），100nmol／l AngⅡ培养24h后，PKB-ser473磷酸化水平进一步降低，较对照组下降了30.2％（P＜0.01）。结论RAS活化与糖尿病发病进程中胰岛β细胞功能损害密切相关，葡萄糖具有浓度及时间依赖性地上调胰岛RAS活性的效应。RAS活化可能通过诱发胰岛炎症反应、氧化应激、局部血流动力学异常、胰岛素受体后信号通路损害及细胞凋亡等机制损伤β细胞存活与功能。早期阻断RAS具有保护胰岛β细胞功能的作用，可以显著降低胰岛素抵抗大鼠STZ致糖尿病的发生率；在糖尿病阶段阻断RAS，对胰岛β细胞存活与功能仍然具有明显的保护效应。阻断RAS可能通过减轻胰岛炎症反应与氧化应激、改善胰岛微血流及β细胞胰岛素信号通路、减少胰岛细胞凋亡等机制保护或改善胰岛β细胞功能。更多还原

【Abstract】 ObjectiveRecently more and more attention has been payed to the relationship between reninangiotensin system（RAS） and type 2 diabetes. The role and its intervention value of RAS inthe development ofβcell dysfunction have become one of the research hot spots. Thisstudy aimed to investigate the effects of RAS blockade on isletβcell function in differentstages of type 2 diabetes, that is insulin resistance stage and overt diabetes stage, and itsimpact on the incidence of STZ-induced diabetes. This study also aimed to investigate themechanisms ofβcell dysfunction induced by RAS activation viaβcell line INS-1 cellscultivation and treatment in vitro.Methods（1） Effects and mechanisms of RAS blockade on isletβcell function in insulinresistance stage: Ninety 8-week male Wistar rats were randomly divided into normalcontrol group（n=15） and high fat diet group（n=75）,which were given high fat diet for 16weeks and then divided into high fat contol group（n=30）, perindopril intervention group（n=15） and telmisartan intervention group（n=30）. Eight weeks later, to investigate theimpact of RSA blockade on the incidence of STZ-induced diabetes, fifteen rats wereselected randomly from high fat contol group and telmisartan intervention grouprespectively as high fat plus streptozotocin（STZ） intraperitoneal injection group（n=15） andtelmisartan plus STZ intraperitoneal injection group（n=15）, which were given STZintraperitoneal injection at the dosage of 20mg/kg and whose random blood glucose was≥16.7mmol/l twice not in one day after one week were considered as diabetes. （2） Effectsand mechanisms of RAS blockade on isletβcell function in overt diabetic stage: Fifty8-week male Wistar rats were randomly divided into normal control group（n=10） anddiabetes model group（n=40）, which were given high fat diet for 8 weeks and after that, STZ were intraperitoneal injected at the dosage of 30mg/kg. The diabetic criteria was asabove. Then the diabetic rats were divided into diabetes group（n=8）, perindoprilgroup（n=10） and valsartan group（n=10）, and treated for 8 weeks.（3）mechanisms ofβcelldysfunction induced by RAS activation:βcell line INS-1 cells were cultivated in vitro andtreated with glucose of different concentration for different time to investigate theexpression of AngiotensinⅡ（AngⅡ） receptor 1 （AT1R）, and with AngⅡof differentconcentration for 24h to investigate its impact on the apoptosis, insulin signal moleculs andfunction of INS-1 cells. In animal experiments, the morphology of islet, insulin contentintraβcell, islet cell apoptosis, inflammation, oxidative stress, islet microvessel densityand RAS expression intra islet were detected by immunohistochemistry or RT-PCR. Theislet function was evaluated by intravenous glucose tolerance test（IVGTT）. In vitro study,the expression of AT1R and insulin was detected by RT-PCR, the cytoactive, apoptosis andinsulin signal molecules were detected by MTT, immunofluorescence and flow cytometry,western-blot respectively.Results1. Effects and mechanisms of RAS blockade on isletβcell function ininsulin resistance stage1.1 insulin sensitivity and islet function of rats with long-term high fat dietIn compared with normal control group（NC, n=15）, the glucose infusion rate（GIR） ofhigh fat control group（FC, n=15） was decreased significantly[（7.80±0.51） mg*kg^-1*min^-1vs （5.32±0.90） mg*kg^-1*min^-1, P＜0.01], indicating that FC group appeared obviousinsulin resistance. The islets of FC group were enlarged with decreased insulin relativeconcentration（IRC） intraβcell （P＜0.01）.The insulin positive nucleus density（ICD） has adecreased tendency, but the variance was not statistically significant. Area under the curveof insulin（AUCI） from 0 to 10 min of FC group was lower in compared with that in NCgroup（P＜0.01）, while the AUCI from 10 to 60 min was increased by 68.8%（P＜0.01）,indicating the deficiency of insulin secretion in the first-stage and an abnormal highsecretion during the second-stage in FC group. 1.2 expression of RAS, inflammatin and apoptosis intra islets of insulin resistance ratsIn compared with NC group, the relative expression of AT1R in FC group wasincreased by 1.16 times（P＜0.01）, the relative expression of IL-1βwas increased by 1.95folds（P＜0.01）, the relative content of NF-KB was increased by 20.5%（P＜0.01）, therelative concentration of Caspase-3 was increased by 19.1%（P＜0.01）, and the number ofapoptotic cells in unit area of islet was increased by 2.43 times（P＜0.01）.1.3 effects of RAS blockade on islet function in insulin resistance ratsAider intervention, the IRC of peridopril group（FP, n=15） and telmisartan group （FT,n=15） were all increased obviously（P＜0.05 or P＜0.01）, the ICD was also increased, butthe variance was not statistically significant, AUCI（0-60） was decreased by 15.6% and17.3% respectively（all P＜0.01）, AUCI（0-10） and AUCI（0-60） were also decreased, but thedifferences were not statistically significant, too. There were no significant differencesbetween FP and FT group.1.4 effects of RAS blockade on inflammation and apoptosis intra islet in insulinresistance ratsIn compared with FC group, the relative expression of AT1R in FP and FT group wasreduced significantly（all P＜0.01）, the relative expression of IL-1βwas decreased by22.4%（P＜0.05） and 28.5%（P＜0.01） respectively, the relative content of NF-KB wasreduced by 20.1%, 22.8%（all P＜0.01）, the relative concentration of Caspase-3 wasdecreased by 15.5%, 17.7%（all P＜0.01）, and the number of apoptotic cells in unit area ofislet was reduced by 60.0%, 67.4%（all P＜0.01）. There were no significant differencesbetween FP and FT group.1.5 effects of RAS blockade in insulin resistance stage on the incidence ofSTZ-induced diabetesIn compared with high fat plus STZ group（FS, n=15）, the incidence of STZ-induceddiabetes of telmisartan plus STZ group（TS, n=15） was reduced signifcantly （P＜0.05）,which were 80%（10/15） and 33%（5/15） respetctively. The difference of fasting bloodglucose between FS and TS group was also significant [（17.5±5.1）mmol/l vs（13.2±4.7）mmol/l, P＜0.05]. 2. Effects and mechanisms of RAS blockade on isletβcell function inovert diabetic stage2.1 islet function of diabetic rats induced by high fat diet plus STZ intraperitonealinjectionIn compared with normal control group（NC, n=10）, the islets of diabetes group （DM,n=8） were enlarged with disarrayed architecture, and IRC was decreased obviously（P＜0.01）. First-phase insulin secretion, which was expressed as AUCI （0-10）, was reducedby 67.0% （P＜0.01） and the early insulin secretion index（EISI） was decreased by 81.8%（P＜0.01）.2.2 expression of RAS, oxidative stress, microvessel density and apoptosis intra islet ofdiabetic ratsThe relative expression of Angiotensinogen（AGT） mRNA of DM group was increasedsignificantly （P＜0.01） in compared with that in NC group. The relative content of inducedNO synthase（iNOS）, which reflected the oxidative stress in islet, was increased by 10.3%（P＜0.01）. The microvessel density（MVD） was decreased by 71.4% （P＜0.01） with theincreased expression of hypoxia inducing factor1α（HIF-1α） by 1.19 folds （P＜0.01）. Thenumber of apoptotic cells in unit area of islet was increased by 2.14 times（P＜0.01）.2.3 effects of RAS blockade on islet function in diabetic ratsAfter intervention, the IRC of ACEI group（AE, n=10） and ARB group（AR, n=10） wasall inreased obviously（P＜0.01） with the islet morphology improved. AUCI（0-10） wasincreased by 41.2% and 33.1% respectively（all P＜0.01）, EISI was increased by 1.84 foldsand 1.74 folds（all P＜0.01）, indicating that the first-phase insulin secretion was partlyrestored. There were no significant differences between AE and AR group.2.4 effects of RAS blockade on oxidative stress, MVD and apoptosis intra islet indiabetic ratsThe relative concentration of iNOS in AE and AR group was decreased by 16.5 % and18.2% （all P＜0.01） respectively in compared with that in DM group, the MVD wasincreased by 62.5% （P＜0.05） and 75.0%（P＜0.01） with the expression of HIF-1αdecreased by 27.2% and 29.0%（all P＜0.01） respectively. The number of apoptotic cells in unit areaof islet was reduced by 29.0% and 36.2% （all P＜0.01） respectively. There were nosignificant differences between AE and AR group.3. mechanisms ofβcell dysfunction induced by RAS activation3.1 expression of AT1R in INS-1 cells under different concentration glucose fordifferent timeIn compared with the expression of AT1R mRNA in INS-1 cells cultivated with 5.6mmol/l glucose for 24h（5.6mG（24h））, there was no significant difference in 5.6mG（48h）group. AT1R expression in 16.7mG（24h） group was increased, but the variance was notstatistically significant, while its expression in 16.7mG（48h） group was increase by 0.72times（P＜0.05）; in 33.3mG （24h） and 33.3mG （48h） group, its expression was inceased by1.33 times and 2.6 times （all P＜0.01） respectively, indicating that with the increament ofglucose concentration and time, the expression of AT1R in INS-1 cells was also increasedaccordingly.3.2 insulin secretion function of INS-1 cells treated with AngⅡat differentconcentration for 24hIn compared with 5.6mG（24h） group, the basic insulin secretion of differentconcentration AngⅡ（0.1/1/10/100nmol/l） treatment group was not changed significantly,but the glucose stimulating insulin secretion（GSIS） was decreased by 7.9%（P＜0.05）,21.1%（P＜0.01）, 26.3%（P＜0.01） and 34.2%（P＜0.01） respectively.3.3 apoptosis of INS-1 cells treated with AngⅡat different concentration for 24hThe apoptosis rate was 5.7% in 5.6mG（24h） group, and 10.3% in the group with 0.1nmol/l AngⅡtreated for 24h, but the variance was not statistically significant. Theapoptosis rate in other groups（1/10/100nmol/l） was all increased significantly with theincreased concentration of AngⅡ.3.4 expression of insulin signal molecules of INS-1 cells treated with AngⅡat differentconcentration for 24hThe expression of insulin receptor substrate serine270（IRS-ser270） phosphorylationwas not detected in the 5.6mG（24h） group, while its expression could be detected after treated with different concentration AngⅡ（0.1/100nmol/l）for 24h, that was 0.31 and0.72（all P＜0.01） respectively. In compared with 5.6mG（24h） group, the expression ofprotein kinase B serine 473（PKB-ser473） phosphorylation was decreased by 20.1% and30.2%（all P＜0.01） after treated with 0.1 nmol/l and 100 nmol/l AngⅡfor 24h respectively.ConclusionsRAS had tight relation with pancreatic isletβcell dysfunction in the development ofdiabetes, glucose could up-regulate the activation of RAS intra islet depending on theconcentration and its duration. The activation of local RAS would provocate inflammation,oxidative stress, hemodynamics abnormality, apoptosis and insulin signal transductionimpairment intra islet, which ultimately induced damage toβcell survival and function. Toblock RAS in insulin resistance stage had protective effects on isletβcell function anddecreased the incidence of STZ-induced diabetes. Even in the stage of overt diabetes, RASblockade also had protective effects on isletβcell function. Its mechanisms may be thedecreased inflammtion and oxidative stress, the improvement of hemodynamics and insulinsignal transduction, and the reduced cell apoptosis intra islet.更多还原