高脂喂养大鼠肾脏NF-κB及炎症因子表达变化及利拉鲁肽的干预研究

发布时间：2018-03-11 09:22

  本文选题：高脂血症　切入点：胰岛素抵抗　出处：《承德医学院》2017年硕士论文　论文类型：学位论文

【摘要】：目的:糖尿病肾病(Diabetic Nephropathy,DN)是糖尿病(DM)常见的慢性并发症,亦是引起终末期肾病(end-stage renal disease,ESRD)的重要原因。其发病机制复杂,近年来发现炎症反应在DN中发挥重要作用。TNF-α及IL-6是主要的炎性标记物,而调控和转录很多炎症因子表达的核转录因子-κB(Nuclear factor-kappa B,NF-κB)是联系肥胖、炎症的重要机制。DN还存在不同程度的糖脂代谢紊乱,常协同致病,其中也与炎症密不可分。脂质代谢紊乱能诱导细胞产生炎症反应,继而发生胰岛素抵抗(insulin resistance,IR),IR可刺激多种炎性细胞因子释放,加重肾小球肥大,此外,IR带来的高胰岛素血症还可使肾小球血流量增加,肾小球毛细血管静水压升高,处于高灌注状态,进而引起蛋白尿。过多的脂质在肾小球沉积,还可导致肾小球硬化。GLP-1受体激动剂(Glucagon like peptide-1 receptor agonist)利拉鲁肽是目前治疗糖尿病的新药,研究发现该药可改善IR、减轻体重,并可降低糖尿病大鼠尿蛋白、减轻肾小球高滤过和肾脏过度肥大从而改善肾功能,但具体机制不详。本研究通过观察高脂喂养IR大鼠肾功能、尿蛋白、血脂,血清及肾组织中NF-κB、TNF-α及IL-6的表达,探讨糖尿病前期IR阶段肾脏炎症因子的表达变化,及与血脂等的相关性,并用利拉鲁肽干预治疗探讨利拉鲁肽对肾脏炎症进程的影响及机制,为其在糖尿病肾病及其他肾脏疾病中的应用提供理论依据。方法:成年雄性Wistar大鼠54只(230-260g),随机分为对照组(C)普通饲料喂养16只与模型组高脂饲料喂养38只,8周后,各组取5只大鼠行高胰岛素-正常葡萄糖钳夹试验计算葡萄糖输注率(glucose infusion rate,GIR),评价胰岛素抵抗,判定造模成功后,将模型组随机分为高脂组(H)、利拉鲁肽低剂量组(Li-1)(100μg/kg/d)和利拉鲁肽高剂量组(Li-2)(200μg/kg/d),每组11只。前两组用生理盐水皮下注射;后两组分别用利拉鲁肽100μg/kg/d及200μg/kg/d皮下注射。干预2周后,每组再取5只大鼠行钳夹试验计算葡萄糖输注率。每组剩余的6只大鼠用代谢笼收集24 h尿液,后腹主动脉放血处死,取肾脏标本行透射电镜观察肾脏病理结构以及应用Real time-PCR技术测定肾组织NF-κB、TNF-α及IL-6m RNA表达,自动生化分析仪检测血肌酐(Scr)、尿素氮(BUN)、总胆固醇(TCH)、甘油三酯(TG)、低密度脂蛋白胆固醇(LDL-C),免疫比浊法测定24h尿微量白蛋白(MAU),Elisa法测定血清NF-κB、TNF-α及IL-6蛋白浓度。结果:1.四组大鼠一般情况比较与对照组相比,高脂组大鼠Scr、BUN、MAU、TCH、TG、LDL-C均明显升高(P0.05);与高脂组相比,利拉鲁肽低剂量组大鼠Scr、BUN、MAU、TG、LDL-C均降低(P0.05),利拉鲁肽高剂量组大鼠Scr、BUN、MAU、TCH、TG、LDL-C明显降低(P0.05);与利拉鲁肽低剂量组相比,利拉鲁肽高剂量组Scr、BUN、MAU、TG、LDL-C进一步降低(P0.05)。2.四组大鼠葡萄糖输注率比较药物干预前:与对照组GIR(30.21±2.51mg/kg.min)相比,高脂组GIR(20.41±3.64mg/kg.min)下降,差异有统计学意义(P0.05)。利拉鲁肽干预2周后:与对照组GIR(30.77±2.37mg/kg.min)相比,高脂组GIR(18.83±1.95 mg/kg.min)下降,差异有统计学意义(P0.05)。与高脂组相比,利拉鲁肽低剂量组GIR(21.55±2.46mg/kg.min)升高(P0.05),与低剂量组相比,利拉鲁肽高剂量组GIR(25.80±1.40mg/kg.min)进一步升高,差异均有统计学意义(P0.05)3.四组大鼠血清NF-κB、TNF-α和IL-6蛋白浓度的比较与对照组相比,高脂组血清NF-κB、TNF-α和IL-6蛋白浓度均升高(P0.05);与高脂组相比,利拉鲁肽干预组NF-κB、TNF-α和IL-6蛋白浓度均降低(P0.05);且利拉鲁肽高剂量组比利拉鲁肽低剂量组,NF-κB、TNF-α和IL-6蛋白浓度进一步降低(P0.05)。4.四组大鼠肾脏组织NF-κB、TNF-α和IL-6 m RNA的比较与对照组相比,高脂组肾脏组织NF-κB、TNF-α和IL-6 m RNA的表达均升高(P0.05);与高脂组相比,利拉鲁肽干预后肾脏组织NF-κB、TNF-α和IL-6 m RNA的表达均降低(P0.05);利拉鲁肽高剂量组与利拉鲁肽低剂量组相比,肾脏组织NF-κB、TNF-α和IL-6 m RNA的表达进一步降低(P0.05)。5.四组大鼠肾脏组织电镜超微结构比较透射电镜结果显示(放大10000倍):对照组大鼠肾小球结构完整,基底膜(GBM)清晰,足突分布整齐、间隙明显;高脂组大鼠肾小球足突肿胀,部分融合、脱落,基底膜增厚;利拉鲁肽干预可改善上述改变,尤以利拉鲁肽高剂量组明显。6.相关性分析研究结果血清TNF-α和IL-6蛋白浓度与MAU呈正相关(r值=0.802、0.748,P0.05);肾组织TNF-αm RNA表达与TCH、TG呈明显的正相关(r值分别为0.878、0.817,P0.05);肾组织IL-6 m RNA表达与TCH、TG呈明显正相关(r值分别为0.806、0.854,P0.05);肾组织NF-κB m RNA表达与TNF-α和IL-6 m RNA表达呈正相关(r值分别为0.810、0.867,P0.05)。结论:1高脂饮食可使大鼠发生脂代谢紊乱及胰岛素抵抗,并可通过增加肾脏NF-κB和炎症因子TNF-α和IL-6表达激活炎症反应,增加微量白蛋白尿,加重肾损伤。2利拉鲁肽可呈剂量依赖性减轻脂代谢紊乱,改善胰岛素抵抗,减少微量白蛋白尿,保护肾功能。3利拉鲁肽可呈剂量依赖性抑制肾脏NF-κB表达,下调肾组织炎症因子TNF-α和IL-6表达,改善肾功能。
[Abstract]:Objective: diabetic nephropathy (Diabetic Nephropathy DN) is a common chronic complication of diabetes mellitus (DM), is also the cause of end-stage renal disease (end-stage renal, disease, ESRD) is an important cause. Its pathogenesis is complex, in recent years found that inflammation play an important role of.TNF- alpha and IL-6 is the main inflammatory markers in DN in the regulation of transcription and expression of many inflammatory factors of nuclear factor kappa B (Nuclear factor-kappa B, NF- K B) is linked to obesity, inflammation is an important mechanism of.DN glucose and lipid metabolic disorder in different degree, which is often synergistic, and inflammation are inseparable. Lipid metabolism can induce cells to produce inflammatory reaction. Then the occurrence of insulin resistance (insulin, resistance, IR, IR) can stimulate a variety of inflammatory cytokines release, glomerular hypertrophy, in addition, hyperinsulinemia caused by IR can make the glomerular blood flow increased, glomerular Capillary hydrostatic pressure, in a high perfusion state, thereby causing proteinuria. Excessive lipid in glomerular deposition, can also lead to glomerular sclerosis.GLP-1 receptor agonist (Glucagon like peptide-1 receptor agonist) is currently the liraglutide treatment of diabetes drugs, the study found that the drug can improve IR, reduce weight, and can reduce the urinary protein in diabetic rats, reduce glomerular filtration and renal hypertrophy and improve renal function, but the specific mechanism is unknown. This study by observing the renal function, IR rats were fed high fat urine protein, blood lipid, blood and kidney tissue of NF- kappa B, expression of TNF- and IL-6, to investigate the expression of inflammatory factors in Kidney Early IR stage of diabetes mellitus and its correlation with serum lipids, and liraglutide treatment to investigate the effects of liraglutide on renal inflammatory processes and mechanisms, as in diabetic nephropathy and other kidney 鐤剧梾涓�鐨勫簲鐢ㄦ彁渚涚悊璁轰緷鎹�.鏂规硶:鎴愬勾闆勬，

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