硫辛酸抑制2型糖尿病大鼠肾脏氧化应激及mTOR信号转导通路激活

发布时间：2018-06-12 14:41

本文选题：硫辛酸 + 雷帕霉素靶蛋白　；参考：《河北医科大学》2017年硕士论文

【摘要】：目的:糖尿病肾脏病(diabetic kidney disease,DKD)是2型糖尿病(type 2 diabetes mellitus,T2DM)常见微血管并发症之一,并已成为导致患者终末期肾脏疾病甚至死亡的主要原因。DKD的发病机制尚未系统阐明,目前认为糖脂代谢紊乱、血流动力学改变、氧化应激、炎症反应、肾脏自噬抑制等与DKD发生发展有着密切联系,其中氧化应激是DKD重要发病机制,还可以介导炎症反应进一步加速肾脏损伤。8-羟基脱氧鸟嘌呤(8-hydroxy-2-deoxygnanosine,8-OHdG)是一种目前反映DNA氧化损伤及体内氧化应激的敏感指标。近年来,随着对雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)信号通路研究的逐渐深入,发现mTOR通路的激活促进肾脏细胞肥大、蛋白质合成增加,参与氧化应激、炎症反应并抑制肾脏细胞自噬,在局灶节段性肾小球硬化、膜性肾病等慢性肾脏疾病的发病中起重要作用。腺苷酸活化蛋白激酶(adenosine5’-monophosphate-activate protein kinase,AMPK)是调控mTOR的上游信号之一,其主要通过抑制mTOR信号通路发挥作用。硫辛酸作为抗氧化剂,可以抑制氧自由基的生成,临床上用于糖尿病周围神经病变治疗收到了良好疗效,但对DKD有无作用尚不明确。本实验通过复制T2DM大鼠模型,测定各组大鼠肾脏中AMPK、p-AMPK、mTOR、p-mTOR的表达情况及体内8-OHdG水平,以探讨mTOR信号通路、氧化应激与T2DM肾脏病的关系及硫辛酸对肾脏mTOR信号通路及氧化应激的影响。方法:22只雄性清洁级Sprague-Dawley(SD)大鼠,鼠龄6周,体重为180g±20g,清洁级环境饲养。应用excel随机数字表法将大鼠随机分为正常对照组(NC组,n=6)、糖尿病组(DM组,n=8)、硫辛酸干预组(LA组,n=8)。正常对照组大鼠继续予以普通饲料喂养,其余两组大鼠予以高糖高脂饲料(蔗糖20%、猪油10%、胆固醇2.5%,胆酸盐1%,基础饲料66.5%)喂养。4周后,糖尿病组及硫辛酸干预组大鼠给予30mg/kg链脲佐菌素腹腔注射,正常组大鼠腹腔注射等体积柠檬酸缓冲液,注射2周后测定大鼠空腹血糖(fastingbloodglucose,fbg),fbg7.8mmol/l视为t2dm造模成功。随后硫辛酸干预组予以100mg/kg硫辛酸腹腔注射,正常对照组和糖尿病组均以等体积生理盐水腹腔注射,每日给药1次,连续给药8周。实验结束前一日收集各组大鼠24小时尿液,用于测定24小时尿蛋白;当日留取血清用于测定肾功能和8-ohdg等指标;取左肾称重后,4%多聚甲醛固定,行he染色和pas染色观察肾脏组织形态学变化,行masson染色观察肾脏组织纤维化情况。免疫组织化学染色法检测大鼠肾脏ampk、p-ampk、mtor、p-mtor的表达。结果:1各组大鼠体重及肾脏肥大指数的比较正常组大鼠体重为(455.17±22.46)g,肾脏肥大指数为(3.65±0.24)mg/g;糖尿病组大鼠体重为(250.67±26.56)g,肾脏肥大指数为(7.12±0.28)mg/g;硫辛酸组大鼠体重为(259.20±15.48)g,肾脏肥大指数为(6.10±0.45)mg/g。与正常组相比,糖尿病组和硫辛酸组大鼠体重减轻,肾脏肥大指数升高(p0.01);与糖尿组相比,硫辛酸组大鼠肾脏肥大指数降低,差异具有统计学意义(p0.01),两组大鼠体重无统计学差异(p0.05)。2各组大鼠生化指标的比较正常组大鼠血糖为(5.17±0.71)mmol/l,尿蛋白为(15.27±1.45)mg/24h,血肌酐为(37.23±4.97)μmol/l,血尿素氮为(6.38±1.18)mmol/l;糖尿病组大鼠血糖为(21.38±1.66)mmol/l,尿蛋白为(314.00±16.14)mg/24h,血肌酐为(36.70±4.80)μmol/l,血尿素氮为(7.52±1.21)mmol/l;硫辛酸组大鼠血糖为(20.60±0.60)mmol/l,尿蛋白为(249.88±28.61)mg/24h,血肌酐(37.80±4.03)μmol/l,血尿素氮(7.76±1.24)mmol/l。与正常组相比,糖尿病组和硫辛酸组大鼠的血糖和24小时尿蛋白均显著升高(p0.01),血肌酐和血尿素氮无明显差异(p0.05);与糖尿病组相比,硫辛酸组大鼠24小时尿蛋白降低(p0.01),血糖、血肌酐和血尿素氮无明显差异(p0.05)。3各组大鼠8-ohdg的比较正常组大鼠8-ohdg为(1.63±0.62)ng/ml,糖尿病组大鼠8-ohdg为(5.09±0.89)ng/ml,硫辛酸组8-ohdg为(2.84±0.64)ng/ml。与正常组相比,糖尿病组和硫辛酸组大鼠的8-ohdg明显升高(p0.05);与糖尿病组相比,硫辛酸组8-ohdg水平明显降低,差异有统计学意义(p0.01)。4各组大鼠肾脏形态学改变肾脏he染色:正常组大鼠肾脏各组织形态正常、结构清晰,无炎性细胞浸润。糖尿病组大鼠肾小球毛细血管袢开放不良,系膜区扩张,部分肾小管细胞出现空泡变性,间质有较多炎性细胞浸润。与糖尿病组相比,硫辛酸组上述病理变化减轻。肾脏pas染色:正常组系膜基质指数为(0.1754±0.0149),糖尿病组系膜基质指数为(0.4052±0.0523),硫辛酸组系膜基质指数为(0.2259±0.0103);与正常组相比,糖尿病组及硫辛酸组大鼠基底膜增厚,系膜基质明显增多(p0.05);与糖尿病组相比,硫辛酸组大鼠系膜基质明显减少,差异有统计学意义(p0.01)。肾脏masson染色:与正常组相比,糖尿病组及硫辛酸组大鼠肾小球蓝色胶原纤维明显增多;与糖尿病组相比,硫辛酸大鼠胶原纤维沉积减少。5免疫组化检测结果各组大鼠肾脏mtor和ampk均有表达,差异无统计学意义(p0.05)。正常组大鼠肾脏p-mtor蛋白少量表达,iod/area为(0.0230±0.0034);糖尿病组和硫辛酸组大鼠肾脏p-mtor蛋白表达明显增加(p0.05),iod/area分别为(0.0547±0.0089)和(0.0316±0.0039);与糖尿病组相比,硫辛酸组大鼠肾脏p-mtor表达减少,差异有统计学意义(p0.01)。正常组大鼠肾脏p-ampk蛋白表达呈棕黄色颗粒沉着,iod/area为(0.0317±0.0040);与正常组相比,糖尿病组大鼠肾脏p-ampk蛋白表达减少,iod/area为(0.0183±0.0024)(p0.01);与糖尿病组相比,硫辛酸组大鼠脏p-ampk表达增加,iod/area为(0.0273±0.0026),差异均有统计学意义(p0.01)。结论:1糖尿病组大鼠肾脏组织出现典型病理改变,肾脏肥大指数和24小时尿蛋白升高,并且8-ohdg水平和mtor蛋白磷酸化明显升高,说明氧化应激增加及mtor信号通路激活与糖尿病肾脏病变有关。2硫辛酸组大鼠肾脏肥大指数和24小时尿蛋白降低,8-ohdg水平明显下降,且肾脏病理改变减轻,说明硫辛酸可以降低糖尿病大鼠体内氧化应激,延缓糖尿病大鼠肾脏病变进展。3硫辛酸组大鼠mTOR蛋白磷酸化水平明显降低,AMPK蛋白活化增加,说明硫辛酸可激活AMPK蛋白,抑制mTOR信号通路,在糖尿病肾脏病变中起到的保护作用。
[Abstract]:Objective: diabetic kidney disease (DKD) is one of the common microvascular complications of type 2 diabetes mellitus (type 2 diabetes mellitus, T2DM), and has become the main cause of end-stage renal disease and death. The pathogenesis of.DKD is not systematically explained. Chemical stress, inflammatory response, and autophagy inhibition are closely related to the development of DKD. Oxidative stress is an important pathogenesis of DKD, and it also mediates the inflammatory reaction to further accelerate renal injury,.8- hydroxyl deoxy guanine (8-hydroxy-2-deoxygnanosine, 8-OHdG), which is a present reflection of DNA oxidative damage and oxidative stress in the body. Sensitive indicators. In recent years, with the research of the mammalian target of rapamycin (mTOR) signaling pathway, the activation of mTOR pathway promotes renal cell hypertrophy, protein synthesis, oxidative stress, inflammatory reaction and inhibition of autophagy of renal cell, in focal segmental glomerulosclerosis and membranous kidney Disease and other chronic renal diseases play an important role. Adenosine5 '-monophosphate-activate protein kinase (AMPK) is one of the upstream signals for the regulation of mTOR. It plays a role mainly by inhibiting the mTOR signaling pathway. As an antioxidant, lipoic acid can inhibit the formation of oxygen free radicals and be used in clinical application. The treatment of diabetic peripheral neuropathy has received good curative effect, but the effect on DKD is not clear. By replicating the T2DM rat model, the expression of AMPK, p-AMPK, mTOR, p-mTOR and the level of 8-OHdG in the kidneys of the rats were measured to explore the relationship between the mTOR signaling pathway, the oxidative stress and T2DM kidney disease and the renal m of the lipoic acid. Methods: the effect of TOR signaling pathway and oxidative stress. Methods: 22 male clean Sprague-Dawley (SD) rats, 6 weeks of age and a weight of 180g + 20g, were kept in a clean environment. The rats were randomly divided into normal control group (NC group, n=6), diabetes group (DM group, n=8), lipoic acid intervention group (LA group, n=8). The normal control group was followed. The other two groups of rats were fed with high sugar and high fat diet (sucrose 20%, lard 10%, cholesterol 2.5%, cholate 1%, and basal diet 66.5%) after.4 weeks, the diabetic group and the rats in the thioctyl acid group were given 30mg/kg streptozotocin intraperitoneal injection, and the normal group rats were injected with equal volume citric acid buffer, after 2 weeks of injection. The rat fasting blood glucose (fastingbloodglucose, FBG) was measured and fbg7.8mmol/l was regarded as a successful model of T2DM. Then 100mg/kg lipoic acid was intraperitoneally injected with lipoic acid in the intervention group. The normal control group and the diabetic group were intraperitoneally injected with equal volume physiological saline, 1 times a day for 8 weeks. The 24 hour urine of rats in each group was collected one day before the end of the experiment. The liquid was used to determine the 24 hour urine protein; on the same day, the serum was used to determine the renal function and 8-OHdG. After the left kidney was weighed, 4% polyformaldehyde was fixed. The changes of renal histomorphology were observed by HE staining and PAS staining. The renal tissue fibrosis was observed by Masson staining. The kidney AMPK, p-ampk, mTOR in rat kidneys was detected by the immunostaining method. P-mTOR expression. Results: 1 the body weight and renal hypertrophy index of rats in each group were compared to (455.17 + 22.46) g and renal hypertrophy index (3.65 + 0.24) mg/g; the weight of the diabetic rats was (250.67 + 26.56) g and the renal hypertrophy index was (7.12 + 0.28) mg/g; the weight of the rats in the sulphur octanic acid group was (259.20 + 15.48) g, and the renal hypertrophy index was the index of renal hypertrophy. (6.10 + 0.45) mg/g. compared with the normal group, the weight loss and renal hypertrophy index of rats in the diabetic group and the lipoic acid group increased (P0.01). Compared with the diabetic group, the renal hypertrophy index of the rats in the sulphur octanic acid group decreased, and the difference was statistically significant (P0.01). The body weight of the two groups was not statistically different (P0.05), the biochemical indexes of the rats in each group of.2 were compared to the normal group. The blood sugar of rats was (5.17 + 0.71) mmol/l, urine protein was (15.27 + 1.45) mg/24h, blood creatinine was (37.23 + 4.97) mu mol/l, blood urea nitrogen was (6.38 + 1.18) mmol/l, blood sugar of diabetic rats was (21.38 + 1.66) mmol/l, urinary protein was (314 + 16.14) mg/24h, serum creatinine was (36.70 + 15.27) mu mol/l, blood urea nitrogen was mmol/l, and lipoic acid group rats Blood sugar was (20.60 + 0.60) mmol/l, urine protein was (249.88 + 28.61) mg/24h, serum creatinine (37.80 + 4.03) mu mol/l, blood urea nitrogen (7.76 + 1.24) mmol/l. compared with normal group, the blood sugar and 24 hourly proteinuria of rats in diabetic group and lipoic acid group increased significantly (P0.01), blood creatinine and blood urea nitrogen had no significant difference (P0.05); compared with diabetic group, The 24 hour urinary protein decreased (P0.01), blood sugar, blood creatinine and blood urea nitrogen (P0.05) in rats of.3, 8-OHdG of rats in each group of 8-OHdG was (1.63 + 0.62) ng/ml, 8-OHdG in diabetic rats was (5.09 + 0.89) ng/ml, and 8-OHdG was (2.84 + 0.64) ng/ml. in the group of lipoic acid (2.84 + 0.64), compared with normal group, diabetic group and lipoid The 8-OHdG of rats in the acid group was significantly increased (P0.05), and the 8-OHdG level of the lipoic acid group was significantly lower than that in the diabetic group. The difference was statistically significant (P0.01) the renal morphological changes of the kidney in each group of.4 rats were changed by HE staining: the normal group of kidney tissues in the normal group was normal, the structure was clear, and the inflammatory cells were not infiltrated. The glomerular capillaries in the diabetic rats The loop opening was bad, the mesangial region expanded, some renal tubular cells appeared vacuolated degeneration, and there were more inflammatory cell infiltration in the interstitium. Compared with the diabetic group, the pathological changes of the lipoic acid group were reduced. The renal PAS staining was (0.1754 + 0.0149), the mesangial matrix index of the diabetic group was (0.4052 + 0.0523), and the mesangial base of the sulphur octanic acid group The mass index was (0.2259 + 0.0103). Compared with the normal group, the basement membrane of the diabetic group and the lipoic acid group was thickened and the mesangial matrix increased significantly (P0.05). Compared with the diabetic group, the rat mesangial matrix of the lipoic acid group was significantly reduced, the difference was statistically significant (P0.01). The renal Masson staining was compared with the normal group, the diabetic and lipoic acid group rats. The glomerular blue collagen fibers increased significantly; compared with the diabetic group, the collagen deposition in the rats with lipoic acid was reduced by.5 immunohistochemistry. The renal mTOR and AMPK were expressed in each group, and the difference was not statistically significant (P0.05). A small amount of p-mTOR protein in the normal group of rats, iod/area was (0.0230 + 0.0034), and the diabetic group and lipoic acid were found. The expression of p-mTOR protein in the kidneys of the rats was significantly increased (P0.05), iod/area was (0.0547 + 0.0089) and (0.0316 + 0.0039), respectively. Compared with the diabetic group, the expression of p-mTOR in the kidney of the rats with lipoic acid was decreased (P0.01). The expression of p-ampk protein in the kidney of the normal group was brown and yellow granules, and iod/area was (0.0317 + 0.0040); Compared with the normal group, the expression of p-ampk protein in the kidney of diabetic rats decreased and iod/area was (0.0183 + 0.0024) (P0.01). Compared with the diabetic group, the expression of p-ampk in the rats of the lipoic acid group increased and the iod/area was (0.0273 + 0.0026). The difference was statistically significant (P0.01). Conclusion: the renal tissue of the 1 diabetic rats showed a typical pathological change of kidney tissue and kidney fertilizer. The high index and 24 hour urine protein increased, and the level of 8-OHdG and the phosphorylation of mTOR protein increased obviously, indicating that the increase of oxidative stress and the activation of mTOR signaling pathway were associated with the decrease of renal hypertrophy index and 24 hour urinary protein in the rats with diabetic renal disease, the level of 8-OHdG decreased significantly, and the renal pathological changes were reduced, indicating thioxin. Acid can reduce oxidative stress in diabetic rats and delay the progression of renal diseases in diabetic rats. The phosphorylation of mTOR protein in.3 thioctanoic acid group is significantly reduced, and the activation of AMPK protein is increased. It shows that thioctanoic acid activates AMPK protein and inhibits the mTOR signaling pathway and plays a protective role in diabetic renal disease.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.2;R692.9

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