黄芪联合葛根素对糖尿病肾病内质网应激相关因子表达的影响

【作者】 崔蕾；

【导师】 王谦；

【作者基本信息】 北京中医药大学 ， 中西医结合基础， 2012， 硕士

【摘要】 糖尿病肾病(diabetic nephropathy, DN)是临床最为常见的糖尿病并发症。DN的实质是糖尿病微血管病变,主要指以血管病变为主的肾小球硬化症,是糖尿病最主要的死亡原因之一。大量实验证实内质网应激(ERS)和糖尿病肾病关系密切。内质网膜上存在3种重要的应激感受蛋白：Ⅰ型跨膜蛋白激酶/核糖核酸内切酶(IRE1)、双链RNA依赖的蛋白激酶样激酶(PERK)和活化转录因子6(ATF6)。这三种跨膜蛋白可以高效的放大应激信号,起始ERS。当未折叠蛋白超负荷时,3种膜蛋白活化,双链RNA依赖的蛋白激酶样ER激酶被激活,作用于真核生物翻译起始因子2a(el F2α)使其磷酸化,进而使新生蛋白合成、转运受阻减少,从而有效缓解了内质网腔蛋白质超负荷。eI F2a磷酸化还可使转录活性因子-4(ATF4)表达增高。ATF4具有转录激活因子作用,诱导凋亡相关因子CHOP表达,从而减轻细胞内蛋白质负荷。PERK的激活和磷酸化的eIF2α可以看作是EIRS存在的核心指示物。中药因其作用机制具有多途径、多靶点、多环节的优势,在针对糖尿病肾病的治疗应用上得到越来越多的认可。本实验从内质网应激角度切入,进一步探讨黄芪注射液和葛根素注射液联合应用治疗DN的机制,为中医药防治糖尿病肾病提供理论依据。研究目的：本实验主要通过探索黄芪注射液和葛根素注射液对KKAγ小鼠DN肾损伤干预效应的作用环节,为明确中药治疗DN的发病机制提供新的理论依据,为糖尿病肾病临床用药提供新的靶点；同时揭示糖尿病肾病病变过程中,内质网应激相关因子PERK、elF2α、ATF4的改变。研究方法：(1)将随机血糖≥13.9mol/L的KKAγ小鼠分为模型组和治疗组(黄芪注射液联合葛根素注射液组)。选取同龄C57BL/6J雄性小鼠作为正常组。从12周开始,治疗组小鼠腹腔注射给药。于20周龄处死各组小鼠,取血清检测空腹血糖、甘油三酯、胆固醇、肌酐、尿素,取肾脏组织,制作病理切片,观察病理形态和超微结构。(2)用Real-time PCR方法,检测各组小鼠肾组织中PERK mRNA、elF2α mRNA、ATF4mRNA的表达。研究结果：(1)与正常组小鼠相比较,模型组小鼠空腹血糖、甘油三酯、胆固醇、肌酐、尿素均显著升高；经过黄芪注射液、葛根素注射液联合用药后,治疗组空腹血糖、甘油三酯、胆固醇、肌酐、尿素较模型组均显著降低。(2)HE染色切片显示正常组小鼠肾小球、肾小管结构正常；模型组小鼠肾脏中肾小管上皮细胞胞浆出现少量空泡,系膜基质增多；治疗组小鼠系膜基质略有增多,肾小管上皮细胞内空泡减少。透射电镜显示,正常组小鼠近曲小管上皮细胞线粒体、内质网结构正常；模型组小鼠肾小球基底膜不规则增厚,足细胞部分足突融合、碎裂；肾小管上皮细胞及足细胞内粗面内质网卷曲成团或扩张、断裂,胶原纤维增多；细胞基质增多；线粒体扩张、空泡变性；治疗组小鼠近曲小管上皮细胞部分线粒体轻度肿胀、卷曲,内质网扩张。(3)20周龄模型组KKAγ小鼠PERK mRNA、elF2α mRNA、ATF4mRNA的表达量与正常组相比明显升高,治疗组较模型组明显降低。研究结论：(1)黄芪注射液与葛根素注射液合用,有降低血糖、血脂,保护肾功能的作用。(2)20周龄KKAγ小鼠肾脏功能损害严重,ERS被激活,内质网应激是糖尿病肾病病情发展的重要因素。黄芪注射液联合葛根素注射液合用可通过调整内质网应激相关因子PERK、elF2α、ATF4mRNA的表达发挥治疗糖尿病肾病的作用。更多还原

【Abstract】 Diabetic nephropathy (DN) is one of the most common clinical diabetic complications. Diabetic microvascular pathological changes are the essence of DN, which refers to the vascular lesions glomerular sclerosis is the main cause of death among diabetes patients. Experiments prove that there is close relationship between the endoplasmic reticulum stress (ERS) and diabetic nephropathy. On the endoplasmic reticulum membrane, there exists important protein: IRE1,PERK,ATF4.The three across membrane proteins can efficiently amplify stress signals, and start ERS. When unfolded protein overloads, the three kinds of membrane protein were activated. When ERS is initiated, the activated PERK can lead to phosphorylation of el F2a, and then prevent the new protein transport into the endoplasmic reticulum cavity, so as to reduce the accumulation of protein. When the EIF2a factor actived, the express of ATF4is also increased. ATF4is revealed to play a role as an activate factor which could induce the apoptosis related factor CHOP expression, and reducing the cellular proteins load. The actived state of el F2α and PERK can be regarded as core indicators of when ERS exists.Due to the Traditional Chinese medicine play a role by multiple mechanism, ways, targets, which advantage is become more and more significantly. This experiment put a perspective on the ERS, discusses the treatment mechanism of Huangqi (Radix Astragali seu Hedysari) injection combined Puerarin injection on DN, look forward to provide basis for traditional Chinese medicine on DN.Objective:Through the exploration of Puerarin injection combine Huangqi injection on KKAy mice’s DN renal injury intervention, look forward to provide new ideas on traditional Chinese medicine therapy for diabetic nephropathy and provide new targets; at the same time reveals the factors changes in ERS.M ethods:(1)Testing the random glucose of KKAγ mice,devide the core≥13.9mmol/l mice into model group and treatment group (Huangqi inject combine Puerarin inject). Selected the same age C57BL/6J mice into the normal group.From12th week, abdominal injection was given to the treatment group, these mice were killed in20th week,take the kidney to observe the pathological change,and take the serum to test biochemical index.(2)Take real-time polymerase chain reaction (PCR) to test the PERK mRNA, elF2α mRNA, ATF4mRNA expression of mice kidney tissues.The results:(1) Compared with the normal group, fasting blood glucose, triglyceride and chol esterol, serum creatinine and urea of model group are significantly increased. The treatment group fasting blood glucose, triglyceride and cholesterol, creatinine, urea in model group were significantly reduced. With HE stained and TEM,we can observe that the renal function of the treatment group is take a turn for better.(2) In the20th week.compared with normal group, model group KKAγ mice PERK mRNA, elF2α mRNA, ATF4mRNA expression are significantly increased; compared with model group,these are reduced in the treatment group.Conclusion:(1) In the DN mice, Astragalus inject combine Puerarin injection can greatly improve the biochemical index and reduce the kidney pathological damage.(2) When KKAγ mice come into diabetic nephropathy stage, the kidney function is seriously damaged and ERS is activated. ERS is an important factor in DN Ⅲness development.Through adjust ERS related factors Huangqi injection combined Puerarin injection play the role of the treatment of DN.更多还原