【作者】 陈凯；

【导师】 张承英；

【作者基本信息】 安徽医科大学 ， 内科学， 2015， 硕士

【摘要】 目的糖尿病肾病(diabetic nephropathy,DN)是糖尿病的常见并发症,同时也是糖尿病患者死亡的一个主要原因,其发病率一直呈逐年升高趋势,目前已成为我国导致终末期肾病的第二大因素。近年的研究认为,DN是一种慢性炎症性疾病,但其炎症反应发生的原因尚未明确。内质网应激(endoplasmic reticulum stress,ERS)已被证实与炎症反应密切相关,可能在DN的炎症反应中起到一定作用。本研究拟通过构建糖尿病大鼠模型,探讨内质网应激及相关炎症反应在糖尿病大鼠肾脏损害中的作用及血管紧张素受体拮抗剂缬沙坦对DN大鼠肾脏中ERS及相关炎症的影响。方法34只SD雄性大鼠,随机分为对照组(Con组,n=10只)、模型组(DM组,n=12只)、缬沙坦治疗组(DM+V组,n=12只)。DM组、DM+V组大鼠腹腔注射STZ40mg/kg,制作糖尿病模型,各组大鼠给予普通饲料喂养,DM+V组每天用缬沙坦灌胃6周(10mg/kg),Con组及DM组只灌等量蒸馏水。实验结束后测量大鼠体重、双侧肾脏重量,应用免疫组化法检测ERS标志蛋白GRP78及中性粒细胞趋化因子1(MCP-1)的蛋白表达及定位,Western blot方法检测ERS相关蛋白P-IRE1α、JNK、P-JNK、NF-κB p65、P-NF-κB p65的蛋白表达水平,实时荧光定量PCR(q RT-PCR)检测肾组织中JNK、NF-κB及炎症因子MCP-1、TNFα、IL-1βm RNA表达变化,同时观察各组大鼠24小时尿蛋白定量、血浆白蛋白(ALB)、尿素氮(BUN)、血肌酐(Scr)等指标的变化。结果1、与Con组相比,DM组大鼠24小时尿蛋白定量、BUN、Scr水平明显升高(P<0.05),ALB明显降低(P<0.05),与DM组相比,DM+V组24小时尿蛋白定量、BUN明显降低(P<0.05),ALB明显升高(P<0.05),Scr较DM组降低但无统计学意义(P>0.05);2、HE染色光镜下Con组肾组织未见明显病理改变,DM组大鼠肾小球系膜细胞增殖肥大,系膜区增宽,系膜基质聚积,肾小球基底膜增厚,肾小管上皮细胞广泛空泡变性,并可见炎细胞浸润,DM+V组与DM组相比,系膜细胞增殖肥大、系膜基质聚积、肾小管上皮细胞广泛空泡变性均减轻;3、免疫组化显示Con组GRP78轻度表达、MCP-1几乎无表达,与Con组相比,DM组GRP78、MCP-1表达明显升高(P<0.05),且肾小管上皮细胞呈强阳性表达,与DM组相比,DM+V组GRP78、MCP-1表达明显降低(P<0.05);4、与Con组相比,DM组P-IRE1α、P-JNK、P-NF-κB p65蛋白表达水平明显升高(P<0.05),与DM组相比,DM+V组P-IRE1α、P-JNK、P-NF-κB p65蛋白表达水平明显降低(P<0.05),且P-JNK、P-NF-κB p65与P-IRE1α蛋白表达程度呈正相关(P<0.05);5、与Con组相比,DM组MCP-1、IL-1β、NF-κB p65、TNFαm RNA表达明显升高(P<0.05),与DM组相比,DM+V组MCP-1、IL-1β、NF-κB p65、TNFαm RNA表达明显降低(P<0.05),3组间JNKm RNA表达变化无明显差异(P>0.05)。结论1、DN中存在ERS的激活,活化的膜蛋白P-IRE1α可能通过激活JNK及NF-κB炎症信号通路引起炎症反应;2、ANGII受体拮抗剂缬沙坦除影响血流动力学因素外,可能通过抑制内质网膜蛋白IRE1介导的JNK及NK-κB信号通路减轻炎症反应,而达到降低蛋白尿,延缓肾功能进展的作用。更多还原

【Abstract】 Objective : As one of the most serious microvascular complications in diabetes,Diabetic nephropathy(DN) has become the second factor leading to end-stage renal disease at present, the incidence of DN is rising year by year. The view that DN is a chronic inflammatory disease has been recognized widely, recently. But the cause of the inflammation reaction is not clear. Endoplasmic reticulum stress( ERS) has been proved to be closely associated with inflammation, and may play an important role in inflammation of the DN. In this experiment, the diabetic rat model was built to study the role of endoplasmic reticulum stress and related inflammation in kidney damage of diabetic rats and the effect of Valsartan.Methods: 34 healthy male SD rats were randomly divided into control group(Con group, n = 10), model group(DM group, n = 12) and valsartan treatment group(DM +V group, n = 12). The rats of DM group and the DM+V group were intraperitoneally injected STZ(40mg/kg) for diabete animal model. Valsartan(10mg/kg) was administered daily by gavage from the next day of the induction to diabetic rat for 6weeks. The rats of DM+V group were intragastrically administered with valsartan(10mg/kg) every day, while Con and DM group were only given the equivalent distilled water for 6 weeks.After the experiment, the weight of the rats and kidney were measured.The expression and distribution of ERS related protein GRP78 and Monocyte chemotactic protein-1(MCP-1) was examined by immunohistochemistry,The expression of ERS related protein P-IRE1α, P-JNK, JNK, NF-κB p65, P-NF-κB p65 was examined by Western blot. Real-time fluorescence quantitative PCR was used to detect the m RNA expressions of JNK, NF-κB and MCP-1, TNFα, IL-1β. 24 hour urine protein excretion, plasma albumin, Scr, BUN were checked meanwhile.Results: ① compared with Con group, 24 hours urinary protein, BUN, Scr of DM group rats were increased significantly(P< 0.05), ALB decreased(P < 0.05) compared with DM group, 24 hours urinary protein, BUN of DM+V group decreased significantly(P < 0.05), ALB increased significantly(P < 0.05), Scr reduced in the DM group,but without statistical significance(P > 0.05); ② Glomerular and tubular in Con group had no significant changes, we found that glomerular extracellular matrix hyperplasia,mesangial matrix markedly increased, basement membrane thicken,mesangium gap widened, renal tubular epithelial vacuoles degeneration with partial stove atrophy, mild interstitial edema, glomerular and interstitial inflammatory cell infiltration in the DM group. compared with DM group, DM+V group appeared the reduction of mesangial cell proliferation, renal tubular epithelial vacuoles degeneration; ③ immunohistochemical appeared that GRP78 was expressed mildly,no expression was found in MCP-1 in the Con group, the expression of MCP-1increased significantly, and strong positive expression in renal tubular epithelial cells,compared with Con group(P<0.05). DM + V group appeared that expression of GRP78,MCP-1 decreased significantly,compared with DM group(P<0.05); ④ Western blot method showed compared with Con group, the expression of P-IRE1α、P-JNK、P-NF-κB p65 was increased significantly in DM group(P < 0.05), compared with DM group,expression of P-IRE1α, P-JNK, P-NF-κB p65 was significantly decreased in DM+V group(P < 0.05), and expression of P-IRE1α, P-JNK, P-NF-κB p65 were positively correlated(P< 0.05); q RT- PCR showed that compared with Con group, the m RNA expression of MCP-1, IL-1β, NF-κB p65, TNFα increased significantly in DM group(P < 0.05),compared with DM group, the m RNA expression of MCP-1, IL-1β, NF-κB p65,TNFαdecreased significantly in DM group(P < 0.05), While there is no significant difference in the expression of JNK m RNA among three groups(P > 0.05).Conclusion: ① ERS and related inflammation was activated in the kidney of DM rats,and the activative membrane protein P-IRE1α can cause inflammation by activating JNK and NF-κB inflammatory signaling pathways; ②ANGII receptor antagonist valsartan may inhibit JNK and NF-κB signaling pathway mediated by membrane protein IRE1α to reduce inflammation and proteinuria, in addition to the factors of influencing the hemodynamics.更多还原