肌苷对糖尿病性膀胱病的保护作用及机制

Inosine protects bladder structure and function in rats with diabetic cystopathy

目的目前针对糖尿病性膀胱病的治疗仍缺乏有效手段,寻找天然抗氧化剂成为当今研究的热点。文中旨在观察肌苷对糖尿病大鼠膀胱的保护作用及探索抗氧化应激机制。方法将60只成年大鼠随机数字表法分为正常对照组、糖尿病组和糖尿病肌苷治疗组,每组各20只,腹腔注射链脲佐菌素(60 mg/kg)诱导糖尿病大鼠模型,1周后取尾静脉血,血糖水平≥16.7 mmol/L定义为糖尿病大鼠。造模成功后肌苷治疗组接受肌苷(75 mg/kg,腹腔注射,2/d治疗,糖尿病组接受同量等渗盐水。干预4周、8周后,各组的两个时间点分别取10只大鼠,取膀胱组织切片做HE染色、用免疫荧光法观察膀胱组织中干细胞因子受体(c-kit)、神经生长因子(NGF)表达情况,电镜下观察各组大鼠膀胱组织的超微结构。同时对大鼠膀胱组织进行TUNEL凋亡分析,并测定膀胱组织中丙二醛(MDA)、超氧化物歧化酶(SOD)及谷胱甘肽(GSH)的含量。结果糖尿病组大鼠膀胱组织HE染色提示黏膜显著增生,肌束排列紊乱,结构松散,肌束断裂,肌束间间隙明显增宽,肌细胞萎缩,淋巴细胞浸润,肌束间胶原纤维填充,血管增生充血,神经束少见;在电镜下观察,可见肌细胞排列紊乱,中间连接消失,肌细胞和间质细胞内线粒体空泡化显著,核膜皱缩,核仁消失,染色质不规则边集,凝聚成块;免疫荧光结果提示NGF和c-kit信号均较正常对照组减弱;4周和8周时,与正常对照组细胞凋亡率[(4.65±3.04、5.48±2.00)%]比较,糖尿病组[(1.68±3.04、10.51±0.90)%]、肌苷治疗组[(7.00±1.72、7.24±1.66)%]凋亡细胞明显增多,与糖尿病组比较,肌苷治疗组凋亡细胞显著减少(P<0.01)。GSH增多(P<0.05),糖尿病组SOD、GSH含量明显增高(P<0.05),MDA含量明显降低(P<0.05);与糖尿病组比较,肌苷治疗组大鼠膀胱组织中SOD含量明显减少(P<0.05),MDA含量明显增加(P<0.05)。8周时,与正常对照组比较,糖尿病组SOD、GSH含量明显降低(P<0.01),MDA含量明显高于正常对照组和肌苷治疗组(P<0.01)。与正常对照组比较,糖尿病组及肌苷治疗组膀胱湿重均明显增加(P<0.01),结论氧化应激在糖尿病性膀胱病的发生和发展中起重要作用,inosine能降低糖尿病大鼠氧化应激水平,减轻糖尿病大鼠膀胱组织的受损程度,保护膀胱的组织结构和功能。

Objective At present, there is still a lack of effective means for the treatment of diabetic cystopathy, and to find natural antioxidants for this purpose has become a hot spot in research. This study is to investigate the protective effect of inosine on the bladder of diabetic rats and its antioxidative stress mechanisms.Methods A total of 60 adult male Sprague-Dawley rats were randomly divided into three groups of equal number： normal control, diabetes mellitus （DM） model control, and inosine intervention. The DM model was made by intraperitoneal injection of streptozotocin at 60 mg/kg. The DM model controls were injected with saline while the model rats in the intervention group with inosine, all at 75 mg/kg, ip, bid. After 4 and 8 weeks of treatment, the bladder tissues were collected from the rats for examination of the structural changes by HE staining, determination of the expressions of c-kit and nerve growth factor （NGF） by immunofluorescence assay, and observation of the ultrastructure of the bladder tissue under the electron microscope, detection of the cell apoptosis by TUNEL, and measurement of the contents of malondialdehyde （MDA）, superoxide dismutase （SOD）, and glutathione （GSH）.Results HE staining indicated significant mucosal hyperplasia, disordered arrangement, loose structure, fracture, expanded intervals and collagen fiber filling of muscle bundles, muscular atrophy, lymphocytes infiltration, vascular hyperplasia and congestion, and few muscle bundles, while electron microscopy manifested disordered arrangement, interrupted connection, mitochondrial vacuolation in muscular and interstitial cells, shrinkage of nuclear membrane, disappearance of nucleoli, and irregular chromatin margination and condensation in the bladder tissues of the DM rat models. Immunofluorescence assay showed that the signals of c-kit and NGF were reduced in the DM models as compared with those in the normal controls. After 4 and 8 weeks of intervention, the cell apoptosis rate was significantly higher in the DM model control （／[1.68±3.04／]% and ／[10.51±0.90／]%） and inosine-treated rats （／[7.00±1.72／]% and ／[7.24±1.66／]%） than in the normal controls （／[4.65±3.04／]% and ／[5.48±2.00／]%）, but remarkably lower in the inosine-treated than in the DM model controls （P〈0.01）. The contents of SOD and GSH were increased （P〈0.05） while that of MDA decreased markedly in the DM models （P〈0.05）, but the former decreased （P〈0.05） while the latter increased significantly in the inosine intervention group as compared with the DM model control group （P〈0.05）. At 8 weeks, the contents of SOD and GSH were remarkably lower in the DM model than in the normal controls （P〈0.01）, while that of MDA markedly higher than in both the normal control and inosine intervention groups （P〈0.01）. The wet weight of the bladder was significantly increased in the DM model and inosine intervention groups in comparison with that of the normal controls （P〈0.01）.Conclusion Oxidative stress plays an important role in the development and progression of diabetic cystopathy. Inosine can protect the bladder structure and function of the DM rat by reducing oxidative stress and injury to the bladder tissue.