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高压氧治疗对2型糖尿病大鼠肾脏的保护作用

聂文洁^*, 曹秀琴, 邵贵强

上海市闵行区吴泾医院，上海 200240

摘要

本研究通过观察高压氧治疗对2型糖尿病Goto-Kakizaki (GK)大鼠肾脏组织结构、细胞凋亡和基质金属蛋白酶表达的影响，探讨高压氧对糖尿病肾脏的保护作用及机制。选用Wistar大鼠8只作为正常对照组(n = 8)，2型糖尿病GK大鼠24只，随机分为模型组(n = 8)、二甲双胍对照组(MH组，n = 8)、高压氧组(HBO组，n = 8)，正常对照组和模型组每天按5 mL/kg灌服纯净水；MH组每天按250 mg/kg二甲双胍混悬液灌胃；HBO组每天5 mL/kg灌服纯净水，并给予0.15 MPa压力，纯氧稳压30 min。大鼠分别治疗三周后测体重，尾部采血测空腹血糖，取肾脏并测肾质量。将肾脏制成石蜡切片并进行HE染色、PAS和Masson染色，用光学显微镜观察各组大鼠肾脏组织病理变化；用TUNEL法检测肾脏细胞凋亡并计算各组肾脏细胞凋亡指数；用免疫组化法对肾脏进行Caspase-3、MMP-2、TIMP-2标记，并测量阳性细胞积分光密度；ELISA方法检测各组大鼠血浆TGF-β1浓度。结果显示模型组大鼠肾小球体积增大，细胞外基质增生(系膜基质增宽、基底膜增厚)，肾小管上皮细胞水肿，HBO组较模型组病变较轻；经高压氧治疗过的HBO组大鼠的肾脏细胞凋亡指数和Caspase-3表达均与正常对照组和MH组无显著性差异(P > 0.05)，而与模型组之间比较差异具有显著性(P < 0.01)。MMP-2、TIMP-2表达正常对照组表达最强，MH、模型组表达最弱，HBO组介于中间，差异具有显著性(P < 0.05)。HBO、MH、模型组血浆TGF-β1浓度较正常对照组都有升高趋势，但各组大鼠血浆TGF-β1浓度差异无统计学上的显著性(P > 0.05)。实验结果表明高压氧治疗可抑制糖尿病大鼠肾脏细胞的凋亡增加，调节MMP-2及其抑制剂的活性，减少细胞外基质的积聚，从而保护肾脏，有利于防治糖尿病肾病的发生和发展。

关键词： 高压氧; 细胞凋亡; 2型糖尿病; Goto-Kakizaki大鼠; 肾脏; Caspase-3; 基质金属蛋白酶

分类号：R3

[Protective effects of hyperbaric oxygen treatment on kidney cells of type 2 diabetic rats.] [Article in Chinese]

NIE Wen-Jie^*, CAO Xiu-Qin, SHAO Gui-Qiang

Wujing Hospital, Shanghai 200241, China

Abstract

The major objective was to explore the effect of early hyperbaric oxygen (HBO) therapy on the tissue structure, apoptosis, and metalloproteinases of kidney cells in Goto-Kakizaki (GK) rats with type-2 diabetes mellitus. GK rats (n = 24) were divided randomly and evenly into model, metformin hydrochloride (MH), and hyperbaric oxygen (HBO) groups, while healthy Wistar rats (n = 8) were used as normal control group. The healthy rats in the normal control group and the GK rats in the model group both intragastrically administered with purified water (5 mL/kg) once per day. Meanwhile, the rats in the MH group received intragastric administration with MH (250 mg/kg) once daily, while the rats in the HBO group inhaled pure oxygen under a constant pressure (0.15 Mpa) for 30 min. After 3 weeks of treatment, the body weight of each rat was measured, and the blood samples were collected from tails. Subsequently, the kidneys of all rats were excised for weighing mass and further examination. For each renal sample, the section was firstly embedded with paraffin and sliced to prepare a histopathologic section stained using HE, PAS and Masson, respectively, for subsequent observation with optical microscopy. Later, the apoptosis of kidney cells was examined using the TUNEL technique by computing the apoptotic index. Furthermore, the histopathologic section were also examined using the immunohistochemistry approach with Caspase-3, MMP-2, and TIMP-2 antibodies, respectively. At the same time, the plasma concentration of TGF-β1 of the rats in each group was detected using ELISA method. These resultant data showed that the pathological changes of the HBO group was less than those of the model group with respect to increased glomerular volume density of mesangial cells, broadening mesangial matrix and thickening basement membrane as well as swelling renal tubular epithelial cell. The index of cell apoptosis and Caspase-3 expression in the HBO group showed no significant difference (P > 0.05) compared with that in the normal control and MH groups respectively, but demonstrated significant difference with that in the model group (P < 0.01). Meanwhile, the MMP-2 and TIMP-2 expressions of the HBO group were stronger than those in the model and MH groups, but weaker than those in the normal control group (P < 0.05). Although the plasma concentration of TGF-β1 in HBO, MH and model groups was greater than that in the normal control group, no significant statistical difference was distinguished among these four groups (P > 0.05). These results indicate that the HBO treatment can inhibit the apoptosis and Caspase-3 expression of renal cells of GK rats, adjust the activity of MMP-2 and its inhibitors, and reduce the accumulation of extracellular matrix. This implies that the HBO treatment might protect renal tissues, thus delaying occurrence and retaining development of diabetic nephropathy.

Key words: hyperbaric oxygen treatment; apoptosis; diabetes mellitus; Goto-Kakizaki rats; kidney; Caspase-3; matrix metalloproteinases-2

收稿日期：2013-07-09　　录用日期：2013-10-21

通讯作者：聂文洁　　E-mail: wenjienie@126.com

引用本文：

聂文洁, 曹秀琴, 邵贵强. 高压氧治疗对2型糖尿病大鼠肾脏的保护作用[J]. 生理学报 2014; 66 (2): 189-194.

NIE Wen-Jie, CAO Xiu-Qin, SHAO Gui-Qiang. [Protective effects of hyperbaric oxygen treatment on kidney cells of type 2 diabetic rats.] [Article in Chinese]. Acta Physiol Sin 2014; 66 (2): 189-194 (in Chinese with English abstract).