氨基胍对大鼠胰岛保护作用的实验研究

Protective Effect of Aminoguanidine on Pancreatic Islet in Rats

目的探讨在细胞因子与大鼠胰岛细胞共同培养过程中,诱导型一氧化氮合酶(i NOS)抑制剂氨基胍对胰岛细胞功能和存活的影响及其机理。方法分离纯化大鼠胰岛,进行胰岛细胞培养。根据培养基中是否加入氨基胍或细胞因子IL-1β和TNF-α,按随机对照原则分为空白对照组(完全培养基)、细胞因子组(加IL-1β和TNF-α)、氨基胍组(加氨基胍)及氨基胍+细胞因子组(加氨基胍及细胞因子)。检测指标包括:培养液中NO水平、胰岛组织中i NOS活性、胰岛细胞存活情况(丫啶橙/溴乙锭染色)、胰岛细胞凋亡情况(TUNEL法)及胰岛功能(胰岛素释放试验)。结果与空白对照组比较,细胞因子组大鼠胰岛组织中i NOS的活性明显提高,培养液中NO的水平明显上升,同时胰岛细胞的存活率下降,大量细胞凋亡,胰岛素分泌明显减少(P<0.01)。与细胞因子组比较,氨基胍+细胞因子组的i NOS的活性〔(3.17±0.51)U/ml比(38.93±4.72)U/ml〕及NO水平〔(50.5±10.4)μmol/L比(313.0±35.4)μmol/L〕明显下降,胰岛细胞存活率活明显升高〔(72.73±3.14)%比(57.07±5.07)%〕,凋亡率明显下降〔(20.11±8.48)%比(41.17±6.87)%〕,胰岛素分泌指数明显升高(3.50±0.27比1.96±0.19),差异均有统计学意义(P<0.01)。结论氨基胍通过抑制i NOS活性,控制NO过量产生,从而减轻细胞因子对胰岛的损害,改善胰岛的存活与功能。

Objective To investigate the effect of inducible nitric oxide synthase （iNOS） inhibitor aminoguanidine on pancreas islets cultured with cytokines TNF-α and IL-1β in rats. Methods Islets isolated from Wistar rats were purified and cultured. According to whether cytokines TNF-α, IL-1β and aminoguanidine were added into the medium respectively or not, islets were divided into 4 groups： cultured with islet only was taken as blank control group, cultured with TNF-α＋IL-1β as cytokine group, cultured with aminoguanidine as aminoguanidine group, and cultured with TNF-α＋IL-1β and aminoguanidine as aminoguanidine＋ cytokine group. NO level in culture medium and iNOS activity in islets tissue （Test Kit）, apoptosis （TUNEL method） and viability of islets cell （acridine orange/ethidium bromide stain）, and the function of islets （insulin release test） were measured. Results Compared with blank control group, the activity of iNOS in islet tissue and level of NO in culture medium increased, and the mass mortality and apoptosis appeared in islet cells, while insulin secretion decreased in cytokine group （P〈0.01）. Compared with cytokine group, the activity of iNOS [（3.17±0.51） U/ml vs. （38.93±4.72） U/ml] and level of NO [（50.5± 10.4）μmol/L vs. （313.0±35.4） μmol/L] decreased, the survival [（72.73±3.14） % vs. （57.07±5.07） % ] increased and the apoptosis rate [（20.11±8.48） % vs. （41.17±6.87） % ] decreased, the insulin secretion （secretion index： 3.50±0.27 vs. 1. 96±0. 19） improved; There were all significant differences in 2 groups （P〈0.01）. Conclusion The iNOS inhibitor aminoguanidine could prevent the islet from the damage of iNOS/NO, alleviate the impairment of cytokines to islets, and ameliorate the survival and function of islets.