牛磺酸降低高糖诱导的视网膜谷氨酸兴奋性及其机制

Effect of taurine on decreasing glutamate excitability induced by high glucose in retina of rats and its mechannism

目的观察牛磺酸对高糖诱导的视网膜谷氨酸兴奋性的影响并探讨其作用的机制。方法体外培养并采用免疫细胞荧光双标鉴定视网膜Müller细胞。实验分10组,分别是:5mmol/L葡萄糖培养组(正常对照组),5mmol/L葡萄糖+0.1mmol/L牛磺酸培养组,5mmol/L葡萄糖+1mmol/L牛磺酸培养组,5mmol/L葡萄糖+10mmol/L牛磺酸培养组,25mmol/L葡萄糖培养组(高葡萄糖组),25mmol/L葡萄糖+0.1mmol/L牛磺酸培养组,25mmol/L葡萄糖+1mmol/L牛磺酸培养组,25mmol/L葡萄糖+10mmol/L牛磺酸培养组,5mmol/L葡萄糖+20mmol/L甘露醇组(甘露醇对照组)。RT-PCR检测谷氨酸转运蛋白(GLAST)和谷氨酰胺合成酶(GS)表达,β-液闪技术检测谷氨酸摄取活性,谷氨酰胺合成酶检测试剂盒检测GS活性。结果 25mmol/L高葡萄糖培养后Müller细胞中GLAST、GS mRNA表达量较正常对照组明显降低(P<0.05),L-[2,3-3 H]-谷氨酸摄取活性由正常对照组的(726±12)cpm/(min.mg protein)降为(352±10)cpm/(min.mg protein),GS活性由(41.1±2.3)μmol/Lγ-谷氨酰羟肟酸(GHA)/(h.mg protein)降为(20.3±2.1)μmol/L GHA/(h.mg protein)(P<0.05)。加入1、10mmol/L牛磺酸干预可明显上调高葡萄糖组Müller细胞中GLAST、GS mRNA表达量,增加L-[2,3-3 H]-谷氨酸摄取活性和GS活性(P<0.05)。加入0.1mmol/L牛磺酸干预可上调高葡萄糖组Müller细胞中GLAST、GS mRNA表达量,增加L-[2,3-3 H]-谷氨酸摄取活性和GS活性,与高葡萄糖组相比差异无统计学意义(P>0.05)。甘露醇对照组Müller细胞中GLAST、GS mRNA表达量、L-[2,3-3 H]-谷氨酸摄取活性和GS活性与正常对照组相比差异无统计学意义(P>0.05)。结论牛磺酸能降低高糖引起的视网膜谷氨酸兴奋性,其作用机制可能通过上调Müller细胞谷氨酸转运和降解能力,从而维持细胞外空间正常的谷氨酸浓度。

Objective To investigate the effect of taurine on decreasing glutamate excitability induced by high glucose in retina of rats and its mechannism. Methods Mtiller cells were cultured in Dulbecco＇s modified Eagle＇s medium（DMEMJin the presence of 5 mmol/L D-glucose（normal glucose）, 5 mmol/L D-glucose plus 0. i mmol/L taurine, 5 mmol/L D-glucose plus 1 mmol/L taurine, 5 mmol/L D-glucose plus 10 mmol/L taurine, 25 mmol/L D- glucose（high glucose）, 25 mmol/L D-glucose plus 0. 1 mmol/L taurine, 25 mmol/L D-glucose plus 1 mmol/L taurine, 25 mmol/L D-glucose plus 10 mmol/L taurine, and 5 mmol/L D-glucose plus 20 mmol/L mannitol for at least three days. The morphologia were identified by confocal microscopy. The expressions of glutamate transporters （GLAST） and glutamine synthetase （GS） were examined by RT-PCR. Glutamate uptake was measured as 3H- glutamate content of the lysates. GS activity was assessed by a spectrophotometric assay. Results At 25 mmol/L high glucose incubation, the expression and the activity of GLAST and GS in M/iller cells were decreased significantly compared with the control（P〈0. 05 ）. 1,10 mmol/L taurine intervention could significantly inhibit the decrease of GLAST and GS in Muller cells induced by high glucose（P〈0. 05）, 0. 1 mmol/L taurine intervention could inhibit the decrease of Glast and GS too but the difference was not significant（P〉0. 05）. The mannitol and taurine did not change the expression and the activity of GLAST and GS in Maller cells comparing to the normal glucose（P〉 0. 05）. Conclusion Neuroexcitability was enhanced by high glucose could be inhibited by taurine via regulating Muller cellsr glutamate uptake and degradation under diabetic conditions.