枸杞多糖对加压诱导凋亡视网膜神经节细胞电生理特性的影响

Effect of LBP on the Electrophysiological Properties in High Pressure-induced Apoptosis of Retinal Ganglion Cells

目的观察枸杞多糖(LBP)对加压诱导凋亡的视网膜神经节细胞(RGCs)钾K^+电流的影响,以探讨枸杞多糖对RGCs的保护作用。方法取SD乳鼠视网膜神经节细胞原代培养,分为对照组,加压组,LBP各剂量组(100,250,500,1 000μg·mL^(-1)),对照组常规培养6 d;加压组常规培养6 d后用自行设计的加压装置加压80 mm Hg,1 h;LBP各组常规培养5 d后,加入不同浓度的LBP共培养24 h后,再加压80 mm Hg,1 h。通过连续波长多功能微孔板检测仪检测各组RGCs线粒体的平均荧光强度值;通过全细胞膜片钳技术观察各组细胞膜电容(membrane capacitance,C_m)和钾(K^+)电流的变化。结果加压组C_m和RGCs线粒体荧光强度低于对照组,差异有统计学意义(P<0.05),LBP 250,LBP 500,LBP 1 000组RGCs线粒体荧光强度和C_m高于加压组(P<0.05,P<0.01)。电流记录显示,与对照组比较加压组外向K^+电流增加,用不同浓度的LBP预处理后,K^+电流增加都有抑制,且呈剂量依赖性(P<0.05,P<0.01)。结论枸杞多糖可抑制加压诱导的视网膜神经节细胞凋亡,其作用可能与抑制K^+电流有关。

Objective To investigate the effect of Lycium bararum polysaccharides （LBP） on the K＋ current （Ik） in high pressure-induced apoptosis of rat retinal ganglion cells （RGCs） using electrophysiologica] method, and to explore the possible mechanisms of LBP on optic nerve protection. Methods The retina of new born Sprague-Dawley （S-D） rats were dissociated into cell suspension with trypsin digestion, cells were cultured and divided into control group, pressure group and LBP groups. Cells in control group were routinely cultured for 6 days; cells in pressure group were cultured routinely for 6 clays and then subject to a pressure of 80 mmHg for 1 hour; cells in LBP groups were cultured routinely for 5 days and different concentrations of LBP was added into the medium 24 hours before cells were pressured 80 mmHg for 1 hour. The rhodamine 123 fluorescence of RGCs mitochondrion in six groups were detected by continuous wavelength muhifunctional microplate detection instrument. The membrane capacitance （Cm） and Ik were recorded from RGCs in the six groups using whole-cell patch-clamp techniques. Results In the pressure group, the Cm and rhodamine 123 fluorescence in RGCs＇ mitochondria were lower than that of control group. These two factors in LBP 250, LBP 500, LBP 1000 groups were significantly higher than that in pressure group according to the concentration increase（P 〈 0.05 ）. The Ik in pressure group was higher than that in the control group. LBP 250, LBP 500, LBP 1000 groups suppressed the current amplitudes of Ik （P 〈 0.05）. At the tested potential ranged from 30 to 60 mV, the current intensities in LBP 250, LBP 500, LBP 1000, and control group were significantly lower than that in pressure group （P 〈 0.05）. Conclusion This study suggested that pressure-induced RGCs apoptosis could be prevented by LBP, the mechanism was linked to Ik currents.