米诺环素抑制谷氨酸诱导的大鼠视网膜神经细胞凋亡

目的：观察米诺环素对谷氨酸诱导的大鼠视网膜神经细胞凋亡的抑制效应及其作用机制。方法:取原代培养的SD乳鼠视网膜神经细胞，随机分为正常对照组、米诺环素对照组（米诺环素20 μmol/L）、谷氨酸组（谷氨酸1 mmol/L）和米诺环素治疗组（米诺环素20 μmol/L＋谷氨酸1 mmol/L）。干预1 h后采用Annexin V/PI流式细胞仪计数凋亡细胞数量，同时检测Rh123以评估线粒体膜电位改变。干预12 h后行MTT检测细胞活性；另取细胞培养上清液做一氧化氮合酶（NOS）的活力单位检测。结果:干预1 h后，流式细胞仪Annexin V/PI检测凋亡细胞比例：正常对照组、米诺环素对照组、谷氨酸组、米诺环素治疗组分别为5.1％、4.3％、15.2％、8.3％。Rh123各组阳性率分别为67.1%、54.2%、27.5%、32.4%。干预12 h后，MTT检测各组细胞吸光度均值分别为： 0.093±0.008、0.099±0.012、0.038±0.008、0.088±0.016。治疗组与正常组之间无显著差异（P>0.05），谷氨酸组与其它各组之间均存在显著差异（P<0.05）。NOS活力单位检测，以正常对照组均数为1，另3组与其比值的均数分别为： 0.987±0.219、1.513±0.472、1.176±0.259。其中谷氨酸组与其它3组之间存在显著差异（P<0.05）。结论:20 μmol/L米诺环素可显著减轻谷氨酸诱导的视网膜神经细胞凋亡，其作用机制与稳定线粒体膜电位，以及抑制NOS活性有关。

Minocycline inhibits rat retinal neural cell apoptosis induced by glutamate

AIM: To investigate the anti-apoptotic effect of minocycline on the rat retinal neural cells. METHODS: Primary cultures of retinal neural cells were prepared from neonatal SD rats. The expressions of NSE (neuron), GFAP (glial cells) and lectin (microglia) were detected by immunocytochemistry. Cultured cells were divided into normal control group (group A), minocycline control group (minocyline 20 μmol/L, group B), glutamate control group (glutamate 1 mmol/L, group C) and minocycline-treating group (minocycline 20 μmol/L+glutamate 1 mmol/L, group D). After 1 h intervention, Annexin V/PI flow cytometry was performed to evaluate the apoptotic cells with Annexin V/PI and mitochondria membrane potential (MMP) with Rh123. After 20 h intervention, MTT was used to test the cell viability, and culture supernatant was collected to test NOS activity. RESULTS: Annexin V/PI testing revealed that the cell apoptotic rate was 5.1％ in group A, 4.3％ in group B, 15.2％ in group C and 8.3％ in group D. MTT showed that mean absorbance was 0.093 in group A, 0.099 in group B, 0.038 in group C and 0.088 in group D. No significant difference between group A and group D was observed. Significant difference between group C and other three groups was found. MMP in group C was reduced compared with group A, which was reversed by minocycline treatment in group D. If NOS activity in group A was 1, that in group B was 0.987±0.219, in group C was 1.513±0.472 and in group D was 1.176±0.259, a significant difference between group C and other three groups was observed. CONCLUSION: Minocycline significantly decreases retinal apoptosis in neural cells treated with glutamate. The mechanism is related to suppressing NOS activity, and stabilizing the MMP in the cells.