State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, 54 Xianlie Road South, Guangzhou 510060, China.
Exp Eye Res. 2010 Mar;90(3):397-404. doi: 10.1016/j.exer.2009.11.017. Epub 2009 Dec 7.
The purpose of the present study was to investigate the effect of Ganoderma spores lipid (GSL) on Bax, Bcl-xl and Caspase-3 expression in damaged retina and to address the effect of GSL on photoreceptor cell lesions induced by N-methyl-N-nitrosourea (MNU). Thirty 50-day-old female Sprague-Dawley rats were divided randomly into five groups to detect the dose-response effect of GSL by electroretinogram (ERG) analysis. Four groups received different daily GSL doses (0.5, 1, 2 and 4 g/kg, respectively) and one control group received no treatment. Sixty rats were divided randomly into an untreated MNU model control group and the GSL treatment group. Retina tissue samples were obtained sequentially 0 h before and 1, 3, 7 and 10 d after MNU injection. Expressions of Bax, Bcl-xl and Caspase-3 were detected by RT-PCR and immunofluorescence assays, then photoreceptor cell apoptosis was confirmed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-digoxigenin nick-end labeling (TUNEL) signals. GSL had a dose-response effect on retina ERG and reversed retinal photoreceptor damage induced by MNU. RT-PCR analysis demonstrated that transcription levels of Bax, Bcl-xl and Caspase-3 in MNU control group and GSL treatment group were all upregulated on 1 d (p < 0.01) and peaked on 3 d (p < 0.01) after MNU injection compared to before MNU injection. GSL treatment significantly decreased mRNA levels of Bax on 1, 3, 7 and 10 d vs. MNU group (all p < 0.01) and mRNA levels of Caspase-3 on 1, 3, 7 d (p < 0.01) and 10 d (p < 0.05) vs. MNU group. Bcl-xl was clearly upregulation on 1, 3, 7 and 10 d vs. MNU group (all p < 0.01). Expression ratios of Bax/Bcl-xl were raised after MNU injection on 1 and 3 d vs. 0 h before MNU (both p < 0.01), peaked on 3 d, then dropped after GSL treatment on 1, 3, 7 and 10 d vs. MNU group (all p < 0.01). Immunofluorescence assays showed GSL decreased Bax and Caspase-3 positive staining levels in retina and increased Bcl-xl level. TUNEL-positive cells were evoked only in the outer nuclear layer and peaked on 3 d in rats receiving MNU (p < 0.01 vs. 0 h before MNU). GSL administration decreased apoptosis levels significantly, and the apoptotic indexes (AIs) of the GSL group were lower than those of MNU group on 1 and 3 d (both p < 0.01). Taken together, these data suggest that GSL may regulate the expressions of Bax, Bcl-xl and Caspases-3, inhibiting MNU-induced rat photoreceptor cell apoptosis and protecting retinal function.
本研究旨在探讨灵芝孢子油（GSL）对受损视网膜中 Bax、Bcl-xl 和 Caspase-3 表达的影响，并探讨 GSL 对 N-甲基-N-亚硝脲（MNU）诱导的光感受器细胞损伤的作用。
将 30 只 50 日龄雌性 Sprague-Dawley 大鼠随机分为五组，通过视网膜电图（ERG）分析检测 GSL 的剂量反应效应。四组分别给予不同的每日 GSL 剂量（0.5、1、2 和 4 g/kg），一组对照组不给予任何治疗。
60 只大鼠随机分为未处理的 MNU 模型对照组和 GSL 治疗组。MNU 注射前 0 小时和注射后 1、3、7 和 10 天分别获取视网膜组织样本。通过 RT-PCR 和免疫荧光检测 Bax、Bcl-xl 和 Caspase-3 的表达，然后通过末端脱氧核苷酸转移酶介导的脱氧尿苷三磷酸-地高辛标记（TUNEL）信号确认光感受器细胞凋亡。
GSL 对视网膜 ERG 具有剂量反应效应，并逆转了 MNU 诱导的视网膜光感受器损伤。RT-PCR 分析表明，与 MNU 注射前相比，MNU 对照组和 GSL 治疗组中 Bax、Bcl-xl 和 Caspase-3 的转录水平在 MNU 注射后 1 天（p<0.01）和 3 天（p<0.01）均上调，并在 3 天达到峰值（p<0.01）。与 MNU 组相比，GSL 治疗组在 1、3、7 和 10 天时 Bax 的 mRNA 水平明显降低（均 p<0.01），在 1、3、7 天时 Caspase-3 的 mRNA 水平降低（p<0.01），在 10 天时 Caspase-3 的 mRNA 水平降低（p<0.05）。Bcl-xl 在 1、3、7 和 10 天时均明显上调（均 p<0.01）。与 MNU 注射前 0 小时相比，MNU 注射后 1 和 3 天时 Bax/Bcl-xl 表达比值升高（均 p<0.01），在 3 天时达到峰值，然后在 GSL 治疗后 1、3、7 和 10 天时均低于 MNU 组（均 p<0.01）。免疫荧光检测显示 GSL 降低了视网膜中 Bax 和 Caspase-3 的阳性染色水平，增加了 Bcl-xl 水平。TUNEL 阳性细胞仅在外核层中出现，在 MNU 注射后 3 天达到峰值（与 MNU 注射前相比，p<0.01）。GSL 给药可显著降低细胞凋亡水平，GSL 组在 1 和 3 天时的凋亡指数（AI）均低于 MNU 组（均 p<0.01）。
综上所述，这些数据表明 GSL 可能通过调节 Bax、Bcl-xl 和 Caspases-3 的表达，抑制 MNU 诱导的大鼠光感受器细胞凋亡，保护视网膜功能。
