牛膝多肽对N-甲基-D-天冬氨酸(NMDA)介导的视网膜光感受器细胞损伤的保护作用

目的　探讨牛膝多肽（ABPP）对N-甲基-D-天冬氨酸(NMDA)介导的光感受器细胞损伤的保护作用，并初步探讨ABPP保护作用的可能机制。方法　在培养的光感受器细胞RGC-5中加入不同浓度的ABPP (0.05 mg·L^-1、0.10 mg·L^-1、0.50 mg·L^-1、1.00 mg·L^-1、5.00 mg·L^-1)作用10 h后，通过NMDA(0.1 mmol·L^-1)介导细胞损伤，同时设立NMDA组、正常组和地卓西平(MK-801)组。倒显微镜下观察各组细胞形态变化； MTT法检测细胞生存率；利用Hoechst 33258/PI双染和流式细胞仪测定细胞凋亡；钙离子成像观察细胞内钙离子变化；实时荧光定量PCR检测Bcl-2和Bax mRNA的变化。结果　NMDA组RGC-5细胞生存率较正常组明显下降（P<0.01);与NMDA组比较，0.10 mg·L^-1ABPP组、0.50 mg·L^-1 ABPP组和1.00 mg·L^-1 ABPP组RGC-5细胞生存率升高，差异均有统计学意义(均为P<0.05)；MK-801组细胞生存率也明显高于NMDA组(P<0.05)，与0.50 mg·L^-1ABPP组比较差异无统计学意义(P>0.05)。因此，在后续的实验中选择0.50 mg·L^-1为ABPP组的有效药物浓度。正常组、ABPP组、MK-801组细胞凋亡率与NMDA组比较差异均有统计学意义(均为P<0.05)。ABPP组、MK-801组细胞内钙离子荧光强度与NMDA组比较差异均有统计学意义（均为P<0.05）。实时荧光定量PCR检测结果显示，与NMDA组比较，正常组、ABPP组、MK-801组细胞内Bcl-2 mRNA表达均增加，Bax mRNA表达均降低，差异均有统计学意义(均为P<0.05)。结论　ABPP可抑制NMDA介导的视网膜光感受器细胞损伤，作用呈一定的浓度依赖性，其机制可能与抑制钙内流以及上调Bcl-2 mRNA表达、下调Bax mRNA的表达有关。

Protective effect of achyranthes bidentata polypeptide on retinal photoreceptor cell injury induced by N-methyl-D-aspartic acid

Objective To investigate the protective effect of achyranthes bidentata polypeptide(ABPP)on retinal photoreceptor cell injury caused by N-methyl-D-aspartic acid(NMDA)and explore its possible mechanisms.Methods Cultured retinal ganglion cell 5(RGC-5)cells were incubated with different concentrations of ABPP(0.05 mg·L^-1,0.10 mg·L^-1,0.50 mg·L^-1,1.00 mg·L^-1,and 5.00 mg·L^-1)for 10 h and then exposed to NMDA(0.1 mmol·L^-1)to induce cell injury.Meanwhile,the NMDA group,normal group,and dizocilpine(MK-801)group were set for comparison.Inverted microscopy imaging was used to observe changes in cell morphology.MTT assay was used to assess cell survival rate.Hoechst33258/PI dual staining and flow cytometry were used to detect cell apoptosis.Calcium ion imaging was used to observe the intracellular calcium concentration.Real-time polymerase chain reaction(RT-PCR)was used to determine mRNA levels of Bcl-2 and Bax in the cells.Results The viability of RGC-5 cells in the NMDA group was significantly lower than that in the normal group(P<0.01).Compared with the NMDA group,the viability of RGC-5 cells in the ABPP(0.10 mg·L^-1,0.50 mg·L^-1,and 1.00 mg·L^-1)groups significantly improved(all P<0.05).Cell viability in the MK-801 group was also significantly higher than that in the NMDA group(P<0.05),but had no statistically significant difference with the ABPP(0.50 mg·L^-1)group(P>0.05).Therefore,in later experiments,0.50 mg·L^-1 was selected as the effective drug concentration for ABPP groups.Cell apoptosis in the normal,ABPP,and MK-801 groups was significantly different from that in the NMDA group(all P<0.05).The intracellular calcium fluorescence intensity in the ABPP and MK-801 groups was significantly different from that in the NMDA group(both P<0.05).RT-PCR results showed that compared with the NMDA group,the mRNA expression levels of Bcl-2 in the normal group,ABPP group,and MK-801 group were significantly elevated,whereas those of Bax were significantly reduced(all P<0.05).Conclusion ABPP can inhibit the retinal photoreceptor cell injury induced by NMDA in a dose-dependent manner.The underlying mechanism may be related to the inhibition of calcium influx,up-regulation of Bcl-2 mRNA,and down-regulation of Bax mRNA.