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  

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

作　　者：张芳玲 章聪 邵蔚 ZHANG Fangling;ZHNAG Cong;SHAO Wei(Department of Ophthalmology,Nanjing Hospital Affiliated to Nanjing University of Chinese Medicine,Nanjing 210003,Jiangsu Province,China;Department of Hepatobiliary Surgery,the Affiliated Suzhou Hospital of Nanjing Medical University,Suzhou 215002,Jiangsu Province,China)

机构地区：[1]南京中医药大学附属南京医院眼科,江苏省南京市210003 [2]南京医科大学附属苏州医院肝胆外科,江苏省苏州市215002

出　　处：《眼科新进展》2022年第2期108-112,共5页Recent Advances in Ophthalmology

基　　金：国家自然科学基金资助(编号81300323)。

摘　　要：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^(-1)ABPP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^(-1)ABPP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。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

关 键 词：牛膝多肽 N-甲基-D-天冬氨酸 视网膜光感受器细胞 凋亡 

分 类 号：R774[医药卫生—眼科]