人参皂苷Rg1治疗高糖损伤大鼠视神经细胞的机制研究:基于生物信息学分析

Mechanism of ginsenoside Rg1 in the treatment of high glucose induced optic nerve cell injury in rats based on bioinformatics analysis

目的通过SwissTarget及PubMed数据库预测人参皂苷Rg1治疗高糖损伤大鼠视神经细胞的潜在靶点及作用机制。方法利用PubMed compound网站获取人参皂苷Rg1的分子结构式并于SwissTarget数据库进行Rg1治疗全部疾病的靶点预测,通过高糖损伤视神经相关PubMed数据库数据集GSE 27382分析视神经损伤患者与健康人员差异基因的表达情况,将SwissTarget数据库的预测结果与PubMed数据库分析结果进行集合,最终筛选Rg1治疗高糖损伤大鼠视神经细胞的潜在靶点IL-2、PTAFR、PSEN2、PSENEN、NCSTN、APH1A、PSEN1、APH1B、HSP90AA1,将获得的靶点进行KEGG及GO富集分析,明确相关的生物过程及分子功能、信号通路,将Notch/MAPK信号通路选定为机制研究的内容。镜下摘取饲养的16只Wistar大鼠双侧视神经,使用含新生胎牛血清的DMEM/F12培养基进行原代培养2周,经过形态学验证后,将细胞分为对照组、高糖损伤组(高糖DMEM/F12培养24 h)、Rg1治疗组(10μg·L^(-1) Rg1及高糖DMEM/F12同时培养24 h)及Notch1抑制剂组(10μg·L^(-1) Tangerintin及高糖DMEM/F12同时培养24 h)。采用CCK-8法检测不同组别大鼠视神经细胞培养24 h、48 h、72 h的增殖能力,采用免疫印迹法验证Notch/MAPK信号通路蛋白表达的变化。结果细胞增殖实验结果显示,与对照组相比,高糖损伤组大鼠视神经细胞培养24 h、48 h、72 h后吸光度均明显下降(均为P<0.05);与高糖损伤组相比,Rg1治疗组大鼠视神经细胞培养24 h、48 h、72 h后吸光度均增加(均为P<0.05)。免疫印记法结果显示,与对照组相比,高糖损伤组大鼠视神经细胞Notch1受体蛋白表达下降,MAPK、JNK、ERK1、P38蛋白表达均增加;而与高糖损伤组相比,Rg1治疗组和Notch1抑制剂组大鼠视神经细胞中Notch1受体蛋白表达均升高,MAPK、ERK1、JNK、P38蛋白表达均下降。结论人参皂苷Rg1通过激活Notch1受体影响Notch/MAPK信号通路,进而缓解高糖引起的大鼠视神经损伤。

Objective To predict the potential target and mechanism of ginsenoside Rg1 in the treatment of high glucose induced optic nerve cell injury in rats by SwissTarget and PubMed data sets.Methods The molecular structure formula of ginsenoside Rg1 was obtained from PubMed compound website,and the targets of Rg1 in the treatment of all diseases were predicted in SwissTarget database.The expression of differential genes between patients with optic nerve injury and healthy people was analyzed by PubMed dataset GSE 27382.Finally,the predicted results of SwissTarget were integrated with PubMed to produce the potential targets of Rg1 in the treatment of high glucose induced optic nerve cell injury in rats,including IL-2,PTAFR,PSEN2,PSENEN,NCSTN,APH1A,PSEN1,APH1B,and HSP90AA1.The obtained targets were enriched with KEGG and GO,and the related biological processes,molecular functions and signaling pathways were identified.The Notch/MAPK signaling pathway was selected as the research objective.The bilateral optic nerves of 16 Wistar rats were harvested under microscope and cultured in DMEM/F12 medium containing fetal bovine serum for 2 weeks.After morphological verification,the cells were divided into control group,high glucose injury group(Cultured in high glucose DMEM/F12 medium for 24 h),Rg1 treatment group(cultured with 10μg·L^(-1) Rg1 and high glucose DMEM/F12 for 24 h)and Notch inhibitor group(cultured with 10μg·L^(-1) Tangerintin and high glucose DMEM/F12 for 24 h).CCK-8 was used to verify the proliferation ability of cells in different groups at 24 h,48 h and 72 h.Western blot was used to verify the protein expression change of Notch/MAPK signaling pathway.Results Compared with the control group,the optical density(OD)values of high glucose injury group at 24 h,48 h and 72 h were significantly decreased(all P<0.05).Compared with the high glucose injury group,the OD values of Rg1 treatment group at 24 h,48 h and 72 h were increased(all P<0.05).The results of Western blot showed that the protein expression level of Notch1 receptor decreased and the expression levels of MAPK,JNK,ERK1 and P38 increased in the high glucose injury group compared with those in the control group;the protein expression level of Notch1 receptor increased while MAPK,ERK1,JNK and P38 protein levels decreased in the Rg1 treatment group and Notch inhibitor group compared with those in the high glucose injury group.Conclusion Ginsenoside Rg1 can alleviate high glucose induced optic nerve injury by activating Notch1 receptor and affecting Notch/MAPK signaling pathway.