摘要
目的:利用串联质谱标记(TMT)定量蛋白质组学技术探究参芎葡萄糖注射液(SGI)拮抗过氧化氢(H_(2)O_(2))诱导H9c2细胞氧化损伤的作用机制。方法:体外培养H9c2细胞,H_(2)O_(2)诱导H9c2细胞建立氧化损伤模型,利用细胞增殖与毒性检测法(MTS)检测细胞存活率,高效液相色谱法(HPLC)进行肽段分级,超高效液相色谱-质谱联用技术检测H9c2细胞的蛋白表达,使用MaxQuant(v1.5.2.8)进行数据检索,高分辨质谱定量分析筛选差异表达蛋白,通过基因本体(GO)和京都基因与基因组百科全书(KEGG)数据库对差异表达蛋白进行生物信息学分析,蛋白免疫印迹法(Western blot)检测细胞内脂质结合蛋白2(Plin2)和原肌球蛋白1(Tpm1)的蛋白表达水平进行验证。结果:通过谱图解析鉴定有48608个特异性肽段,5903个可定量蛋白。与模型组比较,SGI组有82个差异表达的蛋白,其中有22个蛋白上调,60个蛋白下调。GO分析结果显示,差异表达蛋白主要参与程序性细胞死亡调节、细胞增殖调控、心血管系统发育和细胞迁移等生物进程。KEGG分析结果表明,这些蛋白与过氧化物酶体增殖物激活受体(PPAR),黏着斑和磷脂酰肌醇3-激酶/蛋白激酶B(PI3K/Akt),Ras相关蛋白1(Rap1)等通路有关。Western blot结果显示,与模型组比较,SGI能显著增加Plin2蛋白表达水平,显著降低Tpm1蛋白表达水平(P<0.01),与蛋白质组学结果一致。结论:提示SGI可能通过调控PPAR,黏着斑,PI3K/Akt和Rap1等通路抑制细胞凋亡,从而拮抗H_(2)O_(2)诱导细胞氧化损伤,但需要后续实验进一步验证研究。
Objective:To explore the mechanism of Shenxiong glucose injection(SGI)in inhibiting hydrogen peroxide(H_(2)O_(2))-induced oxidative damage in H9c2 cells by tandem mass tags(TMT)-labeled quantitative proteomics.Method:H9c2 cells cultured in vitro were exposed to H_(2)O_(2)for inducing oxidative damage.The cell viability was measured by cell proliferation and cytotoxicity assay(MTS),followed by peptide fractionation by high performance liquid chromatography(HPLC)and protein expression detection in H9c2 cells by ultrahigh performance liquid chromatography-mass spectrometry.MaxQuant(v1.5.2.8)was utilized for data retrieval,and the high-resolution mass spectrometry was conducted to screen out differentially expressed proteins,which were then subjected to gene ontology(GO)and Kyoto encyclopedia of genes and genomes(KEGG)enrichment analysis.The protein expression levels of perilipin 2(Plin2)and tropomyosin 1(Tpm1)in cells were measured by Western blot.Result:The spectral analysis yielded 48608 specific peptide fragments and 5903 quantifiable proteins.Compared with the model group,the SGI group exhibited 82 differentially expressed proteins,of which 22 were up-regulated and 60 were down-regulated.GO analysis results showed that the differentially expressed proteins were mainly involved in biological processes such as programmed cell death regulation,regulation of cell proliferation,cardiovascular system development,and cell migration.As revealed by KEGG analysis,these proteins were mainly related to peroxisome proliferator-activated receptor(PPAR),focal adhesion,phosphatidylinositol 3-kinase/protein kinase B(PI3 K/Akt),and Ras-related protein 1(Rap1)pathways.Western blot results demonstrated that compared with the model group,SGI significantly increased the Plin2 protein expression and decreased the Tpm1 protein expression(P<0.01),consistent with the proteomics results.Conclusion:SGI may inhibit cell apoptosis and antagonize H_(2)O_(2)-induced cell oxidative damage by regulating PPAR,focal adhesion,PI3 K/Akt and Rap1 pathways,which should be further verified by subsequent experiments.
作者
陆定艳
吴忠秀
孙佳
陆苑
王永林
李勇军
郑林
刘亭
LU Ding-yan;WU Zhong-xiu;SUN Jia;LU Yuan;WANG Yong-lin;LI Yong-jun;ZHENG Lin;LIU Ting(Engineering Research Center for the Development and Application of Ethnic Medicine and Traditional Chinese Medicine,Ministry of Education,Stale Key Laboratory of Functions arid Applications of Medicinal Plants,Guizhou Medical University,Guiyang 550004,China;Provincial Key Laboratory of Pharmaceutics in Guizhou Province,Guizhou Medical University,Guiyang,550004,China;School of Pharmaceutical Sciences,Guizhou Medical University,Guiyang 550025,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2022年第1期141-149,共9页
Chinese Journal of Experimental Traditional Medical Formulae
基金
国家自然科学基金项目(81760699,U1812403-05)
贵州省自然科学基金资助项目(黔科合基础[2019]1280号)
贵州省普通高等学校科技拔尖人才项目(黔教合KY字[2021]033)
贵州省优秀青年科技人才项目(黔科合平台人才[2021]5619号)。
