N-甲基小檗胺对细胞内钙稳态的调节作用  

Regulation of N-methyl berbamine on intracellular calcium homeostasis

作　　者：杨东凝 周诗 李玥霖 朱俊蒙 郝丽英 胡慧媛 YANG Dongning;ZHOU Shi;LI Yuelin;ZHU Junmeng;HAO Liying;HU Huiyuan(Department of Pharmaceutical Toxicology,School of Pharmacy,China Medical University,Shenyang 110122,China)

机构地区：[1]中国医科大学药学院药物毒理学教研室,沈阳110122

出　　处：《中国医科大学学报》2025年第2期97-102,共6页Journal of China Medical University

基　　金：国家自然科学基金(81100108);辽宁省教育厅基本科研项目(JYTMS20230130);辽宁省科学技术计划(2022JH2/20200069)。

摘　　要：目的 探讨N-甲基小檗胺(N-MB)对H9c2心肌细胞内钙稳态的调节作用,从而明确N-MB心肌保护作用的可能机制。方法通过MOE软件预测N-MB与Ca_(V)1.2通道的结合并判断二者的结合能力与结合模式。通过全细胞膜片钳技术记录稳定表达hCa_(V)1.2的HEK293细胞内Ca_(V)1.2钙通道电流的变化,观察N-MB (30μmol/L)对Ca_(V)1.2钙通道电流的影响。将Fluo 3-AM荧光探针装载至H9c2心肌细胞中,激光共聚焦显微镜检测N-MB (3、30μmol/L)对心肌细胞内Ca^(2+)浓度的影响。实时定量PCR检测N-MB (3、30μmol/L)对H9c2心肌细胞中Ca^(2+)调控相关基因Cacna1c、Cacnb2、Ryr2、Serca2a、Ncx1表达的影响。结果 经MOE软件预测,N-MB和Ca_(V)1.2通道可以结合,二者相互作用的结合位点主要涉及Phe1191、Thr1420、Asn771等,作用方式有H-donor、pi-pi、pi-H 3种。N-MB(30μmol/L)对Ca_(V)1.2钙通道电流可产生明显抑制作用,其抑制率为(76.09±7.41)%。N-MB (3μmol/L、30μmol/L)作用于H9c2心肌细胞后细胞内钙信号的荧光强度明显增强(P <0.01)。与对照组相比,N-MB (30μmol/L)干预后,H9c2心肌细胞Cacna1c、Serca2a、Ncx1表达差异无统计学意义(P> 0.05),而Cacnb2表达显著减少(P <0.001),Ryr2表达显著增加(P <0.05)。结论 N-MB可以与Ca_(V)1.2钙通道结合。N-MB可能通过抑制Ca_(V)1.2钙通道的表达而抑制钙电流;同时可能通过促进Ryr2表达而升高细胞内Ca^(2+)浓度来参与调节细胞内钙稳态,发挥其心肌保护作用。Objective To explore the regulatory role of N-methyl berbamine(N-MB) in intracellular calcium homeostasis in H9c2 cardiomyocytes,and,thereby,clarify the possible mechanism of the myocardial protective effect of N-MB.Methods Binding of N-MB to Ca_V1.2 channels was simulated using the MOE software,and the binding affinity and binding mode were determined.The hCa_V1.2 gene was transfected into HEK293 cells,and the effect of N-MB(30 μmol/L) on the Ca_V1.2 current was detected using the whole-cell patch clamp technique.In addition,a Fluo 3-AM fluorescent probe was loaded into H9c2 cardiomyocytes,and the effect of N-MB(3,30 μmol/L)on intracellular calcium ion concentration was observed under a laser confocal microscope.The effect of N-MB(3,30 μmol/L) on the expression of Ca~(2+) regulation-related genes Cacna1c,Cacnb2,Ryr2,Serca2a,and Ncx1 in H9c2 cardiomyocytes was examined using real-time quantitative PCR.Results N-MB was predicted to bind to Ca_V1.2 channels.The binding sites mainly involved Phe1191,Thr1420,and Asn771,and the binding modes were H-donor,pi-pi,and pi-H.N-MB(30 μmol/L) significantly inhibited Ca_V1.2 currents,with an inhibition rate of 76.09%±7.41%.The fluorescence intensity of intracellular Ca~(2+) level in H9c2 cardiomyocytes was significantly enhanced with N-MB treatment(3,30 μmol/L,P < 0.01).Compared with the control group,differences in the expression of Cacna1c,Serca2a,and Ncx1 in H9c2 cardiomyocytes were not significant after N-MB(3,30 μmol/L) intervention(P > 0.05),whereas the expression of Cacnb2 significantly reduced(P < 0.001) and the expression of Ryr2 significantly increased(P < 0.05).Conclusion N-MB binds to Ca_V1.2 calcium channels.N-MB may regulate intracellular calcium homeostasis by inhibiting calcium currents by decreasing the gene expression of Ca_V1.2 calcium channels.Additionally,N-MB may also increase intracellular Ca~(2+) concentration by promoting the expression of Ryr2,which could be the mechanism underlying the myocardial protective effect of N-MB.

关 键 词：N-甲基小檗胺 细胞内钙稳态 Ca_(V)1.2钙通道 

分 类 号：R966[医药卫生—药理学]