诃子配伍铁棒锤对戊巴比妥钠诱导的H9c2心肌细胞毒效关系的影响及机制

Influence and Mechanism of Toxicity-efficacy Relationship Between the Combination of Terminalia chebula Retz. and Aconitum pendulum Bush. on Pentobarbital Sodium-induced H9c2 Myocardial Cells

关键词:  铁棒锤  诃子  安全治疗指数  减毒增效  TRP靶点

Key words:  Aconitum pendulum Busch.  Terminalia chebula Retz.  safety treatment index  toxicity-reducing and efficacy-enhancing  TRP targets

摘要:

目的 综合评价诃子(Terminalia chebula Retz.，TCR)-铁棒锤(Aconitum pendulum Busch.，APB)不同配比对H9c2心肌细胞毒效关系的影响，寻找两者的配比规律，并探究TCR-APB药对心肌的保护机制。方法 以APB为参照，按照1∶1，2∶1，3∶1及4∶1的比例配比TCR与APB药材并提取总提物，以戊巴比妥钠构建大鼠H9c2心肌细胞心力衰竭模型，计算4种配比总提物作用于H9c2心肌细胞的IC₅₀与EC₅₀值，比较其安全治疗指数(treatment indices，TI=IC₅₀/EC₅₀)；并进一步探究TCR-APB药对H9c2心肌细胞活力、心肌指标[乳酸脱氢酶(lactate dehydrogenase，LDH)及肌酸激酶(creatine kinase，CK)]水平、线粒体膜电位(ΔΨ)、活性氧(reactive oxygen species，ROS)水平、钙离子(Ca²⁺)浓度及TRP基因、钙调节相关基因的mRNA水平。结果 TI计算结果显示，TCR与APB以2∶1比例配比时安全范围最好；100 ng·mL^–1的APB可显著上调TRPV1、TRPV2及TRPM8 mRNA水平，使TRPV4与TRPA1的mRNA水平显著下调，毒性浓度范围下的调节则相反；分子对接结果发现TRPM8对各成分的亲和力均比其他靶点强；最佳配比提取物及其活性单体均可显著提高细胞活力，改善戊巴比妥钠损伤导致的H9c2心肌细胞LDH渗漏、CK水平上升、ΔΨ降低、细胞内ROS水平上升、胞内Ca²⁺浓度增加以及FKBP1B和TRPM8的mRNA水平下调、RyR2和NOX2的mRNA水平上调等。结论 TCR与APB配伍均可缓解H9c2心肌细胞损伤，起到减毒增效的作用，其最佳配比为2∶1，二者配伍可以通过影响TRP基因，特别是TRPM8，调节肌质网中FKBP1B与RyR2基因以及线粒体中ROS水平，纠正Ca²⁺紊乱，从而保护心肌免受损伤。

Abstract:

OBJECTIVE To comprehensively evaluate the toxicity-efficacy relationship of different proportions of Terminalia chebula Retz.(TCR) and Aconitum pendulum Busch.(APB) on H9c2 myocardial cells, thus investigating the rules of ratio between them, and to explore the protective mechanism of TCR-APB on myocardial. METHODS With APB as the control, TCR was mixed with APB by the ratios of 1∶1, 2∶1, 3∶1 and 4∶1, and the total extracts were obtained. The heart failure H9c2 myocardial cells injury model was induced by pentobarbital sodium, and the IC₅₀ and EC₅₀ of the total extracts of four ratios acting on H9c2 myocardial cells were calculated. Their safety treatment indices(TI=IC₅₀/EC₅₀) were additionally compared. Effect of TCR-APB on H9c2 myocardial cells viability, myocardial index[lactate dehydrogenase(LDH) and creatine kinase(CK)] levels, mitochondrial membrane potential(ΔΨ), reactive oxygen species(ROS) levels, inter-cellular calcium ion(Ca²⁺) concentration as well as TRP and calcium regulatory genes mRNA were measured, respectively. RESULTS According to the TI value of each mixture ratio, it showed the best safety range when the TCR and APB in a ratio of 2∶1; APB(100 ng·mL^-1) could significantly up-regulate the mRNA expression of TRPV1, TRPV2 and TRPM8, and significantly down-regulate the mRNA expression of TRPV4 and TRPA1. While the regulation was opposite at the toxic concentration. The molecular docking results showed that the affinity of TRPM8 to each component was the strongest. The extract of optimal mixture and its active monomer compounds could all significantly increase cell viability, attenuate the pathological injuries such as LDH leakage, CK increase, ΔΨ decrease, intracellular ROS and Ca²⁺ increases, down-regulate mRNA levels of FKBP1B and TRPM8, and up-regulate RyR2 and NOX2 mRNA levels induced by pentobarbital sodium. CONCLUSION The combination of TCR and APB can all attenuate the H9c2 myocardial cells injury and play a role in reducing toxicity and increasing efficacy. The best combination ratio of TCR and APB is 2∶1. The combination of TCR and APB can protect myocardial from injury by affecting TRP genes, especially TRPM8, regulating FKBP1B and RyR2 gene in the sarcoplasmic reticulum and the content of ROS in mitochondria, thereby correcting Ca²⁺ disorders.