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

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 indexlactate 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.