Study On Evaluation Of Active Quality And Toxicity-reducing And Efficacy-enhancing Of Aconitum Pendulum

Objective:In this study,the myocardial protective effect and safety range of aconitum plants were studied by constructing an in vitro heart failure model induced by pentobarbital sodium.And the active quality of aconitum plants was compared with that of the same species,so as to establish an activity-oriented comprehensive analysis method for evaluating the quality of near-source medicinal materials,which provided a theoretical basis for clinical selection of aconitum medicinal materials.H9c2 cell model was used to comprehensively evaluate the toxicity-reducing and efficacy-enhancing of different proportions of Terminalia chebula Retz.（TCR）and Aconitum pendulum Busch.（APB）,and thus decide the best ratio between them.At the same time,their regulation and the mechanism of TRP targets（TRPV1～V4,TRPA1 and TRPM8）,especially TRPM8 on myocardial cells was further explored.Methods:（1）Five kinds of aconitum plants,APB,Aconitum soongaricum Stapf.（ASS）,Aconitum brachypodum Diels.（ABD）,Aconitum contortum Finet&Gagnep.（ACFG）and Aconitum leucostomum Worosch.（ALW）,were collected and extracted.H9c2 cardiomyocytes were used to study the efficacy/toxicity of total alkaloids.MTT and real-time cell analyzer were used to detect cell viability.The ratio of IC₅₀to EC₅₀was calculated and the safety treatment index(TI=IC₅₀/EC₅₀)was compared.Then,grey correlation analysis was used to evaluate the weight of each aconitum alkaloids in the efficacy/toxicity relationship.Finally,the efficacy/toxicity targets of aconitum plants were predicted and verified by network pharmacology.（2）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 extracts of the mixtures were obtained respectively for further study.The efficacy/toxicity of the extracts was studied using H9c2 cells.The ratio of IC₅₀to EC₅₀was calculated through cell viability detection,and TI values were compared to comprehensively sort the toxicity-reducing and efficacy-enhancing of the extracts with different ratios.The dual regulation of TRP targets m RNA expression by APB and the best mixture was explored via q RT-PCR technique.The effects of main active components of the extract on TRP targets were further explored through molecular docking technology.In vitro heart failure model was induced by 8 mg/m L pentobarbital sodium.Cell viability,cardiac function index（LDH and CK）levels,mitochondrial membrane potential（ΔΨ）,reactive oxygen species（ROS）levels,calcium ion(Ca²⁺)concentration and calcium regulatory genes m RNA were measured,respectively.Results:（1）According to the TI values of the five plants,the following active quality ranks were obtained:APB>ALW>ACFG>ABD>ASS,and the TI value of APB was the highest.In the grey correlation analysis of efficacy and toxicity,aconitine had the highest correlation index and the strongest component-effect correlation.After systematic pharmacological analysis,three core targets of AKT1,IL6 and KCNH2 and PI3K/AKT pathways were selected to investigate the mechanism.The result indicated that aconitine and five medicinal extracts could down-regulate the m RNA expression of AKT1,IL6 and KCNH2 at the effective concentration（100 ng/m L）.However,at the toxic concentration（125μg/m L）,aconitine and five medicinal extracts could up-regulate the m RNA expression of AKT1 and IL6 in normal H9c2 cells.（2）According to the TI value of each mixture ratio,the following safety range grades were obtained:mixture ratio of TCR and APB2:1>3:1>4:1>1:1>APB.When the ratio of TCR and APB is 2:1,the TI value is the highest;APB（100 ng/m L）could significantly up-regulate the m RNA expression of TRPV1,TRPV2 and TRPM8,and down-regulate the m RNA expression of TRPV4and TRPA1,while the regulation was opposite at the toxicity concentration.By comparing the affinity of the target to each component,it was found that the affinity of TRPM8 to each component was strongest.The extract of optimal mixture and its active monomer compounds could significantly increase cell viability,attenuate the pathological injuries such as LDH leakage,CK increase,ΔΨdecrease,intracellular ROS and Ca²⁺increases,intracellular ROS and Ca²⁺increases,down-regulat m RNA levels of calcium regulatory genes such as FKBP1B and TRPM8,and up-regulat Ry R2 and NOX2 m RNA levels induced by pentobarbital sodium.Conclusion:The dual effects of aconitum on heart can be evaluated and regulated by TI.Grey correlation analysis provides a relatively fast and accurate research method for the quality and material basis of medicinal materials.Aconitine is one of the main efficacy/toxicity substances of aconitum,and its mechanism may be related to the regulation of AKT1,IL6,KCNH2 genes and PI3K/AKT pathway.The best combination ratio of TCR and APB is 2:1.The combination of TCR and APB can protect myocardial injury by affecting TRP genes,especially TRPM8,regulating FKBP1B and Ry R2 gene in sarcoplasmic reticulum and decreasing mitochondrion ROS release and Ca²⁺disorder.