The Design Of A Combination Drug With Orientin As The Key Ingredient And Its Anti-MIRI Mechanism

ObjectiveThe composition of Chinese medicine is complex and its quality is difficult to control.At present,the major problem in the international traditional Chinese medicine(TCM)is the lack of evidences of pharmacological mechanisms of TCM.With the development of modern TCM,the researches of multi-component Chinese medicine based on the principles of"clarity of material and mechanism " have become known as a new hotspot,and the research mode of component formula based on"components standard,quantitative composition-activity relationship(QCAR),component compatibility and optimization design"has been initially established.Previous studies have confirmed that orientin could reduce the damage of myocardial ischemia reperfusion injury(MIRI).In this study,orientin was used as the key to component design,explore the method of optimization——experimental design,model establish model,multi-objective optimization.How to component design,whether it is better than single component to reduce the damage of MIRI,are the main contents discussed in this paper.METHODS AND RESULTS1.Screening compounds for component optimizationFirstly,previous studies on plants containing orientin were carried out,and the selection conditions of constrained components were as follows:(1)flavonoid;(2)coexistence with orientin in the same plant;(3)research reports on antioxidant and anti-inflammatory effects.Five components of quercetin,vitexin,quercetin,luteolin and isoorientin were selected.In the second step,H9c2 cell viability were evaluated by CCK-8 assay.Thirdly,the hypoxia/reoxygenation(H/R)injury model of H9c2 cells was established by tri-gas incubator.The effects of various compounds on the survival rate and leakage rate of H9c2 cells were evaluated by lactate dehydrogenase(LDH)and CCK-8.The results showed that the proliferation of H9c2 cells was not significantly affected by the incubation time of orientin(0?200?M),quercitrin(0?200?M),vitexin(0?80?M),and isoorientin(0?k200?M);besides isoorientin,orientin,quercitin and vitexin could significantly reduce the cell injury rate(P<0.05).Therefore,onentin(0?50?M),quercitrin(0?40?M)and vitexin(0?24?M)are the most suitable components and concentration range for follow-up uniform test design.2.Study on the best combination of components based on the method of UD-SR-SAIn this experiment,different combinations of orientin,quercitrin and vitexin were obtained by uniform design(UD).The following pharmacology indicators were tested:1)Assay of CCK-8 was used to detect the effects of different combinations on the cell survival rate of H9c2 hypoxia/reoxygenation injury model;2)Assay of LDH was used to detect the cell leakage rate;3)nitric oxide(NO)was determined by NO assay kit.Based on the above pharmacodynamic experimental results,a stepwise regression model was established by R program,and the equation between the comprehensive pharmacodynamics and the dosage of orientin,quercetin and vitexin was obtained.Based on the theory of simulated annealing algorithm,we calculated the dosage of each compound by python,when the comprehensive efficacy is the best.Finally,pharmacodynamic experiments were carried out to verify the best combination.The results showed that the best combination could significantly improve the survival rate of H9c2 myocardial cells,reduce the cell damage rate and NO content,and the best combination had more obvious effect on reducing the cell damage rate than the single compound of orientin,quercitin and vitexin(P<0.05).This indicated that the eutherapeutic combination(OQV-e)had the optimum anti-MIRI effect than the single compound.3.Study on the mechanism of OQV-e on H/R injury of H9c2 cellsThe morphology of the damaged H9c2 cells was observed under optical microscope.It was found that OQV-e could significantly improve the damaged H9c2 cells.Assays of western blot was used to detect the expression of p-JNK,JNK,Beclin 1 and LC3 in H9c2 cells,and to evaluate the effect of the best ratio on the JNK pathway and autophagy in H9c2 cells damaged by hypoxia/reoxygenation.The results showed that compared with the model group,the ratio of p-JNK/JNK decreased,the ratio of LC3-II/LC3-I decreased significantly,and the expression le'vel of Beclin 1 increased significantly(P<0.05).It indicated that OQV-e could inhibit JNK signaling pathway and autophagy,thus protecting myocardium from H/R injury.Conclusion:1.Orientin(0-50 ?M),quercitrin(0-40?M)and vitexin(0-24?M)were the most suitable components and concentration ranges for follow-up uniform design.2.The optimum combination of components based on UD-SR-SA can protect the damaged cardiac myocytes from H/R,and can significantly reduce the damage rate of H/R cells than single compound.3.OQV-e on H/R cells may protect H/R H9c2 cells through inhibiting the JNK signaling pathway and inhibiting the excessive autophagy,thus achieving the purpose of protecting the injured cells from H/R.