Study On The Mechanism Of Sanao Decoction And Its Compatibility Of Components In The Intervention Of Asthma By TRPV1

Objective:In order to explore the effect and mechanism of Sanao Decoction on asthma through TRPV1 from the perspectives of in vivo,in vitro,and drug receptor molecular interactions,as well as to elucidate the scientific compatibility connotation of Sanao Decoction,Sanao Decoction whole formula and its decomposed formula were used to intervene with the OVA compound PM2.5 induced asthma mouse models,Sanao Decoction whole formula and main components alone and in combination were used to intervene with IL-4 induced 16HBE cell,molecular docking computer simulation technology and genetic modification technology were used to predicte possible target site.Method:1.In order to explore the mechanism of Sanao Decoction in the effect of asthma through TRPV 1,and the contribution of each disassembled formula,Sanao Decoction and its disassembled formula were used to intervene OVA compound PM2.5-induced asthma mouse model.2.In order to investigate the effect of Sanao decoction on Ca2+influx through TRPV 1 and elucidate its compatibility,we analyzed the main components of Sanao decoction through mass spectrometry,and then used computer simulation technology to analyze the binding ability of each component with TRPV1.We detected the Ca2+influx of 16HBE cells through TRPV1 that were intervened by Sanao decoction and its main components alone or in combination.3.Through computer simulation technology,we investigated the binding strength and mode of the main components of Sanao decoction with TRPV 1,and predicted the molecular mechanism by which the main components in Sanao decoction inhibit Ca2+influx through the TRPV1 channel.4.Based on computer simulation analysis,we mutated the amino acid sites of the main components in Sanao decoction that bind to TRPV 1.The mutations were as follows:the amino acid E at position 211 of the ATP binding region was mutated to A,the D mutation at position 509 of the conformational region was mutated to A,and the D mutation at position 647 of the extracellular Ca2+binding region was mutated to A.The Ca2+influx of the mutated cells through the TRPV1 channel was detected,confirming the main sites of the main components in Sanao decoction that bind to TRPV1,and elucidating the molecular mechanism involved,and the significance of the compatibility of the main components of Sanao decoction.Result:1.Through the study of the effects and mechanisms of Sanao Deoction on asthma mice,we found that the levels of eosinophils in peripheral blood and BALF increased,lung tissue pathological damage,airway mucus hypersecretion,airway hyperresponsiveness,immune factors IL-4 and IL-13 levels increased in BALF,and neuropeptide SP and PGE2 levels increased.The pathological symptoms of asthma in mice were improved to varying degrees after the intervention of Sanao Decoction and its disassembled formula.The effect of Sanao Decoction,the combination of Ephedra and Almond,and the combination of Ephedra and Licorice was better,while the effect of the combination of Almond and Licorice was relatively weaker.2.After administering TRPV1 inhibitors to intervene in asthma mice,good effects were achieved,confirming once again that TRPV1 is likely involved in the process of asthma.Both immunohistochemistry and western blot experiments confirmed that TRPV1 was upregulated in asthma mice and localized on the surface of the bronchial airway.After the intervention of Sanao decoction,disassembled formula and TRPV1 inhibitor,the expression of TRPV1 was significantly reduced.3.According to the results of the Ca2+influx experiments through TRPV1,we found that freeze-dried powder of Sanao decoction could effectively inhibit the Ca2+influx through TRPV1,and the effect was very stable.By detecting the effects of gradient concentrations of Ephedrine,Amygdalin and Enoxolone alone or in combination on Ca2+influx through TRPV1,it was confirmed that within a certain effective concentration range,Ephedrine,Amygdalin and Enoxolone alone all have a significant inhibitory effect on Ca2+ influx;Within the entire gradient concentration range selected in this study,the combination of Ephedrine and Amygdalin,and the combination of Ephedrine and Amygdalin and Enoxolone significantly inhibited Ca2+influx,and the effect was very stable;Within a certain effective concentration range,the combination of Ephedrine and Enoxolone,and the the combination of Amygdalin and Enoxolone also has a significant inhibitory effect on Ca2+ influx.4.Molecular docking computer simulation was performed on Ephedrine,Amygdalin and Enoxolone with TRPV1,and it was found that the binding strength of these three components with TRPV1 was consistent with their inhibition of Ca2+influx through TRPV1.Due to its structural characteristics,Ephedrine can form strong ionic interactions with TRPV 1,and there are also many possibilities for binding sites.At the same time,it does not form competitive binding with Amygdalin.Amygdalin mainly binds to the transmembrane region of TRPV1 with strong binding ability,and can form a synergistic effect with Ephedrine to enhance the effect.However,Enoxolone binds weakly to TRPV1 and binds to Amygdalin at the same binding site,with steric hindrance and cannot form a synergistic effect.5.Compared with wild-type cells,16HBE cells with E211 mutation showed no change in Ca2+ influx through TRPV1,however,16HBE cells with D509 mutation and 16HBE cells with D647 mutation showed a significant decrease in Ca2+ influx through TRPV1,indicating that the D509 and D647 sites may play an important role in Ca2+ influx through TRPV1.Ephedrine,Amygdalin and Enoxolone can all significantly inhibit Ca2+ influx in 16HBE cells with E211 site mutations,but cannot further inhibit Ca2+influx in 16HBE cells with D509 or D647 site mutations.This suggests that D509 and D647 sites are likely binding sites for Ephedrine,Amygdalin,and Enoxolone to exert their effects.Conclusion:1.The effect of Sanao decoction on asthma may be related to TRPV1.The combination of Ephedra and Almond,and the combination of Ephedra and Licorice has a better effect,followed by the combination of Almond and Licorice.2.Sanao decoction,along with its main components Ephedrine,Amygdalin and Enoxolone,all have an inhibitory effect on Ca2+influx through TRPV1.In the component compatibility,the combination of Ephedrine and Amygdalin and Enoxolone,and the combination of Ephedrine and Amygdalin both has a better effect,followed by the combination of Ephedrine and Enoxolone and the combination of Amygdalin and Enoxolone.3.Molecular docking computer simulation predicts that Ephedrine can form strong ionic interactions with TRPV1,with more possibilities for binding sites and no competitiveness with Amygdalin.Amygdalin has a strong binding ability with TRPV1 and can form a synergistic effect when combined with Ephedrine.The binding ability of Enoxolone to TRPV1 is weak,and it binds to the same binding site of TRPV 1 with Amygdalin,which has steric hindrance and cannot form a synergistic effect.4.The D509 and D647 sites may play an important role in the Ca2+influx through TRPV1.The main binding sites of Ephedrine,Amygdalin and Enoxolone to TRPV 1 may be the D509 and D647 sites,which can be directly bound to the extracellular Ca2+binding region of the TRPV1 receptor,or by changing the conformation of TRP V1,to achieve the effect of inhibiting Ca2+influx,reflecting the connotation of the synergistic effect of multiple components and targets after the compatibility of traditional Chinese medicine.