The Mechanism Of Analgesic Effect For Cinnamon-dried Ginger Herb Pair Based On Network Pharmacology

Objective: In this study,the effect and mechanism of cinnamon-dried ginger herb pair on resistance of cold pain were explored through in vivo experiment,in vitro experiment and network pharmacology,which laid a theoretical foundation for the development of drugs to improve cold pain.Methods:1.Effect of cinnamon-dried ginger herb pair on pain behavior of miceFoot pain and abdominal pain by cold were simulated by cold plate experiment and0.75% mustard oil.The first occurrence time of pain behavior and the total number of pain behavior within a certain period of time were observed.The pathological changes of colonic tissue.2.Prediction of mechanism of cinnamon-dried ginger herb pair for cold pain treatment based on network pharmacologyThe effective components were screened by TCMSP database,and the target of effective components were predicted by SEA,SWISS and STITCH database.Disease targets of cold pain were collected by Gene Cards and OMIM database using "cool and pain" as search terms.The intersection of the two targets was used to obtain the common target.Construction of component-target network and PPI network rendering using common targets and visualization using Cytoscape3.9.1;Score the parameters Betweenness,Closeness and Degree of nodes in the two networks respectively.Codes whose scores are greater than the average are taken in each network,and the intersection is the predicted core target or components.GO function and KEGG pathway enrichment analysis were performed on the co-acting targets.Finally,the core target was verified by Western Blot.3.Effects of Cinnamomum Cinnamomi and Ginger on the function of ion channels in primary dorsal root ganglion cells and its preli minary mechanism.The calcium ion imaging technique was used to investigate:(1)the effects of different concentrations of 6-gingerol and cinnamaldehyde on the activities of TRPA1 and TRPV1 channels of DRG cells before and after cold stimulation;(2)The desensitization of cinnamaldehyde and 6-gingerol on TRP channels after cold stimulation of DRG cells was investigated under the condition of simultaneous ad ministration of TRPA1 and TRPV1 antagonists;(3)Through the joint intervention of PLC and CaN,the key factors in the channel desensitization process,the role of PLC and CaN in the regulation of TRP channel by 6-gingerol and cinnamaldehyde was investigated.At the same time,the Western Blot experiment was used to investigate the effect of each group’s intervention treatment on the expression of TRPA1,TRPV1 and CGRP,the key proteins of the channel,under cold stimulation.Results:1.Compared with the cold stimulation group,the ad ministration group could signifi CaNtly prolong the time of the first occurrence of pain behavior and reduce the frequency of pain behavior.In addition,cinnamon-dried ginger herb pair CaN improve the change of colonic tissue morphology and reduce the expression of inflammatory factors IL-6 and TNF-α in cold abdo minal pain model mice.2.A total of 50 components of cinnamon and 58 components of dried ginger were obtained,and a total of 1948 component targets and 85 disease targets were collected.After intersection,39 common targets were obtained.9 core targets and 11 core components were predicted by the composition-target network,and 11 core targets were predicted by PPI network.After intersection,the results were TRPA1,TRPV1 and SLC6A4.At the same time,molecular docking results showed that the docking effect of6-gingerol with TRPV1 was good;The docking result of cinnamaldehyde and TRPA1 is good.The enrichment of KEGG pathway was mainly concentrated in calcium ion signaling pathway and inflammatory regulation TRP pathway.The results of GO enrichment analysis mainly focused on pain perception,ion channel regulation and temperature stimulus response.Western blot analysis showed that cinnamomum dried ginger could reduce the expression of TRPA1,TRPV1 and CGRP in colonic tissue of cold abdo minal pain model mice3.(1)The combination of 6-gingerol and cinnamaldehyde at different concentrations can reduce the change of calcium fluorescence intensity before and after cold stimulation;(2)The 6-gingerol+cinnamaldehyde intervention group and the TRPA1inhibitor+TRPV1 inhibitor intervention group can reduce the change of calcium fluorescence intensity after cold stimulation,and down-regulate the expression level of TRPA1,TRPV1,CGRP protein.The 6-gingerol,cinnamaldehyde,TRPA1,TRPV1 inhibitor intervention group has no significant difference compared with the model group;(3)There was no significant difference between the 6-gingerol+cinnamaldehyde intervention group pretreated with PLC inhibitor and/or CaN inhibitor and the model group.The 6-gingerol+cinnamaldehyde intervention group pretreated with PLC inhibitor and/or CaN inhibitor and the TRPA1 inhibitor+TRPV1 inhibitor intervention group decreased the change of calcium ion fluorescence intensity and the expression of TRPA1,TRPV1 and CGRP protein after cold stimulation;There is no significant difference between TRPA1 inhibitor and TRPV1 intervention group and model group.Conclusion:(1)cinnamon-dried ginger herb pair could enhance the cold-pain resistance of mice,improve the pathological changes of colonic tissue.(2)The effects of improve cold pain by cinnamon-dried ginger herb pair drug pairs are achieved through two key targets,TRPA1 and TRPV1.The possible mechanism is through calcium ion signaling pathway,PLC and CaN regulating TRPA1 and TRPV1 signaling pathway.(3)6-gingerol and cinnamaldehyde CaN cross-desensitize TRPA1 and TRPV1,and this process is regulated by PLC and CaN.