Study On The Mechanism Of Evodiamine To Improve Acute Lung Injury And Construction Of Spiro-oxindole Precursor

Objective: Acute lung injury(ALI)is a serious systemic inflammatory syndrome,and there is still a lack of effective treatments.Evodiamine is a class of indole alkaloids with significant anti-inflammatory activity.Studies have shown that evodiamine can improve ALI,but the research on its mechanism of action is still relatively limited.We find that Apelin may be a key molecule in the anti-inflammatory effect of evodiamine by transcriptome screening and bio-information analysis,Therefore,we took this as an entry point to study the mechanism of evodiamine in improving ALI through in vivo and in vitro pharmacological experiments to provide new ideas for the prevention and treatment of ALI.In addition,with the help of drug design ideas,the diversified synthesis of the indole skeleton has important research value for the development of new drugs.In this study,we combined the spiro-oxindole and sesamol through [4+2] cyclization according to the combination principle in medicinal chemistry to construct a class of spiro-oxindole alkaloid analogs to provide compound sources for lead compounds.Method: 1.In vitro,RAW264.7 cells were used as the research object.First,the cytotoxicity of evodiamine to RAW264.7 cells was detected by the MTT method,then the inflammation model was established with LPS+Nigericin,and the Annexin VFITC/PI double staining flow cytometry and LDH method were used to detect The effect of evodiamine on the apoptosis of RAW264.7 cells induced by LPS+Nigericin;Transcriptome sequencing(RNA-seq)was used to screen the differentially expressed genes(DEGs)of RAW264.7 cells under different intervention conditions,and then bioinformatic analysis methods such as cluster analysis,GO,KEGG and GSEA enrichment analysis were used to study the molecules and signal pathways that may regulate the anti-inflammatory effects of evodiamine under the stimulation of LPS+Nigericin;Real-time PCR and Western-blotting were used to verify the effects of evodiamine on key factors and related cytokines found by RNA-seq;ELISA was used to detect the influence of exogenous Apelin-13 on related cytokines.In vivo,establish ALI mouse model through LPS,and observe the effects of evodiamine on cytokines in mouse lung lavage fluid by ELISA;H&E and Masson’s staining were used to observe the effects of evodiamine on LPS-induced lung tissue damage;Immunohistochemistry was used to evaluate the effect of evodiamine on the expression level of related factors in lung tissue.2.Select biologically active isatin and sesamol as starting materials,and form intermediates through aldol condensation.The intermediate and 2-ethynylphenol were catalyzed by bimetal [4+2] cyclization to construct a class of spiro-oxindole alkaloid analogs,and the structure is determined by NMR,HRMS and other methods.Result: 1.(1)Through MTT method,LDH assay and flow cytometry,it was found that evodiamine showed low toxicity to RAW264.7 cells,and evodiamine significantly inhibited the cell apoptosis caused by LPS+Nigericin.(2)Through transcriptome screening and bioinformatics analysis,evodiamine can change the expression pattern of DEGs in cells before and after LPS+Nigericin stimulation,and regulate the Apelin signal transduction pathway;RNA-seq show that evodiamine after inflammation stimulation Alkaline treatment can significantly up-regulate the expression of Apelin and reverse the expression of multiple cytokines.(3)Real-time PCR and Western-blotting verified that evodiamine can up-regulate the expression of Apelin,and can significantly inhibit the expression of IL-6 and CCL17 after inflammatory stimulation;exogenous Apelin-13 can inhibit the release of IL-6 and CCL17 and reverse the decline of EGF.(4)In the LPS-induced ALI mouse model,evodiamine treatment can promote the secretion of Apelin in the lungs,partially reverse the increase of IL-6 and CCL17;improve the degree of lung tissue damage and fibrosis induced by LPS;up-regulate the expression of Apelin in lung tissue partially reversed the increase of IL-6,CCL17 and Col IV expression levels.2.A spirooxindole-pyran skeleton was constructed by [4+2] tandem reaction,with a yield of 95.26%,and the universality of the substrate was investigated.It was found that the reaction universality is poor,and currently only 4 extended compounds with low yield have been obtained.Conclusion: 1.In vitro,evodiamine can protect RAW264.7 cells from LPS+Nigericin-induced inflammatory damage by inhibiting cell apoptosis in a concentration-dependent manner,inhibiting the release of inflammatory cytokines and activating Apelin pathway.In addition,it was also found that Apelin-13 could inhibit the release of IL-6 and CCL17,and up-regulate EGF to protect lung injury,indicating that evodiamine may play an anti-inflammatory role by regulating Apelin.In vivo,evodiamine can improve the inflammatory injury and fibrosis of lung tissue by upregulating Apelin to inhibit the expression of cytokines and profibrotic factors.This is the same as the results of in vitro studies.It can improve LPS-induced lung inflammation and fibrosis by activating Apelin pathway,thereby protecting ALI.2.This reaction can successfully synthesize the target product,but the universality of the reaction is poor,which is not conducive to the expansion of the compound.The design of the reaction route still faces many challenges.The influence of electronic effect and steric hindrance on the synthesis effect is the focus of the subsequent experimental design improvement.