Anti-Inflammatory Mechanism Of Compound Iodinephomachalasin Based On Proteomics Technology

Cytochalasin compounds are a class of compounds produced by fungal metabolism with amino acid and polyketone condensed skeleton structure.These compounds have attracted attention for their anti-inflammatory,antibacterial,antiviral,and anticancer activities.Our research group screened the anti-inflammatory activity of cytochalasin compounds and found that the compound IPC(Iodinephomopchalasin)had good anti-inflammatory activity,but its anti-inflammatory mechanism was not clear.Therefore,in this thesis,LPS-induced RAW264.7cells were used to establish inflammation models.Using the active compound IPC as guidance,proteomics and cell transfection techniques were used to search for the potential active targets and anti-inflammatory mechanisms of IPC.Firstly,the MTT method,Griess method,ELISA,Western Blot,and RT-PCR were used to study the anti-inflammatory mechanism of IPC.The results showed that IPC at the concentrations of 20,10,and 5 μM could inhibit the secretion of NO,IL-6,and TNF-α,as well as the mRNA and protein expression of i NOS and COX-2 in LPS-induced RAW264.7 cells.But IPC does not play its anti-inflammatory role through the classical NF-κB/MAPK signaling pathway.TMT labeled proteomics was used to screen different groups of differential proteins in RAW264.7 cells.With Qvalue < 0.05 and Foldchange > 1.5 as screening conditions,it was found that 362 proteins were up-regulated and 321 proteins were down-regulated in the LPS group compared with the Control group.Compared with the LPS group,196 proteins were upregulated and 124 proteins were down-regulated in the LPS＿IPC group.There were 102 different proteins in LPS-VS-Control and LPS＿IPC-vs-LPS groups.These proteins are involved in the regulation of inflammation.KEGG pathway enrichment revealed that these common differential proteins were mainly related to the JAK/STAT signaling pathway.PPI correlation analysis showed that UPP1 and STAT1/2 interacted,suggesting that UPP1 might be involved in the regulation of STAT1/2,and IPC might play an anti-inflammatory role by mediating UPP1 to regulate STAT1/2.To verify the results of the above speculation,we transfected RAW264.7 cells with UPP1 si RNA and overexpressed plasmid.The results showed that after the knockdown of UPP1 and LPS stimulation,the secretion of NO,IL-6,and TNF-α were decreased;the m RNA and protein expression levels of i NOS and COX-2 were also significantly down-regulated;the total protein level and phosphorylated protein expression level of STAT1/2 were decreased.It can also inhibit the nuclear translocation of STAT1/2.After UPP1 overexpression,the secretion of NO,IL-6,TNF-α,the mRNA,and protein expression levels of iNOS and COX-2 were significantly increased.The total protein and phosphorylated protein expression levels of STAT1/2 were increased,and the nuclear translocation of STAT1/2 was promoted.These results indicate that UPP1 plays a certain proinflammatory function.However,UPP1 overexpression was followed by 20 μM IPC,and it was found that the compound IPC reversed this cellular inflammation caused by UPP1 overexpression.In summary,UPP1 can regulate the expression of total protein and phosphorylation level of STAT1/2,and promote nuclear translocation and inflammation.Compound IPC exerts antiinflammatory activity by down-regulating UPP1 to inhibit total protein and phosphorylation levels of STAT1/2,and inhibit nuclear translocation of STAT1/2.UPP1 may be a potential target for the treatment of inflammation.