The Protective Effect And Mechanism Of Non-steroidal Organic Selenium Compounds On Inflammation In LPS-activated RAW264.7 Cells

As a common disease of digestive system,the incidence of inflammatory bowel disease(IBD)has increased significantly in China and even the world in the past 20 years.At present,immunosuppressive agents and anti-inflammatory drugs are mainly used to relieve IBD in clinical practice,but long-term use of these therapeutic drugs has relatively large side effects.The modified compounds based on the parent nuclear structure of non-steroidal compounds are currently the focus of research,because of their excellent biological activities such as antioxidation,anti-inflammation,immune regulation and so on.In this study,through the establishment of lipopolysaccharides(LPS)stimulated mouse peritoneal macrophages(RAW264.7)inflammatory bowel disease IBD cell model,from the two aspects of oxidative stress and inflammatory response,explore the protective effects and molecular mechanisms of 9 new non-steroidal organic selenium compounds on inflammatory bowel disease.The results of the study are as follows:1.The protective effects of 9 non-steroidal organic selenium compounds on LPS-activated RAW264.7 cells(1)The content of Nitrite was detected by Griess method.The results showed that the 9 non-steroidal organic selenium compounds significantly reduced the Nitrite content of RAW264.7 cells activated by LPS,indicating that the 9 non-steroidal organic selenium compounds had an inhibitory effect on the inflammation of RAW264.7 cells.The CCK8 method was used to detect cell viability,and the results showed that 9 non-steroidal organic selenium compounds had no significant effect on cell viability.(2)The mRNA expression levels of iNOS,p47 phox and inflammatory factors(IL-1β,IL-18)were detected by fluorescence quantitative PCR.The results showed that the 9 compounds significantly reduced the expression levels of IL-1β mRNA;Compounds SLL-1-43,SLL-1-44,SLL-1A-40 and SLL-1A-46 significantly reduced the expression levels of iNOS,p47 phox and IL-18;Compounds SLL-1A-39 and SLL-1-39 significantly reduced the expression levels of iNOS mRNA;The compound SLL-1-40 significantly reduced the expression level of p47 phox mRNA;The compound SLL-1A-49 significantly reduced the expression level of IL-18 mRNA.The above research results show that: 9 kinds of non-steroidal organic selenium compounds can reduce the production of iNOS,p47 phox and inflammatory factors.Based on the above results,the most effective compounds SLL-1-43 and SLL-1-44 were selected for further study.(3)After LPS-activated RAW264.7 cells were treated with compounds SLL-1-43 and SLL-1-44 for 24 h.The DCFH-DA method was used to detect ROS levels in RAW264.7 cells.The results showed that compounds SLL-1-43 and SLL-1-44 could significantly reduce the level of ROS in RAW264.7 cells.2.The study on the docking and dynamics of nine non-steroidal organic selenium compounds with NOX2 subunit p47phoxAutodock Vina and Desmond software were used to conduct molecular docking and kinetic simulation of p47 phox with 9 kinds of non-steroidal organic selenium compounds.The docking results showed that: 9 compounds interacted with p47 phox protein.The compounds SLL-1-43 and SLL-1-44 were selected for subsequent molecular docking and kinetic simulation.The docking and kinetic simulation results showed that the compound SLL-1-43 interacted with p47 phox protein amino acid residues TRP193 and TRP263 to form π-π interaction and hydrogen bond with SER208,the affinity value was-6.6Kcal/mol.The compound SLL-1-44 forms a π-πinteraction with p47 phox amino acid residues TRP193 and TRP263 with an affinity value of-6.9Kcal/mol.These results indicate that p47 phox protein is a potential target of the compounds SLL-1-43 and SLL-1-44.3.The molecular mechanism of compounds SLL-1-43 and SLL-1-44 in inhibiting inflammation in LPS-activated RAW264.7 cellsThe Western blot results showed that compounds SLL-1-43 and SLL-1-44 treated LPS-activated RAW264.7 cells for 24 hours,and Compounds SLL-1-43 and SLL-1-44 significantly down-regulated the protein expression levels of p47 phox,p-p65,NLRP3,and p-p38.In summary,the compounds SLL-1-43 and SLL-1-44 can inhibit the release of ROS and inflammatory factors via regulating the expression of proteins related to the NOX2/p47 phox and the downstreaam inflammatory-related signaling pathways,thereby achieving IBD protection.It is of great significance to further study its targeting NOX2 protein inhibition in the future,and to conduct in-depth research on SLL-1-43 and SLL-1-44 as potential drugs for IBD.