The Role Of MiR-181a In Regulating The Inflammatory Pathway Of NLRP3 In Human THP-1-Derived Macrophages Stimulated By Ox-LDL

Objective: Atherosclerosis is a chronic inflammatory response characterized by lipid deposition and foam cell formation in blood vessel walls,and macrophages play a key role in the occurrence and development of this chronic inflammation.Innate immunity is involved in this chronic inflammatory process,and NLRP3 inflammatory pathway is an important link in innate immunity.More and more studies have shown that micro RNA is involved in the regulation process of inflammation.Among numerous micro RNAs,the role of mi R-181 family in chronic inflammatory response has been widely concerned.However,the effect of mi R-181 a on NLRP3 has not been studied in relevant literature,our experiment aims to investigate whether mi R-181 a has a regulatory effect on the inflammatory pathway of NLRP3 in the human THP-1-derived macrophage inflammation model stimulated by ox-LDL and explore its possible mechanism.Materials and methods: Using PMA(phorbol myristate acetate)stimulate human THP-1cells 24 hours to make it into attached macrophages,then THP-1 derived macrophages were stimulated by ox-LDL with time gradient and concentration gradient,respectively.The expression level of mi R-181 a was detected,according to the results of qrt-PCR,12 h,50ug/ml were selected as appropriate stimulation time and concentration of ox-LDL.After cells were treated with 50ug/ml ox-LDL for 12 h,the expressions of mi R-181 a and proteins of MEK/ERK/NF-?B and NLRP3 inflammatory pathways were detected.To further investigate whether mi R-181 a has an effect on the expression of proteins related to the inflammatory pathway of NLRP3,we divided the cells into 6 groups: Control group,ox-LDL group,ox-LDL + mi R-181 a mimics group,ox-LDL + mi R-181 a mimics negative control group,ox-LDL + mi R-181 a inhibitors group,ox-LDL + mi R-181 a inhibitors negative control group,the proteins expression levels of MEK/ERK/NF-?B and NLRP3 inflammatory pathways were detected.Target gene of mi R-181 a was predicted and searched by Target scan7.1 and dual luciferase reporter analysis.To investigate whether mi R-181 a regulates the NLRP3 inflammatory pathway by targeting MEK1,we divided the cells into 6 groups: Control group,ox-LDL group,ox-LDL + mi R-181 a inhibitors+U0126 group,ox-LDL + mi R-181 a inhibitors group,ox-LDL+mi R-181 a inhibitors negative control+U0126 group,ox-LDL + mi R-181 a inhibitors negative control group.The expressions of mi R-181 a in each group were detected by qrt-PCR,and the expressions of inflammatory molecules related to the inflammatory pathways of MEK/ERK/ NF-?B and NLRP3 were detected by Western blot.Result: Compared with the control group(blank group),the expression level of mi R-181 a decreased significantly after THP-1 macrophages were stimulated by ox-LDL,while the activation of MEK/ERK/NF-?B pathway and the expression of NLRP3 related proteins(NLRP3,CASPASE1,IL-18,IL-1?)were upregulated.When the cells were exogenous overexpressed of mi R-181 a and then stimulated by ox-LDL,the activation level of the MEK/ERK/NF-?B inflammatory pathway in THP-1 macrophages decreased,and the expression level of proteins related to the inflammatory pathway of NLRP3(such as NLRP3,CASPASE1,IL-18,IL-1?,etc.)was down-regulated.Exogenous knockdown of mi R-181 a showed the opposite results to the overexpression group.Using double luciferase reporter analysis,we verified that MEK1 is a direct target gene of mi R-181 a,and mi R-181 a can inhibit the expression of MEK1 directly.To further verify that mi R-181 a affects the activation of NLRP3 inflammatory pathway by targeting MEK1,we knocked down mi R-181 a and then treated cells with U0126 before ox-LDL stimulation,we found that U0126 reversed the increased activation of the MEK/ERK/NF-?B pathway and upregulation of NLRP3 inflammasome-related proteins(NLRP3,CASPASE1,IL-18,IL-1?)that resulted from mi R-181 a knockdown.In other words,U0126 can simulate the role of mi R-181 a at a certain level.Conclusion: Our results suggest that mi R-181 a affect the activation of the MEK/ERK/NF-?B pathway by targeting MEK1 and then regulate the NLRP3 inflammatory pathway in human THP-1-derived macrophage stimulated by ox-LDL.