The Role And Mechanism Of MKP5 On Free Fatty Acid-induced Inflammatory Response In Macrophages

Obesity,as a disease,has increasingly attracted people's attention.Obesity can induce chronic inflammation in adipose tissue,which deteriorate may result to insulin resistance,and then induce many metabolic diseases such as type II diabetes,lipid metabolism disorder.Therefore,it is emergency to research the chronic inflammation in adipose tissue which induced by obesity.There are four main mechanisms contributed to chronic inflammation,including adipocyte hypertrophy,local hypoxia,macrophage infiltration and endoplasmic reticulum stress.Macrophages infiltration and adipocyte hypertrophy are closely related to macrophages.This experiment studied inflammation with macrophage as the experimental objective.Chronic inflammation is closely related to MAPK pathway,which included three members,JNK,ERK,P38.Active the MAPK pathway was realized by phosphorylate MAPK members,and DUSP is a dual specificity phosphatase which can induce MAPK inactivation.MKPs is one of the members in DUSP family,this study aim to explore the effect of MKP5 on adipocyte tissue chronic inflammation induced by obesity,and the relationship between MKP5 and MAPK pathway.The MKP5 stably overexpressed mouse RAW264.7 cell line was constructed to explore the function of MKP5 on macrophage inflammation induced by palmitic acid(PA),the potential molecular mechanism was further researched.Purposes:(1)To study the function of MKP5 on the macrophage inflammatory response induced by PA;(2)To explore the relationship between MKP5 regulation of PA induced RAW264.7 cell inflammation and MAPK signaling pathway.Methods and Results:(1)MKP5 was closely related to macrophage inflammationRAW264.7 cells were treated with 500 ?M PA for 0,1,3,6,9 hours.As the results showed by Real-Time PCR,inflammatory factors IL-6,IL-1? and IL-12 and MKP5 were significantly up-regulated with time after PA treated(P<0.05),which implied that MKP5 was related to inflammation.(2)MKP5 inhibited macrophage inflammation induced by PAMKP5 stably overexpressed RAW264.6 cell line was constructed and treated with 500 ?M PA for 3 hours.The Real-Time PCR result indicated that the expression of inflammatory factors in PA treated group were obviously up-regulated compared to the control(P<0.05),verified that the model of PA induced inflammation was available.However,the expression of inflammatory factors were down-regulated in RAW-MKP5 group compared to RAW-PC group(P<0.05),indicated that MKP5 can relieve the inflammation induced by PA.(3)MKP5 can induce the macrophage M1 phenotype converted to M2 phenotypeTreated RAW-PC and RAW-MKP5 cells with LPS and IFN-? for 12 hours to induce the M1 phenotype,and treated with IL-13 for 12 hours to induce M2 phenotype,extracted total RNA after fast for 6 hours and tested the gene expression of IL-6,IL-12 or Arg-1 by Real-Time PCR.The results showed that cells presented inflammatory phenotype,indicated that M1 phenotype was induced successful,and IL-6,IL-12 in RAW-MKP5 was down-regulated compared to RAW-PC(P<0.05).The results showed that M2 phenotype was also induced successful,and the anti-inflammatory factor Arg-1 up-regulated more significant in RAW-MKP5(P<0.05).All the results indicated that MKP5 can induce the macrophage M1 phenotype converted to M2 phenotype,then relieve the inflammation.(4)MKP5 inhibits inflammatory response in macrophage-adipocytes interaction induced by PA.The cells were co-cultured for 24 h followed by 12 h palmitate stimulation.We observed significant increase in the levels of inflammatory cytokines,including IL-6,MCP-1 in macrophage-adipocyte coculture without palmitate stimulation.PA stimulation further increased the production of IL-6,MCP-1.Importantly,overexpression of MKP5 in macrophages greatly suppressed the production of inflammatory mediators,including IL-6,MCP-1 with or without palmitate stimulation(5)MKP5 regulate MAPK pathway to inhibit macrophages inflammatory response induced by PA.Treated RAW-PC and RAW-MKP5 cells with PA for 0,0.5,1,3,6 h.As the results showed by Western Blot,we found that the activation of all the three groups of MAPK was induced at 6h after PA stimulation.Overexpression of MKP5 significantly inhibited the activation of JNK,p38 and ERK.Taken together,the results suggest that MKP5 may inhibit macrophage inflammatory response by inhibiting JNK,p38 and ERK activation.(6)Construction of obese mouse model4-5 weeks male C57BL/6J mice were fed with either HFD(n=16)or Chow(n=15)for 20 weeks.The results of body weight and blood glucose showed that there was a significant difference in body weight and blood glucose between the HFD group and the Chow group,which proved that the obese mouse model was successfully constructed.(7)MKP5 inhibits the inflammatory response of SVCs from WAT.After successful modeling of obese mice,the SVCs in mouse white adipose tissue were isolated and cultured.The SVCs of Chow group and HFD group were infected with Ad-MKP5 and Ad-GFP.The macrophage marker F4/80 and chemokine MCP-1,TNF-?,IL-6 gene expression were detected by Real-time PCR.The results showed that in the Ad-GFP group,the expression of F4/80,MCP-1,TNF-?,and IL-6 genes in SVCs was significantly higher in the HFD group than the Chow group,demonstrating that the inflammation in SVCs from WAT was significantly aggravated;In the HFD group,the expression levels of F4/80,MCP-1,TNF-?,and IL-6 in SVCs infected wiyh Ad-MKP5 were significantly higher than those in infected with Ad-GFP.Overexpression of MKP5 can significantly inhibit the inflammatory response of macrophages in WAT of obese mice.Conclusion:(1)MKP5 can effectively alleviate the inflammation of macrophages induced by PA;(2)MKP5 regulates the inflammatory response of macrophages by the JNK,P38 and ERK pathways.