The Role And Mechanism Of FFA In Promoting The Expression Of IL-6 In Adipocytes By Promoting KLF7 Overexpression

Objective The increase of free fatty acids(FFA)levels in obese state leads to an increase in the expression and release levels of inflammatory factors such as interleukin-6(IL-6)in adipocytes.The specific mechanism is not yet very clear.Based on the culture of adipocytes in vitro,this study explored the role of KLF7 in the expression of inflammatory factor IL-6 induced by FFA,and attempted to elucidate the possible molecular mechanisms to provide new targets for the treatment of obesity,inflammation and related metabolic diseases.Methods(1)Adipocytes cultured in vitro were stimulated with different concentrations of palmitic acid(PA).Using qRT-PCR and Western blot to detect the mR NA and protein expression levels of inflammatory factors such as KLF7 and key factors of TLR4/NF-κB/IL-6 inflammatory signaling pathway and human monocyte chemoattractant protein-1(MCP-1),interleukin 1β(IL-1β)and so on.(2)The expression of KLF7 or TLR4 in adipocytes was upregulated or downregulated,after which the mRNA and protein expression levels of these key factors were detected.KLF7 expression was downregulated while PA stimulated adipocytes,and then the mRNA and protein expressions of KLF7/p65 and downstream inflammatory cytokine IL-6 were detected.(3)The NCBI and UCSC databases were used to query key genes in human IL-6.A luciferase reporter plasmid inserted into the key gene of the IL-6 promoter region was constructed.The luciferase reporter assay was used to determine whether KLF7 had a transcriptional activation effect on IL-6.(4)Using NCBI and UCSC databases to search for key genes of human IL-6promoter regions,and construct luciferase plasmids carrying key genes of IL-6 promoter region.The luciferase reporter assay detects whether KLF7 has transcriptional activation on IL-6.(5)CHIP-PCR assay detects the ability of KLF7 to bind to the IL-6 promoter region.Results1)High concentration of PA stimulates adipocytes and promotes the expression of inflammatory factors such as TLR4,KLF7 and IL-6 in adipocytes.Mouse fibroblasts differentiated into mature adipocytes,and a large number of lipid droplets were identified by oil red O staining,indicating that the induction was successful.After high concentration of PA stimulated adipocytes for 48 hours,the mRNA and protein expressions of inflammatory factors such as TLR4,KLF7,pp65 and IL-6 in adipocytes were significantly increased(P<0.05).2)TLR4 can positively regulate the expression of KLF7 and IL-6 in adipocytes.After up-regulating TLR4 expression in adipocytes,the mRNA and protein expression levels of KLF7 and IL-6 were significantly increased(P<0.05).After down-regulating TLR4 expression,the mRNA and protein expression levels of KLF7 and IL-6 were significantly decreased(P<0.05).3)KLF7 can positively regulate p65 phosphorylation and IL-6 expression in adipocytes.After up-regulating KLF7 expression in adipocytes,the mR NA and protein expression levels of pp65 and IL-6 were significantly increased(P<0.05).After down-regulation of KLF7 expression,the mRNA and protein expression levels of pp65 and IL-6 were significantly lower(P<0.05).4)High concentration of PA can promote the expression of IL-6 by up-regulating the expression of KLF7 in adipocytes.PA stimulated adipocytes and downregulated the expression of KLF7.The mR NA and protein expression levels of IL-6 were significantly lower than those in the PA-stimulated group(P<0.05).5)KLF7 can transcriptionally activate IL-6 expression.Using the NCBI and UCSC databases,the key gene sequence of the IL-6 promoter region was2001 bp.In 293 T cells,luciferase reporter gene experiments showed that the luciferase activity was significantly increased after transfection of KLF7 with the pGL-3-Basic 2001 luciferase reporter gene plasmid containing the IL-6 promoter region.(P<0.05).Different IL-6 truncated luciferase reporter plasmids were co-transfected into 293 T cells with KLF7 overexpression plasmid,respectively.And,the results showed that the luciferase activity value was not different from the control group after transfecting pGL-3-Basic1501(excluding 5’-1900~1400 3’bp).The result suggested that the promoter region 5’-1900~1400 3’bp is a key sequence for KLF7 transcriptional activation of IL-6.6)KLF7 can bind to the 5’-1696~1386 3’bp and 5’-354~104 3’bp sequences of the IL-6 promoter region.After overexpressing KLF7 in 293 T cells,the results of Chromatin Immunoprecipitation(CHIP)showed that the sequence of the promoter region of IL-6 amplified by KLF7 antibody-bound protein-DNA complex was 5’-1696~1386 3’ bp and 5’-354~104 3’ bp.Conclusion1)High levels of free fatty acids in obese conditions promote KLF7 and IL-6 expression via TLR4.2)KLF7 can directly bind to the IL-6 promoter region sequence between 5’-1696~1386 3’ bp and5’-354~104 3’ bp activates IL-6 and exerts a pro-inflammatory effect.