The Inhibitory Effect And Mechanism Of TLR9/KLF4 In NF-?B Inflammatory Signal Pathway Of Adipocyte

Object: On the basis of human omental tissue study,high-fat diet induced obese rat model and adipocyte culture in vitro,the research intends to observe the high FFA effects on TLR9/KLF4 expression and the downstream key factors of NF-?B inflammatory signal pathway,exploring the mechanism of inflammatory response suppressed by TLR9/KLF4.Methods: Selecting 50 cases of normal weight Uygur individuals?NC?and 45 cases of obese weight individuals?OB?to detect the general data and biochemical indicators.32 healthy male Wistar rats were divided into two group,and fed normal diet and high fat diet respectively to construct the obese rat model.In the 4thand 10 thweek,we detect the levels of blood lipid and glucose.In vitro,culturing pre-adipocyte 3T3-L1 to differentiate into mature adipocyte which is identified by red oil O.Under the palmitic acid?PA?of different concentrations?0 ?M,0.2?M,2 ?M,20 ?M,100 ?M,200 ?M?,using si-RNA method to down-express TLR9/KLF4,using p IRES2-KLF4-Zs Green1 to over-express KLF4,and using virus infection to over-express TLR9,respectively.The q RT-PCR and Western Blot were used to detect m RNA and protein expression levels of TLR9,KLF4 and key inflammatory-related factors in the cell and omental adipose tissue.Elisa was used to detect release levels of inflammatory cytokines.The BSP technology was used to detect KLF4 gene DNA methylation in omental adipose tissue of obese individuals.Results:1.Human tissue experiment?1?The general information and levels of plasma blood lipid and inflammatory factors The body weight,WC,HC,WHR,BMI,TG,LDL and TNF-? levels in the OB group were significantly higher than those in the NC group,while the level of APN was significantly lower than those in the NC group,the difference was statistically significant?P<0.05?.?2?The m RNA and protein expression levels of key genes in omental adipose tissue In OB group,TLR9 and KLF4 m RNA and protein expression levels were significantly lower than NC group?P<0.05?,and APN m RNA and protein expression levels were lower than NC group;The P65,TNF-? and IL-6 m RNA and protein expression levels were significantly higher than NC group?P<0.05?.?3?The relationship among the expression levels of TLR9/KLF4 and key genes of lipid metabolism/inflammatory signaling pathway TLR9 m RNA expression level was significantly positive correlated with HDL,MYD88,SRC and KLF4?P<0.05?;KLF4 m RNA expression level was significantly negative correlated with BMI,TG and TNF-??P<0.05?,while positive correlated with NF-?B?P<0.01?.?4?Comparison of DNA methyltransferase expression and the positive rate of KLF4 gene methylation in omental adipose tissue In OB group,the m RNA expression levels of DNMT1,DNMT3 a and DNMT3 b were significantly higher than the NC group?P<0.05?.In OB group,the methylation positive rate of KLF4 gene promoter region?10.429%?was significantly higher than NC group?5.714%?,in which the methylation rate of Cp G₃3 and Cp G₃4 were significantly higher than that of NC group?P<0.05?.2.Rat experiment?1?Construction of the obese rat model In the 4thweek after high-fat diet feeding,the weight of the high fat diet?HFD?group was significantly higher than that of the normal control?NC?group?P<0.05?.In the 10 th week,the weight of the HFD group did not appear to increase,but the weight was still higher than that of the NC group?P<0.01?.Compared with the weight of the NC group?318.50 ± 38.07?,the HFD group?403.00 ± 50.38?was 20% higher.The Lee's index?337.10 ± 4.72?of the HFD group was significantly higher than that of the NC group?323.42 ± 4.72??P<0.01?.The waist circumference was significantly higher in the HFD group?19.88±0.48 vs 17.58±0.30,P<0.01?,and visceral fat mass was higher in the HFD group with the fatty liver formation.?2?Comparison of the levels of blood glucose,blood lipid,adipocytokines and inflammatory factors in two groups In the 4thweek after high-fat diet feeding,the levels of FFA,TG,TC and LDL in the HFD group were significantly higher than those in the NC group?P<0.05?.In the 10 th week,the levels of Glu,TG,FFA,and TNF-? in the HFD group were significantly higher than that in the NC group?P<0.05?,while the levels of LPT and APN were significantly lower than those in the NC group?P<0.05?.?3?The key genes m RNA expression levels of inflammatory signaling pathway in omental adipose tissue In HFD group,the m RNA expression levels of TLR9,MYD88,KLF4 and NF-?B were lower than the NC group;while,SRC and IL-6 were higher than the control group.?4?Correlation of key genes of inflammatory signaling pathway The level of plasma FFA was significantly positive correlated with TNF-??P<0.05?,while negative correlated with TLR9 m RNA expression level?P<0.05?;TLR9 m RNA expression level was significantly positive correlated with KLF4?P<0.05?.3.Experiments in vitro?1?Culture and differentiation of 3T3-L1 adipocytes The 3T3-L1 cell is fusiform and has no lipid droplet exist;Differentiation to the 8thday,there were more than 90% mature adipocyte,gathering more lipid droplet.The oil red O staining results showed that the lipid droplet was dyed red.?2?Detecting regulatory effect of KLF4 on downstream inflammatory factors Down-regulated KLF4 can significantly increase the expression levels of IL-6 and MCP-1,while suppress the expression level of the APN?P<0.01?.Up-regulated KLF4 can significantly suppress the expression levels of P65,TNF-?,IL-1? and MCP-1?P<0.05?,while increase the expression level of APN?P<0.05?.?3?Detection of TLR9 regulation on KLF4 Compared with Mock and Negative Control group,up-regulated TLR9 can significantly increase the m RNA and protein expression levels of KLF4?P<0.05?,and the m RNA expression level of P65 and MCP-1 are significantly lower than the negative control?P<0.05?while the APN is significantly higher.Down-regulated TLR9 can significantly decrease the expression m RNA and protein expression levels of KLF4?P<0.05?,and the m RNA expression level of P65,IL-6 and MCP-1 are significantly higher than the negative control?P<0.05?while the APN is significantly lower?P<0.05?.?4?Under different concentrations of PA stimulate,the expression levels of inflammatory cytokines were detected With the increase of PA concentration,the protein expression level of TLR9 had no statistical significance,while the levels of KLF4 were increased after the first reduce trends.When the PA concentration is 20 ?M,the KLF4 expression level was the highest,the difference was statistically significant compared with NC group?P<0.05?.Under the 100 ?M and 200 ?M PA stimulate,the m RNA and protein expression levels of KLF4 were significantly decreased compared with 20 ?M PA stimulate?P<0.05?.In the 20 ?M PA stimulate group,the m RNA expression levels of IL-6,MCP-1 and APN and the release expression level of IL-6 were significantly higher than the 0 ?M PA stimulate group;In the200 ?M PA stimulate group,the m RNA expression levels of P65,IL-6,MCP-1 and IL-1? and the release expression levels of IL-6 and MCP-1 were significantly higher than the 0 ?M PA stimulate group;In the 200 ?M PA stimulate group,the m RNA expression level of IL-6 and the release expression levels of IL-6 and MCP-1 were significantly higher than the 20 ?M PA stimulate group,while the m RNA expression level of APN was significantly lower than the 20?M PA stimulate group,and the above differences were all statistically significant?P<0.05?.?5?Under the 20 ?M PA stimulate,down-regulating KLF4 to detect the expression of downstream inflammatory factors Under the 20 ?M PA stimulate,Down-regulated KLF4 can increase the m RNA expression levels of MCP-1 and IL-1? and the release expression level of IL-6?P<0.05?,while inhibit the m RNA expression level of the APN?P<0.01?.?6?Under the 200 ?M PA stimulate,up-regulating KLF4 to detect the expression of downstream inflammatory factors Under the 200 ?M PA stimulate,Up-regulated KLF4 can significantly suppress the expression levels of P65,TNF-? and IL-1?,while increase the m RNA expression level of APN?P<0.05?.?7?Detecting the KLF4 effects on glycolipid metabolism ability In culture medium,up-regulated KLF4 can significantly suppress the level of TG and GLU?P<0.05?,and down-regulated KLF4 can significantly increase the level of TG?P<0.05?.Under the 20 ?M PA stimulate,Down-regulated KLF4 can significantly increase the level of TG and GLU?P<0.05?.Conclusions:?1?In mature adipocyte,TLR9 can promote KLF4 expression,inhibiting the expression of P65 of NF-?B active submit to exert the anti-inflammatory function.?2?The low concentration of PA can promote KLF4 expression,improving the glycolipid metabolism ability to exert the anti-inflammatory function;while,the high concentration of PA can suppress the KLF4 expression,inhibiting the anti-inflammatory function to aggravate inflammation.?3?The lower expression of KLF4 of omental adipose tissue may be related to the KLF4 gene promoter region methylation.