Effects Of CXCR2 Expression On Insulin Signaling Pathway Of Hepatocyte Induced By Different Reginal Adipose Tissue

Objectives:The cytokines secreted by different regions of adipose tissue(AT)are different,and visceral adipose tissue(VAT)is more secretive of interleukin-8(IL-8)than subcutaneous adipose tissue(SAT).The biological effect of IL-8 depends on binding to its specific receptor CXCR1/2.Recent studies have found that CXCR2 is more closely related to metabolic diseases.The aim of this study is to explore the effect of hepatocyte CXCR2 expression on insulin signaling pathway induced by different regional AT using RNA interference technology.Methods:Firstly,the quantitative real-time PCR(qPCR)method was used to detect the expression of CXCR2 in HepG2 cells and screen CXCR2-specific small interfering RNA(siRNA)for subsequent experiments.Secondly,the qPCR method has also been used to detect the level of CXCR2 in HepG2 cells,HepG2 cells transfected with CXCR2-siRNA or CXCR2-overexpression plasmids co-cultured with different regional fat depot.Thirdly,after 48 hours of co-cultivation,AT and Transwell chamber were removed,and the LDH activity in the supernatants of each group was measured.Last but not least)in order to investigate the effect of hepatocyte CXCR2 expression on IRS2/PI3K/Akt2 signaling pathway induced by different regional AT,we collected HepG2 cells from each co-culture group under basic conditions and insulin stimulation,and the expression of IRS2,p-IRS2(S731),PI3K-p85,Akt2 and p-Akt2(S473)protein were detected by Western blot(WB)method.Results:(Ⅰ)Screening results of CXCR2-siRNA:(1)CXCR2 were expressed in HepG2 cells;(2)Compared with siRNA2 and siRNA3,siRNA 1 significantly reduced CXCR2 expression in HepG2 cells.(Ⅱ)Verification results of hepatocyte CXCR2 expression under fat depot-specific differences:(1)Compared with the HepG2 group,the expression levels of CXCR2 in HepG2 cells co-cultured with AT were significantly increased;(2)Compared with the HepG2+SAT group,the expression of CXCR2 in the HepG2+VAT group increased significantly;(3)Compared with the group of HepG2 cells co-cultured with AT,the expression of CXCR2 in HepG2 cells transfected with CXCR2-siRNA induced by AT was significantly reduced,while the expression of CXCR2 in HepG2 cells transfected with CXCR2-overexpressing plasmid induced by AT was significantly increased;(4)Compared with the HepG2&CXCR2 plasmid+SAT group,the expression level of CXCR2 in the HepG2&CXCR2 plasmid+VAT group was significantly increased.(Ⅲ)LDH activity in the supernatant:(1)Compared with the HepG2 group,LDH activity in the supernatant of HepG2 cells co-cultured with AT was increased;(2)LDH activity of HepG2+VAT group was significantly higher than that of HepG2+SAT group;(3)Compared with the group of HepG2 cells co-cultured with AT,LDH activity of supernatant in HepG2 cells transfected with CXCR2-siRNA co-cultured with AT was reduced,and LDH activity of supernatant in HepG2 transfected with CXCR2-overexpressed plasmid co-cultured with AT was elevated.(Ⅳ)Effect of CXCR2 expression on key proteins of insulin signaling pathway:(1)Compared with the HepG2 group,the expression of IRS2,PI3K-p85,and p-Akt2(S473)proteins in HepG2 cells cultured with AT were reduced,while the expression of p-IRS2(S731)protein was increased.In addition,VAT has a more significant effect on protein expression than SAT;(2)Compared with the group of HepG2 cells cultured with AT,the expression levels of IRS2,PI3K-p85 and p-Akt2(S473)protein in HepG2 cells transfected with CXCR2-siRNA co-cultured with AT were up-regulated,but p-IRS2(S731)protein expression was down-regulated;(3)Compared with the group of HepG2 cells co-cultured with AT,the expression levels of IRS2,PI3K-p85 and p-Akt2(S473)protein in HepG2 cells transfected with CXCR2-overexpressing plasmid co-cultured with AT were down-regulated,but p-IRS2(S731)protein expression was up-regulated;(4)There was no statistical difference in the expression level of Akt2 protein in HepG2 cells between the experimental groups.Conclusions:(1)HepG2 cells can express CXCR2.(2)siRNA1 has the best CXCR2 interference effect on HepG2 cells.(3)Adipose tissue can up-regulate CXCR2 levels in hepatocytes,and VAT can promote the expression of CXCR2 in HepG2 cells more than SAT.(4)CXCR2-siRNA and CXCR2-overexpression plasmids are effective in interfering with CXCR2 expression in HepG2 cells.(5)There may be a positive feedback activation mechanism between VAT and the up-regulation of CXCR2 expression in hepatocytes.(6)Hepatocytes survival may be affected by VAT,SAT and CXCR2.(7)AT may affect the insulin signaling pathway of hepatocytes.In addition,VAT can inhibit the expression of key proteins in the hepatocytes more than SAT.(8)CXCR2 negatively regulates IRS2,PI3K-p85 and p-Akt2(S473)protein expression in hepatocytes,suggesting that CXCR2 is an important participant in AT-mediated insulin resistance in hepatocytes.Increased expression of CXCR2 in hepatocytes induced by AT may interfere the normal transdaction of hepatocyte insulin signaling pathways.(9)CXCR2 positively regulates the expression of p-IRS2(S731)protein in hepatocytes.The p-IRS2(S731)protein may be involved in hepatic insulin resistance.(10)The mechanism of insulin resistance induced by AT in hepatocytes may not be related to Akt2 protein,and CXCR2 may not affect the expression of Akt2 protein in hepatocytes.