The Regulatory Effects Of Chemerin/ChemR23 On High Glucose-induced Inflammation Of Glomerular Endothelial Cells

BackgroundDiabetic nephropathy(DN)has become one of the important causes of end-stage renal disease(ESRD)worldwide.Glomerular endothelial cell(GEnC)is an important component of glomerular filtration barrier,which play important roles in limiting proteinuria production and maintaining normal renal function.A study found that high glucose environment can induce endothelial cells in the obvious inflammatory response and dysfunction.To explore the causes of endothelial cell inflammatory response is very important to understand the pathogenesis of diabetic nephropathy and to find effective clinical treatment.Chemerin is a discovered adipocytokine that is expressed in a variety of cells in the body in 1997.Its secretion into the blood after the regulation of systemic and local inflammatory response and induced leukocyte chemotaxis.ChemR23 is the most important receptor for recognition of Chemerin.Studies have confirmed that Chemerin through ChemR23 activation of a variety of intracellular signal pathway,causing downstream transcription of inflammatory factors,resulting in tissue inflammatory injury.But so far,the function of Chemerin and ChemR23 in glomerular endothelial cell inflammatory response in diabetic nephropathy has not yet clear.Objective1.To observe the changes of Chemerin,ChemR23 and TNF-α and IL-6 in mouse GEnCs under high glucose environment.2.After gene silencing the expression of ChemR23,to observe the expression of inflammatory factor induced by Chemerin.3.After gene silencing the expression of ChemR23,to observe the expression of inflammatory factor induced by high glucose and the effect on activating the signal pathway protein p38 MAPK.4.To inhibit the activation of p38 MAPK,and observe the expression of inflammatory factors.Methods The condition of immortalized mouse GEnCs was studied.1.Mouse GEnCs were cultured in vitro and divided into four groups: normal control group(glucose: 5.6mmol/L),20mmol/L high glucose group,40mmol/L high glucose group and osmotic pressure control group(5.6mmol/L glucose +34.4mmol/L mannitol),24 hours after the different medium for testing.Western blot and qRT-PCR were used to detect the expression of Chemerin,ChemR23,TNF-α and IL-6.ELISA was used to detect the expression of TNF-α and IL-6 in the culture supernatant.2.The lentiviral vector for ChemR23 was constructed.The lentiviral vector and empty vector were transfected into GEnCs induced by Chemerin.The GEnCs were divided into four groups: normal control group,Chemerin-stimulated group,Chemerin+empty vector group and Chemerin+shRNA group.After a certain period of time to carry out the test.The expression of TNF-α and IL-6 were detected by ELISA and qRT-PCR.3.The lentiviral vector and empty vector were transfected into GEnCs cultured in high glucose group.The GEnCs were divided into four groups: normal control group,high glucose group,high glucose+empty vector group and high glucose + shRNA group.The expression of TNF-α and IL-6 were detected by ELISA and qRT-PCR.The expression of p38 MAPK and phosphorylated-p38 MAPK(p-p38 MAPK)were detected by Western blot.4.Mouse GEnCs were cultured in vitro,which were divided into three groups: normal control group,high glucose group and high glucose+inhibitor group.Before adding high glucose stimulation,p38 MAPK inhibitor SB203580 was added.After a certain period of time to carry out the test,the levels of TNF-α and IL-6 were detected by ELISA and qRT-PCR.Results1.Changes of Chemerin,ChemR23 and inflammatory factors IL-6 and TNF-α in GEnCs in high glucose environment:Compared with the control,the expression of IL-6 and TNF-α was significantly increased in high glucose group(P<0.05).The expression of Chemerin was also elevated(P<0.05).However,the expression of ChemR23 did not change(P>0.05).2.The expression of IL-6 and TNF-α in GEnCs induced by Chemerin were observed by lentiviral vector interfering with ChemR23 expression:Lentivirus baring shRNA could efficiently suppress ChemR23 expression.The IL-6 and TNF-α levels in Chemerin+shRNA group were significantly decreased compared with Chemerin+empty vector group(P<0.05).3.The expression of IL-6 and TNF-α in GEnCs induced by high glucose and the activation of p38 MAPK signaling pathway were observed by lentiviral vector interfering with ChemR23 expression:Compared with high glucose+empty vector group,and the expression of IL-6 and TNF-α in high glucose+shRNA group were significantly decreased(P <0.05).Western blot analysis showed that compared with the control group,the high glucose group could significantly increase the phosphorylation level of p38 MAPK(P <0.05),leading to its activation.The level of p-p38 MAPK in the high glucose+shRNA group was significantly lower than that in the high glucose +empty vector group(P <0.05).4.The expression of IL-6 and TNF-α in mouse glomerular endothelial cells was detected by using p38 MAPK specific inhibitor SB203580:The levels of IL-6 and TNF-α in high glucose+SB203580 group were significantly lower than those in high glucose group(P <0.05).Conclusions1.High glucose stimulation can induce Chemerin expression in GEnCs.2.By binding to ChemR23,Chemerin activates p38 MAPK signaling pathway,and then promotes expression of IL-6 and TNF-α.These inflammatory cytokines aggravate inflammation of endothelial cells.3.Targeting ChemR23 is a potential way to manipulating high glucose-induced endothelial inflammation.