Mechanism Of ANG ? Regulating HMGB1 Secretion In Macrophage And Its Effect On Macrophage Polarization

ObjectiveRAW264.7 macrophage cells were stimulated with Angiotensin II to investigate their effects on macrophage reprogramming and secretion of high mobility group protein B1?HMGB1?,and the mechanism was further studied.Method?1?The effect of ANG II on the secretion of HMGB1The RAW264.7 macrophage cells were treated with different concentrations of ANG II?0,10nmol/L,100nmol/L,1?mol/L,10?mol/L?for the corresponding time?0,3h,6h,9h,12h?and total RNA and protein were extracted.RT-qPCR and western blot were used to measure the HMGB1 mRNA and protein expression level,respectively.Immunofluorescence was used to detect the shuttle of HMGB1 in macrophage after treatment by ANG II.Meanwhile,the expression of HMGB1 in cell culture supernatants was detected by ELISA.?2?Molecular mechanism of ANG II-induced HMGB1 secretionCo-immunoprecipitation assay was used to detect acetylation of HMGB1 and the level of SIRT1 protein after ANG II treatment.The phosphorylation levels of JAK2/STAT3 was analyzed by western blot.Before stimulated with ANG II,the cells were pretreated with Niclosamide and HBC for 2 h,western blot was used to detect the phosphorylation of JAK2,STAT3 the level of HMGB1 protein expression was also detected by western blot.?3?The role of extracellular HMGB1 induced by ANG II on macrophage polarizationThe protein level of iNOS after ANG II and HMGB1 stimulation in macrophage was detected by western blot,ELISA detection of cytokine levels.The cells were divided into different groups:control group,EP+ANG II group,SB+ANG II group,and ANG II group,then detect the HMGB1 and iNOS expression.Using HMGB1 siRNA to specifically inhibit HMGB1,ANG II culture supernatant and HMGB1 siRNA cell culture supernatant were collected and the experiment was divided into:control group,ANG II culture supernatant and macrophage co-culture group,ANG II culture supernatant+losartan and macrophage co-culture group,HMGB1 siRNA culture supernatant and macrophage co-culture group,HMGB1 siRNA culture supernatant+losartan and macrophage co-culture,then detect i NOS protein by western blot.?4?The effect of HMGB1 on the expression of ANG II and AT₁RMacrophages and primary monocytes were treated with different redox forms of HMGB1.The expression of AT₁R protein was detected by western blot,and the expression of ANG II in cell culture supernatant was measured by ELISA.Results?1?ANG II can promote the secretion of HMGB1 in a dose-and time-dependent manner,and 10?mol/L ANG II treatment for 12 hours had the best effect.Immunofluorescence showed that ANG II treated for 9 hours showed significant nuclear plasma translocation.The ELISA results showed that HMGB1in the cell culture supernatant was increased significantly.?2?The data of co-immunoprecipitation showed that ANG II can promote the dissociation of HMGB1 and SIRT1 and promote the acetylation of HMGB1.The phosphorylation levels of JAK2/STAT3 was increased after treated with ANG II in macrophages.The phosphorylation levels of JAK2/STAT3 and the expression of HMGB1 were decreased after pretreatment with Niclosamide and HBC for 2 h.?3?In the EP+ANG II group,and SB+ANG II group,the expression of HMGB1in macrophages was significantly decreased,and the iNOS protein level was also decreased;which indicated that EP and SB weakened the ANG II-induced polarization of macrophage.Compared with the ANG II culture supernatant,no HMGB1 was detected in the culture supernatant in the HMGB1 siRNA treated group.The protein levels of iNOS in HMGB1 siRNA culture supernatant and macrophage co-culture group and HMGB1 siRNA culture supernatant+Losartan and macrophage co-cultureg roup were significantly reduced.?4?HMGB1 has the ability to promote the secretion of ANG II and its receptor AT₁R expression,especially disulfide-HMGB1.Conclusion?1?ANG II can induce macrophage polarize to M1 by promoting secretion of HMGB1 in macrophage,while secreted HMGB1 can promote the expression of ANG II and its receptor AT₁R by positive feedback.?2?ANG II can promote the dissociation of HMGB1 and SIRT1,increase the acetylation of HMGB1,and regulate the secretion of HMGB1 through the JAK/STAT signaling pathway.