Paeonol Inhibits STAT3-medied Inflammatory Injury Of Endothelial Cells By Up-regulating Lipopolysaccharide Stimulated Monocytes-derived Exosomal MiR-223

Atherosclerosis（AS）is a chronic inflammatory process happened in artery which involved in various cells,and the inflammatory reaction run though the hole process.The injury of vascular endothelial cells（VECs）is the initial step of AS which will lead to abnormal secretion of inflammatory factors and immune cells infiltration they are the important mechanism of AS.Lipopolysaccharide（LPS）,as the main inducement of AS,can activate the innate immunity of Monocyte/macrophage（M/MΦ）,resulting in VECs damage.Exosomes involve in regulation of important physiological activities of cells and play an important role in the formation and development of AS by transmiting proteins and miRNAs from cell to cell.M/MΦderived exosomes are dispatched in response to injury,infection and inflammation.Exosomes released by monocytes contain a high amount of miR-223 that is shuttled into the endothelial cells in a biologically anti-inflammation effect.MiRNA-223 may inhibit the development of AS by regulating its downstream Signal transducers and activators of transcription 3（STAT3）and other inflammatory target genes.Therefore,as a regulatory factor in the upstream of STAT3,miRNA-223 may be a new target for the treatment of AS.Paeonol（Pae）was one of the active ingredients which isolated from dried root bark of Paeonia Suffruticosa Andr.and dried root and rhizome of Cynanchum Paniculatum（Bge.）Kitag.Pae has various functions such as anti-inflammatory,reducing blood fat,blood activating and stasis eliminating extensive pharmacological effects.Our recent studies have confirmed that Pae can has certain protective effect on the the damage of VECs,but further exploration is necessary to explain while the protective effect is related to the regulation of exosomal miR-223 in the VECs.In this study,we first observed the effects of Paeonol on the STAT3 pathway and miR-223expression in endothelial cells of aorta of high fat fed ApoE^-/-mice.In vitro,LPS was used as an inflammatory inducer to activate monocytes.The effect of LPS induced THP-1 cells derived exosomes in VECs was observed.The effect of Pae inhibits VECs inflammation by regulating THP-1 cells derived exosomes was elucidated from the gene level,which provides a new target and scientific basis for the application of Pae to the treatment of AS.OBJECTIVETo explore the effect of Pae on the inflammation of AS mice and the expression of miR-223 and p-STAT3,the effect of Pae on LPS stimulated monocyte derived exosomal miR-223 and its negative regulation of target protein STAT3 in VECs,it provided a new way to reveal the anti-AS drug from the potential target of monocyte derived exosomal miR-223.METHODSApoE^-/-mice were fed with high fat diet to replicate the AS model.HE staining was used to observe the pathological changes of the aorta in mice.The expression of IL-1βand IL-6 was detected by ELISA.Western blot was used to detect the expression of STAT3,pSTAT3,ICAM-1and VCAM-1.The expression of CD68 and p-STAT3 in aorta of each group was detected by immunohistochemistry.qPCR was used to detect the expression of miR-223.The THP-1/HUVEC co culture system was established.Detection of adhesion rate of HUVEC cells by Dil staining.The morphology of exosomes was observed by transmission electron microscope.NanoZS and NTA were used to detect potential and particle size distribution of exosomes.Western blot method was used to detect marker protein of exosomes.Observation of HUVEC uptake exosomes by laser microscopy and flow cytometry.Detection of miR-223 target gene by double luciferase gene report test.RESULTS1 Pae inhibits the formation of plaque and the expression of inflammatory mediators in ApoE^-/-miceWe established the ApoE^-/-AS model mice by feding high cholesterol diet,Pae can significantly reduce AS lesions and reduce plaque;decrease ApoE^-/-mice serum inflammatory cytokine of IL-1β,IL-6 levels;decrease ApoE^-/-mice aortic adhesion molecule VCAM-1 and ICAM-1 levels;Pae inhibited the adhesion and infiltration of aortic macrophages and inhibited the expression of pSTAT3 in ApoE^-/-mice,and it significantly increased the miR-223 content in aorta of ApoE^-/-mice.2 Pae inhibits HUVEC inflammatory response in co-cultured with LPS stimulated THP-1 cellsTo determine the best time and concentration of Pae effecting on THP-1 cells,we treated THP-1cells stimulated by LPS with different concentrations of Pae.Pae could inhibit the proliferation of THP-1 cells at 24h,60 M and Pae increases the expression of miR-223 in LPS-stimulated THP-1cellls.Then we establish a co-culture system of THP-1 and HUVEC,and we found that THP-1 can transfer miR-223 to HUVEC in the co-culture system.Pae increases the expression of miR-223 in HUVEC co-cultured with LPS stimulated THP-1 cells.Pae,exosomes secretion inhibitor GW4869and exosomes uptake inhibitor Heparin will decrease the level of IL-1β,IL-6,VCAM-1 and ICAM-1 in in HUVEC co-cultured with LPS stimulated THP-1 cells..3 The effects of Pae on the biological morphology of the exosomesIn order to verify the exosome function,we firstly use centrifugal ultrafiltration extraction of exosomes under different conditions,and characterize the exosomes by a variety of methods.exosomes were characterized by transmission electron microscopy.The exosomes has a kind of saucer clear membrane structure;Western blot were detected marker protein expression of exosomes CD9,LAMP2,CD63 CD54,Alix,TSG101,HSP70 was significantly higher than that of the THP-1 cells and the negative control Calnexin expression in exosomes;NanoZS and NTA showed that each exosomes overall particle peak at about 105 nm,the potential value about-10,no significant differences between the groups.4 Pae-exo decreased inflammatory response of HUVECIn order to exclude the interference of the Pae contained in the exosomes derived from THP-1cells treated with Pae on downstream experiments,we detected the content of Pae in the exosomes under different concentrations of Pae by HPLC.We found that the content of Pae in the exosomes increased with the increase of Pae treatment concentration.A trace of Pae（0.5,2,8 nM）in exosomes had no effect on the activity of HUVEC and have therapeutic effects on LPS-exo stimulated HUVEC.LPS-exo can significantly reduce the HUVEC activity,but Pae-exo was not obviously reduce the HUVEC activity.Increase the expression of miR-223 in HUVEC after uptake of exosomes.Pae-exo can increase the level of miR-223 in HUVEC.Pae-exo decreases the level of IL-1β,IL-6,ICAM-1,VCAM-1 in HUVEC and the adhesion of HUVEC to THP-1 cells,which can reduce the level of STAT3,pSTAT35 Pae inhibits STAT3 pathway by increasing THP-1 derived exosomal miR-223 in HUVECThe double luciferase gene report test showed that the miR-223 target was STAT3,After transfection of miR-223 mimic,the content of miR-223 in exosomes and HUVEC which uptake exosomes could be increased and the expression of STAT3 and p-STAT3 were reduced.Inhibition of the level of IL-1β,IL-6,ICAM-1,VCAM-1 and the adhesion of HUVEC to THP-1 cells.However,miR-223 inhibitor can reverse the effect of Pae,increase the expression of STAT3 and p-STAT3,and increase the level of IL-1β,IL-6,ICAM-1,VCAM-1 and the adhesion of HUVEC.CONCLUSION1.The mechanism of Pae resistance to AS in ApoE-/-mice may be related to the inhibition of STAT3 inflammatory pathway,the expression of IL-1β,IL-6,ICAM-1,VCAM-1,and the inhibition of monocyte adhesion.2.Pae can increase the expression of miR-223 in exosomes derived from THP-1 cells,increase the content of miR-223 in HUVEC after uptake of exosomes,decrease the expression of STAT3,p-STAT3,inhibit the expression of HUVEC IL-1β,IL-6,ICAM-1,VCAM-1,and decrease the adhesion of monocyte,while miR-223 inhibitor can reverse the action of Pae.