Effect Of Exosomes From M1 Macrophage On Proliferation Of Vascular Smooth Muscle Cells And Intervention Of Icariin

Objective: To investigate the effect of exosomes derived from M1 macrophages activated by LPS on the proliferation of rat VSMCs,and to explore the intervention effect of ICA on the proliferation of VSMCs.Methods:(1)Culture and identification of macrophages and VSMCs: rat peritoneal macrophages and rat VSMCs were extracted,and the surface markers CD68 of macrophages and α-SMA of VSMCs were identified by immunofluorescence technique.(2)M1-type macrophages were induced by LPS: Macrophages were randomly divided into Control group and LPS(100 ng/m L)group.The Control group was given constant volume medium for 24 h.The m RNA expression levels of inflammatory cytokines IL-1β,IL-6 and TNF-α in macrophages were detected by RT-PCR,and the protein levels of i NOS and TNF-α were detected by Western Blot.The surface marker CD86 of M1 type macrophages was detected by immunofluorescence technique.(3)Exosome extraction and identification: Exosomes in the culture medium of the Control group(Cexo)and the LPS group(LPSexo)were extracted by ultra-high speed centrifugal in method(2).Exosomes morphology was observed by transmission electron microscopy,the particle size range of exosomes was detected by particle size analysis,and the surface marker protein CD63 and TSG101 of exosomes were detected by Western Blot.(4)Effects of LPSexo on the proliferation of VSMCs: VSMCs were randomly divided into Control group,Cexo group and LPSexo group.Exosomes extracted from(3)were added into the culture medium of VSMCs.After 24 h,the proliferation of VSMCs was detected by MTT colorimetry,Western Blot and flow cytometry.Di I labeled exosomes,and the uptake of VSMCs exosomes was observed under fluorescence microscope.Exosome inhibitor GW4869 was used to intervene the proliferation of VMSCs induced by LPSexo.Highthroughput sequencing of mi RNAs in exosomes and VSMCs was performed.(5)Intervention effect of ICA on LPSexo-induced VSMCs proliferation: VSMCs were randomly divided into Control group,VSMCs+ ICA(1 μmol/L)group,LPSexo group,ICA1μmol/L group,ICA0.1 μmol/L group,ICA0.01 μmol/L group,and DMSO group.The same volume of serum-free medium and various concentrations of ICA were pretreated for 2 h,and LPSexo was added to induce VSMCs.After 24 h of treatment,the viability of VSMCs was detected by MTT colorimetry,the proliferation of VSMCs was detected by Western Blot and flow cytometry.Exosome uptake by VSMCs after the action of ICA was also observed under microscope.Results:(1)Culture and identification of macrophages and VSMCs: Macrophage marker protein CD68 and VSMCs marker protein α-SMA were positive,and the cell purity was 90%and 100%,respectively.(2)LPS-induced M1-type macrophages: 24 h after LPS(100 ng/m L)treatment,the m RNA expression levels of IL-1β,IL-6 and TNF-α were significantly increased,which were 7,8and 4 times of the Control group,respectively.The protein levels of i NOS and TNF-α were also significantly higher than that of the Control group.A large amount of marker CD86 was expressed on the surface of M1 type macrophages,indicating that M1 type macrophages were successfully induced.(3)Identification of exosomes: under electron microscope,the shape of exosomes was halfsunken spherical or saucer-shaped,and the particle size ranged from 50 to 150 nm.A large number of CD63 and TSG101 proteins were expressed on the surface of exosomes,indicating that the exosomes was extracted successfully.(4)The effect of LPSexo on the proliferation of VSMCs: compared with the Control group,the proliferation of VSMCs in the Cexo group was not significant,but the proliferation of VSMCs treated with LPSexo was significant,and the effect could be antagonized by GW4869.LPSexo can be taken up by VSMCs into cells and concentrated around the nucleus.Compared with Cexo group,the expression level of rno-mi R-450a-5p was significantly down-regulated in LPSexo group,and the expression level of rno-mi R-5132-5p＿L-2R+2was significantly up-regulated.Compared with the VSMCs+ LPSexo group,the expression level of rno-mi R-450a-5p was significantly down-regulated.In addition,the expression levels of mi RNAs such as rno-mi R-466C-5P＿R + 1＿1SS18CT in exosomes and VSMCs also changed accordingly.(5)Intervention of ICA on LPSexo-induced VSMCs proliferation: ICA(0.01,0.1,1 μmol/L)could significantly inhibit LPSexo-induced VSMCs proliferation in a concentrationdependent manner.Conclusion: LPS-activated macrophage derived exosomes can induce the proliferation of VSMCs,and ICA can reduce the proliferation effect.