The Effect Of Exosomes Derived From Bone Marrow Mesenchymal Cells On Macrophage Polarization

Objective:Sepsis is defined as life-threatening organ dysfunction caused by a dysregulatedhost response to infection.Severe sepsis can cause multiple organ dysfunction,meanwhile lung dysfunction can lead to acute lung injury(ALI)or more worse to acute respiratory distress syndrome(ARDS).The treatment of sepsis mainly relies on anti-infection,organ support.Currently,no specific therapeutic drugs are approved to use in sepsis treatment.The mechanism of sepsis induced ARDS is mainly the imbalance of pro-inflammatory and anti-inflammatory responses,including the release of pro-inflammatory cytokines,which stimulates the corresponding signal transduction system in cells and induces apoptosis,thus aggravating ARDS.The polarization of macrophages plays an important role in regulating the imbalance of inflammation.Macrophages can be differentiated into M1 type and M2 type.M1 can secrete a large number of pro-inflammatory cytokines,such as tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),producing pro-inflammatory effects and exerting the autoimmune function of the body.M2 secretes anti-inflammatory factors such as interleukin 10(IL-10)and arginase 1(Arg-1),which are involved in tissue repair and wound healing.According to different stimulating factors,M1/M2 can reciprocally transform,and the polarization of macrophages determines the result of inflammation.Previous studies have shown that exosomes can reduce the permeability of pulmonary capillaries,inhibit inflammatory responses,reduce oxidative stress injury,reduce the severity of lung injury,and improve the survival rate of septic induced ALI mice.In addition,umbilical mesenchymal stem cells can promote the transformation of macrophages into M2 subtypes and repair myocardium.Based on the above cognition and existing research,we hypothesized that exosome originating from bone marrow mesenchymal stem cells may have influence on the polarization of macrophages.We use LPS and exosome induced bone marrow-derived macrophage,detect cytokines in the supernatant macrophage at different time points,judge macrophage polarization.Methods:Part One:Rat bone marrow mesenchymal stem cells were cultured,and the supernatant was collected after the cells were attached to the wall and co-culture with esoxome-deleted culture medium for 24-48h.Exosomes were obtained by overspeed centrifugation.Western Blot was used to identify them,and BCA protein assay was used to detect their concentration.Part Two:Extract and culture murine bone marrow-derived macrophages,adjust the cell concentration to 1×106/m L,and incubate them in 12-well plates.PBS control group,lipopolysaccharide(100 g/L)group,lipopolysaccharide(100 g/L)+exosome(10mg/L)group were set,and 3 Wells were repeated in each group.After cell attachment,the intervention began.PBS,lipopolysaccharide and exosomes were added at the same time.The cell supernatant was collected at 6h,12h,24h and 48h after intervention,and the concentration changes of TNF-α,IL-6,IL-10 and Arg-1 in the supernatant of macrophages were detected by ELISA.Then sterile cell slide was placed in a 12-well plate,wherein macrophages were incubated with 1×10~6/m L,and the grouping method was the same as above.It was then fixed with paraformaldehyde,washed with PBS,incubated with TNF-and arg-1 antibodies,put in the refrigerator overnight at 4℃,incubated with secondary antibodies,washed with PBS,sealed with DAPI,and perform immunofluorescence experiment.Results:1.Western Blot:show the expression of exosome specific membrane protein markers CD9 and CD63.2.Macrophages derived from bone marrow of mice grew adherent to the wall,showing the cells branching out into antennae and with high cell transmittance,and were successfully cultured as steady macrophages.3.Morphology of macrophages after LPS stimulation:the cells were oblate with short tentacles and poor transmittance,which were morphological characteristics of M1.The morphology of macrophages after exosomes were added:the cells were cordlike,antenna elongated and had high transmittance,which was the typical morphology characteristics of M2.4.ELISA results:At the same time point,the levels of TNF-αand IL-6 secreted by macrophages in the lipopolysaccharide group were significantly higher than those in the control group and the lipopolysaccharide plus exosome group.At the same time point,the levels of IL-10 and Arg-1 secreted by macrophages in the lipopolysaccharide plus exosome group were significantly higher than those in the control group and the LPS group.5.Immunofluorescence:The expression of TNF-αin macrophages in the lipopolysaccharide group was high,while the expression of TNF-αwas significantly down-regulated in the lipopolysaccharide plus exosome group,and the fluorescence intensity of Arg-1 protein was up-regulated.Conclusion:Bone marrow mesenchymal derived exosomes can inhibit the polarization of M1,promote the activation of macrophages to M2,reduce the generation of pro-inflammatory factors and promote the generation of anti-inflammatory factors.Exosomes can maintain the balance of M1/M2 macrophages by influencing the polarization of macrophages,so as to develop the pro-inflammatory response to the anti-inflammatory response and improve the inflammatory response.