MiR-1246 In Exosomes Of Gingival Mesenchymal Stem Cells Regulates M2-type Macrophages In The Treatment Of Multiple Sclerosis Through STAT3 And NF-κB Pathway

Objective:Studies have shown that in the early stage of Multiple sclerosis（MS）and experimental autoimmune encephalomyelitis（EAE）,macrophages infiltrate the central nervous system（CNS）and destroy nerve cells.Mesenchymal stem cells derived exosomes（MSC-exo）are implicated in EAE,Systemic lupus erythematosus,Systemic lupus erythematosus,SLE),periodontitis,colitis and other diseases can reduce inflammation by promoting the polarization of M2 macrophages;Sequencing analysis showed that miR-1246 was the miRNA with the highest content in Gingival mesenchymal stem cell（GMSC）.Therefore,this study aims to explore whether Gingival mesenchymal stem cell derived exosomes（GMSC-exo）can treat EAE by targeting macrophages with miR-1246 and its mechanism of action.Methods:1.Identification of GMSC:Their multidirectional differentiation potential was confirmed by inducing osteogenic and lipogenic differentiation of GMSC,and the expressions of stem cell surface markers（CD34,CD45,CD73,CD90 and CD105）of GMSC were detected by flow cytometry.2.Identification of GMSC-exo:GMSC-exo was extracted using a kit combined with ultrafiltration method.Transmission electron microscope（TEM）was used to observe the ultrastructure of GMSC-exo,NTA was used to detect the particle size distribution,and WB was used to detect the expression of exosome markers（CD9,CD63 and CD81 proteins）.3.miRNA inhibition of GMSC:LV3-hsa-miR-1246 inhibitor and LV3-NC were transfected into GMSC to extract exosomes,so as to achieve the purpose of inhibiting the expression of miR-1246 in GMSC.Gene detection of miR-1246 was performed on exosomes to evaluate the transfection effect of lentivirus.4.GW4869 prestimulates GMSC:GW4869 is co-cultured with GMSC to inhibit the secretion of exosomes by GMSC.As a control,the effects of GMSC and GMSC-exo are verified.In vivo experiment:1.Establishment of EAE model:The EAE model was induced by MOG_35-55,and the EAE mice were observed daily and their weight and clinical scores were recorded.2.Therapeutic intervention:EAE mice were randomly divided into 6 groups:PBS group,GW4869-GMSC group,GMSC group,GMSC-exo group,GMSC^miR-1246IN-exo group and GMSC^NC-exo group.The mice in each group were treated on the 18^th day after immunization（Day 18）.3.Pathological test:Day 40,mice were sacrificed,and the spinal cord of mice was stained with HE and LFB to observe the inflammatory cell infiltration and demyelination.4.Changes in M1/M2 macrophage ratio:The proportion of M1 macrophages（F480⁺CD11b⁺CD86⁺）and M2 macrophages（F480⁺CD11b⁺CD206⁺）in spleen of mice was detected by flow cytometry.The levels of CD86,i NOS,CD206 and Arg-1 genes in spinal cord of mice were detected by q RT-PCR.5.Changes in levels of inflammatory cytokines:ELISA was used to detect changes in serum levels of inflammatory cytokines（TNF-α,IL-12,TGF-βand IL-10）in mice.6.Action pathway of miR-1246 in EAE:The expressions of p-STAT3,STAT3,p-P65,P65,TERF2IP and IκBαin the spinal cord of mice were detected by WB to investigate whether miR-1246 could affect the NF-κB pathway and improve EAE by targeting TERF2IP protein.In vitro experiment:LPS+IFN-γwas used to induce the polarization of M0 RAW264.7 into M1 macrophages to simulate the inflammatory environment.7 groups were set up:M0group,M1 group,M1+GW4869-GMSC group,M1+GMSC group,M1+GMSC-exo group,M1+GMSC^miR-1246IN-exo group,M1+GMSC-exo group,q RT-PCR（CD86,i NOS,CD206and Arg-1）and WB（p-STAT3,STAT3,p-P65,P65,TERF2IP,and IκBα）were detected by direct co-culture method.Cell supernatants（TNF-α,IL-12,TGF-βand IL-10）were collected and detected by ELISA.The above methods were used to explore whether miR-1246 in GMSC-exo can affect the NF-κB pathway and STAT3 pathway to improve EAE by targeting TERF2IP protein in macrophages.Results:1.GMSC is consistent with the basic characteristics of MSC:it has the potential of multidirectional differentiation;CD105,CD73 and CD90 were expressed,but CD34and CD45 were not expressed.2.GMSC-exo conforms to the basic characteristics of exosomes:GMSC-exo is a spherical vesicle with bilayer membrane structure;Expression of CD63,CD9 and CD81;The average particle size was 106±3.15 nm,which was consistent with the diameter range of exosomes.3.Lentivirus was successfully transfected into GMSC,and miR-1246 in GMSC^miR-1246IN-exo was inhibited.4.In vivo experiment results proved that the EAE model was successfully constructed by MOG_35-55;Compared with the GMSC-exo group,the GMSC^miR-1246IN-exo group showed decreased body weight and increased clinical scores.Inflammatory cell infiltration and demyelination were aggravated.The proportion of M2 macrophages decreased and the proportion of M1 macrophages increased.The levels of M1 macrophage-related gene CD86 and i NOS increased,and the levels of M2 macrophage-related gene CD206 and Arg-1 decreased.Inflammatory cytokines TNF-αand IL-12 were increased,while anti-inflammatory cytokines TGF-βand IL-10 were decreased.The expressions of TERF2IP,p-P65 and P65 proteins were increased,while the expressions of IκBα,p-STAT3 and STAT3 proteins were decreased.Compared with GW4869-GMSC group,inflammatory cell infiltration,M2 macrophage proportion,anti-inflammatory factor and TERF2IP protein expression were decreased in GMSC-exo group,indicating that the inhibitory effect of GW4869 on exosome secretion inhibited the immunomodulatory effect of GMSC.Compared with GMSC-exo group,the proportion of M1 macrophages,i NOS m RNA expression,IL-10 cytokine and p-STAT3 protein expression were decreased in GMSC-exo group.No statistical significance was found in other indicators,indicating that GMSC also has an immunomodulatory effect,but GMSC-exo has the best therapeutic effect.5.In vitro experiment results showed that flow cytometry showed that the proportion of M1 macrophages after LPS+IFN-γcombined stimulation was 88.2%,indicating that M1macrophages were successfully induced.Compared with M1+GMSC^miR-1246IN-exo group,the proportion of M1 macrophages increased and the proportion of M2 macrophages decreased in M1+GMSC-exo group.The levels of CD86 and i NOS increased,the levels of CD206 and Arg-1 decreased,the levels of TNF-αand IL-12 increased,the levels of TGF-βand IL-10 decreased,the protein expressions of TERF2IP,p-P65 and P65 increased,and the protein expressions of IκBα,p-STAT3 and STAT3 decreased.Compared with M1+GW4869-GMSC group,the expressions of pro-inflammatory and anti-inflammatory factors were decreased in M1+GMSC-exo group and increased in M1+GW4869-GMSC group.Compared with M1+GMSC-exo group,CD206,Arg-1 and p-STAT3 in M1+GMSC-exo group were significantly increased,while no statistical difference was found in other indicators,indicating that GMSC-exo had a better effect on inhibiting inflammatory response than GMSC.Conclusion:1.Both GMSC and GMSC-exo have immunomodulatory effects,which can inhibit inflammation and reduce demyelination,thus improving EAE symptoms,but GMSC-exo has the best therapeutic effect.2.GMSC-exo and its miR-1246 can inhibit M1 macrophages and promote M2macrophages,that is,GMSC-exo and miR-1246 can inhibit inflammation by promoting the polarization of M2 macrophages.3.GMSC-exo and miR-1246 can further inhibit the NF-κB signaling pathway and promote the STAT3 signaling pathway to improve EAE by inhibiting the expression of TERF2IP.In conclusion,GMSC-exo can treat EAE by targeting macrophages with miR-1246.The mechanism of action is that miR-1246 inhibits the expression of TERF2IP,low expression of TERF2IP can inhibit the NF-κB signaling pathway and promote the STAT3 signaling pathway.