Study Of The Mechanism By Which Human Adipose-Derived Mesenchymal Stem Cell-derived Exosomes Promote Macrophage Polarization In The Treatment Of Facial Nerve Injury

Objective:The normal function of facial expression muscles is known as a second life of human being.The facial nerve(FN)is an important cranial nerve in the field of otology and the lateral skull base.FN injury can cause facial movement disorders and seriously affect the life of patients.However,even with surgical treatment,the patient’s treatment effect is still unsatisfactory,non-surgical treatment techniques after FN injury will be an important adjuvant therapy.Studies suggest that mesenchymal stem cell-derived exosomes play an important role in regulating the immune microenvironment and tissue repair and regeneration,but there is no research on the repair effect of stem cell-derived exosomes on facial nerve injury.The purpose of this study was to investigate the therapeutic effect of human adipose-derived mesenchymal stem cell-derived exosomes(MSC-Exos)on facial nerve injury and the role of macrophage polarization in MSC-Exos improving the local immune microenvironment and promoting facial nerve regeneration.To further clarify the mechanism by which MSC-Exos regulate macrophage polarization and provide new ideas for the clinical treatment of FN injury.Method:(1)Human adipose-derived mesenchymal stem cells(hAdMSCs)were isolated,the expression of surface marker molecules was identified by flow cytometry,their multiple differentiation potentials were identified by inducing osteogenic and adipogenic differentiation,MSC-Exos were separated by ultracentrifugation,and the morphology of exosomes was observed by transmission electron microscopy(TEM).The particle size was calculated by nanoparticle tracking analysis(NTA),and the expression of exosome markers was detected by western blotting technology.(2)The rat FN injury model was constructed,and 45 rats were randomly divided into 3 groups:sham operation group,local injection PBS model group and local injection MSCExos treatment group.The facial expression function of rats was evaluated at 1,3,7,and 14 days after the operation.The repair effect of MSC-Exos on FN injury was evaluated by HE staining,tissue immunofluorescence staining and TEM.(3)Rat facial nerve and peripheral venous blood were extracted on the 7th day after FN injury.Western blotting,real-time fluorescence quantitative polymerase chain reaction(qRT-PCR)and enzyme-linked immunosorbent assays were used to extract the blood.An enzyme-linked immunosorbent assay(ELISA)was used to detect the M2 polarization and systemic inflammatory response of macrophages in facial nerve tissue.After coculturing MSC-Exoss with macrophages differentiated from a human monocytic leukemia cell line(THP-1)for 48 hours,lipid polysaccharide(LPS)-stimulated macrophages were used to simulate the inflammatory microenvironment after FN injury.Flow cytometry,qRT-PCR and ELISA were used to detect the polarization of macrophages and the secretion of inflammatory factors.(4)By miRNA sequencing analysis of MSC-Exos(n=3),the co-expressed miRNAs were subjected to bioinformatics analysis to identify the key pathway by which MSC-Exos regulate macrophage polarization,and then Western blotting was performed in vivo and in vitro.Finally,through a rescue experiment in recipient cells,flow cytometry and qRTPCR were used for further verification to verify the mechanistic pathway by which MSCExoss regulate macrophage M2 polarization.Results:(1)Flow cytometry showed that hAdMSCs had positive expression of CD73,CD90,CD44,etc.,on the surface,but they did not express CD 106,CD34,CD45 or other molecules;osteogenic and adipogenic induction experiments showed that the isolated hAdMSCs had multidirectional differentiation potential.The extracted MSC-Exos conformed to the definition of exosomes in terms of morphology,particle size and expression of marker molecules.(2)In the rat FN injury model,it was confirmed that MSC-Exos can promote axonal and myelin regeneration,reduce the inflammatory response,and significantly improve FN function in FN-injured rats.(3)Both in vitro and in vivo experiments confirmed that MSC-Exos can up regulated the expression of M2 macrophage-related genes while inhibiting the expression of M1 macrophage-related genes,promoting the polarization of macrophages to the M2 type and regulating the inflammatory response.(4)For the top 100 co-expressed miRNAs in MSC-Exos,KEGG protein pathway enrichment analysis indicated that the MAPK signaling pathway was significantly enriched.Combined with the previous literature,the MAPK/NF-κb pathway was selected for verification.In vivo and in vitro Western blot results showed that MSC-Exos inhibited the activation of the MAPK/NF-κb signaling patfhway,among which the p38 MAPK/NFκb signaling pathway was downregulated most significantly.After adding this pathway agonist into macrophages,flow cytometry and qRT-PCR results further confirmed that MSC-Exos promoted M2 polarization of macrophages mainly by inhibiting the p38MAPK/NF-κb signaling pathway.Conclusion:This study confirmed that MSC-Exos can reduce the inflammatory response after FN injury,promote axon and myelin regeneration,and improve facial nerve function;MSCExos can improve the local immune microenvironment after FN injury by promoting the M2 polarization of macrophages and promote axonal regeneration;and under inflammatory microenvironmental conditions,MSC-Exos can promote the polarization of macrophages to the M2 type with tissue repair function by inhibiting the p38 MAPK/NFκb signaling pathway and reduce the proinflammatory phenotype of M1-type macrophage cells.