| 【Purpose】This study explored the mechanism of electroacupuncture(EA)by regulating the release of exosomes and mediating miR-21 transmission in micro RNA(miRNA)to accelerated nerve regeneration,and launched experiments in vivo and in vitro.Enriching the mechanism of acupuncture and moxibustion in the treatment of peripheral nerve injury(PNI)from a new perspective of exosomes-mediated miR-21 transmission of information,providing experimental evidence for the clinical promotion of EA treatment of PNI and the exploration of new targets and theoretical basis for the treatment of PNI.【Methods】1.The effect of EA on the release and delivery of miR-21 from rat exosomes of sciatic nerve injury(SNI)model rats: SNI rat models were prepared,and after EA treatment for 1 week,the serum exosomes were extracted by ultracentrifugation.The expression of CD9 and CD63 was identified by WB to detect the exosomes.The expression of miR-21 in the serum exosomes was detected by RT-q PCR.The exosomes marker p CT-CD63-GFP was transfected with lentivirus,and the transfer and internalization of Schwann cells(SC)exosomes to axons were observed in vivo.2.The effect of exosomal miR-21 on EA’s promotion of nerve regeneration: rat model of SNI was constructed,the exosome release inhibitor GW4869 was used to inhibit the release of exosomes in the circulation,and the sponge RNA(miR-21-5p-sponge)lentiviral damage local transfection was used to inhibit local miR-21 expression,and explores the mechanism of exosomal miR-21 in EA promoting SNI.Experimental grouping: Model group(Model),electroacupuncture group(EA),EA+GW4869 group(EG),EA+miR-21-5p-sponge group(ES).3 weeks later observation of the general situation of rats,detection of serum exosomal miR-21 expression in Model and EA.The nerve conduction velocity(NCV)was measured to evaluate the neuromuscular conduction.the sciatic functional index(SFI)was measured to reflect the recovery of sciatic nerve function,and the wet weight ratio of gastrocnemius muscle(WWRG)was measured from the perspective of neurotrophic muscle,to comprehensively reflects the recovery of sciatic nerve function.Immunofluorescence staining observation(axonal remyelination,SC proliferation,neurotrophic factors(NTFs)expression).RT-q PCR detection of damage local miR-21,rat sprouty homolog 2(SPRY2),growth-associated protein-43(GAP43),DNA Methyltransferase 3A(DNMT3A),to clarify the effect of exosomal miR-21 on EA’s promotion of nerve regeneration..3.The transfer of exosomal miR-21 from SC to neurons and its influence on the growth of neurites: The transfer of exosomal miR-21 from SC to axons was verified in vitro.The internalization of the co-culture between SC and neurons was assessed by a classic Boyden chamber(BD Falcon,0.4 μm pore size).Co-culture SC and NG108-15 in Boyden chamber.NG108-15 was inoculated on the lower layer,and the SC expressing CD63::GFP was on the upper layer.By immunofluorescence in miR-21 situ hybridization,the delivery of exosomes was traced under the microscope.In order to study the effect of exosomal miR-21 delivered by SC on the growth of neuronal processes and cell viability,NG108-15 cells were divided into 4 groups: NC stands for control group,NC+EXO for exosome group,IN for miR-21 inhibitor group,and IN+EXO means miR-21 inhibitor + exosome group.After NG108-15 was planted in a confocal culture dish,the next day,adding PBS(NC),3 μg exosomes(NC+EXO),10 m M miR-21 inhibitor(IN),10 m M miR-21 inhibitor and 3 μg Exosomes(IN+EXO)separately,after 24 hours of treatment,immunofluorescence staining of βIII-Tubulin was performed,and neurons activity was observed by cell counting kit-8(CCK-8)..【Results】1.The effect of EA on the release and delivery of miR-21 in SNI model rats: After 1week,the miR-21 expression of serum exosomes was detected after treatment and was found elevated in Model vs.Sham(**P<0.01);Expression of miR-21 in EA was reduced vs.Model(##P<0.01).The results of the internal reference U6 and the external reference miR-39 were consistent.Total RNA of injured local nerve was extracted,and miR-21 in Model was reduced vs.Sham through RT-q PCR(*P<0.05),and escalated in EA(*P<0.05).miR-21 was extraordinary exaltation in EA vs.Model(##P < 0.01).After labeling the exosomal surface marker CD63 with green fluorescence,neurofilament-200(NF200)(red)and GFP(green)immunofluorescence staining were co-localized by a confocal microscope,and there were sites that express both NF200 and GFP.2.The effect of exosomal miR-21 on EA’s promotion of nerve regeneration: 3 weeks after modeling,there was no significant improvement or difference in the general state of rats in Model and EA,while the plantar ulcers of the rats in EG and ES were significantly worse than those in the previous two groups.exosomes expression of miR-21 in serum was found significantly went up in EA vs.Model after 3 weeks(**P<0.01).Detection of nerve function recovery indicators: NCV,SFI,WWRG rised in EA vs.Model(**P<0.01).The number of axons increased remarkably in EA and EG vs.Model group(**P<0.01,*P<0.05),and slight elevated in ES but no statistical difference(P>0.05).EA MBP expression was clearly higher vs.Model,and the number and integrity of myelin sheath were significantly improved(**P<0.01),the myelin sheath in EG also increased(**P<0.01),but a large number of deformities.There were significantly more S-100α positive cells in EA and EG with a rise in AOD vs.Model(**P<0.01).Three types of NTFs were detected by immunofluorescence on the cross-section of injured local nerves: nerve growth factor(NGF),brain-derived neurotrophic factor(BDNF)and glial cell-derived neurotrophic factor(GDNF).Expressions of these three NTFs were clearly up in EA vs.Model,(**P<0.01),and the upregulation of NGF was statistically significant(*P<0.05)and GDNF(**P<0.01)in EG,up-regulation of GDNF in ES(**P<0.01).Significant improvement of miR-21 was found in EA vs.Model(**P<0.01),while SPRY2 was obviously decased in injured local nerves in EA vs.Model(**P<0.01),and EG was also down-regulated(*P<0.05),and there was an obvious negative linear relationship between the two.GAP43 and DNMT3 A in EA were obviously improved vs.Model(**P<0.01),while a slight increase of GAP43 in ES(*P<0.05).It was found that miR-21 has an obvious positive linear relationship with the two.3.The transfer of exosomal miR-21 from SC to neurons and its influence on the growth of neurites: immunofluorescence double staining observed the transfer of CD63::GFP exosomes from SC to NG108-15 soma and its neurites.Through miR-21 in situ hybridization combined with immunofluorescence double staining,it was detected that exosomes miR-21 could be transported from SC to NG108-15 and its protrusions.WB detection of CD9 and CD63 verified that ultracentrifugation can remove fetal bovine serum(FBS)exosomes and extract exosomes from cell supernatants.NC+EXO had longer average protrusion length and longest protrusion length vs.NC(**P<0.01),and the average protrusion length of IN was shorter(**P<0.01),the longest protrusion length was also shorter but there is no statistical difference,IN+EXO average protrusion length was longer(**P<0.01),and the longest protrusion length had no statistical difference.The average protrusion length and longest protrusion length of IN and IN+EXO were shorter vs.NC+EXO(##P<0.01).The average protrusion length of IN+EXO(&&P<0.01)and the longest protrusion length(&P<0.05)increased vs.IN.Through cell counting kit-8(CCK-8),it was found that the neuronal cell viability of NC+EXO was higher vs.NC(**P<0.01),while IN and IN+EXO were lower(**P<0.01).Cells in liveliness in IN+EXO increased vs.IN(&&P<0.01).【Conclusion】1.In the early stage of SNI,EA reduces serum exosomes miR-21 and increases miR-21 in injured local nerves.SC exosomes can be transmitted to axons.2.For long-term SNI,EA increases serum exosomal miR-21 and injured local nerve miR-21.Exosomal miR-21 participates in positively regulating EA to promote SNI repair.3.Exosomal miR-21 can be transferred from SC to NG108-15 cell body and its protrusions.SC-derived exosomal miR-21 can promote the growth of NG108-15 protrusions and cell viability. |
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