| Objective:Diabetic peripheral neuropathy(DPN)is one of the most common complications of diabetes and the leading cause of non-traumatic amputations,there is no ideal treatment for DPN.Multiple cell-derived exosomes,including Schwann cells and mesenchymal cell-derived exosomes,have been reported to improve DPN progression and promote neuropathy recovery.There have been multiple reports of hemotherapy having powerful restorative effects in multiple regenerative areas.Young blood is rich in a variety of substances that are completely different from aging,which can play a role in rejuvenating aging individuals.However,it is not clear whether there are differences in plasma-derived exosomes between diabetic individuals and age-matched healthy individuals,and whether plasma-derived exosomes from age-matched healthy individuals and ageing individuals can also improve DPN in diabetic individuals and related molecular mechanisms remains to be explored.Therefore,the main objective of this study was to compare the difference of plasma-derived exosomes between DPN rats and age-matched healthy rats and to investigate whether plasma-derived exosomes of healthy rats(hplasma-exos)and aging rats(ageing-exos)can ameliorate DPN.The main micro RNA(miRNA)in plasma-derived exosomes that play a role in improving DPN were identified.The signaling pathway of DPN improvement was revealed to provide a reliable molecular target for clinical DPN intervention.Methods:1.Plasma-derived exosomes were collected from DPN rats and age-matched healthy rats,and the extracted exosomes were characterized by transmission electron microscopy(TEM),particle size analysis and Western blot;2.The differential expression of miRNA in the above two types of plasma-derived exosomes was analyzed by miRNA sequencing technology.3.DPN rats were injected with 300μl(100μg)hplasma-exos intravenously twice a week for four consecutive weeks,and mechanical threshold was measured by Von Frey test,the thermal threshold was measured by hot plate test,and motor ability was measured by Rotarod test.Sensory and motor nerve conduction velocity were measured by electrophysiological tests to assess the recovery of nerve function.4.The sciatic nerve of DPN rats receiving hplasma-exos injection was observed by immunofluorescence staining and TEM to evaluate the myelin changes of sciatic nerve.The nerve fibers in the intraepidermal of the foot pad were stained with PGP9.5to evaluate the severity of small fiber neuropathy.The blood perfusion of sciatic nerve and plantar skin was analyzed by Di I perfusion and Laser Doppler Flow imaging.NF-L + SYN/a-BTX staining was performed on extensor digitorum longus(EDL)muscles to observe the innervation of the neuromuscular junction(NMJ)and muscle spindle.5.FITC-CTB was injected into the tibialis anterior muscle and the proportion of cells retrograde traced to the dorsal root ganglion(DRG)was counted;the recovery of muscle innervation was evaluated by measuring the wet weight of gastrocnemius muscle.6.The differentially expressed miRNA in the sequencing data were analyzed,sciatic nerves of rats in different groups were sampled,and the expression of miRNA in sciatic nerves was verified by RT-PCR.7.Through bioinformatics analysis,the miRNA(miR-20b-3p)that may play an important role in hplasma-exos was selected,and the exogenously synthesized miRNA analogue(miR-20b-3p agomir)were used for intrathecal injection of DPN rats.The recovery of neurological function was evaluated according to the above methods.8.Schwann cells(RSC96)were cultured with high glucose(100m M)to simulate the diabetes microenvironment in vivo.After exogenous addition of hplasma-exos,the expression level of autophagy related proteins was verified by Western blot,and the changes of autophagy organelles in different groups were observed by TEM.9.Transfected miRNA inhibitor(miR-20b-3p inhibitor)into Schwann cells under different stimuli,and the changes of autophagy level of Schwann cells were detected by Western blot and TEM.10.Western blot analysis was performed to detect the expression changes of target genes and autophagy related proteins after treatment with miRNA mimic(miR-20b-3p)alone or combined with target gene agonists(Colivelin TFA).11.Dual luciferase gene reporting experiment verified the regulation of miRNA(miR-20b-3p)on the target gene(STAT3).12.The DPN rats were injected with 300μl(100μg)ageing-exos through the tail vein twice a week for four consecutive weeks and the neurological recovery was evaluated according to the above methods.Results:1.There are differences in miRNA in plasma-derived exosomes between DPN rats and age-matched healthy rats.2.Hplasma-exos can improve the nerve function of DPN rats,the behavior and nerve conduction velocity of DPN rats can be restored to a certain extent.3.Hplasma-exos alleviated peripheral nerve injury in DPN rats,after hplasma-exos injection,sciatic nerve demyelination in DPN rats was improved,and the intraepidermal nerve fiber density(IENFD)of the foot pad of DPN rats was also improved.4.Hplasma-exos improved blood perfusion in sciatic nerve and plantar skin of DPN rats.5.Hplasma-exos augmented the motor and sensory innervation of the targets,after hplasma-exos injection,the innervation of NMJ and muscle spindle was improved,the proportion of FITC-CTB-positive DRG cells was increased,and the wet weight of gastrocnemius muscle was increased.6.Treatment with miR-20b-3p agomir can improve peripheral neuropathy in diabetic rats.7.MiR-20b-3p down-regulated the expression of STAT3 and promoted autophagy in high-glucose stimulated Schwann cells.8.Ageing-exos had no positive effect on the improvement of DPN.Conclusions:1.MiRNA in plasma exosomes was different between DPN and age-matched healthy rats.2.MiR-20b-3p was enriched in hplasma-exos,and both of which can alleviate peripheral neuropathy in DPN rats.3.By targeting STAT3,miR-20b-3p promoted autophagy of Schwann cells in pathological state,thereby inhibiting the progression of DPN.4.Ageing-exos were not found to improve DPN. |
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