BMSC-Exos Promotes M2 Polarization Of Microglia Through PPARγ Pathway And Modulate Inflammation In Spinal Cord Injury

Spinal cord injury causes motor and other physiological disorders of the nervous system.Inflammatory reaction is an important pathological process after spinal cord injury,in which microglia plays a major role.The microglia has two polarized phenotypes,M1 pro-inflammatory and M2 anti-inflammatory.Inflammatory response and microglia phenotype are regulated by PPARγ.Bone marrow Mesenchymal stem cell exosomes may play a protective role in spinal cord injury,but the mechanism remains to be studied.Therefore,it is an important medical problem to study the pathophysiology of spinal cord injury and find specific intervention targets to alleviate spinal cord injury and promote repair.Objective:1.To determine the effect of BMSC-Exos on lipopolysaccharide-induced microglia inflammation.2.To investigate the effect and mechanism of BMSC-Exos on the repair of spinal cord injury.Methods:PartⅠ:BMSC-Exos inhibits reduces microglia inflammationQ-PCR was used to detect the expression of IL-1β,IL-6 and TNF-αafter LPS stimulation of BV2 microglia The concentration of BMSC-Exos was determined by CCK-8,and the activation of NF-κB pathway and the expression of inflammatory factors were detected by immunofluorescence and Western blotting after LPS stimulation of BV2 microglia.PartⅡ:BMSC-Exos activates the PPARγpathway and promotes the transformation of microglia from M1 to M2The expression of i NOS and CD206,the microglia microglia markers of i NOS and CD206,PPARγ,i NOS and CD206 were detected by Western blotting,and the expression of CD206 and PPARγwas detected by Western blotting,observe the co-bid situation.To demonstrate that BMSC-Exos activates the PPARγpathway and microglia from M1 to M2.PartⅢ:BMSC-Exos stimulates PPARγpathway to promote spinal cord injury repairThe expression of PPARγ,i NOS and CD206 in the spinal cord was detected by Western blotting,and the activation of NF-κB pathway and the expression of inflammatory factors in the spinal cord were detected by immunofluorescence and Western blotting,respectively.The expression of PPARγ,i NOS and CD206 in the spinal cord was detected by Western blotting,and the expression of PPARγ,i NOS and CD206in the spinal microglia was detected by Western blotting.These results suggest that BMSC-Exos can change the microglia from M1 proinflammatory to M2anti-inflammatory by activating PPARγpathway,which can reduce the inflammatory reaction after spinal cord injury and promote the repair of spinal cord injury.Results:1.The relative viability of BV2 microglia stimulated with LPS at a concentration of100 ng/m L for 24 h was most suitable for subsequent experiments,the expression of IL-1β,IL-6 and TNF-αin BV2 cells were significantly higher than that in normal BV2cells after LPS stimulation with 100 ng/m L for 24 H.The optimal concentration of BMSC-Exos was 2×10~7patricles/m L.Subsequent experiments showed that BMSC-Exos could inhibit the activation of NF-κB pathway and down-regulate the expression of IL-1β,IL-6 and TNF-α,but this effect was partially counteracted by the inhibition of PPARγpathway.2.BMSC-Exos intervention can activate PPARγpathway,reduce the expression of i NOS,a microglia marker of M1,and increase the expression of CD206,a microglia marker of M2.CD206 was co-labeled with PPARγimmunofluorescent staining.These effects were partially offset by inhibition of PPARγpathway by GW9662.3.After female SD rats were hit at T10 with HI-0400 Spinal Cord Hammer,the motor function of both hind limbs was lost.The recovery of motor function of rats treated with BMSC-Exos was the best,but the recovery of motor function of rats treated with BMSC-Exos and GW9662 was significantly worse than that of rats treated with BMSC-Exos.The recovery of the striking site of the spinal cord in each group was observed on the 28th day after operation.The results of HE staining showed that BMSC-Exos could promote the repair of the injured spinal cord,and inhibition of PPARγpathway could weaken the repair function of BMSC-Exos.Three days after operation,the activation of NF-κB inflammatory pathway and the expression of IL-1β,IL-6 and TNF-αin the spinal cord were detected.It was found that BMSC-Exos could inhibit the activation of NF-κb pathway,down-regulated the expression of IL-1β,IL-6 and TNF-αprotein.The expression of NF-κB P65 and inflammatory factors increased after GW9662injection.The expression of PPARγ,i NOS and CD206 in the spinal cord was detected.The results showed that the expression of PPARγwas significantly increased,the expression of CD206 was increased and the expression of i NOS was decreased in the BMSC-Exos treated group,however,the trend of expression of BMSC-Exos and GW9662 was opposite.Conclusions:1.BMSC-Exos inhibited the activation of NF-κB pathway and decreased the expression of microglia inflammatory factors after LPS stimulation.2.BMSC-Exos stimulates the PPARγpathway to polarize the microglia toward M2,which reduces inflammation after spinal cord injury and promotes spinal cord injury repair.