The Research Of Macrophage Inflammation Inhibition And The Sciatic Nerve Recovery By Neural Stem Cell Conditioned Medium

Objectives To investigate the repair effect of neural stem cell conditioned medium(NSC-CM)in sciatic nerve crush injury,and and to observe its effect on inflammation and macrophage infiltration in rats,also to further clarify the effect and mechanism of NSCCM on the inflammatory status of macrophages in vitro.Methods(1)Establishment of sciatic nerve crush injury model in SD rats;(2)The sciatic nerve function index,footprint analysis,and gastrocnemius wet weight were observed to investigate the effects of NSC-CM on the recovery of behavioral function after sciatic nerve crush injury in rats;(3)The effects of NSC-CM on neurofilament and myelin regeneration were analyzed by HE staining and Transmission electron microscope;(4)The effects of NSC-CM on inflammation and macrophage infiltration after crush injury were analyzed by immunohistochemical staining;(5)Extract and culture rat peritoneal macrophages in vitro,and use LPS to establish a macrophage inflammation model;(6)The effects of NSC-CM on the expression of pro-inflammatory factors and mechanism on macrophages were analyzed by RT-qPCR,Western blot,and ELISA.Results(1)Compared with PBS,the NSC-CM treatment significantly improved the sciatic nerve function index and toe extension of the injured side after crush injury,and improved the gastrocnemius atrophy(P < 0.05).(2)Compared with PBS,NSC-CM treatment promoted axon regeneration and myelin regeneration after sciatic nerve crush injury(P < 0.05).(3)Compared with PBS,NSC-CM treatment reduced the expression of cytokines(IL-6,IL-1?,TNF-?)and the infiltration of inflammatory macrophages(CD68)on the 14 th day after injury(P < 0.05).But in the early stage of injury,that is,on the third day after injury,there was no statistical difference in cytokines and macrophages in the NSC-CM treatment group compared with PBS(P > 0.05).(4)Compared with the control group,NSC-CM significantly inhibited the expression of macrophage pro-inflammatory related proteins(IL-6,IL-1?,TNF-?,iNOS)induced by LPS(P < 0.05).(5)Compared with the control group,NSC-CM significantly activated Sirt-1 protein expression in macrophages(P < 0.05),and regulated HIF-1? and NF-?B signal molecules(P < 0.05).Treatment with Sirt-1 specific inhibitor EX 527 can partially reduce the anti-inflammatory effect of NSC-CM on macrophages.Conclusions(1)NSC-CM improved the functional recovery of rats after sciatic nerve crush injury and promotes axon and myelin regeneration.(2)NSC-CM reduced the expression of inflammatory factors and the infiltration of macrophages in the later stage of injury(3)NSC-CM significantly inhibited the expression of macrophage proinflammatoryrelated proteins induced by LPS in vitro.(4)NSC-CM inhibits macrophage inflammation by activating Sirt-1 signaling pathway.