The Clinical And Experimental Study Of MSCs Promoting Neurological Repair After TBI By Regulating Th17/Treg Immune Homeostasis

Objective:To investigate the effect of mesenchymal stem cells(MSCs)on Th17-Treg cells transdifferentiation and the balance of Th17-Treg after traumatic brain injury(TBI),and the effects of this process on inflammatory response and neurofunctional prognosis after TBI were studied.Methods:(1)The blood of patients who meet the criteria of TBI group and control group were collected,and the changes in the balance of Th17/Treg in peripheral blood of the two groups were compared by flow cytometry.(2)Th17 cells were preconditioned with 1ug/mL lipopolysaccharide(LPS),and randomly divided into three groups: Th17 group,Th17 + MSCs group and Th17 + SIS3 + MSCs group.Collection the cells for flow cytometry and Western Blotting to explore if the Th17 cells could transdifferent into Treg cells and its mechanisms a total of 8 days after co-culture.(3)Adult Sprague-Dawley(SD)rats were randomly divided into the sham-injured group,TBI group and TBI+MSCs group.The rats in TBI group were received hydraulic percussion brain damage,the combat pressure was 2.0 atm.And the rats in TBI+MSCs group were received MSCs transplantation in the center and surrounding area of injured lesion after injury immediately.Spatial learning and memory was measured by the test of Morris water maze.The modified neurological severity scores(mNSS)of rats was evaluated to detect functional recovery of nervous system.Immunofluorescence staining was used to observe the neurogenesis in dentate gyrus(DG)of injured hippocampus at acute and chronic stage after injury.HE staining was used to compare the inflammatory response of injured lesions at acute stage after injury.Flow cytometry was examined to compare the changes of the balance of Th17/Treg in peripheral blood and brain tissues after injury.And Western Bloting was used to detect the anti-inflammatory and immunomodulatory mechanism of MSCs after TBI.Results:(1)Compared with control group,the T lymphocyte subgroup in the peripheral blood was shifted to the pro-inflammatory Th17 cells within 1 week after TBI,and the ratio of Th17/Treg was significantly increased.(2)Compared with Th17 group,Th17 cells were significantly decreased in Th17+MSCs group,and the number of Treg cells were increased significantly,the expression of Smad3 was increased,and the expression of NF-?B was decreased.Compared with Th17+MSCs group,Th17 cells in Th17+SIS3+MSCs group were significantly increased,as the Treg cells were significantly decreased,the expression of Smad3 was decreased,and the expression of NF-?B was increased.(3)Compared with Sham group,the escape latency in TBI group was significantly longer,the number of platform crossings and the time stayed in the target quadrant was decreased,and the score of mNSS was increased.Howere,compared with TBI group,the escape latency in TBI+MSCs group was significantly shorter,the number of platform crossings and the time stayed in the target quadrant was increased,and the score of mNSS was decreased.The rats in TBI group showed greater cell injury and interstitium edema compared with TBI+MSCs group 1 day,3 days and 7 days after injury,and the number of inflammatory cells were significantly decreased in TBI+MSCs group.Compared with Sham group,BrdU/DCX double labeled cells in rat hippocampal DG were significantly increased at 1 week after injury both in TBI group and TBI+MSCs group.And the double labeled cells in TBI+MSCs group were markedly increased in comparison to TBI group.Meanwhile,the BrdU/NeuN double-labeled cells in hippocampal DG were significantly increased at 1 month and 2 month after injury both in TBI group and TBI+MSCs group,and the double labeled cells in TBI+MSCs group were markedly increased in comparison to TBI group.Besides,the ratio of Th17/Treg in peripheral blood and brain tissue was significantly increase in TBI group in comparision to Sham group,while decrease in TBI+MSCs group in comparision to TBI group.Furthermore,the expression of NF-?B was significantly increased in TBI group compared with Sham group,while compared with TBI group,the expression of Smad3 was significantly increased and the expression of NF-?B was significantly decreased in TBI+MSCs group.Conclusion:MSCs could induce the proinflammatory Th17 cells transdifferent into the anti-inflammatory Treg cells by TGF-?/Smad3/NF-?B signaling pathways,regulate the inflammatory response and the balance of Th17/Treg after TBI both in central and peripheral system,improve the hippocampal neurogenesis after TBI,thereby promoting the functional recovery of nervous system and improving the neural functional outcomes after TBI.