Effects And Mechanisms Of Human Umbilical Cord Mesenchymal Stem Cells Transplantation In Reducing LPS-induced Neuroinflammation In The Brain Of Mice

Objective:Neuroinflammation is an inflammatory reaction that occurs in the central nervous system.Neuroinflammation is the innate and adaptive immune responses that are initiated toward a variety of harmful insults through the release of inflammatory mediators by various immune cells.Neuroinflammation in the initial stage is mainly beneficial and protective;Chronic neuroinflammation causes neuronal damage and synaptic loss,leading to memory impairment.Many neurological disorders and age-related neurodegenerative diseases（such as AD,PD,ALS）are accompanied by neuroinflammation.No curative treatment is available for neurodegenerative diseases.Therefore,inhibiting the occurrence of neuroinflammation provides a new research direction for exploring the treatment of neurodegenerative diseases.MSCs can synthesize and secrete many bioactive substances,such as trophic factors and cytokines,may have immunosuppressive and immunomodulatory effects.Because of evoked only minimal immune reactivity,MSCs transplantation will open up interesting new possibilities for cell therapy and regenerative medicine.The reviewed data demonstrate that MSCs have a significant therapeutic potential in treating neuroinflammation-related cognitive disfunctions including age-related neurodegenerative diseases.But the mechanism by which it works is unclear.Therefore,we explore the effect and mechanism of transplanted hUC-MSCs on LPS-induced neuroinflammation in this project.Methods:1.Evaluation of safety on mesenchymal stem cells derived from hUC-MSCs1)Short-term toxicity test of hUC-MSCs:Forty C57BL/6 mice were divided into hUC-MSCs-treated groups and 0.9%Na Cl solution treated groups.After injection,the mice were observed for 14 days,the weight of mice was recorded,and after 14 days,the mice were sacrificed,and the quality of organs was weighed and recorded;2)Hemolysis test:In order to prepare an erythrocyte suspension,fresh blood was collected from a healthy rabbit.A 2%suspension was added to five concentration gradients of hUC-MSCs;Meanwhile,1.5m L distilled water and saline was respectively as positive and negative control.The mixed liquids were blended gently and incubated at 37 ℃ for 3 hours.Centrifuge the supernatant after the end of the incubation and determine the OD₅₇₀ value;3)Vascular stimulation test:New Zealand white rabbits were infused with hUC-MSCs via the ear marginal vein.A 0.9%Na Cl solution was used as a control.Any abnormal changes occurring in vascular and surrounding tissues were observed to evaluate the influence of hUC-MSCs treatment.After 48 hours of the treatment,vascular and surrounding tissues were collected and stained with H&E.2.HUC-MSCs relieve cognitive impairment of LPS-induced inflammation models1)LPS-induced inflammation models:The C57BL/6 mice were named as control and LPS groups.The i.p.injection of LPS or saline was daily administered for7 days.Subsequently,the behavioral tests of cognitive capacity were assessed using Y maze and Novel object recognition;2)Effects of hUC-MSCs on cognitive impairment in LPS-induced neuroinflammatory models:C57BL/6 mice were named as three groups:saline group,LPS group and LPS+hUC-MSCs group.The behavioral tests of cognitive capacity were assessed using Y maze and Novel object recognition after the injection.3.Effect of hUC-MSCs on LPS-induced neuroinflammation1)Effects of hUC-MSCs on LPS-induced neuroinflammation in BV2 cells:The BV2 cells were divided into three groups:PBS group,LPS group,LPS+hUC-MSCs group,the concentrations of TNF-αand IL-6 in the culture supernatants were determined using TNF-αand IL-6 ELISA kits after treatment;M1 polarization of BV2cells after treatment was detected using immunofluorescence methods;2)Effects of hUC-MSCs on LPS-induced neuroinflammation mice:The C57BL/6mouses were named as three groups:saline group,LPS group and LPS+hUC-MSCs group.Subsequently,the behavioral tests of cognitive capacity were assessed using Y maze and Novel object recognition after injection;The concentrations of TNF-αand IL-6 in the cortex and hippocampus were determined using TNF-αand IL-6 ELISA kits;M1 polarization of microglia in the cortex and hippocampus after treatment was detected using immunofluorescence methods.4.The role of rh TSG-6 secreted by hUC-MSCs in alleviating LPS-induced neuroinflammation1)We search the GEO datasets in order to find the differentially expressed genes in LPS-induced hUC-MSCs.Perform GO analysis on the target database.Combined with the literature reports,we identified the next experimental research subjects;2)The concentrations of TSG-6 in the culture supernatants of hUC-MSCs were determined using human TSG-6 ELISA kits;3)Effect of rh TSG-6 on LPS-induced BV2 cells:The BV2 cells were designed into three groups:PBS group,LPS group and LPS+rh TSG-6 group,the concentrations of TNF-αand IL-6 in the culture supernatants were determined using TNF-αand IL-6ELISA kits after treatment;M1 polarization of BV2 cells after treatment were detected using immunofluorescence methods;4)Effects of rh TSG-6 on LPS-induced neuroinflammation mice:The C57BL/6mouses were designed into three groups:saline group,LPS group,LPS+rh TSG-6group.Subsequently,the behavioral tests of cognitive capacity were assessed using Y maze and Novel object recognition after injection;The concentrations of TNF-αand IL-6 in the cortex and hippocampus were determined using TNF-αand IL-6 ELISA kits;M1 polarization of microglia in the cortex and hippocampus after treatment were detected using immunofluorescence methods.Results:1.The hUC-MSCs used in this study do not cause toxicity,immune response,hemolytic and vascular irritation after in vitro and in vivo experiments,suggesting that they have biological safety;2.The treatment of hUC-MSCs alleviates the behavioral disorder of LPS-induced mouse model;3.HUC-MSCs significantly attenuated expression of pro-inflammatory cytokines TNF-α,IL-6.In addition,hUC-MSCs administration inhibited the microglial M1polarization;4.Rh TSG-6 significantly attenuated expression of pro-inflammatory cytokines TNF-αand IL-6.In addition,rh TSG-6 administration inhibited the M1 polarization of microglial and cognitive impairment in LPS-induced mouse model.Conclusion:The hUC-MSCs have biological safety.Intravenous injections of hUC-MSCs can relieve neuroinflammation and cognitive impairment in LPS-induced mice,which may be achieved through the release of TSG-6 by hUC-MSCs.