Effects And Mechanism Of Exosomes Derived From Bone Marrow Mesenchymal Stem Cells On Neuroinflammation After Traumatic Brain Injury

The purpose of this study was to explore the effect and the possible mechanism of exosomes(Exosome,Exo)derived from bone marrow mesenchymal stem cells(bone mesenchymal stem cells,b MSCs)on neuroinflammation after traumatic brain injury(Traumatic brain injury,TBI).Firstly,the effects of b MSCs and b MSCs-Exosomes on the polarization phenotype of microglia-like BV2 cells were studied by a transwell co-culture system in vitro.And then the traumatic brain injury was made in mouse by lateral fluid percussion injury model.Then the exosomes derived from bone marrow mesenchymal stem cells were injected into the caudal vein to verify the effects of exosomes on neuroinflammation after traumatic brain injury in vivo.Furthermore,through mi RNA sequencing and literature review,a variety of neuroinflammation related mi RNAs were selected to be research,as well as the relevant pathways.The relevant mi RNAs were verified to determine the target mi RNA in present study.Finally,the mi RNA down-regulated and over-expressed mice were constructed with lentivirus.Then the TBI model was made to verify the influence and the mechanism on the process of neuroinflammation after traumatic brain injury.The results of this study showed that b MSCs-Exosome can promote the polarization of microglia to anti-inflammatory(M2)phenotype and inhibit neuroinflammatory response both in vitro and in vivo.In addition,b MSCs-Exosomes can reduce neuronal apoptosis and protect damaged brain tissue,in which the mi R181b-IL-10/STAT3 pathway may play a major role.PART 1 Effects of b MSCs and b MSCs-derived exosomes on the polarization of microglia-like BV2 cells in vitroObject:To explore the effects and possible mechanism of b MSCs and b MSCs derived exosomes on the polarization phenotype of microglia like BV2 cells in vitro.Methods:BV2 cells in resting state were inoculated in 12-well Transwell co-culture plate.2.0×105 cells were inoculated and cultured in 10% fetal bovine serum(FBS)and DMEM medium in each well.Lipopolysaccharide(LPS)(100ng/ml)was added to each well and co-cultured with BV2 cells for 24 hours.Then they were divided into three groups: MG group(MG group),b MSCs and MG co-cultured group(b MSCs+MG group)and b MSCs-exosome and MG co-cultured group(b MSCs-Exo+MG group).In the MG group,only 500 ul 10% exosome-deleted serum and DMEM was added in the inserted chamber;in the b MSCs+MG group,5.0×104 b MSC cells were inoculated in the inserted chamber,and then supplement the medium(10% exosome-deleted serum + DMEM)to500 ul;in the b MSCs-Exo+MG group,100?l Exosomes were inoculated in the inserted chamber and then supplement the medium to 500?l using the same medium.Refresh the culture medium of the sub-chamber using 10% exosome-deleted serum and DMEM.Then site the transwell plates into the cell incubator.The co-culture system was taken out 48 hours later,and the cells of each group were collected for immunofluorescence detection,flow cytometry detection and RT-q PCR detection.The proportion and polarization phenotype of M1/M2 as well as the expression levels of pro-inflammatory and anti-inflammatory factors were observed respectively.Results:The results of immunofluorescence showed that the amount of Arg1+ cells in b MSCs+MG group and b MSCs-Exo+MG group were significantly higher than that in MG group.The results of flow cytometry showed that the proportion of Q3 region(Iba-1+/CD206+)cells in b MSCs+MG group(9.39 ±1.91%)and b MSCs-Exo+MG group(16.07 ±2.87%)was significantly higher than that in MG group(4.25 ±1.41%)(P<0.05),while the proportion of Q3 region(Iba-1+/CD206+)cells in b MSCs-Exo+MG group was much higher than that in b MSCs+MG group(P<0.05).The results of RT-q PCR showed that the expression of IL-1?,IL-6 and TNF-? in b MSCs+MG group and b MSCs-Exo+MG group was inhibited compared with MG group,while the expression of IL-10 and TGF-? was significantly increased.The relative expression level of IL-10 and TGF-? was significantly higher in b MSCs-Exo+MG group than that in b MSCs+MG group.Compared with b MSCs,b MSCs-Exo played a stronger role in regulating the expression of inflammatory factors.Conclusions:Both b MSCs and exosomes derived from b MSCs can promote the polarization of activated BV2 microglia to anti-inflammatory phenotype(M2-type).The use of exosomes derived from b MSCs induce microglia transform into anti-inflammatory phenotypes(M2);b MSCs and exosomes derived from b MSCs can inhibit the expression of pro-inflammatory cytokines such as IL-1?,IL-6 and TNF-?,and promote the expression of anti-inflammatory cytokines such as IL-10 and TGF-?.And b MSCs-Exo played a stronger role in regulating the expression of inflammatory factors.PART 2 Effects and mechanism of b MSCs-derived exosomes on neuroinflammation after traumatic brain injury in miceObject:To observe the effects of b MSCs-derived exosomes on neuroinflammation and neuronal apoptosis in mice after traumatic brain injury,as well as the relevant mi RNAs and pathways involved in the process.Methods:C57BL/6 mice(male,6-8W)were divided into four groups: sham group(sham group,bone window was made only),traumatic brain injury group(TBI group),normal saline injected group(TBI+Saline group)and exosome injected group(TBI+b MSCs-Exo group).On the 1st,3rd and 7th day after the traumatic brain injury,five mice in each group were sacrificed and stored in the refrigerated at-80 ? for following detection.All the remaining mice were sacrificed on the 7th day after TBI,and the brain tissue was collected to make paraffin sections.We use Nissl staining to observe the injured area.TUNEL staining was used to observe neuronal apoptosis and immunofluorescence staining was used to observe the expression of Arg1 in the injured area,Western Blot was used detect the expression of Arg1 and i NOS in the injured brain tissue while RT-q PCR was used to detect the expression of pro-inflammatory and anti-inflammatory factors on the 1st,3rd and 7th day after TBI.The expression of several proteins relevant to neuroinflammatory process was detected as well.According to the results of mi RNA sequencing and literature review,several possible mi RNAs and its relevant signal pathways were selected and then verified by q PCR.Results:Nissl staining showed that the area of cortical injury in TBI+b MSCs-Exo group(0.509 ±0.177)was significantly smaller than that in TBI group(0.697 ±0.39)and TBI+Saline group(0.709 ±0.196)(P<0.05).The TUNEL staining showed that there were neuronal apoptosis in TBI group,TBI+Saline group and TBI+b MSCs-Exo group,but the number of apoptotic neurons in TBI+b MSCs-Exo group(22.2 ± 3.03)was significantly lower than that in TBI group(42.1±4.30)and TBI+Saline group(40.8±5.26)(P<0.05).The immunofluorescence staining showed that Arg1 positive cells were expressed in TBI group,TBI+Saline group and TBI+b MSCs-Exo group,but the proportion of Arg1 positive cells in TBI+b MSCs-Exo group was higher than the other two group(P<0.05).The results of Western Blot showed that the content of Arg1 in the injured brain tissue of TBI+b MSCs-Exo group was higher than that of TBI group and TBI+Saline group(P<0.05),and the content of i NOS was relatively lower(P<0.05).The results of quantitative Real-time PCR showed that the injection of b MSCs-derived exosome could inhibit the expression of pro-inflammatory cytokines such as IL-1? and TNF-? and the activity of NF?B was inhibited too.The expression of IL-10 and TGF-?were up-regulated as well as the expression of STAT3.In addition,after the injection of b MSCs-derived exosome,the expression of mi R181 b was relatively higher among the mi RNAs.Conclusions: The injection of b MSCs-derived exosome can reduce the neuronal apoptosis in the cortical area.The neuroinflammatory was inhibited and the transformation of microglia to anti-inflammatory phenotype(M2 type)was promoted.Among the mi RNAs,mi R181 b pathway may be involved in the process via IL-10/STAT3 pathway.PART3 The effects and mechanism of mi R181 b on neuroinflammation after traumatic brain injuryObject : To study the effect and mechanism of mi R181 b on neuroinflammatory response after traumatic brain injury.Methods : Mmu-mi R181b-up-regulation and mmu-mi R181b-inhibitor lentiviruses were constructed and then injected into the right parietal cortex of mice to construct brain-specific up-regulation and down-regulation mice.The mice were divided into three groups: TBI group(the TBI model was made in normal mice,),TBI-up group(TBI model was made in mi R181 b up-regulated mice)and TBI-down group(TBI model was made in mi R181b-inhibited mice).Then the TBI model was made by lateral fluid percussion instrument.On the 1st,3rd and 7th day after the establishment of the model,five mice in each group were sacrificed.Their brains were removed and stored in the refrigerator at-80? for following detection.All the remaining mice were sacrificed on the 7th day after TBI,and the brains were collected to make paraffin sections.We used TUNEL staining to observe the apoptosis of neurons in the injured area.Western Blot was used to detect the expression of Arg1,i NOS and STAT3 in the brain of each group on the 7th day after TBI.RT-q PCR was used to detect the expression of pro-inflammatory and anti-inflammatory factors on the 1st,3rd and 7th day after TBI.Results:On the 7th day after TBI,the number of TUNEL positive cells in the injury area of TBI-up group(24.8±1.94)was significantly lower than that of the other two groups(P<0.05).The expression of Arg1 and STAT3 in TBI-up group was significantly higher than that of TBI group and TBI-down group on the 7th day after TBI(P<0.05),while the expression of i NOS was lower than the other two groups(P<0.05).The expression of IL-10 and TGF-? in TBI-up group increased continuously after TBI,and the relative expression level was significantly higher than that in the other two groups on the 1st,3rd and 7th day after TBI(P<0.05).The expression of IL-1? was lower than that in the other two groups on the 1st,3rd and 7th day while the expression of TNF-? was lower than that in the other two groups on the 3rd and 7th day after TBI(P<0.05).No significant difference was found on the expression level of IL-6among the three groups at each time point(P>0.05).Conclusion:The overexpression of mi R181 b can effectively reduce the neuronal apoptosis and neuroinflammatory response after traumatic brain injury.The mi R181 b can also promote the transformation of microglia to the anti-inflammatory phenotype(M2 type).These effects of mi R181 b are achieved by activating the IL-10/STAT3 pathway.