Protective Effects Of MG53 Combined With HUC-MSCs Transplantation In A Mouse Model Of Traumatic Brain Injury

Traumatic brain injury(TBI)is a common traumatic disease caused by external force,which causes brain nerve dysfunction and nerve cell death.It can also cause severe physical,psychological and cognitive impairments,therefore a new therapy for TBI is in critical need.The emergence of stem cells and regenerative medicine will provide new ideas for the treatment of TBI.Several experimental and clinical studies have shown that stem cell transplantation exerted beneficial effects in TBI,via paracrinefactors and cell replacement.Human umbilical cord derived mesenchymal stem cells(h UC-MSCs)have fast rate of self-renewal,low immunogenicity,and low risk of teratoma formation,supporting their therapeutic value.However,the migration and survival of transplanted stem cells are often hindered because of the pathology and cell death that accompanies TBI caused by excessivereactive oxygen species generation associated with tissue ischemia and inflammation.Mitsugumin53(MG53)protein is a member of the tripartite motif(TRIM)family proteins.It has an essential role in cell membrane repair and recovery.MG53 protein can protect various cell types against membrane disruption when applied to the extracellular environment and ameliorate the pathologies associated with muscular dystrophy,acute lung injury,myocardial infarction,and acute kidney injury in rodent and large animal models of these diseases.Additionally,intravenous delivery of MG53 was able to permeate through the blood-brain barrier to protect against ischemic brain injury.However,there is no report about the research of MG53 protein combined with h UC-MSCs in the treatment of TBI.In addition,the protective effect of MG53 protein on damaged neuron cells has not been reported.Objective In the present study we aim to explore the protective effects of MG53 protein on injured neuron cells in vitro and the protective effects of MG53 combined with h UC-MSCs transplantation in a mouse model of traumatic brain injury in vivo.Part I: To determine the HT22 cell damage model of hippocampal neurons induced by Lipopolysaccharide and to explore the effect of MG53 on HT22 cell proliferation,apoptosis,cycle,migration and its molecular mechanism.Part II: To investigate the protective effects of MG53 in combination with h UC-MSCs as well as its underlying biological mechanisms in vivo.Methods Part Ⅰ Protective effect of MG53 protein on HT22 cells damaged by LPS 1.CCK-8 assay was used to detect cell proliferation and determine the optimal concentration and time of LPS to induce HT22 cells injuried.The experiment was divided into normal control group(NC group),MG53 group,LPS group and MG53+LPS group.2.After LPS treatment,cell cycle was measured by flow cytometry and Annexin V-FITC/PI method to detect cell apoptosis.3.Scratching experiment was used to detect the migration distance of cells.ELISA kit was used to detect changes in cell SOD activity and MDA content.4.Quantitative real time PCR were used to detect the m RNA levels of the TNF-α,IL-6,IL-1β,TLR4,My D88 and NF-κB expression.5.Western Blot was used to detect the expression of Bax,Bcl-2,cyclin D1 and the activity of TLR4/NF-κB signaling pathway.Part Ⅱ MG53 protein combined with h UC-MSCs in the treatment of TBI and its molecular mechanism 1.C57BL/6 mice subjected to TBI were random Ly divided into 4 groups: TBI group,MG53 group,MSCs group and MG53+MSCs group.2.At day 3 post TBI,PI staining was used to detect the damage of cortical cell death;brain edema was detected by dry wet method.3.Western Blot was used to detect the expression of Bax,Bcl-2,MG53 and pro-survival pathway related protein p-Akt1,expression of p-GSK3β and p-ERK1/2.4.Immunofluorescence detection of hippocampal MAB1281 expression;SOD activity and MDA content in mice serum were tested by ELISA.5.Between 1 and 28 days,the m NSS score was used to evaluate the recovery of neurological function in mice;Between 21 and 28 days,the Morris water maze test to determine the learning and memory ability of mice;At day 28 post TBI,the new recognition test,sucrose preference test and forced swimming test to determine the degree of depression of mice.6.CV staining was used to detect the volume of brain injury in mice;RT-PCR were used to detect the m RNA levels of the BDNF and NGF expression;At days 14 and 28,mices were sacrificed and brain tissue was prepared to assess neurogenesis by immunofluorescent staining of DCX and Neu N,respectively.Results Part Ⅰ Protective effects of MG53 protein on HT22 cells damaged by LPS and its mechanism 1.LPS stimulation had a concentration and time dependent effect on the proliferation of HT22 cells.That was,low concentration level promoted cell proliferation,and high concentration inhibited cell proliferation and induced cell apoptosis(P<0.05).CCK-8 results showed that after 500mg/L LPS induced HT22 cells 48 h,the cell growth inhibition rate was close to 50%,which could be used for subsequent experimental models.2.Compared with LPS group,cell survival rate increased,apoptosis rate decreased,G0/G1 phase decreased,S phase increased and migration capacity increased(P<0.05)in MG53+LPS group.Western results showed that compared with LPS group,the expression of Bcl-2 protein expression increased,Bax protein expression decreased,Cyclin D1 expression increased in MG53+LPS group(P<0.05).3.ELISA results showed that compared with the NC group,the SOD activity decreased and the MDA content increased in LPS group.Compared with the LPS group,the SOD activity increased and the MDA content decreased in the MG53+LPS group,and the difference was statistically significant(P<0.05).4.Western Blot results showed that compared with the NC group,the TNF-α,IL-6,IL-1β,TLR4,p-NF-k Bp65,p-IKBα protein expression increased(P<0.05),the total NF-κBp65 protein expression was not significantly changed(P>0.05),and the IKBα protein expression decreased in LPS group(P<0.05).Compared with the LPS group,the TNF-α,IL-6,IL-1β,TLR4,p-NF-k Bp65,p-IKBα protein expression decreased(P<0.05),the total NF-κB p65 protein expression was not significantly changed(P>0.05),and the IKBα protein expression increased in LPS+MG53 group(P<0.05).The result of q RT-PCR is consistent with the result of Western Blot(P<0.05).Part Ⅱ MG53 protein combined with h UC-MSCs in treatment of TBI and its molecular mechanism 1.After operation of 3 days,compared with the TBI group,the brain water content of mice in treatment groups was significantly lower,and the MG53+MSCs group was the lowest(P<0.05),while there was no significant difference in the contralateral brain(P>0.05).Compared with the TBI group mices,the PI positive cells in the cerebral cortex of each treatment mice decreased,the expression of Bcl-2 protein increased,Bax protein decreased,and the change of the MG53+MSCs group was the most obvious(P<0.05).2.Immunofluorescence showed that MAB1281 positive cells could be detected in MSCs and MG53+MSCs groups,but no MAB1281 positive cells were found ingroup TBI and MG53 groups.Compared with the TBI group,the SOD activity of mice in each treatment groups increased,the MDA content of mice in each treatment groups decreased,and the effect of the MG53+MSCs group was better(P<0.05).3.Western Blot results showed that MG53 protein could be detected in MG53 and MG53+MSCs groups,while MG53 was not detected in TBI and MSCs groups.Compared with the TBI group,the expression of p-Akt1 and p-ERK1/2 increased significantly in MG53 group(P<0.05),and there was no significant change of p-GSK3β in MG53 group(P>0.05).The expressions of p-Akt1,p-GSK3β and p-ERK1/2 were significantly increased in MSCs and MG53+MSCs groups,and the changes in the MG53+MSCs group were more obvious.It is suggested that MG53 and h UC-MSCs may play an essential role in brain damage mice by regulating PI3K/Akt-GSK3β and ERK1/2 signaling pathways.4.After operation of day 1 and 3,the weight of mice in each group decreased,and the weight began to increase from day 7.After 7 days,compared with the TBI group,the body weight of mice in each treatment group increased,the m NSS score decreased,and the MG53+MSCs group was more significant(P<0.05).Compared with the TBI group,the number of cross platform in each treatment group increased,the resting time of the platform increased,the escape latency shortened,the suspension time decreased,the discrimination index and the sugar water preference increased significantly(P<0.05),while the effect of the MG53+MSCs group was better(P< 0.05).5.Compared with the TBI group,the expression of neurotrophic factor BDNF and NGF m RNA in each treatment groups increased(P<0.05),while the effect of MG53+MSCs group was better(P<0.05).The results of immunofluorescence showed that compared with the TBI group,the positive cells of DCX and Neu N in the hippocampus of each treatment groups increased(P<0.05),while the DCX and Neu N in the MG53+MSCs group were more significant(P<0.05).Conclusion 1.MG53 protein can promote the survival of HT22 cells damaged by LPS,inhibit cell apoptosis,promote cell operation from G0/G1 to S phase,improve cell migration and antioxidant capacity,inhibit TLR4/NF-k B signaling pathway,and reduce LPS induced inflammatory response.2.MG53 protein and h UC-MSCs can migrate through the blood-brain barrier to the injured part of TBI mices,increase the weight of the mices,improve the cognitive function,enhance the learning and memory ability,improve the nerve recovery,reduce the depression,inhibit the apoptosis,promote the nerve regeneration and antioxidant level,while the MG53+MSCs treatment is more obvious than the single treatment.In addition,MG53 protein and h UC-MSCs may play a protective role in TBI mices by activating PI3K/Akt-GSK3β and ERK1/2 signaling pathways.