The Experimental Study Of Resveratrol Combined With Bone Marrow Mesenchymal Stem Cells In The Treatment Of Spinal Cord Injury In Rat

Spinal cord injury is a serious central nervous system injury.Most patients lose the ability of movement and self-care after spinal cord injury,which often brings serious burden to their families and society.After spinal cord injury,a series of pathophysiologic changes caused by the changes of spinal cord microenvironment often lead to "waterfall" effect,which leads to disastrous consequences and brings difficulties and obstacles to the treatment of spinal cord injury.Because of the various physiological characteristics,such as proliferation,multi-directional differentiation and self-renewal,stem cells are the research focus of spinal cord injury treatment in recent years.Among them,bone marrow mesenchymal stem cells are widely used for research because of their easy availability,immunological characteristics and ethical rationality.However,the pathological and physiological reactions in the local microenvironment,such as inflammation,apoptosis and oxidative stress,often lead to the decline of the survival rate of stem cell transplantation,and ultimately lead to the decline of the neuroprotective function,resulting in the poor therapeutic effect of spinal cord injury.Resveratrol,due to the discovery of "French paradox",has been widely studied for its outstanding anti-inflammatory,anti-aging and anti-oxidative stress.Resveratrol has been proved to be able to up regulate sirt-1 signaling pathway and this is related to its potential effects.NF-?B is one of the important signaling pathways to activate inflammation,while Sirt-1 is the upstream pathway of NF-?B.Sirt-1 and inflammatory changes after spinal cord injury and whether resveratrol can protect bone marrow mesenchymal stem cells in inflammatory environment and its mechanism have not been studied.Whether resveratrol combined with bone marrow mesenchymal stem cells can improve the therapeutic effect of spinal cord injury has not been studied also.Therefore,the study will verify the effect and mechanism of resveratrol combined with bone marrow mesenchymal stem cells in the treatment of spinal cord injury in rats through the following three parts:1.Changes of Sirt-1expression after spinal cord injury and its relationship with changes of inflammatory response after spinal cord injury in rats.2.Bone marrow mesenchymal stem cells of primary rats was cultured in vitro to make inflammatory model,and the effect and mechanism of resveratrol on the inflammatory model of bone marrow mesenchymal cells were explored.3.To verify the effect and mechanism of resveratrol/bone marrow mesenchymal stem cells in the treatment of spinal cord injury by rat spinal cord injury model.Purposes: The changes of Sirt-1 signaling pathway and inflammation after spinal cord injury,the reaction mechanism of bone marrow mesenchymal stem cells in inflammatory environment,the effect and mechanism of resveratrol on the inflammatory model of bone marrow mesenchymal stem cells,the effect and mechanism of resveratrol and bone marrow mesenchymal stem cells transplantation after spinal cord injury were studied and analyzed.Aims to provide theoretical and experimental basis for guiding the clinical application of resveratrol and bone marrow mesenchymal stem cells transplantation in spinal cord injury,and to provide new methods and new ideas for the treatment of spinal cord injury.Method:Part ?: The 6-8-week-old female healthy SD rats weighing 200-220 g were randomly divided into sham group(Sham operation group)and spinal cord injury group.According to the time setting after spinal cord injury,the sham operation group was divided into five subgroups: 1 d,3 d,5 d,7 d and 14 d after spinal cord injury.According to Allen's method,the model of spinal cord injury was made in rats.In Sham group,only the corresponding vertebral lamina was removed,and no operation was performed.Western Blot was used to detect the changes of Sirt-1 protein expression in the spinal cord of each group.The expression of IL-1 ? and IL-6 in the spinal cord of each group was detected by Elisa.The expression of Sirt-1 and TNF-?in the spinal cord tissues of sham group and 3d spinal cord injury group were detected by immunofluorescence staining.Part ?: Bone marrow mesenchymal stem cells were cultured in vitro from suckling rats.And their growth morphology was observed under light microscope.Flow cytometry was used to test the expression of CD34/CD45/CD29/CD90 antigen on the surface of primary cells.MTT assay was used to verify the change trend of cell survival rate with time in the inflammatory model of bone marrow mesenchymal stem cells induced by TNF-?(20 ng/ml).MTT was used to test the effect of resveratrol at different concentrations on the cell survival rate in the inflammatory model of bone marrow mesenchymal stem cells induced by TNF-?(20 ng/ml).Bone marrow mesenchymal stem cells were randomly divided into three groups: control group,TNF-? group,TNF-?+resveratrol group.The expression of IL-1? and IL-6 in the supernatant of each group was detected by Elisa.Western Blot was used to detect the changes of Sirt-1 and NF-?B protein expression and the changes of the expression of Cleaved Caspase-3 and Caspase-9.Then,bone marrow mesenchymal stem cells were randomly divided into two groups: TNF-?+resveratrol group,TNF-?+resveratrol+EX527 group.Real-time PCR was used to detect the changes of Sirt-1 and NF-?B gene expression in the two groups.The expression of IL-1 ? and IL-6 in the two groups were detected by Elisa.Flow cytometry(Annexin V-FITC-PI)was used to examine the changes of apoptosis in the two groups.Part ?: 6-8-week-old female healthy SD rats weighing 200-220 g were randomly divided into four groups: sham group,spinal cord injury group,spinal cord injury+BMSCs transplantation group,spinal cord injury+BMSCs+resveratrol transplantation group.According to Allen's method,the model of spinal cord injury was made in rats.In sham group,only the corresponding vertebral lamina was removed,and no operation was performed.BBB score was used to detect the changes of motor function in four groups.Nissl staining was used to detect the number of neurons in the anterior horn of spinal cord.Western blot was used to detect the changes of sirt-1/NF-?B,IL-1?/IL-6 protein expression and apoptosis related protein Cleaved Caspase-3/Caspase-9 expression.The difference of PKH26 and Neu N positive cells in spinal cord tissue between spinal cord injury+BMSCs transplantation group and spinal cord injury+BMSCs+resveratrol transplantation group was detected by fluorescence microscope.The content of BDNF in spinal cord of four groups was detected by Elisa.Results:Part ?: Western Blot showed that the expression of Sirt-1 changed with the time after spinal cord injury.The expression of Sirt-1 was significantly higher on the first day after spinal cord injury than that in the sham operation group.The expression of Sirt-1 reached the highest on about three days,and then gradually decreased and became stable.The results of Elisa showed that the expression of inflammatory factors(IL-1?,IL-6)also changed with the time after spinal cord injury.The expression of IL-1?,IL-6 in the first day after spinal cord injury was significantly higher than that in the sham operation group.The expression of inflammatory factors reached the highest level about three days after spinal cord injury,and then gradually decreased and became stable.The expression of Sirt-1 and TNF-? was up-regulated,according to the results of immunofluorescence double staining in sham group and SCI 3d group.Part ?: Under the light microscope,the rat bone marrow mesenchymal stem cells were cultured and proliferated in vitro for 3 days in the shape of long fusiform or polygonal,showing the growth trend of fibroblast like colonies.The results of flow cytometry showed that the cultured cells had the surface antigen characteristics of CD34(-)CD45(-)CD29(+)CD90(+)and the primary bone marrow mesenchymal stem cells had stem cell phenotype.It was confirmed that the primary bone marrow mesenchymal stem cells had stem cell phenotype.There was statistical significance in12 h with control group,and the decreasing trend of cell survival rate gradually slowed down as time continued to increase.Different concentrations of resveratrol have different effects on cell survival in bone marrow mesenchymal stem cells inflammatory model.The cell survival rate in the 1 ? mmol/L group was significantly higher than that in the inflammatory group.The results of Elisa showed that the expression of inflammatory factors in bone marrow mesenchymal stem cells was significantly higher than that in control group after inflammatory stimulation,while that in resveratrol intervention group was significantly lower than that in inflammatory model group.Western Blot analysis showed that the expression of Sirt-1 and NF-?B in bone marrow mesenchymal stem cells after inflammatory stimulation was higher than that in control group,while the expression of Sirt-1 and NF-?B in TNF-?+resveratrol group was significantly higher than that in TNF-? group,and the corresponding expression of NF-?B was significantly decreased.After inflammatory stimulation,the expression of Cleaved Caspase-3/Caspase-9 was higher than that of control group.The expression of apoptotic protein in TNF-?+resveratrol group was significantly lower than that in TNF-? group.Real-time PCR results showed that the expression of Sirt-1 in TNF-?+resveratrol+EX527 group was significantly lower than that in TNF-?+resveratrol group,and the corresponding expression of NF-?B was significantly higher.The results of Elisa showed that the expression of inflammatory factors in TNF-?+resveratrol+EX527 group was significantly higher than that in TNF-?+resveratrol group.Flow cytometry(Annexin V-FITC-PI)showed that the apoptosis of TNF-?+resveratrol+EX527 group was significantly higher than that of TNF-?+resveratrol group.Part ?: The results of BBB showed that the rats had obvious motor dysfunction after spinal cord injury.Both bone marrow mesenchymal stem cells transplantation and bone marrow mesenchymal stem cells+resveratrol combined transplantation showed the effect of promoting the recovery of motor function.The BBB score of bone marrow mesenchymal stem cells+resveratrol combined transplantation group was higher than that of bone marrow mesenchymal stem cells alone transplantation group.Nissl staining showed that after spinal cord injury,the number of neurons in the anterior horn of the spinal cord was significantly lower than that in the sham operated group.bone marrow mesenchymal stem cells transplantation and bone marrow mesenchymal stem cells+resveratrol combined transplantation increased the number of neurons in the anterior horn of the spinal cord.The average number of neurons in the bone marrow mesenchymal stem cells+resveratrol combined transplantation group was higher than that in the bone marrow mesenchymal stem cells alone transplantation group.However,no significant difference were detected between the two groups.Western Blot showed that the expression of Sirt-1,NF-?B and inflammatory factors were significantly higher in the bone marrow mesenchymal stem cells group than in the sham group.At the same time,after spinal cord injury,the expression of apoptotic protein Cleaved Caspase-3/Caspase-9 was significantly higher in bone marrow mesenchymal stem cells group than in sham group.There was no significant difference between bone marrow mesenchymal stem cells group and SCI group in the corresponding index changes.However,the expression of apoptotic protein in resveratrol+bone marrow mesenchymal stem cells group was significantly lower than that in SCI group and bone marrow mesenchymal stem cells group.After spinal cord injury,PKH26 positive rate and Neu N positive rate were significantly higher in resveratrol+bone marrow mesenchymal stem cells group than in bone marrow mesenchymal stem cells group.The results of Elisa showed that BDNF expression in bone marrow mesenchymal stem cells transplantation group and resveratrol+bone marrow mesenchymal stem cells combined transplantation group was significantly up-regulated after spinal cord injury,while BDNF expression in resveratrol+bone marrow mesenchymal stem cells combined transplantation group was slightly higher than that in bone marrow mesenchymal stem cells transplantation group.However,no significant difference was detected between the two groups.Conclusion: Part ?: After spinal cord injury,the expression of Sirt-1 was consistent with that of inflammatory factors.In addition,Sirt-1 is related to the expression of inflammatory factors after spinal cord injury.Sirt-1 may be involved in inflammatory response after spinal cord injury.Part ?: Inflammatory environment can affect the survival of bone marrow mesenchymal stem cells.Resveratrol of appropriate concentration can increase the cell survival rate of bone marrow mesenchymal stem cells in inflammatory environment and play a protective role.Resveratrol can up regulate the expression of Sirt-1,inhibit NF-?B,inhibit the inflammatory response,reduce the apoptosis of bone marrow mesenchymal stem cells in the inflammatory environment,and realize the protection of bone marrow mesenchymal stem cells in the inflammatory environment.Part ?: After spinal cord injury,resveratrol combined with bone marrow mesenchymal stem cells had better effect on motor function recovery than bone marrow mesenchymal stem cells alone.Resveratrol and bone marrow mesenchymal stem cells combined transplantation can inhibit apoptosis of spinal cord tissue by up regulating Sirt-1 expression,inhibiting NF-?B expression and inflammatory response,but bone marrow mesenchymal stem cells transplantation has no obvious effect.Resveratrol combined with bone marrow mesenchymal stem cells can increase the survival of bone marrow mesenchymal stem cells and the number of neurons in the spinal cord.Bone marrow mesenchymal stem cells transplantation or resveratrol and bone marrow mesenchymal stem cells combined transplantation can significantly increase the expression of BDNF.