| BackgroundSpinal cord injury(SCI)is a common disease in clinical spine surgery,and its sensation and motor function are impaired due to severe trauma.Spinal cord injury can lead to dysfunction of the spinal cord,which in turn impairs the normal physiological function of the spinal cord,including sensation,movement,autonomy,and reflex.With the rapid development of the economy and the improvement of people's living standards,the transportation industry and the construction industry have developed rapidly compared with the past,but the number of patients with spinal cord injuries has increased.Due to the high disability rate of spinal cord injury,it causes great pain to the patients,and also brings a heavy economic burden to the family and society.Medication and surgery are traditional methods of treating spinal cord injury.Although these methods can alleviate the pain of patients to a certain extent,the curative effect is still limited and cannot solve the problem from the fundamental upside.Nerve repair is a key problem in the treatment of spinal cord injury,and cell transplantation provides a new method for the treatment of spinal cord injury.Mesenchymal stem cells are pluripotent adult stem cells that can differentiate into mesoderm cells and partially differentiate into non-mesodermal lineages,and thus are trophic factors for tissue repair and regeneration.Bone marrow-derived mesenchymal stem cells(BMSCs)have high self-renewal activity and differentiation potential,and can be induced into a variety of cells under different conditions,including nerve cellsAt present,the inducers for inducing BMSCs to differentiate into neural cells at home and abroad are mainly retinoic acid,dimethyl sulfoxide,?-mercaptoethanol,glycerol,etc.,but the neuron-like cells induced by the above inducer have a shorter survival time in vitro.Some of the inducers also have certain toxicity and cannot be applied in clinical practical treatment.In the final analysis,the ultimate goal of inducing BMSCs to differentiate into neural cells in vitro is to apply and clinically practical treatment,so finding new effective and harmless inducers is one of the most interesting issues at present.Traditional Chinese medicine has the advantages of wide source and relatively low side effects.It has been proven to promote cell growth and differentiation,and is non-toxic,thus facilitating cell transplantation after differentiation.A large number of studies have shown that a variety of Chinese medicines have outstanding performance in inducing stem cell differentiation.Tetramethylpyrazine(TMP)is an alkaloid which is one of the active constituents extracted from the traditional Chinese medicine Chuanxiong.It has anti-oxidation,prevents calcium overload overload in cells,anti-neuronal apoptosis,and protects nerve cell function.Therefore,it is widely used in the treatment of various ischemic and traumatic diseases of neurology.Studies have found that tetramethylpyrazine combined with BMSCs transplantation can repair spinal cord injury and induce differentiation of the latter into bone marrow stromal cells,but the specific mechanism of action is not well understood,and there are no studies on BMSCs combined with tetramethylpyrazine and BMSCs alone.The effects of treatment on neurological recovery were compared.ObjectiveIn this study,the rat spinal cord injury model was used as the research object.The recovery of neurological function after spinal cord injury was treated with BMSCs transplantation,tetramethylpyrazine and BMSC transplantation.The related mechanisms were discussed in order to apply tetramethylpyrazine to spinal cord injury.Treatment provides theoretical and experimental evidence.Method(1)The fetus and tibia of SD rats born for 3 days were collected,and the cells in the bone marrow cavity were collected and cultured.The surface markers of BMSCs were detected by flow cytometry,such as CD34,CD44,CD45,CD90,CD11b and CD 106.The cultured BMSCs were isolated.A model of spinal cord injury in Wistar rats was established.Wistar rats were divided into control group,model group,BMSCs group and BMSCs+TMP group.Rats in each group were treated with slanting plate test,modified Tarlov classification and Basso Be-attie Bresnahan(BBB)score for each group.The motor function of the rats was evaluated;the sensory evoked potential(SEP)and the motion evoked potential(MEP)of the rats were detected.The spinal cord tissues of each group of rats were collected and paraffin sections were prepared.The glial fibrillary acidic protein(GFAP)and Nerve growth factor(NGF)in the spinal cord of each group were detected by immunohistochemistry.expression.(2)Rat spinal cord tissue was collected and spinal cord neurons were isolated and cultured.The rat spinal cord neuron cells were treated with H2O2 to establish a damaged spinal cord neuron cell model.The rat spinal cord neuron cells were randomly divided into control group,model group,BMSCs group and BMSCs+TMP group.The cells in each group were treated according to the grouping requirements.After 48 hours of culture,the apoptosis and proliferation activity were detected.The cell culture supernatant was used to detect the expression of TNF-a in the supernatant;the expression of caspase-3,GFAP and NGF protein in each group was detected.Results(1)Morphological observation and identification of BMSCs showed that after 24 hours of culture,some cells began to adhere to the bottom of the flask,and most of them appeared in pairs,and the shape was elliptical and had obvious light transmission;continued culture After 72 h,spherical and fusiform cells were observed in the field of view;on the 5th day of culture,fusiform and polygonal cells were observed.Under the microscope,the number of BMSCs in the culture flask was significantly increased.The cells of the first to third generations have a good growth state and a strong proliferative activity,and the cells are mainly monolayer cells.On the first day and the second day of culture,the cells were flat,spindle-shaped and irregular,and the nuclei were clearly visible.The third generation cells are more uniform in shape.The surface markers of rat BMSCs were detected by flow cytometry.The results showed that the third generation BMSCs positively expressed CD44,CD90 and CD 106,while CD11b,CD34 and CD45 were negative.The above results confirmed that BMSCs have been successfully isolated and cultured.The results of the recovery of motor function in each group showed that the critical angle,BBB score and Tarlov score of the model group,BMSCs group and BMSCs+TMP group at 2 weekends were significantly higher than those at 1 week after surgery.Increased(P<0.05).Compared with the control group,the critical angle,BBB score and Tarlov score of the other groups were significantly decreased(P<0.05).Compared with the model group,the critical angle of the slanting plate,BBB score and Tarlov score of BMSCs group and BMSCs+TMP group increased significantly,and the critical angle,BBB score and Tarlov score of sloping plate were significantly increased(P<0.05);Compared with the group,the critical angle of the sloping plate of BMSCs+TMP group,BBB score and Tarlov score increased significantly,and the critical angle of sloping plate,BBB score and Tarlov score were significantly increased(P<0.05).The results of postoperative SEP test in each group showed that SEP was detected in each group 2 weeks after surgery to observe the recovery of neurophysiological response in rats.Compared with the control group,the SEP latency of other groups of rats increased significantly,and the amplitude decreased significantly(P<0.05).Compared with the model group,the SEP latency of BMSCs group and BMSCs+TMP group was significantly decreased,and the amplitude was significantly increased(P<0.05).Compared with the BMSCs group,the SEP latency of the BMSCs+TMP group was significantly decreased,and the amplitude was significantly increased(P<0.05).The postoperative MEP results of the rats in each group showed that MEP experiments were performed on the rats in each group 2 weeks after the operation,and the latency and amplitude of MEP were observed and recorded.Compared with the control group,the MEP latency of the other groups was significantly increased,while the amplitude was significantly decreased(P<0.05).Compared with the model group,the MEP latency of the BMSCs group and the BMSCs+TMP group decreased significantly,and the amplitude increased significantly(P<0.05).Compared with the BMSCs group,the MEP latency of the BMSCs+TMP group was significantly lower than that of the BMSCs group(P<0.05).The results of immunohistochemistry showed that compared with the control group,the positive expression rates of GFAP and NGF protein in the spinal cord tissues of other groups were significantly decreased(P<0.05);compared with the model group,BMSCs group and BMSCs+TMP The positive expression rates of GFAP and NGF protein in the spinal cord of rats were significantly increased(P<0.05).The positive expression rates of GFAP and NGF protein in spinal cord tissue of BMSCs+TMP group were significantly higher than those of BMSCs group.<0.05).The above results indicate that the combination of tetramethylpyrazine injection and BMSCs has a significant effect on the recovery of spinal cord injury.(2)Apoptosis test results showed that compared with the control group,the apoptosis rate of the other groups was significantly increased(P<0.05);compared with the model group,the BMSCs group and the BMSCs+TMP group were withered.The mortality rate was significantly decreased(P<0.05).Compared with the BMSCs group,the apoptosis rate of BMSCs+TMP group was significantly lower(P<0.05).The above results indicate that the combination of tetramethylpyrazine and BMSCs can more effectively protect spinal cord neurons damaged by H2O2 and inhibit their apoptosis.The expression of Caspase-3 protein showed that the expression of caspase-3 protein was significantly up-regulated in the other groups compared with the control group(P<0.05).Compared with the model group,the cells in BMSCs group and BMSCs+TMP group were compared.The expression of caspase-3 protein was significantly decreased(P<0.05).Compared with BMSCs group,the expression of caspase-3 protein in BMSCs+TMP group was significantly decreased(P<0.05).The above results indicate that the combination of tetramethylpyrazine and BMSCs can inhibit the expression of caspase-3,which plays a role in the apoptosis of spinal cord neurons induced by H2O2 injury.The cell viability assay showed that the activity of the other groups was significantly lower than that of the control group(P<0.05).Compared with the model group,the activities of BMSCs group and BMSCs+TMP group were significantly increased(P<0.05);compared with the BMSCs group,the activity of BMSCs+TMP cells increased significantly(P<0.05).The above results indicate that the combination of oxazide and BMSCs can effectively increase the activity of H2O2 injured spinal cord neurons and promote their proliferation.The results of TNF-? level in the cell culture supernatant showed that compared with the control group,the TNF-a levels in the supernatants of the other groups were significantly higher(P<0.05);compared with the model group,BMSCs group and BMSCs The level of TNF-? in the cell culture supernatant of TMP group was significantly decreased(P<0.05).Compared with BMSCs group,the level of TNF-? in BMSCs+TMP group was significantly decreased(P<0.05).After spinal cord injury,nerve cells can produce a large amount of TNF-?-induced spinal cord tissue ulceration and necrosis.The results of this study indicate that the combination of tetramethylpyrazine and BMSCs can inhibit the release of TNF-?from spinal cord neurons injured by H2O2,thereby protecting nerve cells.The expression of GFAP and NGF in each group showed that compared with the control group,the expression levels of GFAP and NGF protein in the other groups were significantly lower(P<0.05).Compared with the model group,the BMSCs group and The expression levels of GFAP and NGF protein in BMSCs+TMP group were significantly increased(P<0.05).Compared with BMSCs group,the expression levels of GFAP and NGF protein in BMSCs+TMP group were significantly increased(P<0.05).The above results indicate that the combination of tetramethylpyrazine and BMSCs can induce the expression of GFAP and NGF,and the activation and growth of spinal cord neurons damaged by H2O2,and finally play a role in protecting neurons and promoting the recovery of neuronal function.Conclusion(1)Tetramethylpyrazine combined with BMSCs transplantation can significantly improve the neurological function of rats with spinal cord injury.(2)Tetramethylpyrazine combined with BMSCs transplantation can effectively inhibit spinal cord neuron damage caused by H2O2,maintain its activity,inhibit apoptosis,its mechanism and inhibition of apoptosis-related protein caspase-3 expression,up-regulate the neuro-activated protein GFAP and promote nerve The expression of the growth-related protein NGF may be related. |
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