| Objective:Previous experimental research found that cerebral ischemia can stimulate endogenous neurogenesis to a certain extent and participate in spontaneous repair after ischemia,but endogenous neurogenesis cannot produce a sufficient number of mature neurons and Glial cells,so the attention and research on neural stem cells(NSCs)transplantation has gradually increased.The previous research of the research group showed that Tetramethylpyrazine(TMP)has a significant promotion effect on the neurogenesis of middle cerebral artery occlusion(MCAO)model rats and NSCs toward ischemic injury area directional migration.SDF-1/CXCR4 is a classic regulatory pathway that promotes stem cell migration.As a positive regulator,Nrf2 can promote the transcription of SDF-1 receptor CXCR4 to promote bone marrow stem cell migration.In this study,the effect of TMP combined with transplantation of NSCs on MCAO model rats was observed,and the mechanism of TMP combined with transplantation of NSCs on ischemic stroke was explored based on stem cell biological behavior regulation.Method:The MCAO model was established by thread embolization method,and thenreperfused after ischemia for 90 minutes.Rats with successful modeling were randomly divided into model group(Model),Tetramethylpyrazine(TMP)group,neural stem cell(NSCs)transplantation group,TMP combined with NSCs transplantation group.At the same time set up asham operation(Sham)group as a control.TMP and TMP+NSCs transplantation group received intraperitoneal injection of TMP 40mg/kg,once a day after the operation;NSCs transplantation group and TMP+NSCs transplantation group transplanted NSCs 3*107 to the ischemic striatum 3 days after the surgery.50 mg/kg BrdU was injected intraperitoneally 3 days after surgery and 3 days before draw materials.Perform neurological function score(mNSS),muscle strength test,grid walking,open field test and forced swimming test to investigate the improvement of TMP on the neurological function and mobility of rats;observe the proliferation,migration and differentiation of NSCs on the distribution of BrdU+,BrdU+/DCX+,BrdU+/NeuN+BrdU+/GFAP+cells by immunofluorescence detection;PKH26+staining was used to detect the survival and migration of transplanted NSCs.Western blot was used to detect the protein expression of SDF-1 and the protein expression of signaling pathways Nrf2,KEAP1,HO-1,and NQO1.Real-time fluorescence quantitative PCR detection of mRNA levels of SDF-1,CXCR4,VEGF and NGF genes,and to investigate the mechanism of NSCs migration.Result:1.Evaluation of the effect of TMP combined with NSCs transplantation on the nerve repair of MCAO model rats:Compared with the sham operation group,the weight,muscle strength and open field mobility of MCAO model rats after ischemia were significantly reduced,and neurobehavioral scores were significantly increased.The index of missteps on the contralateral forelimb of ischemic lesions increased,and the resting time of swimming became longer,indicating that the successful replication of MCAO model.After TMP,NSCs transplantation and TMP+NSCs transplantation treatment,the neurological behavioral score of rats decreased,muscle strength recovered,mobility improved,wrong step index decreased,and swimming time increased,especially in the TMP+NSC transplantation group..2.The effect of TMP combined with NSCs transplantation on the survival,migration and differentiation of exogenous NSCs in MCAO model rats:PKH26-positive cells were observed by immunofluorescence detection,indicating that NSCs were successfully injected into the striatum and survived;and except for the transplantation site,PKH26-positive cells can also be observed in the vicinity,suggesting that a certain range of migration of surviving NSCs occurs,and that the number of migrated cells in the TMP+NSCs transplantation group is more and the migration distance is further.At the same time,we also tested PKH26 and NeuN,GFAP double-labeled cells,and did not find double-labeled positive cells,suggesting that NSCs transplanted into the brain within the current detection range,found no differentiation into neurons and astrocytes.3.The effect of proliferation,migration and differentiation on the TMP combined with NSCs transplantation to the endogenous NSCs:after ischemia,compared with the sham group,the number of BrdU+,BrdU+/DCX+,BrdU+/NeuN+,BrdU+/GFAP+ cells in the model group increased.Compared with the model group,the number of BrdU+,BrdU+/DCX +,BrdU+/NeuN+,BrdU+/GFAP+ cells in TMP and NSCs transplantation alone treatment and the combination group were significantly increased,and effect of the combination treatment group was the most significant.4.The effect of TMP and NSGs transplantation on the Nrf2/HO-1/CXCR4 pathway:the expression of SDF-1 protein in the TMP group,NSCs transplantation group and TMP+NSC transplantation group were increased,while the protein expression of Nrf2,HO-1 and NQO1 was up-regulated in the three treatment groups,and the expression of KEAP1 protein was decreased.Compared with the model group,TMP group,NSGs transplantation group and TMP+NSCs transplantation group can up-regulate SDF-1,CXCR4,VEGF,NGF genes.Conclusion:1.TMP,NSCs transplantation and TMP combined with NSCs transplantation can improve the neurobehavioral changes of MCAO model rats,especially TMP combined with NSCs transplantation has the best effect;2.Improves the survival,migration and differentiation of NSCs transplanted into the MCAO model brain,which is one of the ways of TMP combined with NSCs transplantation to treat MCAO model rats;3.Promotes the secretion of neurotrophic factors in the brain area,improve the brain microenvironment,and promote the proliferation,migration and differentiation of endogenous NSCs,which is another important way for TMP and joint NSCs transplantation to exert neuroprotection on MCAO model rats;4.Activating the Nrf2/HO-1/CXCR4 pathway may be part of the mechanism of TMP combined with NSCs transplantation to promote the survival and migration of endogenous neurogenesis and exogenous transplanted NSCs. |
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