| Background:Worldwide,stroke is the main cause of death and neurological dysfunction.The regeneration of the central nervous system after stroke is extremely limited,and the recovery of spontaneous function mainly depends on the internal repair mechanism of the brain,including neuroprotection,nerve regeneration,structural remodeling of axons and dendrites,etc.Mesenchymal stem cells are pluripotent stem cells isolated from mesenchymal tissue.Exosomes have been shown to be the main paracrine effector of mesenchymal stem cells and which can be released by mesenchymal stem cells to mediate intercellular communication to maintain a dynamic and stable microenvironment for tissue repair.In addition,many studies have found that various exosomes from MSCs can promote nerve regeneration,and this phenomenon is likely to be related to exosome miRNA.MicroRNAs(miRNAs)are small non coding RNAs that play a role by fine-tuning the expression of specific neurogenic gene targets at the post transcriptional level and regulate the development of mature neurons of neural progenitor cells.The latest research found that miR-17-92 cluster can promote neurogenesis and axonal growth in vitro,and customized exosomes rich in miR-17-92 cluster activate m TOR and inhibit GSK3β by activating it to regulate axonal regeneration of the central nervous system.Our team previously studied that SNX intervention in BMSCs conditioned medium can promote the axonal growth of primary cortical neurons,and the intervention of Tetramethylpyrazine and ferulic acid monomer ratio in BMSCs conditioned medium can also promote the axonal growth of neurons.In view of these,whether Xionggui formula can work with BMSCs exos to improve the brain microenvironment to stimulate the growth of neuronal axons,and its specific role is worth further discussion.Objective:To explore whether Xionggui recipe and exosomes combined treatment can promote the brain microenvironment after ischemic brain injury,so as to more effectively stimulate the growth of neuronal axons,and whether it can increase neural plasticity and functional recovery through the mechanism target of miR-17-92/PTEN signaling pathway.Methods:1.Select 220-250 g rats,first isolate and culture bone marrow mesenchymal stem cells,collect cell culture medium through multiple passages,and finally extract BMSCs exosomes by ultra-high speed centrifugation,and identify exosomes by flow cytometry;2.According to the longa score,the rats were scored for neurological impairment,and the rats with behavior score of 1-3 were successfully included in the group.The rat model of t MCAO was established by thread occlusion method;Experimental rats were used to splite into six groups:(1)model group(t MCAO group): rats were given 1 × PBS caudal vein injection;(2)Xionggui prescription group(t MCAO+SNX group): rats were given shunaoxin dropping pills(dissolved in normal saline,45mg/kg/d per rat by gavage,24 h after t MCAO,for 7 consecutive days);(3)Exosomes group(t MCAO+exos group): rats were injected with BMSCs exosomes 400 μg/kg via caudal vein(24h,3D,5D,7d after t MCAO);(4)Synergetic group(t MCAO+SNX+exos group): intervene shunaoxin dropping pills and BMSCs exos according to the above methods;(5)Sham operation group(sham group),1 × PBS were injected for rats.3.The modified neurological deficit assessment MNSs was evaluated on the 1st,7th and21 st days after modeling;The brain was perfused into the heart on the 7th and 21 st days after modeling,the pathological changes of brain tissue can be survey by HE staining in each group.The protein expressions of MBP and NG2 in the infarcted cortex of rats on the 21 st day were detected by immunofluorescence staining and Westernblot immunoblotting to detect the differentiation and migration of oligodendrocytes and evaluate axonal myelin regeneration;4.The m RNA expression level of miR-17-92 cluster was detected by real-time fluorescence quantitative method;5.Western blot was used to detect the downstream target proteins PTEN and GSK3 of miR-17-92 cluster β、 The expression level of axon growth protein GAP-43;Results:1.The modified nervous system severity score(m NSS)score data indicated that the neurological deficit score of sham operated rats on the 1st,7th and 21 st days after cerebral ischemia injury was 0,and there was no neurological deficit;On the 7th and 21 st days after cerebral ischemia-reperfusion,compared with sham operation,the m NSS score of each group decreased.On the 7th day after ischemia.The difference of t MCAO score was obvious significant to compared with sham group;The m NSS score in the the SNX and exosomes combined treatment group was lower than the t MCAO group,and the difference was significant;There was no significant difference between t MCAO+SNX group and t MCAO+exos groupto compared with t MCAO+SNX+exos group.On the 21 st day,compared with t MCAO group,the scores of t MCAO+SNX group,t MCAO+exos group and t MCAO+SNX+exos group declined,and there was marked significant;The score of t MCAO+SNX+exos group.By contrast,t MCAO+EXOS group and t MCAO+SNX group.There is a downward trend,no statistical difference.2.He staining method was used to observe the morphology of neurons in the penumbra of rats in each group.The results displayed that the sham group had uniform staining,full and explict structure,round or cone-shaped distribution and uniform arrangement of neuronal cells,large and central nuclei,and obvious nucleoli.In the t MCAO group,the brain tissue structure in the cerebral cortex was broken,the nerve cells were arranged irregularly,the neurons were obviously swollen,the nucleolus was not clear,nuclear pyknosis occurred,there were vacuolar changes of different sizes in the intercellular space,and interstitial edema occurred.Compared with the t MCAO group,the morphological manifestations of neuronal necrosis and tissue edema were less in the t MCAO+SNX group,t MCAO+exos group and t MCAO+exos+SNX group.Compared with t MCAO+SNX group and t MCAO+exos group,the pathological damage of necrotic neurons and brain edema in t MCAO+exos+SNX group was less.The results indicated that SNX combined with BMSCs exos could reduce neuronal injury in cerebral cortex after cerebral ischemia-reperfusion and reduce the recovery of brain damaged tissues.3.The semi dark oligodendrocyte precursor cell markers were observed by NG2 immunofluorescence staining after ischemia-reperfusion in the brain tissue of rats on the 7th day.Compared with the t MCAO group,the number of positive cells in t MCAO+SNX group,exosome group and the SNX and exosomes combined treatment group increased,and there was marked significant.The number of positive cells in the t MCAO group increased than sham operation group,,and there was marked significant;Compared with exosome group and traditional Chinese medicine group,the number of positive cells in the SNX and exosomes combined treatment group increased,but there was not marked significant.The myelin basic protein MBP,a marker of semi dark mature oligodendrocytes after cerebral ischemia-reperfusion in rats on the 7th day,was observed by Westernblot.The results are shown in the figure.Compared with the t MCAO group,the expression level of MBP protein in t MCAO+SNX group,exosome group and the SNX and exosomes combined treatment group increased,and there has statistical significance.Compared with t MCAO+SNX group and the exosome group,the number of positive cells in the the SNX and exosomes combined treatment group increased,but there was not marked significant.4.according to the results of PCR detection of cerebral cortex of rats with ischemia-reperfusion for 7 days,the results are shown in the figure.In mir17,compared with the t MCAO group,the m RNA expression of exosomes group and the SNX and exosomes combined treatment group increased,and there was marked significant,while there in traditional Chinese medicine group was not marked significant.In mir18 a,compared with the t MCAO group,the m RNA expression of traditional Chinese medicine group,the SNX and exosomes combined treatment group and exosome group increased,and there was marked significant.In mir19 a,mir19b and mir20 a,compared with the t MCAO group,the m RNA expression in the the SNX and exosomes combined treatment group increased with statistical significance,while between the exosome group and t MCAO+SNX group the difference is not significant;Compared with the exosome group,the m RNA expression in the the SNX and exosomes combined treatment group increased,and there was marked significant.In mir19 b and mir20 a,compared with t MCAO+SNX group,the m RNA expression in the the SNX and exosomes combined treatment group increased,and there was marked significant.In mir92 a,there was no significant change among the groups.5.The Westernblot results of cerebral cortex in ischemic penumbra area indicated that the expression level of PTEN protein in exosome group decreased with statistical significance to compared with the t MCAO group,and while there was no statistical significance between traditional Chinese medicine group and synergistic group;The expression level of PTEN protein in the the SNX and exosomes combined treatment group decreased to compared with t MCAO+SNX group,and the difference was marked significant.Westernblot test results show that GSK3βThere was no significant difference to compared with the t MCAO group in protein expression level;Westernblot test results indicated(FIG.13,14),the expression level of gap43 protein in t MCAO+SNX group and the exosome group increased to compare with the model group,and there was obvious significant.Compared with the exosome group and t MCAO+SNX group,the expression level of gap43 protein in the the SNX and exosomes combined treatment group increased,but the difference was not obvious significant.Conclusion:1.The combined intervention of Xionggui formula and BMSCs exosomes in rats with cerebral ischemia-reperfusion injury can improve their neurological defects and pathological damage of brain tissue,and promote the differentiation of oligodendrocytes and myelination in the recovery period of ischemic stroke;2.Xiongguifang and BMSCs exosomes synergistically intervene in rats after ischemia-reperfusion brain injury by regulating axonal regeneration through mir-17-92/pten signaling pathway. |
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