| Background:Stroke is a complex and life-threatening disease and one of the main causes of death and disability worldwide.The recovery of stroke requires a series of precise interaction between neural stem cells and neurovascular units,so it is the first choice to recover the blood supply of ischemic area as soon as possible after symptoms appear,but the effect of existing treatment methods is not ideal.According to traditional Chinese medicine,ischemic stroke is fan Tao of apoplexy,which is caused by the disorder of Qi and blood circulation.Although its etiology and pathogenesis are complex,it is no more than Qi and blood.The Xionggui Prescription,which is composed of chuanxiong and Danggui,is a common prescription for treating ischemic cerebrovascular disease.In the early stage of the team,Shunaoxin pills,a Chinese patent medicine developed by Xionggui Prescription,was used for relevant research,which showed that Shunaoxin pill could promote angiogenesis,neuron regeneration and brain tissue reconstruction in rats with ischemic brain injury.In addition,modern medical research has found that stem cells have the clinical potential to treat stroke,but there are also many risks;some studies have shown that exosomes play a leading role in the paracrine effect of stem cells,and can avoid many risks of stem cell therapy,but the efficacy is still insufficient.In addition,PI3K/Akt / mTOR signaling pathway plays an important role in promoting cell cycle,cell proliferation and angiogenesis.Objective:To explore whether Xionggui Prescription can regulate the microenvironment and improve the effect of exosomes derived from bone marrow mesenchymal stem cells(BMSCs-exosomes) on angiogenesis in the recovery period of ischemic stroke;to analyze whether Xionggui Prescription and BMSCs-exosomes can regulate angiogenesis in the recovery period of ischemic stroke through mTOR/p70S6K signal pathway.Methods:1.Culture and identification of primary rat BMSCs.BMSCs were isolated from rats and identified by cell flow cytometry.Exosomes in serum were removed by centrifugation of 100000 g for 12 hours,and 10% non exosomes serum medium was prepared to culture BMSCs.2.Isolation and identification of BMSCs-exosomes.BMSCs-exosomes were obtained by centrifugation of BMSCs culture supernatant,ultracentrifugation,and stored in -80° with 5005?g/ml suspension in 1×PBS.Western blot and transmission electron microscopy were used to identify the morphology.3.Using thread embolism method to make transient middle cerebral artery occlusion ischemia(tMCAO) model of SD rats.The experimental animals were randomly divided into six groups,and the rats after successful modeling were divided into model group(tMCAO group),Xionggui Prescription(t MCAO+SNX group),exosome group(tMCAO+exos group),and synergy group(tMCAO+SNX+exos group),inhibitor group(tMCAO+SNX+exos+RAPA group)and sham operation group(Sham group),of which Xionggui Prescription was given Shunaoxin dripping pills(dissolved in physiological saline,Each rat was intragastrically administered at 45mg/kg/d,administered 24 h after t MCAO and continued for seven days),the exosome group was injected with BMSCs-Exosomes 400?g/kg tail vein(administered 24 h,3d,5d,7d after tMCAO)In the synergistic group,Shunaoxin dripping pills and BMSCs-exosomes were intervened according to the above method,while in the inhibitor group,RAPA was given before the administration of Shunaoxin dripping pills and BMSCs-exosomes(2 mg/kg/d intraperitoneal injection per rat),24 h after tMCAO and continued for 7 days),the sham operation group and the model group were given an equal volume of 1×PBS tail vein injection.Rats were scored for neurological deficits,foot failure tests,and dropout tests on days 1,3,and 7 after modeling.The laser Doppler cerebral blood flow monitor-DRT4 was used to monitor the change of cerebral blood flow at 21 days after tMCAO.After 21 days of feeding,the brain was perfused and the brain infarct area of each group was observed by HE staining.4.Western blot was used to detect the expression of PI3K,Akt,mTOR,P70S6 K,p-pi3 k,p-Akt,p-mTOR,p-p70s6k and VEGF in PI3K/Akt/mTOR/P70S6K signal pathway.Results:1.The neurological deficit score of rats in each group showed that the MNSs score of each group was decreased on the 3rd day after ischemia-reperfusion,and the MNSs score of synergetic group was significantly lower than that of model group(P<0.05);compared with the synergetic group,the difference between exosomes group and inhibitor group was not statistically significant(P>0.05).On the 7th day after ischemia-reperfusion,the MNSs score of each group was significantly lower than that of the model group,and the difference was statistically significant(P<0.05);compared with the synergistic group,there was no significant difference in the exosomes group(P>0.05),but the difference was statistically significant in the inhibitor group(P<0.05).2.The drop line test of rats in each group showed that there was no significant differencebetween the model group and the other groups on the third day after ischemia-reperfusion(P>0.05);compared with the synergetic group,there was no significant difference in the exosomes group and the inhibitor group(P>0.05).On the 7th day after ischemia-reperfusion,there was no significant difference between the two groups(P>0.05),but there was no significant difference between the two groups(P>0.05).3.After 3 days of ischemia-reperfusion,there was no significant difference in the scores of other groups compared with the model group(P>0.05);compared with the synergetic group,there was no significant difference in the exosomes group and the inhibitor group(P>0.05).On the 7th day after ischemia-reperfusion,the foot dysfunction test showed that compared with the model group,the score of synergetic group was significantly decreased(P<0.01);compared with the synergetic group,there was no significant difference in exosomes group(P>0.05),and the score of inhibitor group was significantly higher than that of synergy group(P<0.05).4.The results of cerebral blood flow monitoring showed that compared with the model group,the differences of Xionggui Prescription group,exosomes group and synergetic group were statistically significant(P<0.05);compared with the synergetic group,there was no significant difference in the exosomes group(P>0.05),but the cerebral blood flow in the inhibitor group was lower(P<0.05).5.Cerebral infarction volume comparison results showed that compared with the model group,the differences of Xionggui Prescription group,exosomes group and synergetic group were statistically significant(P<0.05);compared with the synergetic group,there was no significant difference in exosomes group(P>0.05),but there was significant difference in inhibitor group(P<0.05).6.Western blot showed that there was no significant difference in the expression levels of PI3K,Akt,P70S6K among the groups(P>0.05);compared with the synergistic group,the expression of m TOR protein in the inhibitor group was significantly lower(P<0.05).Compared with the model group,the protein expression levels of p-pi3k,p-mTOR,p-p70s6k and VEGF in the Xionggui Prescription group were increased(P<0.05);the protein expression levels of p-pi3k,p-mTOR,p-p70s6k and VEGF in the exosomes group were increased(P<0.05);the protein expression levels of p-pi3k,p-Akt,p-p70s6k and VEGF in the synergetic group were increased(P<0.05),and the protein expression of p-m TOR was increased(P<0.01).Compared with the synergistic group,the protein expression levels of p-pi3k,p-Akt,p-mTOR,p-p70s6k and VEGF in the inhibitor group were significantly decreased(P<0.05,P<0.01);the protein expression levels of p-Akt,p-mTOR,p-p70s6k and VEGF in the exosomes group weresignificantly decreased(P < 0.05).Conclusion:1.Xionggui Prescription can regulate the microenvironment,improve the effect of BMSCs-exosomes on angiogenesis in the recovery period of ischemic stroke,and improve the brain tissue damage and nerve function defect of t MCAO rats.2.Xionggui Prescription and BMSCs-exosomes can regulate the angiogenesis of tMCAO rats through PI3K/Akt/mTOR/p70S6K signal pathway and promote the repair of neural function after ischemic brain injury. |
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