Intervention Effect And Mechanism Of Exercise And HWTX-? On Chronic Cerebral Ischemia In Mice

Chronic cerebral ischemia is caused by a variety of causes of long-term cerebral blood flow insufficiency,leading to persistent or progressive cognitive and neurological dysfunction ultimately,which has a severe impact on the quality of life of the patient.In recent years,the pathogenesis,pathological changes and prevention of cerebral ischemia of more and more experimental study has been made,The study found that ischemic brain injury and Ca²⁺signal transduction abnormalities lead to increased cytosolic Ca²⁺concentration is closely related.Selenocosmia huwena is Rare spider specie,which mainly distributed in Yunnan and Yunnan provinces,The most abundant is the huwen toxin-I?HWTX-??,biological activity studies showed that HWTX-? selectively acts on the N-type calcium channel,which inhibits neuronal presynaptic membrane release of neurotransmitters and blocks nerve conduction,is a new type of N-type calcium channel blockers,and it has been found that HWTX-? has a strong neuroprotective effect in the rat global cerebral ischemia-reperfusion model.Sport has a unique role in preventing disease and improving quality of life,the early research group showed that:moderate-intensity aerobic exercise as a functional rehabilitation treatment,in a variety of chronic disease animal models,has significant anti-organ damage protection.In this study,a mouse model of chronic cerebral ischemia was established,aerobic exercise and HWTX-? tail vein injection were used to separate and in combination intervention,to investigate the possible molecular mechanism of Notch signaling pathway-related factors,calcium overload and expression of neural repair-related factors,and to provide theoretical and experimental basis for the effective treatment of cerebral ischemic diseases.Meanwhile,we compared the mRNA expression of brain tissue and blood-related factor,discussed the mRNA expression of two different tissues,and mRNAs in brain tissue were sequenced and analyzed in order to provide a new idea for the monitoring and treatment of related diseases.Objective:To investigate the effect of aerobic exercise and HWTX-? on chronic cerebral ischemia injury and differential expression of Notch channel related cytokine Notch1,Jagged1,NICD,sodium calcium exchange protein NCX1,SYP and calcium binding protein CaBP-D28k mRNA in brain and blood of mice,differential analysis of mRNA in brain and the relationship to chronic cerebral ischemia.Methods:Chronic cerebral ischemia injury model was constructed in70 male KM mice and they were randomly divided into 7 groups:model group?M?,HWTX-? group?vena caudalis injection of HWTX-? after ischemia,H?,aerobic exercise treadmill group?TM?,HWTX-? combined with aerobic exercise treadmill group?H+TM?,aerobic exercise swimming group?SW?,HWTX-? combined with aerobic exercise swimming group?H+SW?,sham operation group?only surgery without ligation of the right common carotid artery,SO?.Group TM,H+TM,SW and H+SW doing aerobic exercise 6 times a week for 5 weeks after 3 days of grouping,group H,H+TM and H+SW was given vena caudalis injection of HWTX-??0.05ug/g BW?for 30 days of injection 15 times,Clark neurological function score was taken after grouping and at the end of exercise.At the end of the experiment,the mice's eyes were removed and the brain tissues were isolated.The focal neurological function and general neurological function of mice were detected by Clark's neurological score.The appearance of ischemic brain atrophy and ischemic focus was observed by whole-brain anatomy.Nissl staining was used to observe the changes of morphology.The concentration of serum NSE and S100B in mice were detected by enzyme linked immunosorbent assay?ELISA?.The expressions of Notch1,Jagged1,NICD,NCX1,SYP and CaBP-D28k protein in brain tissue were detected by immunohistochemistry.The expression of Notch1,Jagged1,NICD,NCX1,SYP and CaBP-D28k mRNA in brain tissue and blood were detected by Real time PCR.The high-throughput sequencing technique was used to detect brain tissue mRNAs,screening mRNA molecules that specifically differ in brain tissue during exercise.Results:1)Nissl staining morphology showed that nerve cells in cerebral cortex of mice of group M were trachychromatic,appeared a large number of karyopyknosis,obvious vacuoles,nuclear necrosis,obvious loss of neurons in group M;Vacuoles were not obvious,part of the nucleuses and Nissl bodies were visible in cerebral cortex nerve cells of group H;Cerebral cortex of mice of group TM have obvious loss of neurons and obvious vacuoles but better than the group M;The vacuoles were less in cortical nerve cells of group H+TM,the caryons were light-stained and less neuronal loss;nerve cells in cerebral cortex of mice of group SW appeared obvious vacuoles,but less than the group M,trachychromatic,a few karyopyknosis,obvious loss of neurons in group SW,but better than the group M;nerve cells in cerebral cortex of mice of group H+SW appeared much vacuoles,obvious karyopyknosis,part trachychromatic,but the nucleus is clear,Nissl body is clear and edge set,trachychromatic,less neuronal loss.2)The right side of the brain atrophy was obvious in the group M,two modes of exercise have a positive effect on the increase in cerebral ischemic blood flow in mice,toxin and exercise combined intervention effect is more obvious;Toxin combined with exercise have delayed the formation of ischemic macula.3)The neurological function scores of each intervention group were better than the group M.4)Compared with group M,there were significant differences in serum NSE protein content between group TM,group SW and group H+SW?P<0.01?.The difference of serum S100B protein in group H+SW was statistically significant?P<0.01?.5)Immunohistochemistry and Real-time PCR results showed that Compared with group M,brain tissue's expression of Notch1,Jagged1 mRNA were up-regulated in group H,group H+TM and group H+SW?P<0.05 or P<0.01?,The expression of Notch1,Jagged1 and NICD protein was up-regulated compared with the model group?P<0.05 or P<0.01?,However,there was no significant difference in the expression of protein and mRNA between group TM and group SW compared with the model group?P>0.05?.Compared with group H,the expression of Notch1 and Jagged1 protein in group H+TM was up-regulated?P<0.01?,the expression of Jagged1 and NICD protein in group H+SW was up-regulated?P<0.01?,while the expression of Notch1 and Jagged1mRNA in group H+SW was up-regulated?P<0.05?.The expression of synaptophysin mRNA in group H,group TM,group H+TM and group H+SW was significantly higher than that in group M?P<0.05 or P<0.01?.Compared with the group M,the expression of synaptophysin was up-regulated in all the intervention groups,the difference of synaptophysin protein content in group H and group TM was statistically significant?P<0.05?,in group H+TM and group H+SW was statistically significant?P<0.01?.The expression of NCX1 mRNA and protein in group H+TM,group H+SW and group H were higher than those in group M?P<0.05 or P<0.01?.The expression of CaBP-D28k mRNA in group H+TM and group H+SW was higher than that in group M?P<0.05?,the expression of CaBP-D28k protein in group H+TM,group H+SW and group H was higher than that in group M?P<0.05?.6)the expression of Notch1,Jagged1and NCX1 mRNA in brain and blood have a moderate degree of correlation?r>0.5?,7)A total of 18714 genes were obtained by high-throughput sequencing.There were 650 significant differences in the swimming group compared with the model group,with a decrease of 467and a increase of 183.According to the function and the relationship with cerebral ischemia and exercise,it is divided into five categories:channel related factors,Signal transmission related factors,metabolic related factors,inflammation related factors and stress related factors.Conclusion:1)Aerobic exercise and HWTX-? have neuroprotective effects on chronic cerebral ischemic injury.2)The neuroprotective mechanism of HWTX-? can be mediated by calcium-binding protein-D28k,Synaptophysin and the classical Notch signaling pathway.The neuroprotective mechanism of aerobic exercise is to promote the occurrence and remodeling of synapses after cerebral ischemia by upregulating SYP expression.3)Notch1,Jagged1,Ncx1 factors and 650different genes may be a new target and/or marker for chronic cerebral ischemic therapy.