Study On The Protective Effect Of Catalpol On "Neurovascular Unit"

Stroke is the second-largest disease leading to death. There is only tPA approved to applied in clinic,but the time window is 4.5h that is so short for therapeutic.What’s more,the use of tPA might increase the risk of hemorrhage.As the consequence of the fact,this drug can’t be widly applied. Meanwhile,the use of thrombolytic can’t notablly ameliorate the symptoms of ischemic stroke.Thus,it’s an urgent problem for scientists to search and develop the potential durgs for stroke.Luckily,the concept of "the neurovascular unit" offerd an dierection.Objectives:To study whether the catalpol can protect "Neurovascular unit" in vitro and in vivo. Besides,Using the cell of PC 12 to expore the potential mechanism of the effetion of catalpol on neuron.Part I The research of the protective effet of catalpol on the major components of "neurovasular unit" in vitro.Method:The major components of "neurovascular unit" primary cells were cultured.1.Neurons were cultured and different concentration of glutamate(4mM,8 mM,16mM,32mM) were added to induce neurotoxicity,while different doses of catalpol(0.3μM,3μM,30μM,300μM)were added to the middium 2h before glutamate to explore their effects.2.Astrocytes were cultured and different concentration of hydrogen peroxide (62.5μM,125μM,250μM,500μM,1000μM) were added to induce neurotoxicity, while different doses of catalpol (10μM,30μM,100μM,300μM) were added to the middium 2h before hydrogen peroxide to explore their effects.3 ECs were culture different concentration of SNP (125μM,250μM,500μM,1000μM,2000μM) were added to induce neurotoxicity, while different doses of catalpol (3.33μM,10μM,30μM) were added to the middium 2h before SNP to explore their effects.All the three primary cells viability were measured by MTT assay after instrument drugs added to induce cell injury as the same treament as catalpol.Finally,the effect of catalpol on BDNF secretion of ECs were measured by BDNF assay with or without SNP.Results:Glutamate induced neurons injury in a dose-dependent manner from 4mM to 32mM.0.1μg/mL catalpol and1μg/mL catalpol could inverse glutamate-induced neurotoxity. Hydrogen peroxide induced astrocytes injury in a dose-dependent manner from 62.5μM to 1000mM.30μM,100μM catalpol and 300μM catalpol could inverse hydrogen peroxide-induced neurotoxity. SNP induced astrocytes injury in a dose-dependent manner from 500uM to 2000mM.3.3μM catalpol and 10μM catalpol could inverse SNP-induced cell injury.We found that catalpol not only could increase the BDNF secretion of ECs in the absence of SNP,but also could improve the concentration of BDNF secretion after SNP induced cell injury.Conclusion:Catalpol could protect "Neurovascular unit" in vitro and the mechanism might be related to BDNF.Part II The research of the protective effect of different concentration of catalpol on tMCAO rats.Method:Study took advantage of suture in preparation of rats model of tMCAO,which the time is 2h.24h after operation,nervous funtion grades were carried out with the method of Bederson score.Besides,the neuromuscular function and corner test were applied.TTC staining was used to calculate the infarct volumes. "The neurovasular unit" space was build by gelatin-Indian ink reperfution, GFAP staining and nissl body staining to explore the effect of catalpol on ischemic cerebral rats.Results:The result of Bederson score indicate that the catalpol can’t ameliorate nervous function.While the result of neuromuscular function as well as corner test indicated that the high doses of catalpol(10mg/kg) could significant ameliorate nervous function. The TTC staining also indicated the high doses of catalpol could notabaly reduce the infarct volumes.According the result of the built space of "Neurovascular unit" dipalyed that catalpol could ameliorate the injured organisation.No matter the group of middle doses or high doses of catalpol could increase the number of vasculars,astrocytes and neurons versus the group of model at the injured sides.Conculusion:Afer cerebral ischemic for 2h and reperfusion 24h,the group of high doses of catalpol could significantly ameliorate the nervous function and infarct volumes.Besides it,catalpol also could prevention from "Neurovascular unit" injure.Part III The research of catalpol on the hydrogen peroxide-induced PC 12 neurotoxicity:with emphasis on autophagy and apoptosisMethod:Different concentration of hydrogen peroxide(62.5μM,125μM,250μM, 500μM,1000μM were added to the culture medium for 3h,12h,24h,48h,and the cell viabiliy was measured by MTT assay.Different doses of catalpol(0.01mM,0.1 mM, 1mM) were added before hydrogen peroxide, and cell viabiliy was measured by MTT assay.Bcl-2, Bax and cleaved-Caspase3 raleted to apoptosis were mesured by Westen Blot.Beside it,LC3Ⅱ,Beclinl and P62 related to autophgy were alse mesured by Western Blot.Results:Different concentration of hydrogen peroxide induced PC 12 injury in a time-dependent manner in 24h.When different concentration of hydrogen peroxide induced PC 12 injury for 12h,the cell viability of 250μM hydrogen peroxide was 81.78%.Different concentration of catalpol could significantly increase the number of PC 12 alone.When hydrogen peroxide were added,catalpol could significantly ameliorate cell injury and effect of catalpol was in a manner of dose-dependent.From the result of Westen blot,we found that catalpol could inhibit the apoptosis by reduce the expression of Bax,Bcl2 and cleaved-Caspase3.Besides,the level of LC3II and Beclinl were increased after high dose of catalpol(1mM),in simultaneously, the level of P62 were down-regulation.Conclusion:The results suggested that the inhibiton of apoptosis and activation of autophagy could be responsible for the neuroprotective effects of catalpol in hydrogen peroxide-induced cell injury.Up-regulation of Bcl2/Bax and down-regulation of cleaved-Caspase3 might play an important roles in inhibition of apoptosis.While down-regulation of P62 and up-regulation of Beclinl might play an important roles in this process.