Investigations On Therapeutic Effects And Mechanism Of Ergothioneineine Against Cerebral Ischemic Stroke

Objective:1.To explore the pharmacokinetics of ergothioneine(EGT),the distribution across the blood-brain barrier and the possible mechanism across the blood-brain barrier.2.To explore the neuroprotective effect of EGT in photochemically induced ischemic stroke model(Photochemical Ischemic,PI)and middle cerebral artery occlusion model((Middle cerebral artery occlusion,MCAO)induced by thread occlusion in mice.3.To explore the mechanism of oxygen free radical injury and inflammatory injury in the neuroprotective effect of EGT and its relationship with PI3K/Akt-Nrf2 signal pathway in two CIS(Cerebral Ischemia Stroke)models,so as to provide theoretical basis for the clinical application of EGT.Methods:1.Determination of drug concentration-time curve of EGT in rats and distribution of EGT across the blood-brain barrier in mice:male and female SD rats were selected and sampled at different time points after a single administration of EGT(25mg/kg).The drug concentration-time curve was drawn and the pharmacokinetic parameters were calculated by non-compartment model.Male and female C57/BL6 mice were injected through tail vein and sanpled at different time points after a single administration of EGT(40mg/kg),and the concentration of EGT in brain tissue was determined.The transcriptional level of Octn-1 was detected by RT-PCR method.2.Study on the neuroprotective effect of EGT on CIS model mice:the CIS model induced by PI and MCAO was established.The volume of cerebral infarction was measured by TTC method.The relative blood flow of cerebral cortex was detected by laser speckle flow meter.(rCBF),was used to detect the changes of glial cells and neurons around the infarct focus.The changes of motor function in PI-induced and MCAO-induced CIS models were detected by fatigue bar test and neurobehavior respectively.The differerences of EGT drug group,Model group and positive drug EDV group were compared,and the neuroprotective function of EGT was evaluated.3.Study on the mechanism of EGT participating in neuroprotective effect of ischemic stroke:the mouse model of MCAO was established.The activity of SOD and the content of MDA in the brain were detected by SOD and MDA kits,and the transcriptional levels of TNF-α,IL-1β and IL-6 in the brain were detected by RT-PCR.After administration of PI3K inhibitor,the volume of cerebral infarction was measured by TTC,and the protein expressions of PI3K,Akt,p-PI3K,p-Akt,Keapl,Nrf2,HO-1 and TNF-α in the brain of model mice were analyzed by Western Blot method to explore the mechanism of neuroprotective effect of EGT through PI3K/Akt-Nrf2.Results:1.The plasma concentration-time curve of EGT showed that there was no significant difference in plasma concentration-time curve between male and female rats during i.v.administration,and the T1/2 was about 20.9512.59h;Vd was about 3.53±0.40L/kg;CL was about 0.16±0.05L/h/kg;During i.g.administration,the blood concentration-time curves of rats of different genders were similar,but the blood concentration of males was higher than that of females.and the peak time Tmax was about 6.20±0.89h,the T1/2 was about 40.50±45.97h;Cmax was about 7454.75±3340.69μM/L;Vd was about 3.43±1.54L/kg;CL was about 0.11±0.08L/h/kg;AUC(0-24)was about 105226.96±45749.84 μM/h/kg;AUC(0-∞)was about 315757.41±247205.11μM/h/kg.The results of tissue concentration of EGT at different times showed that EGT could be distributed in the brain through the blood-brain barrier of mice,and the content of EGT in the brain of male and female mice was the highest at 0.5h.RT-PCR analysis showed that EGT cou-ld increase the transcription level of Octn-1 in the surroundi-ng tissues o-f cerebral ischemia in mice.2.EGT has neuroprotective effect on CIS model mice induced by PI and MCAO:compared with Model group,EGT can reduce the volume of cerebral infarction(P<0.01),increase the relative blood flow of cerebral cortex(P<0.01),effectively reduce the activation number of astrocytes and microglia around the infarct focus of the two models(P<0.01),and improve the motor function of PI model mice,and MCAO model mice motor coordination function and perception function.3.The antioxidant and anti-inflammatory effects of EGT may be involved in the neuroprotective effects of EGT on PI and MC AO-induced CIS model mice:compared with Model group,EGT can increase the activity of SOD and reduce the production of MDA(P<0.05).By up-regulating the phosphorylation levels of PI3K and Akt,reducing Keapl,activating Nrf2 into the nucleus,promoting the expression of HO-1 and reducing the expression of TNF-α protein around the infarct focus(P<0.05).After administration of PI3K inhibitor,there was no significant difference in cerebral infarction volume between EGT group and Model group(P>0.05),but the expression of PI3K,p-Akt,Nrf2 and HO-1 was down-regulated(P<0.05),and the activation of this signal pathway was inhibited.Therefore,EGT may play a neuroprotective role through PI3K/Akt-Nrf2.Conclusion:1.After injection of EGT through i.v.,the peak concentration was quickly reached in blood and brain tissue.EGT has a long half-life and slow clearance in vivo,and is mostly distributed in tissues,indicating that it has high bioavailability and can meet the requirements of drug evaluation in the acute stage of ischemic stroke2.EGT alleviates the brain function damage caused by CIS by reducing the cerebral infarction volume,increasing the cerebral cortical blood flow value and inhibiting the activation of glial cells,so as to exert the neuroprotective function.3.EGT may regulate oxygen free radical injury and acute inflammation by activating PI3K/Akt-Nrf2 signal pathway,and participate in the neuroprotective role in the process of CIS.