Neuroprotective Effects And Its Mitochondrial Mechanisms Of Gallic Acid On Ischemic Stroke Injury

Object:Ischemic stroke is a kind of cardiovascular disease which has serious harm to human health.A large number of studies have shown that mitochondria plays an important role in the process of programmed neurological cell death during ischemia-reperfusion,which has become a new target for effective intervention in stroke injury.Gallic acid?GA?is a kind of polyphenols derived from plants such as palm leaf rhubarb,Eucalyptus and dogwood,and it has significant antioxidant effect.This study aims to further investigate whether GA has an effect on ischemic stroke and demonstrate whether its effect is related to mitochondria,and this study will provide a new theoretical basis for experimental application of GA in ischemic stroke.Methods:In order to study the pharmacodynamic effect of GA on ischemic stroke,the experimental model of ischemic stroke were established by using middle cerebral artery occlusion?MCAO?in the first part of the study.?MCAO?,MCAO+GA25mg/kg group,MCAO+GA50mg/kg group and MCAO+CSA10mg/kg group.The activity of SOD in ischemic penumbra neurons was detected by colorimetric method.H&E staining was used to detect brain tissue changes,Cerebral infarct volume was detected by TTC method.Apoptosis of neurons was detected by TUNEL staining.The release of Cyt C was detected by Western blot.In vitro,Na₂S₂O₄ was used to construct the hypoxia / reoxygenation injury cell model.The cells were pretreated with different concentrations of GA?0.1,1,10?mol / L?,and the cell viability was detected by MTT assay.DCFH-DA fluorescence probe was used to detect ROS content,Mitosox red fluorescence probe method was used to detect the content of mitochondrial ROS,Clark oxygen electrode measured cell oxygen consumption,luciferase assay was used to detect intracellular ATP content.The anti-ischemic stroke of GA was evaluated by the above trial system,and discuss a preliminary discussionon mitochondrial function protection.In order to further clarify the related sites of mitochondrial function of GA,the open cell mPTP model was constructed by H₂O₂ method in the second part of in vitro experiment.Cell viability was detected by MTT assay.Apoptosis was detected byAnnexin V-FITC/PI flow cytometry.JC-1 staining,Calcein AM-CoCl₂ and Cyt C immunostaining were used to evaluate the openness of mPTP.The activity of SOD and the content of MDA were measured by colorimetry.The levels of cleavable caspase-3,9 and Cyt C were detected by Western blot.In vitro,the mitochondria of the isolated liver and the Calcium Green5 N fluorescent marker were used to detect calcium retention capacity by the transient steady-state fluorescence spectrometer.Through the above tests,the effect of GA targeting mPTP on mitochondrial function was systematically evaluated.To elucidate the mechanism of GA on mPTP,the mitochondria of isolated mice liver were extracted in the third part,Ca²⁺ stimulates exfoliated mitochondria,and the degree of binding between CypD and ANT-1 was detected by immunoprecipitation method.The effect of GA on the activity of aminoproline cis-trans isomerase activity in CypD was established.The MCAO model was established,the infarct volume was measured by TTC.The open model of mPTP was established by H₂O₂ method.Cleaved caspase-3,CypD and p-ERK,Calcein AM-CoCl₂ staining was used to evaluate the openness of mPTP.Finally,ERK pathway inhibitor U0126 was used to verify the above-mentioned GA effect from both in vivo and in vitro.Results:In the first part,the study group found that GA can effectively against MCAO and Na₂S₂O₄-induced neuronal hypoxia-reoxygenation injury.Compared with MCAOgroup,the infarct volume,the number of TUNEL positive cells and the Cyt C release in the GA50 mg/kg group were significantly decreased.In addition,the levels of SOD in the brain cells of GA rats were significantly increased after GA treatment,these results suggest that GA has aeffective effect against cerebral ischemia-reperfusion injury.It is important that these neuroprotective effects of GA may be closely related to their protective effect on mitochondrial function.Compared with the model group,GA can significantly inhibit the decrease of mitochondrial membrane potential,the decrease of ATP content and the decrease of oxygen consumption,and the increase the level of total ROS and mitochondrial ROS.These results indicate that GA has the effect of anti-ischemic stroke and it is closely relatedto its function of protecting mitochondria of nerve cells.In the second part,the study found that GA protects mitochondria against hypoxia damage were associated with its effect on mPTP and increased mitochondrial calcium threshold levels.Compared with the control group,the level of Calcein AM decreasedwith the treatment of 500?mol/L H₂O₂,which indicates that the mPTP in SH-SY5 Y cells were open.The level of membrane potential decreased,the expression level of Cleaved caspase-9,-3 increased and the release of Cyto C increased,indicating H₂O₂ induce apoptosis.Pretreatment with GA can significantly reverse the H₂O₂-induced mPTP openingin SH-SY5 Y cell,promote cell survival,inhibit the occurrence of apoptosis.Compared with the control group,the mitochondrial calcium retention capacity of GA was significantly higher than that of the control group.The results showed that mPTP was one of the targets of GA protecting mitochondrial function.In the third part,the study found that the activity of cis-trans isomerase in CypD was significantly higher than that in the control group,whereas the level of complex CyPD-ANT-1 in the mouse liver mitochondria was significantly decreased.Interestingly,the expression level of CypD in SH-SY5 Y cells was significantly decreased with the increase of ERK phosphorylation level under EGF-induced and MCAO injury stimuli,and U0126 inhibited ERK phosphorylation,and the effect of GA against H₂O₂-induced MPTP opening and MCAO-induced cerebral ischemic injury were reversed.Compared with GA group,the levels of Calcein AM in GA combine with U0126 groups were significantly decreased,the expression of Cleaved caspase-9,-3,cerebral infarction Volume significantly increased.These results indicate that GA directly affects the activity of CypD amino-proline cis-trans isomerase,inhibits its binding to ANT-1,on the other,it inhibits the expression of CyPD by acting on ERK phosphorylation level and increases the opening of mPTP to play its neuroprotective effect.Conclusion:Conclusion : GA can directly inhibit the expression of CypD and increase themitochondrial mPTP threshold by directly affecting the binding of CypD to ANT-1and affecting the phosphorylation of ERK,and effectively protect neuronal injury during ischemic stroke.In view of the key role of mPTP in a variety of oxidative stress injury processes,GA may be a potential therapeutic agent for such diseases,and its potency is worthy of further explore.