P38 MAPK-PPARγ Signaling Pathway Participates In The Autophagy During The Induction Of Brain Ischemic Tolerance By CIP

Ischemic stroke is one of the main causes of human death and disability.Although the global diagnosis and treatment technology for ischemic stroke has continued to improve the prognosis of patients to a certain extent,once an ischemic stroke occurs,the progress of the pathological process cannot be reversed,bringing serious sequelae to the patients.Therefore,exploring the mechanism of cerebral ischemia and trying to improve the tolerance of neurons to ischemic damage is an important issue to be solved urgently.A large number of studies have shown that cerebral ischemic preconditioning can induce brain ischemic tolerance,in other words,giving animals a slight transient cerebral ischemia in advance can protect neurons against subsequent fatal cerebral ischemic damage.However,the mechanism of cerebral ischemic preconditioning to induce brain protection still needs further exploration.Autophagy is the process by which lysosomes degrade and utilize the material components in cells.Under the action of autophagy-inducing signals,cells form a double-layer membrane cup-shaped partition,which surrounds the degraded product to form pro-autophagosomes.After that,the pre-autophagosomes gradually extend,completely enveloping the cytoplasmic components to be degraded to form a hermetic autophagosome.Then,the outer membrane of the autophagosome combines with the lysosomal membrane to form an autophagolysosome,and the content is degraded by lysosomal enzymes.Autophagy is a double-edged sword.Cells can initiate moderate autophagy to clear damaged organelles and abnormally folded proteins,which plays an important role in protecting cells;however,incomplete or excessive autophagy may lead to cell death.Neonatal rat ischemia and hypoxia can cause cognitive and memory deficits by activating autophagy.Acidic post-conditioning protects neurons against focal cerebral ischemia by enhancing mitochondrial autophagy and prolongs the reperfusion duration.In the event of local cerebral ischemia,intermittent fasting protects the brain by reducing autophagy.Therefore,trying to regulate autophagy may be beneficial to the prevention and treatment of cerebral ischemic diseases.In recent years,studies have found that peroxisome proliferators-activated receptor(PPARγ)is closely related to autophagy.For example,cerebral ischemia-reperfusion in rats can cause severe brain damage.Pre-administration of PPARγ agonist rosiglitazone can reduce neuronal autophagic death.PPARγ agonist 15d-PGJ2 can reduce neuronal death caused by cerebral ischemia by inhibiting autophagy.Our previous studies have proved that PPARγ participates in the cerebral ischemic tolerance induced by CIP.However,whether PPARγ induces brain ischemic tolerance through regulating neuronal autophagy has not been reported yet.p38 MAPK is a subtype of the MAPK family discovered in 1994.During cerebral ischemia,the p38 MAPK signaling pathway is mainly activated by glutamate,hydrogen peroxide,and inflammatory factors(such as TNF-α,IL-1).After p38 MAPK is activated,it can enter the nucleus and activate downstream kinases or a variety of transcription factors to exert effects.Studies have confirmed that rattlesnake toxin protein activates human lung cancer cell apoptosis and autophagy through the p38 MAPK signaling pathway.p38 MAPK is involved in the death and autophagy of human gingival fibroblasts mediated by endoplasmic reticulum stress.Oleic acid inhibits rapamycin-induced hepatocyte autophagy through p38 MAPK.This article aims to investigate whether the p38 MAPK-PPARγ signaling pathway participates in brain ischemic tolerance induced by cerebral ischemic preconditioning by regulating autophagy.Part one Autophagy was involved in the induction of brain ischemic tolerance induced by CIPObjective:Using a rat four-vessel occlusion model of global cerebral ischemia to observe whether CIP induces brain ischemic tolerance by activating neuronal autophagy in the hippocampal CA1 area.Methods:1.To observe the effect of CIP on the activation of autophagy in hippocampal CA1 area of rats,healthy male Wistar rats were randomly divided into 2 groups(n=5):(1)VAO group: only bilateral common carotid arteries were exposed,but without occluding the blood flow;(2)Cerebral ischemic preconditioning(CIP)group: Bilateral common carotid arteries were clamped for 3 minutes,then restored with blood flow.The CIP group was further devided into 7 time points,namely 0min,3h,6h,12 h,24h,48 h,72h after ischemia.The rats were decapitated and the brain tissue was taken at the specified time point.The expression of autophagy-related proteins p62,Beclin1 and LC3 were detected by western blot;the expression of LC3 in pyramidal neurons in the hippocampal CA1 area was detected by immunofluorescence;the autophagosome expression in the CA1 hippocampus was detected by transmission electron microscopy.2.3-MA obliterates the brain ischemic tolerance induced by CIP.Healthy male Wistar rats were randomly divided into 6 groups(n=5):(1)VAO group;(2)CIP group: the bilateral common carotid arteries were clamped for 3minutes and then reperfused with blood flow;(3)Ⅱ group: the bilateral common carotid arteries were clamped for 8minutes and then reperfused with blood flow;(4)CIP+Ⅱ group: the rats were first subjected to a CIP for 3 min then reperfused,2 day-interval,a lethal ischemic insult for 8 min was given then reperfused with the blood flow;(5)3-MA+CIP+Ⅱ group: the rats were injected with autophagy inhibitor3-MA into the lateral ventricle 30 minutes before CIP,the other process was the same as the CIP+Ⅱ group.According to the different concentration of3-MA,the 3-MA+CIP+Ⅱ group was divided into 3 subgroups(each subgroup n=5);(6)DMSO+CIP+Ⅱ group: the rats were injected with 3% DMSO as a solvent of 3-MA into the lateral ventricle 30 minutes before CIP,and the other process was the same as that CIP+Ⅱ group.All the rats were scarificed at 7 d after the VAO operation or the last time of ischemia for neuropathological evaluation by thionin staining.Results:1.Western blot showed that compared with the VAO group,the expression of p62 has no difference at 0min time point after CIP.Compared with the 0min time point of CIP group,the expression of P62 increased significantly from 3h to 72 h,and reached the peak value at 48 h after CIP(P<0.05).Compared with the VAO group,the expression of Beclin1 has no difference at 0min time point after CIP.Compared with the 0 min time point of CIP group the expression of Beclin1 was significantly up-regulated from 3 h to 72 h and reached peak value at time point of 48 h after CIP(P<0.05).Compared with the VAO group,the expression of LC3Ⅱ/LC3 I has no difference at 0min time point after CIP.Compared with the 0 min time point of CIP group,LC3Ⅱ/LC3 I increased significantly from 6h to 72 h,and reached the peak value at 48 h after CIP(P<0.05).Immunofluorescence results showed in the VAO group,the LC3 immunoreactivity was extremely low in neurons of CA1 hippocampus,while the LC3 immunoreactivity in CIP group was relatively high.Electron microscope results showed that neurons in the VAO group appeared to be normal with fairly healthy endoplasmic reticulum,mitochondria,lysosomes and the nuclei.In the hippocampal CA1 neurons of the CIP group,organelles were intact,some autophagosomes,double-membrane structures and engulfment of cytoplasmic materials by autophagosome could be observed.2.3-MA obliterates the brain ischemic tolerance induced by CIPThionin staining showed that in rats subjected to VAO operation,the pyramidal neurons in the CA1 hippocampus were unaffected.No significant neuronal damage or loss was detected 7 days after the CIP.However,obvious neuronal destruction or loss in the CA1 hippocampus was observed 7 days after Ⅱ.Neuronal density was significantly decreased compared with that of the VAO group.When the animals were pretreated with the CIP 2 days before the lethal ischemic insult,the above injured changes were prevented clearly,which indicate that the CIP protected the pyramidal neurons in the CA1 hippocampus against the delayed neuronal death induced by the lethal ischemic insult.To investigate the role of autophagy in brain ischemic tolerance induced by CIP,the rats were injected with autophagy inhibitor 3-MA before CIP.Thionin staining showed the neurons in DMSO+CIP+Ⅱ group were untouched,with regular cell arrangement and clear nucleoli.In the 100nmol3-MA+CIP+Ⅱ group,some cells lost and necrotic,and the cell arrangement was relatively regular.Compared with the CIP+Ⅱ group,there was no significant difference in ND.In the 200 n M 3-MA+CIP+Ⅱ group,obvious neuronal loss or pyknosis death was observed.Compared with the CIP+Ⅱ group,ND was significantly decreased.A large number of cell degeneration and necrosis were observed in the 400 n M 3-MA+CIP+Ⅱ group,and the normal neurons were rare.Compared with the CIP+Ⅱ group,ND was significantly reduced.These results suggest 3-MA dose-dependently inhibited the neuroprotection induced by CIP in the CA1 hippocampus.Summary:Autophagy activation is involved in the induction of brain ischemic tolerance by CIP.Part two PPARγ regulates autophagy during the induction of brain ischemic tolerance by CIPObjective: To explore whether PPARγ participates in regulateing autophagy during the induction of brain ischemic tolerance by CIP.Methods:1.To observe the effect of CIP on the expression of PPARγ in hippocampal CA1 area of rats,healthy male Wistar rats were randomly divided into 2 groups(n=5):(1)VAO group: only bilateral common carotid arteries were exposed,but without occluding the blood flow;(2)CIP group: Bilateral common carotid arteries were clamped for 3minutes,then restored with blood flow.The CIP component was devided into7 time points,namely 0min,3h,6h,12 h,24h,48 h,72h after surgery.The rats of the above groups were decapitated at specified time points,and the expression of PPARγ in hippocampal CA1 area was detected by western blot.2.Inhibition of PPARγ signaling pathway affects CIP-induced neuronal autophagy,healthy male Wistar rats were randomly divided into 4 groups(n=5):(1)VAO group;(2)CIP group;(3)DMSO+CIP group: the rats were injected with 3% DMSO as a solvent of GW9662 into the lateral ventricle 30 minutes before CIP,and the other process was the same as that CIP group.(4)GW9662+CIP group: the rats were injected with autophagy inhibitor GW9662 into the lateral ventricle 30 minutes before CIP,the other process was the same as the CIP group.According to the different concentration of GW9662,the GW9662+CIP group was divided into 3 subgroups(n=5 for each subgroup).The rats of the above groups were decapitated at 48 h time point after CIP,and the expressions of autophagy-related proteins p62,Beclin1,and LC3 were detected by western blot;the expression of LC3 in pyramidal neurons in the hippocampal CA1 area was detected by immunofluorescence;Autophagosome expression in the CA1 hippocampus was detected by transmission electron microscopy.3.The effect of inhibiting PPARγ on brain ischemic tolerance,induced by CIP,healthy male Wistar rats were randomly divided into 3 groups(n=5):(1)CIP+Ⅱ group;(2)DMSO+CIP+Ⅱ group;(3)GW9662+CIP+Ⅱ group.Neuropathological evaluation for all groups was performed at time point of 7 days after the VAO operation or the last time of ischemia.Results:1.CIP up-regulated the expression of PPARγ in the CA1 hippocampusWestern blot results showed that compared with the VAO group,there was no significant difference in PPARγ expression at 0 min after CIP.Compared with the 0min time point,PPARγ increased from 3h after CIP,reached the peak at 48 h time point,and continued to 72h(P<0.05).The results showed that CIP caused the activation of PPARγ.2.The effect of inhibiting the PPARγ signaling pathway on neuronal autophagy induced by CIPWestern blot results showed that compared with the CIP group,the expression of Beclin1,P62 and LC3Ⅱ/LC3Ⅰ protein in the 2.5nmol GW9662+CIP group and 5nmol GW9662+CIP group was down-regulated(P<0.05).Compared with the CIP group,the DMSO+CIP group and 1.25 nmol GW9662+CIP group showed no significant changes in the expression of Beclin1,P62 and LC3Ⅱ/LC3Ⅰ(P>0.05).Immunofluorescence results showed that,compared with the CIP group,preadministration of the PPARγ inhibitor GW9662 in the lateral ventricle,and the expression of LC3Ⅱ in the pyramidal neurons of the hippocampal CA1 area was significantly reduced.Compared with the CIP group,the expression of LC3Ⅱ in the DMSO+CIP group did not change significantly.Transmission electron microscopy results showed that compared with the CIP group,the number of autophagosomes in the GW9662+CIP group was significantly down-regulated.Compared with the CIP group,the number of autophagosomes in the DMSO+CIP group did not change significantly.The above results indicate that inhibition of PPARγ can block CIP induced autophagy3.Inhibition of PPARγ can block the brain ischemic tolerance induced by CIPThe results of thionin staining showed that the neurons in the hippocampal CA1 area of the rats in the CIP group were neatly arranged,and the cell morphology was intact.Compared with the CIP+Ⅱ group,the neuron morphology of the DMSO+CIP+Ⅱ group had no significant changes,and there was no significant difference in ND(P>0.05).Compared with the CIP+Ⅱ group,a large number of cell degeneration and necrosis were obsvered in the5 nmol GW9662+CIP+Ⅱ group,normal neurons were almost invisible,and ND was significantly decreased(P<0.05).The above results indicate that inhibition of PPARγ can block the brain ischemic tolerance induced by CIP.Summary:PPARγ participates in regulateing autophagy during the induction of brain ischemic tolerance by CIP.Part three p38 MAPK-PPARγ signaling pathway participates in the autophagy during the induction of brain ischemic tolerance induced by CIPObjective:To explore whether p38 MAPK-PPARγ signaling pathway participates in the autophagy during the brain ischemic tolerance induced by CIP.Methods:1.To observe the effect of CIP on the expression of p-p38 MAPK in the hippocampal CA1 region of rats,healthy male Wistar rats were randomly divided into 2 groups(n=5):(1)VAO group: only bilateral common carotid arteries were exposed,but without occluding the blood flow;(2)CIP group: Bilateral common carotid arteries were clamped for 3minutes,then restored with blood flow.The CIP component was devided into7 time points,namely 0min,3h,6h,12 h,24h,48 h,72h after surgery.The rats of the above groups were decapitated at the specified time points,and the expression of p-p38 MAPK in hippocampal CA1 area was detected by western blot.2.The effect of SB203580 on the activation of PPARγ and autophagy.Healthy male Wistar rats were randomly divided into 4 groups(n=5):(1)VAO group;(2)CIP group;(3)DMSO+CIP group: the rats were injected with 3% DMSO as a solvent of SB203580 into the lateral ventricle 30 minutes before CIP,and the other process was the same as that CIP group.(4)SB203580+CIP group: the rats were injected with autophagy inhibitor SB203580 into the lateral ventricle 30 minutes before CIP,the other process was the same as the CIP group.According to the different concentration of SB203580,SB203580+CIP group was divided into 3 subgroups(n=5 for each subgroup)The rats of the above groups were decapitated at 48 hours after CIP,and the expressions of PPARγ and autophagy-related proteins p62,Beclin1 and LC3 were detected by western blot;the expression of LC3 in pyramidal neurons of hippocampal CA1 area was detected by immunofluorescence;expression of autophagosomes in hippocampal CA1 region was detected by transmission electron microscopy.3.The effect of SB203580 on CIP induced brain ischemic tolerance.Healthy male Wistar rats were randomly divided into 3 groups(n=5):(1)CIP+Ⅱ group;(2)DMSO+CIP+Ⅱ group;(3)SB203580+CIP+Ⅱ group.Neuropathological evaluation for all groups was performed at time point of 7 days after the VAO operation or the last time of ischemia.Results:1.CIP up-regulated the expression of p-p38 MAPK in the CA1hippocampusWestern blot results showed that compared with the VAO group,the expression of p-p38 MAPK has no significant difference in the 0 min of CIP group(P>0.05).Compared with the immediate time point of the CIP group,the expression of p-p38 MAPK increased from the 3h time point and lasted to72 h,and reached the peak at the 6h time point(P<0.05).2.The effect of SB203580 on the activation of PPARγ and autophagy2.1 The effect of SB203580 on the activation of PPARγWestern blot results showed that compared with the CIP group,in the SB203580+CIP group,as the dose increased,the expression of PPARγ protein was down-regulated in a dose-dependent manner after CIP(P<0.05).Compared with the CIP group,the expression of PPARγ in the DMSO+CIP group did not change significantly(P>0.05).The above results indicate that inhibition of p38 MAPK can block the activation of PPARγ.2.2 The effect of SB203580 on CIP-induced autophagyWestern blot results showed that compared with the CIP group,in the SB203580+CIP group,with the increase of the dose,the expression of Beclin1,P62 and LC3Ⅱ/LC3Ⅰ protein was down-regulated in a dose-dependent manner after CIP(P<0.05).Compared with the CIP group,the DMSO+CIP group showed no significant changes in the expression of Beclin1,P62 and LC3Ⅱ/LC3Ⅰ(P>0.05).The immunofluorescence results showed that,compared with the CIP group,the p38 MAPK inhibitor SB203580(5nmol)was given through the lateral ventricle in advance,and the expression of LC3Ⅱ in the hippocampal CA1 pyramidal neurons was significantly reduced.Compared with the CIP group,the expression of LC3Ⅱ in the DMSO+CIP group did not change significantly.Transmission electron microscopy results showed that compared with the CIP group,the number of autophagosomes in the SB203580+CIP group was significantly down regulated.Compared with the CIP group,the number of autophagosomes in the DMSO+CIP group did not change significantly.The above results indicate that inhibition of p38 MAPK can block CIP induced autophagy.3.SB203580 blocked the brain ischemic tolerance induced by CIPThe results of thionin staining showed that the neurons in the CA1 region of the hippocampus of rats in the CIP+Ⅱ group were arranged neatly,and the cell morphology was intact.Compared with the CIP+Ⅱ group,the morphology of DMSO+CIP+Ⅱ cells did not change significantly,and there was no significant difference in ND(P>0.05).Compared with the CIP+Ⅱ group,the SB203580+CIP+Ⅱ group showed a large number of neuronal degeneration and necrosis,almost no normal neurons were seen,and the ND decreased significantly(P<0.05).The above results indicate that inhibition of p38 MAPK can block the brain ischemic tolerance induced by CIP.Summary:p38 MAPK participates in the activation of autophagy via activating PPARγ during the induction of brain ischemic tolerance by CIP.Conclusions:(1)Autophagy activation is involved in the brain ischemic tolerance by CIP.(2)PPARγ participates in regulating autophagy during the induction of brain ischemic tolerance induced by CIP.(3)p38 MAPK participates in the activation of autophagy via activating PPARγ during the induction of brain ischemic tolerance by CIP.The above results indicate that p38 MAPK-PPARγ signaling pathway participates in the autophagy during the induction of brain ischemic tolerance by CIP.