| Temporal lobe epilepsy(TLE)is the most frequent and intractable epilepsy.50%of patients with TLE suffer from one or more cognitive deficits,which seriously affects the quality of life of patients with TLE and aggravates the burden of home caregivers.But there is no effective treatment for the cognitive comorbidity of epilepsy.Therefore,it is urgent to find or develop therapeutic drugs for the cognitive comorbidity of epilepsy to meet clinical needs.It is currently believed that hippocampal neuronal injury and synaptic plasticity disorder play the causal role in cognitive comorbidities of TLE.A large amount of evidence showed that there are a lot of intersections between programmed cell death of neurons and the regulation of synaptic plasticity,such as the activation of P38 MAPK signaling pathway.Inhibition of neuronal cell death is often accompanied by an improvement in synaptic plasticity.As a result,neuroprotective therapy has become a hot topic in the prevention and treatment of cognitive comorbidities of TLE.Ferroptosis is a programmed cell death mediated by iron-driven lipid peroxides overaccumulation,which is closely related to cognitive dysfunction.However,whether ferroptosis is involved in the cognitive comorbidities of TLE has not been reported.The key elements triggering and executing the ferroptosis processes included iron overload,glutathione(GSH)depletion or glutathione peroxidase 4(gpx-4)inactivation,lipid peroxidase accumulation.Studies have shown that iron deposition,lipid peroxides accumulation,GSH depletion and abnormal expression of synaptic plasticity proteins were found in the hippocampus of rats with kainic acid(KA)induced TLE,a classic TLE model.Based on this,ferroptosis may occur in hippocampal neurons of rats with KA induced TLE.Studies have shown that iron-driven lipid oxidative stress can mediate the activation of P38 MAPK.Activation of P38 MAPK can impair the cognitive function of Alzheimer's disease mice by down-regulating the expression of hippocampal synaptic plasticity protein markers,such as synaptophysin and postsynaptic density protein 95.Ferrostatin-1(fer-1),a specific inhibitor of ferroptosis,can mitigate hippocampal neuron injury and improve cognitive dysfunction in the disease models,such as Alzheimer's disease,ischemic stroke,Parkinson's disease.The possible mechanisms include inhibiting lipid oxidative stress and reducing excessive iron accumulation in the brain.This study intends to verify whether ferroptosis occurred in hippocampus of rats with KA induced TLE,fer-1 can improve the cognitive function of rats with KA induced TLE,and further to explore whether fer-1 inhibits hippocampal neuronal damage or restore the expression of synaptic proteins.This study would provide a new therapeutic target for cognitive comorbidities of TLE.Part one:ferroptosis occurred in hippocampus of rats with KA induced TLEObjective:To whether ferroptosis occurred in hippocampus of rats with KA induced TLE.Methods:24 male SD rats were randomly divided into sham group(n=12)and KA group(n=12).Temporal lobe epilepsies was induced in the rats by stereotaxic intrahippocampal administration of KA.At the end of fifth week after model made,Y maze,novel object recognition experiment and water maze experiment were used successively to evaluate the cognitive functions of rats in each group.45 days after model made,the pathological features of ferroptosis in hippocampal neurons of rats with KA induced TLE were observed by transmission electron microscope.Nissl staining and HE staining were used to evaluate hippocampal neuron damage in rats.Perls blue staining was used to detect hippocampal iron deposition in rats of each group.Glutathione(GSH)kit was used to detect GSH levels in the hippocampus of rats in each group.The expression of glutathione peroxidase 4(GPX-4)in the hippocampus of rats was determined by immunohistochemistry.Malondialdehyde(MDA)kit was used to detect the level of MDA in the hippocampus of rats in each group.Results:(1)The data of the Y-Maze test showed that the spontaneous alternation rate of rats in the KA+vehicle group decreased significantly compared with the sham+vehicle group of rats(P<0.05).(2)The Novel object recognition test showed that the discrimination index of KA group decreased significantly compared with the sham group(P<0.05).(3)The results of spatial navigation tests showed that the escape latency and the swimming distance decreased progressively in experimental rats of all groups during the five training days.From the second day onwards,the escape latency and the swimming distance of the KA group were higher than that of the sham group(P<0.05).There was no significant difference(P>0.05)in the average swimming velocity between all groups of rats.In the spatial probe test,the number of platform crossings of KA group was less than that of sham groups(P<0.05).The number of platform crossings was significantly increased in the KA+fer-1 group compared to KA group(P<0.05).There was no significant difference(P>0.05)in the number of platform crossings between sham group and sham+fer-1 group.(4)The results of HE staining indicated that the number of cells in CA1 and CA3 regions of the hippocampus decreased remarkably in the KA+vehicle group as compared to the sham+vehicle group(P<0.05).Treatment with Fer-1resulted in significant attenuation of prominent neuronal loss in CA1 and CA3regions of hippocampus of KA induced TLE rats(P<0.05).There was no significant difference(P>0.05)in the number of cells in CA1 and CA3 regions of hippocampus between sham+vehicle group and sham+fer-1 group.(5)The results of Nissl staining indicated that the number of Nissl stained cells in CA1 and CA3 regions of the hippocampus decreased remarkably in the KA+vehicle group as compared to the sham+vehicle group(P<0.01).Treatment with Fer-1 resulted in significant attenuation of prominent neuronal loss in CA1 and CA3 regions of hippocampus of KA induced TLE rats(P<0.01).There was no significant difference(P>0.05)in the number of Nissl stained cells between sham+vehicle group and sham+fer-1 group.(6)The occurrence of ferroptosis in the hippocampus of KA treated rats was measured by TEM.Our results showed that the average mitochondrial area of the hippocampus neuron of KA group was smaller than that of sham+vehicle group(P<0.05).(7)The results of Perls'staining showed that the average percentages of the iron positive area in CA1 and CA3 regions of the hippocampus increased significantly in the KA group compared with the sham group(P<0.05).(8)The number of GPX4 immunoreactive cells in the hippocampus was compared by immunohistochemistry.The results showed that the number of GPX4 immunoreactive cells in CA1 and CA3 regions of hippocampus decreased significantly in the KA group compared to the sham group(P<0.05).(9)Compared to the sham group,the levels of GSH in the hippocampus decreased significantly in the KA+vehicle group(P<0.05).(10)Compared to the sham group,the levels of MDA in the hippocampus increased significantly in the KA group(P<0.05).Conclusion:Ferroptosis occurred in neurons in the hippocampal CA1 region of rats with KA induced TLE.Ferroptosis processes might be involved in the mechanism of hippocampal neuronal injury and cognitive impairment.Part two:Ferrostatin-1 improved cognitive function in rats with kainic acid induced temporal lobe epilepsy by inhibiting ferroptosis processesObjective:To explore the mechanism of ferrostatin-1 inhibiting hippocampal injury and improving cognitive function in rats with KA-induced TLE.Methods:This experiment was divided into two parts:(1)Ferrostatin-1improved cognitive function in rats with kainic acid induced temporal lobe epilepsy by inhibiting ferroptosis processes.24 male SD rats were randomly divided into sham group(n=12)and KA group(n=12).Temporal lobe epilepsies was induced in the rats by stereotaxic intrahippocampal administration of KA.45 days after model made,video was used to monitor the spontaneous seizures of rats in each group.At the end of fifth week after model made,Y maze,novel object recognition experiment and water maze experiment were used successively to evaluate the cognitive functions of rats in each group.At sixth week after model made,the pathological features of ferroptosis in hippocampal neurons of rats with KA induced TLE were observed by transmission electron microscope.Nissl staining and HE staining were used to evaluate hippocampal neuron damage in rats.Perls blue staining was used to detect hippocampal iron deposition in rats of each group.Glutathione(GSH)kit was used to detect GSH levels in the hippocampus of rats in each group.The expression of glutathione peroxidase 4(GPX-4)in the hippocampus of rats was determined by immunohistochemistry.Malondialdehyde(MDA)kit was used to detect the level of MDA in the hippocampus of rats in each group.(2)Effects of fer-1 on the expression of p-p38 in the hippocampus in rats with KA induced TLE.48 male SD rats were randomly divided into sham+vehicle group(n=12),sham+fer-1 group(n=12),KA+vehicle group(n=12)and KA+fer-1group(n=12).Rats were given intraperitoneal injection of fer-1 or vehicle for14 days,depending on the group.The rats were given intraperitoneal injection of fer-1 or vehicle for 14 consecutive days according to different groups.45 days after model made,the expression of P38 and p-p38 in the hippocampus of rats in each group were detected by western blot.Results:(1)In contrast,70%of rats in the KA+vehicle group and 50%of rats in the KA+fer-1 group had spontaneous seizures.There was no significant difference between the KA+vehicle group and KA+fer-1 groups(x~2=0.833,P=0.361).(2)The data of the Y-Maze test showed that the spontaneous alternation rate was significantly improved in the KA+fer-1 group compared to the KA+vehicle group(P<0.05).(3)The Novel object recognition test showed that the discrimination index was significantly improved in the KA+fer-1 group compared to KA+vehicle group(P<0.05).(4)The results of spatial navigation tests showed that the escape latency and the swimming distance decreased progressively in experimental rats of all groups during the five training days.From the third day onward,the escape latency and the swimming distance were significantly(P<0.05)improved in the KA+fer-1 group compared to the KA+vehicle group.There was no significant difference(P>0.05)in the average swimming velocity between both groups of rats.In the spatial probe test,the number of platform crossings was significantly increased in the KA+fer-1 group compared to KA+vehicle group(P<0.05).(5)The results of HE staining indicated that treatment with fer-1 resulted in significant attenuation of prominent neuronal loss in CA1 and CA3 regions of hippocampus of KA induced TLE rats(P<0.05).(6)The results of Nissl staining indicated that treatment with fer-1 resulted in significant attenuation of prominent neuronal loss in CA1 and CA3 regions of hippocampus of KA induced TLE rats(P<0.05).(7)The results of Perls'staining showed that treatment with fer-1 deceased prominently the average percentages of the iron positive area in the hippocampus of KA-treated rats(P<0.05).(8)The number of GPX4 immunoreactive cells in the hippocampus was compared by immunohistochemistry.The results showed that compared to the KA+vehicle group,the number of GPX4 immunoreactive cells in CA1 and CA3 regions of hippocampus increased significantly in the KA+fer-1 group(P<0.05).(9)Treatment with fer-1 attenuated the reduction of GSH in the hippocampus of KA-treated rats significantly(P<0.05).(10)Treatment with fer-1 attenuated the increases of MDA in the hippocampus of KA-treated rats significantly(P<0.05).(11)Compared to the sham+vehicle group,the ratio of p-P38/P38 in the hippocampus increased significantly in the KA+vehicle group(P<0.01).Compared to the KA+vehicle group,the ratio of p-P38/P38 in the hippocampus reduced significantly in the KA+fer-1 group(P<0.05).There was no significant difference(P>0.05)in the ratio of p-P38/P38 in the hippocampus between sham+vehicle group and sham+fer-1 group.Conclusion:(1)Fer-1 improved the cognitive function of rats with KA induced TLE through mitigating hippocampal neuronal injury.(2)Fer-1 alleviated hippocampal neuronal injury by inhibiting ferroptosis processes.Part three:Ferrostatin-1 mitigates synaptic protein injury of epileptic rats by inhibiting P38 MAPK activationObjective:To investigate the mechanism of ferrostin-1 mitigating synaptic protein injury and improving cognitive function in rats with KA-induced TLE.Methods:This experiment was divided into two parts:(1)Effects of fer-1 on the expression of SYN and PSD95 in the hippocampus in rats with KA induced TLE.48 male SD rats were randomly divided into sham+vehicle group(n=12),sham+fer-1 group(n=12),KA+vehicle group(n=12)and KA+fer-1 group(n=12).Rats were given intraperitoneal injection of fer-1 or vehicle for 14 days,depending on the group.The rats were given intraperitoneal injection of fer-1 or vehicle for 14 consecutive days according to different groups.45 days after model made,the expression of SYN and PSD95 in the hippocampus of rats in each group were detected by immunohistochemistry and western blotting.(2)Effects of SB203580,a specific inhibitor of P38 MAPK,on the cognitive dysfunction and the expression of p-p38,SYN and PSD95 in the hippocampus in rats with KA induced TLE.48 male SD rats were randomly divided into sham+Vehicle group(n=12),sham+SB203580 group(n=12),KA+Vehicle group(n=12)and KA+SB203580 group(n=12).Rats were given intraperitoneal injection of SB203580 or Vehicle for 14 days,depending on the group.At the end of fifth week after model made,Y maze,novel object recognition experiment and water maze experiment were used successively to evaluate the cognitive functions of rats in each group.45 days after model made,the expression of SYN and PSD95 in the hippocampus of rats in each group were detected by immune-histochemistry and western blotting.The expression of P38and p-p38 in the hippocampus of rats in each group were detected by western blot.Results:(1)Compared to the sham+vehicle group,the expression of SYN in the hippocampus decreased significantly in the KA+vehicle group(P<0.05).Compared to the KA+vehicle group,the expression of SYN in the hippocampus increased significantly in the KA+fer-1 group(P<0.01).There was no significant difference(P>0.05)in the expression of SYN in the hippocampus between sham+vehicle group and sham+fer-1 group.(2)Compared to the sham+vehicle group,the expression of PSD95 in the hippocampus decreased significantly in the KA+vehicle group(P<0.05).Compared to the KA+vehicle group,the expression of PSD95 in the hippocampus increased significantly in the KA+fer-1 group(P<0.05).There was no significant difference(P>0.05)in the levels of PSD95 in the hippocampus between sham+vehicle group and sham+fer-1 group.(3)The data of the Y-Maze test showed that the spontaneous alternation rate of rats in the KA+Vehicle group decreased significantly compared with the sham+vehicle group of rats(P<0.05).The spontaneous alternation rate was significantly improved in the KA+SB203580 group compared to the KA+vehicle group(P<0.05).There was no significant difference(P>0.05)in the spontaneous alternation rate between sham+Vehicle group and sham+SB203580 group.(4)The Novel object recognition test showed that the discrimination index of KA+Vehicle group decreased significantly compared with the sham+Vehicle group(P<0.05).The discrimination index was significantly improved in the KA+SB203580 group compared to KA+Vehicle group(P<0.05).There were not significantly different between the sham+Vehicle group and sham+SB203580 group.(5)The results of spatial navigation tests showed that the escape latency and the swimming distance decreased progressively in experimental rats of all groups during the five training days.From the second day onwards,the escape latency and the swimming distance of the KA+Vehicle group were higher than that of the sham+vehicle group(P<0.05).From the third day onward,the escape latency and the swimming distance were significantly(P<0.05)improved in the KA+SB203580 group compared to the KA+vehicle group.There was no significant difference(P>0.05)in the escape latency and swimming distance between sham+Vehicle group and sham+SB203580 group.There was no significant difference(P>0.05)in the average swimming velocity between all groups of rats.In the spatial probe test,the number of platform crossings of KA+Vehicle group was less than that of sham+Vehicle groups(P<0.01).The number of platform crossings was significantly increased in the KA+SB203580group compared to KA+Vehicle group(P<0.05).There was no significant difference(P>0.05)in the number of platform crossings between sham+Vehicle group and sham+SB203580 group.(6)Compared to the sham+vehicle group,the ratio of p-P38/P38 in the hippocampus increased significantly in the KA+Vehicle group(P<0.01).Compared to the KA+Vehicle group,the ratio of p-P38/P38 in the hippocampus reduced significantly in the KA+SB203580 group(P<0.01).There was no significant difference(P>0.05)in the ratio of p-P38/P38 in the hippocampus between sham+Vehicle group and sham+SB203580 group.(7)Compared to the sham+vehicle group,the expression of SYN in the hippocampus decreased significantly in the KA+Vehicle group(P<0.01).Compared to the KA+Vehicle group,the expression of SYN in the hippocampus increased significantly in the KA+SB203580 group(P<0.05).There was no significant difference(P>0.05)in the expression of SYN in the hippocampus between sham+Vehicle group and sham+SB203580 group.(8)Compared to the sham+vehicle group,the expression of PSD95 in the hippocampus decreased significantly in the KA+Vehicle group(P<0.01).Compared to the KA+Vehicle group,the expression of PSD95 in the hippocampus increased significantly in the KA+SB203580 group(P<0.05).There was no significant difference(P>0.05)in the levels of PSD95 in the hippocampus between sham+vehicle group and sham+SB203580 group.Conclusion:Fer-1 could up-regulate the expression of SYN and PSD95 in the hippocampus of rats with KA induced TLE by inhibiting p38 MAPK activation,thereby improve the cognitive function of rats with KA induced TLE.Conclusion of all textFerroptosis occurred in neurons in the hippocampal CA1 region of rats with KA induced TLE.Ferroptosis processes was involved in the mechanism of cognitive impairment of rats with KA induced TLE.Ferrostatin-1 could significantly improve the cognitive function of rats with KA induced TLE.The mechanism was through attenuating hippocampal neuronal loss by inhibiting ferroptosis processes as well as restoring the expression of synaptic proteins by suppressing P38 MAPK activation. |
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