| Temporal lobe epilepsy（TLE）is one of the most common epileptic syndromes.Nearly half of TLE patients have cognitive dysfunction,which seriously affects their quality of life and physical or mental health,but there is still a lack of effective treatment.Therefore,it is of great theoretical value and practical significance to develop new therapeutic targets and carry out research on the pathogenesis of cognitive dysfunction in TLE.In our previous studies,we found that the mechanism of cognitive impairment in TLE comorbidity is related to ferroptosis in neurons in the hippocampus and other brain regions.Therefore,neuroprotective therapy has become a research hotspot in the prevention and treatment of cognitive dysfunction in TLE in recent years.Ferroptosis is a kind of iron-dependent non-apoptotic programmed cell death discovered in recent years,and it is closely related to cognitive dysfunction of TLE.The key factors that trigger ferroptosis procedure are excessive accumulation of iron(Fe2+),glutathione（GSH）consumption or glutathione peroxidase 4（GPX-4）inactivation,lipid peroxide accumulation,and intracellular iron(Fe2+)overload is the key factor triggering ferroptosis.Studies have shown that membrane iron transporter,ferroportin（FPN）is the key protein to regulate the level of intracellular Fe2+and maintain intracellular iron homeostasis.The activation of nuclear factor erythroid 2-related factor 2（Nrf2）is involved in the process of inhibiting the damage of neurons and improving cognitive function,and also can regulate the expression of FPN.In recent years,it has been found that klotho protein has neuroprotective effect and anti-cognitive impairment effect in neurodegenerative diseases,and the down-regulation of klotho expression in hippocampus may be related to cognitive impairment of TLE comorbidity,but its specific effect and mechanism are not clear.In this study,we will first determine the expression and distribution of klotho in the hippocampus of TLE rats induced by lithium chloride-pilocarpine（LiCl-Pilo）.Secondly,we will explore the improvement of cognitive impairment by klotho through reducing the damage of hippocampal neurons induced by LiCl-Pilo in TLE rats.Finally,from the point of view that activating Nrf2 can inhibit iron death and up-regulate the level of Fe2+in hippocampal neurons regulated by FPN,the mechanism of klotho in improving TLE cognitive impairment is revealed,so as to find a new therapeutic target for TLE cognitive impairment.Part I:The expression and distribution of klotho in the hippocampus of TLE rats induced by LiCl-PiloObjective To determine the expression and cellular localization of klotho in the hippocampus of TLE rats induced by LiCl-Pilo,and to explore whether there was a correlation between the level of klotho and cognitive impairment of TLE.Method Adult male SD rats were randomly divided into Con group and LiCl-Pilo group.The LiCl-Pilo group was divided into four subgroups:3d group,2w group,4w group and 6w group.There were 20 rats in each group.The TLE rat model was induced by LiCl-Pilo,and the behavior of epileptic seizures was observed.After modeling,some rats of each group were deeply anesthetized according to different time points,and the klotho expression and cellular localization of klotho in hippocampal tissue of TLE rats were detected by immunoblotting and double immunofluorescence respectively.The cognitive function of rats in Con group and 6w group was evaluated by water maze test.Nissl staining and HE staining were used to evaluate the damage of hippocampal neurons in each group.Results（1）Behavioral results:No seizures occurred in the Con group.After inducing SE by LiCl-Pilo,all the rats in the 4 subgroups of SE group had Racine grade 4 or more,the SE duration was about within 30-40 minutes,and the mortality rate was 13.75%（11/80）.（2）The results of klotho expression detected by Western blotting:compared with the Con group,the klotho expression level of the model group at each time point began to decrease on the 3rd day after administration of LiCl-Pilo,but there was no significant difference in statistics.It continued to decrease from 2w to 6w,and reached the lowest level at 4w.（3）The results of double immunofluorescence detection:klotho was co-expressed with neuron specific marker Neun,but not with astrocyte marker GFAP in both Con group and LiCl-Pilo group.（4）Study on cognitive behavior of rats:the results of water maze test showed:compared with Con group,the average latency of finding latent platform in LiCl-Pilo group was longer than that in Con group,and the number of times crossing the platform and the proportion of swimming time in target quadrant decreased significantly in LiCl-Pilo group.There was no significant difference in escape latency and average speed in water maze visual platform test among all groups（P>0.05）.（5）Study on the injury of hippocampal neurons in rats:the results of HE staining showed that the cell count in CA1 area of LiCl-Pilo group was significantly lower than that of Con group,and the cell staining became lighter and blurred,and the difference was statistically significant（P<0.001）.The results of Nissl staining showed that compared with the Con group,the number and volume of Nissl positive neurons in the CA1 area of the LiCl-Pilo group were significantly lower than those of the LiCl-Pilo group（P<0.01）.Conclusion（1）Klotho was expressed in rat hippocampal neurons but not in astrocytes.（2）The down-regulation of Klotho in hippocampal tissue of TLE rats induced by LiCl-Pilo may be related to hippocampal neuronal damage and cognitive impairment in TLE rats.Part II:Effects of klotho on behavior and hippocampal neuronal damage in LTE rats induced by LiCl-PiloObjective To investigate the role of klotho in LiCl-Pilo-induced LTE rats.Method The experiment was divided into two sections.（1）Study on transfection efficiency of over-expressed adeno-associated virus klotho（AAV-KL）in hippocampus of rats:healthy adult male SD rats were randomly divided into AAV-KL group and AAV-NC group with only empty adeno-associated virus vector.There were 20 rats in each group.Immunofluorescence and Western blotting were used to detect the transfection and protein expression of klotho in the hippocampus of rats at 3 and 9 weeks after injection of AAV-KL.（2）Effects of klotho on susceptibility to seizures,cognitive function and hippocampal neuronal damage in LTE rats induced by LiCl-Pilo:the expression of endogenous klotho in hippocampus was up-regulated by stereotaxic injection of AAV-KL into hippocampus.Healthy adult male SD rats were randomly divided into Con group,Saline group,AAV-NC group and AAV-KL group,with 20 rats in each group.TLE rat model was induced by LiCl-Pilo.The latency of epilepsy and the spontaneous times of chronic epilepsy in each group were recorded（5 weeks after modeling）.Water maze test was used to evaluate the cognitive function of rats in each group from the 6th week after modeling.Nissl staining and HE staining were used to evaluate the damage of hippocampal neurons in each group.The pathological characteristics of ferroptosis of hippocampal neurons in each group were observed by transmission electron microscope.Western blotting was used to detect the expression of klotho and GPX-4 in hippocampus.GSH kit and ROS kit to detect the expression of GSH and ROS in hippocampus,and Iron Assay kit to detect the level of Fe2+in hippocampus.Results（1）AAV virus vector transfection:immunofluorescence staining showed that AAV virus vector was successfully transfected into hippocampal neurons at3 and 9 weeks after injection of AAV virus vector.（2）Klotho expression:at 3 and 9 weeks after injection of AAV virus vector into the hippocampus,the results of WB assay showed that the expression of klotho protein in the hippocampus of AAV-KL group was significantly higher than that of AAV-NC group,and there was significant difference between the two groups（P<0.001）.（3）Behavioral results:after SE was induced by LiCl-Pilo,the rats in Saline group,AAV-NC group and AAV-KL group all had seizures of Racine grade 4or above,the attack time was controlled at about 30 minutes,and the mortality was 13.33%（8/60）.There was no significant difference in the latency from the injection of LiCl-Pilo to the onset of grade 4 or more epileptic seizures in all groups.In the fifth week after injection of LiCl-Pilo,90%,80%and 90%of rats in Saline group,AAV-KL group and AAV-NC group had spontaneous seizures,respectively,but there was no significant difference in the number of spontaneous seizures among the three groups.（4）Study on cognitive behavior of rats:the results of water maze test showed:compared with Con group,the average latency of Saline group was significantly longer than that of AAV-NC group（P<0.001）,and the average latency of AAV-KL group was significantly shorter than that of AAV-KL group（P<0.05）.In the spatial search experiment,the times of crossing the platform in Saline group were significantly lower than those in Con group（P<0.001）,and the times of crossing platform in AAV-KL group were significantly higher than those in AAV-NC group（P<0.001）.In the spatial search experiment,the proportion of swimming time across the target quadrant in the Saline group was significantly lower than that in the Con group（P<0.001）,while that in the AAV-KL group was significantly higher than that in the AAV-KL group（P<0.001）.There was no significant difference in escape latency and average speed in visual platform test among all groups（P>0.05）.（5）Study on the injury of hippocampal neurons in rats:the results of HE staining showed that the cell count in CA1 area of Saline group was significantly lower than that of Con group（P<0.001）,and that of CA1 area of AAV-KL group was significantly higher than that of AAV-KL group（P<0.01）.The results of Nissl staining showed that the number of Nissl positive neurons in CA1 area in Saline group was significantly lower than that in Con group（P<0.001）,and the number of Nissl positive neurons in CA1 area in AAV-KL group was significantly higher than that in AAV-NC group（P<0.001）.（6）Study on the pathological characteristics of ferroptosis in rat hippocampal neurons:the results of transmission electron microscope showed that the mitochondrial ridge in hippocampal CA1 region of TLE group decreased and the adventitia ruptured.The results of transmission electron microscope showed that the area of mitochondria in CA1 region of Saline group was smaller than that of Con group,and the mitochondrial area of CA1 region of AAV-KL group was significantly higher than that of AAV-NC group.（7）The expression levels of klotho and GPX-4 in hippocampus of rats in each group were detected by Western blotting:compared with Con group,the expression levels of klotho and GPX-4 in hippocampus of Saline group were significantly down-regulated,and the expression levels of klotho and GPX-4 in hippocampus of AAV-KL group were significantly higher than those of AAV-NC group.（8）The expression levels of GSH and ROS were detected by GSH kit and ROS kit:compared with Con group,the expression of GSH in hippocampal neurons of Saline group was significantly down-regulated and ROS expression was significantly up-regulated,and compared with AAV-NC group,the expression of GSH and ROS in hippocampal neurons of AAV-KL group were significantly up-regulated and down-regulated.（9）The level of Fe2+in hippocampus of rats was detected by Iron Assay kit:compared with Con group,the level of Fe2+in hippocampal neurons in Saline group was significantly higher than that in AAV-NC group,and the level of Fe2+in hippocampal neurons in AAV-KL group was significantly lower than that in AAV-KL group.Conclusion（1）Klotho did not affect seizure susceptibility and did not attenuate spontaneous seizures in LiCl-Pilo induced TLE rat models.（2）The improvement of cognitive impairment induced by LiCl-Pilo in TLE rats by Klotho is related to the reduction of hippocampal neuronal damage.（3）Klotho can inhibit the ferroptosis of hippocampal neurons and reduce the damage of hippocampal neurons by reducing the iron deposition and ROS levels in the hippocampus and up-regulating the levels of GSH and GPX-4 in the hippocampus of TLE rats induced by LiCl-Pilo.Part III:The molecular mechanisms of klotho improves cognitive impairment in TLE rats induced by LiCl-PiloObjective To explore the molecular mechanism of klotho improves the cognitive impairment in TLE rats induced by LiCl-Pilo.Method The expression of endogenous klotho in hippocampus was up-regulated by stereotaxic injection of AAV-KL into hippocampus.Healthy adult male SD rats were randomly divided into Con group,AAV-NC group,AAV-KL group and AAV-KL+ML385 group,with 20 rats in each group.The rats in AAV-KL+ML385 group were given intraperitoneal injection of Nrf2 specific inhibitor（ML385,30mg/kg）for 7 days,and TLE rat model was induced by LiCl-Pilo.From the 6th week after modeling,the cognitive function of rats in each group was evaluated by water maze test,the damage of hippocampal neurons was evaluated by Nissl staining and HE staining,the pathological characteristics of iron death of hippocampal neurons in each group were observed by transmission electron microscope,the expression levels of Nrf2,FPN and GPX-4 in hippocampus were detected by Western blotting,and the expression levels of GSH and ROS in hippocampus were detected by GSH kit and ROS kit.The level of Fe2+in hippocampus of rats was detected by Iron Assay kit.Results（1）Behavioral results:the rats in AAV-NC group,AAV-KL group and AAV-KL+ML385 group all had seizures of Racine grade 4 or above after SE was induced by LiCl-Pilo.The attack time was controlled at 30 minutes,and the mortality was 15.00%（9/60）.（2）Study on cognitive behavior of rats:the results of water maze test showed that the average latency of AAV-NC group was significantly longer than that of Con group（P<0.001）,and that of AAV-KL group was significantly shorter than that of AAV-NC group（P<0.001）,and that of AAV-KL+ML385group was significantly longer than that of AAV-KL+ML385 group（P<0.001）.Compared with Con group,the times of crossing platform and the proportion of swimming time in target quadrant were significantly decreased in AAV-NC group（P<0.001）,and the proportion of swimming time in target quadrant in AAV-KL group was significantly higher than that in AAV-NC group（P<0.01,P<0.001）.Compared with the AAV-KL group,the times of crossing the platform and the proportion of swimming time in the target quadrant were significantly reduced in the AAV-KL+ML385 group.There was no significant difference in escape latency and average speed in visual platform test among all groups（P>0.05）.（3）Study on the injury of hippocampal neurons in rats:the results of HE staining showed that the cell count in CA1 region of hippocampus in AAV-NC group was significantly lower than that in Con group（P<0.001）,and the cell count in CA1 region in AAV-KL group was significantly higher than that in AAV-KL group（P<0.001）.The cell count in hippocampal CA1 region of AAV-KL+ML385 group was significantly lower than that of AAV-KL group（P<0.05）.The results of Nissl staining showed that the number of Nissl positive neurons in hippocampal CA1 region in AAV-NC group was significantly lower than that in AAV-NC group（P<0.001）,and the number of Nissl positive neurons in AAV-KL group was significantly higher than that in AAV-NC group（P<0.001）.Compared with AAV-KL group,the number of Nissl positive neurons in hippocampal CA1 region of AAV-KL+ML385 group was significantly decreased（P<0.05）.（4）Study on the pathological characteristics of ferroptosis in rat hippocampal neurons:the results of transmission electron microscope showed that the mitochondrial area of hippocampal CA1 region in AAV-NC group was significantly lower than that in Con group（P<0.001）,and that in AAV-KL group was significantly higher than that in AAV-NC group（P<0.001）.Compared with AAV-KL group,the area of mitochondria in hippocampal CA1region of AAV-KL+ML385 group decreased significantly（P<0.05）.（5）The expression of Nrf2,FPN and GPX-4 was detected by Western blotting:compared with the Con group,the protein expression of Nrf2,FPN and GPX-4N in the hippocampus of the AAV-NC group was significantly lower than that of the AAV-NC group,and the protein expression of Nrf2,FPN and GPX-4 in the hippocampus of the AAV-KL group was significantly higher than that of the AAV-NC group.Compared with AAV-KL group,the protein expression of Nrf2,FPN and GPX-4 in hippocampal tissue of AAV-KL+ML385group was significantly down-regulated（P<0.05）.（6）The expression levels of GSH and ROS were detected by GSH kit and ROS kit:compared with Con group,the expression of GSH in hippocampal neurons of AAV-NC group was significantly down-regulated and ROS expression was significantly up-regulated（P<0.001）,and the expression of GSH and ROS in hippocampal neurons of AAV-KL group were significantly higher than those of AAV-NC group（P<0.001）.Compared with AAV-KL group,the expression of GSH in hippocampal neurons of AAV-KL+ML385group was significantly down-regulated and ROS expression was significantly up-regulated.（7）The level of Fe2+in hippocampus of rats was detected by Iron Assay kit:the level of Fe2+in hippocampal neurons of AAV-NC group was significantly higher than that of Con group（P<0.001）,and the level of Fe2+of hippocampal neurons of AAV-KL group was lower than that of AAV-NC group（P<0.001）.The level of Fe2+in hippocampal neurons in AAV-KL+ML385 group was significantly higher than that in AAV-KL group（P<0.05）.Conclusion（1）Klotho inhibits ferroptosis of hippocampal neurons and alleviates hippocampal neuronal damage by activating Nrf2 to down-regulate ROS levels and up-regulate GSH and GPX-4 levels in hippocampus.（2）Klotho inhibits ferroptosis of hippocampal neurons and reduces the injury of hippocampal neurons by activating Nrf2 to up-regulate FPN expression and down-regulate Fe2+level.Conclusion of all textKlotho can significantly improve the cognitive impairment induced by LiCl-Pilo in TLE rats by reducing hippocampal neuronal injury.The mechanism is related to the inhibition of ferroptosis of hippocampal neurons by activating Nrf2 to down-regulate the level of ROS and up-regulating the levels of GSH and GPX-4 in the hippocampus,and down-regulating the level of Fe2+by activating Nrf2 to up-regulate the expression of FPN. |
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