| Objective: vps13 d was knockdown to observe the behavioral changes of pilocarpine acute epilepsy rat model,to detect the effects of vps13 d gene knockdown on the expression of DRP1,LC3 II and P62,and to explore whether VPS13 D participates in rat epilepsy and its related mechanisms by regulating mitochondrial fission and mitophagy.Methods: Healthy adult male SD rats were selected and vps13 d gene knockdown virus was built.vps13 d gene knockdown virus was injected into the hippocampus of rats with brain stereotaxic apparatus to construct vps13 d gene knockdown rat model by brain stereotactic locator.The rat model of DRP1 inhibitor was established by intraperitoneal injection of mdivi-1.After intervention,the rats were injected with lithium chloride and pilocarpine to establish the acute epilepsy model.The experimental rats were randomly divided into six groups: control group(n=8),epileptic model group(n=8),vps13d-kd group(n=8),empty virus group(n=8),mdivi-1 group(n=8)and DMSO group(n=8).During the experiment,the latency period of the first seizure and the number of seizures per unit time(Racine score level 4 and above)were observed to evaluate the severity of seizures in the rats of each group by statistical analysis.Morphological changes of rat hippocampal cells were observed by HE staining.Ultrastructural changes of hippocampal mitochondria were observed by transmission electron microscopy.Immunohistochemistry and immunofluorescence were used to detect the cellular localization and the expression of VPS13 D in the hippocampus of control group and epileptic model group.Immunohistochemistry and Western Blot were used to detect the effects of vps13 d gene knockdown on the expression of mitochondrial power-related protein DRP1,autophagy marker protein LC3 II and autophagy adaptor-protein P62.Results:1.HE staining was used to observe the morphological changes of nerve cells in hippocampal tissues of rats in the control group and epileptic model group.The result showed that nerve cells in the hippocampal CA1,CA3 and DG regions of the control group were even distributed,regular in shape and arranged in order.while those in the epileptic model group were uneven distributed,irregular morphology,disordered arrangement and swelling.2.Immunohistochemistry and immunofluorescence were used to detect the localization and expression of VPS13 D in brain tissues of the control group and the epileptic model group.2.1 Immunohistochemical results: VPS13 D was main expressed in the cytoplasm of rats hippocampal neuronal cells,and Semi-quantitative analysis results showed that the optical density of VPS13 D positive cells decreased in the epilepsy model group,with statistically significant difference(P<0.05).2.2 Immunofluorescence results: VPS13 D was expressed in hippocampal CA1,CA3 and cortical areas of rats,VPS13 D was co-expressed with the neuronal dendritic marker MAP2,and was not expressed with the glial cell marker GFAP.The results of semi-quantitative analysis showed that the absolute value of fluorescence signal intensity of VPS13 D positive cells in the epilepsy model group was reduced,and the difference was statistical significant(P<0.05).3.Behavioral results: there was no seizure in the control group,and the first seizure latency was statistical analyzed in the other groups.The results showed that the latency of epileptic seizure in the vps13 d gene knockdown group was significant shortened,while the latency of epileptic seizure in the Mdivi-1 group was significantly prolonged(P<0.05).The number of seizures per unit time in vps13 d gene knockdown group increased significantly,and the number of seizures per unit time in mdivi-1 group decreased obviously(P < 0.05).4.Changes of mitochondrial ultrastructure: In the control group,the mitochondrial structure in the CA1 area of the hippocampus was intact without mitochondrial membrane or crest damage,and no mitochondrial morphological abnormalities were found.In the epileptic model group,mitochondria were damaged in different degrees,including irregular mitochondrial morphology,fuzzy edge,damaged mitochondrial cristae,obvious edema around mitochondria,mitochondrial fission and formation of mitochondrial autophagy bodies.5.Western blot analysis of the effect of vps13d-kd intervention on drp1 expression.The results indicated that DRP1 expression was lower in the hippocampus of normal control rats,slightly increased in the epileptic model group,and significantly increased in the vps13d-kd group(P < 0.05).6.Localization and quantitative expression of autophagy marker protein LC3 II and autophagy adaptor protein P62 in hippocampal tissues of rats in each group.6.1 Immunohistochemical examination showed that autophagy marker protein LC3 II and autophagy adaptor protein P62 were most located in the cytoplasm of rats hippocampal nerve cells.6.2 Western Blot results showed that the expression of LC3 II protein was lower in the control group and slightly increased in the epilepsy model group(P<0.05).LC3 II expression was significantly decreased in vps13 d gene knockdown group(P<0.05).The expression of LC3 II was slightly decreased in the Mdivi-1 group,compared with the epilepsy model group(P < 0.05),but there was no statistical significance compared with the control group(P>0.05).The expression of P62 protein was lower in the hippocampus of the control group,but increased moderately in the epilepsy model group(P<0.05).Compared with the epilepsy model group,the expression of P62 protein was significantly increased in vps13 d gene knockdown group(P < 0.05),and the expression level was slightly increased in the Mdivi-1 group(P < 0.05),while the difference was not statistical significantly compared with the control group(P>0.05).Conclusion: VPS13 D may be involved in the regulation of epileptic seizures in rats,and its mechanism may be related to the promotion of excessive mitochondrial fission and the excessive inhibition of mitophagy. |
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