| Epilepsy is a group of heterogeneous chronic neurological diseases characterized by diverse types of epileptic seizures.There is a lot of social discrimination because patients have abnormal excitatory or synchronized neuronal activity in the brain,which leads to seizures.According to the data released by the World Health Organization(WHO)in 2001,neurological diseases account for 30.8% of the total number of years of disability,while epilepsy accounts for 0.5% of the total disease burden,with a global prevalence of 1.5 to 14 per 1000 people.Epilepsy occurs in all age groups,but the most common is children.Ideal and reasonable treatment is of great significance for reducing brain damage caused by epilepsy and reducing the global burden of disease.About 30% of patients with epilepsy are intractable epilepsy,which is difficult to control with drugs.Epilepsy surgery is currently the most effective treatment for long-term seizure relief in patients with partially drug-resistant focal epilepsy.However,due to the high risk of surgery,the consequences are difficult to reverse and other reasons,it is not widely used.Currently,based on a large number of gene studies,molecular targeted drug therapy for epilepsy has attracted a lot of attention in the academic world.Therefore,it is of great significance to explore the underlying molecular pathological mechanism of epilepsy for the development of new and more effective therapeutic targets.Mi RNA play a regulatory role on target m RNA by destroying the stability of target m RNA and inhibiting the translation of target m RNA.At present,a large number of studies have found that the abnormal expression of micro RNAs is closely related to neurological damage and degenerative diseases,including epilepsy.It has been reported that within 30 minutes after seizure,more than 200 miRNAs show different expressions in the serum of patients.Studies have reported that a large number of miRNAs show abnormal expression levels in the hippocampus of patients with temporal lobe epilepsy and epileptic animals,and miRNAs play a variety of regulatory roles in the pathological process of temporal lobe epilepsy.mmu-miR-330-5p(miR-330-5p)was reported to be decreased in pilocarpine induced mouse epilepsy model,suggesting that miR-330-5p may be involved in the pathological mechanism of epilepsy.Therefore,in-depth study of the role of miR-330-5p in epilepsy has a certain theoretical basis and is conducive to the development of new therapeutic targets.In recent years,the analysis of brain tissue of patients with refractory epilepsy indicates that inflammatory process and neuroinflammation play an important role in the pathogenesis of epilepsy.Toll-like Receptors(TLRs)activation plays an important role in these processes.A large number of studies have shown that TLR4 expression is elevated in epileptic patients and animals and is involved in the pathogenesis of epilepsy.Notably,through bioinformatics analysis,miR-330-5p can target binding to the 3 ’non-coding region of TLR4.Therefore,we hypothesized that the dysregulation of miR-330-5P expression may play a regulatory role in the development of epilepsy through targeted regulation of TLR4.The therapeutic effect of ketogenic diet on epilepsy has been reported and confirmed by a large number of literatures.In addition,ketogenic diet has been reported to regulate miRNA expression.In view of this,this study established an epileptic mouse model to explore the effects of miR-330-5p /TLR4 axis on hippocampal tissue damage,nerve cell apoptosis and neuroinflammation in epileptic mice and related molecular mechanisms.In addition,whether ketogenic diet regulates epileptic mice through miR-330-5p /TLR4 pathway was further explored.Methods: 1.In this study,epileptic seizures were induced by intraperitoneal injection of pilocarpine in C57BL/6 male mice.Racine’s standard scoring system was used to score epileptic mice for behavior.Electroencephalogram(EEG)detection was performed 1 hour after pilocarpine injection,and the amplitude and peak frequency were quantitatively analyzed.2.The Target Scan website predicted the targeted binding sites of miR-330-5p and TLR4 in 293 T cells,and the double luciferase assay was used to verify the targeted binding of miR-330-5p and TLR4 in 293 T cells.3.Cell experiments verified the targeting relationship between miR-330-5p and TLR4: 293 T cells were transfected with mimics NC,miR-330-5p mimics,inhibitor NC,miR-330-5p inhibitor.After 48 h of transfection,q PCR was used to detect the levels of miR-330-5p in mimics NC and miR-330-5p mimics groups to verify whether the transfection was successful.TLR4 protein levels in the above groups were detected by Western blot.4.HE staining was used to observe the morphology of hippocampal CA1 region of mice in each group.The results showed that neurons in the hippocampal CA1 region of mice in the control group were clearly arranged with normal morphology and no neuron damage was observed,while neurons in the hippocampal CA1 region of mice in the epilepsy group were obviously lost.Compared with the model group,hippocampal CA1 region neuron damage was significantly reduced in mice injected with miRNA-330-5p mimics.However,the injection of miR-330-5p NC had no significant effect on neuron injury in hippocampal CA1 region.5.Morphological changes of hippocampal tissues(CA1 regions)were detected by HE staining.6.To verify the expression levels and targeting relationship of miR-330-5p and TLR4 in mice of each group: The expression levels of miR-330-5p and TLR4 in hippocampus of each group were detected by q PCR;TLR4 levels were detected by Western blot.TLR4 expression in hippocampus was detected by immunohistochemistry.7.To verify the effect of miR-330-5p overexpression on inflammatory response in epileptic mice: The levels of IL-1β,IL-6 and IL-18 in blood of mice in each group were detected by ELISA kit.8.To verify the effect of miR-330-5p overexpression on neuronal apoptosis in epileptic mice: Western blot was used to detect the protein levels of cleaved caspase-3,Bcl-2,Bax and p53 in hippocampus of each group.9.To verify the effect of overexpression of miR-330-5p on NF-κB pathway activation in epileptic mice: The expression levels of IκBα(NF-κB inhibitory protein A),P-IκBα(phosphorylated IκBα),NF-κB P65 in cytoplasm and nucleus of each group were detected by Western blot.10.Ketogenic diet feeding and modeling: C57BL/6 mice were randomly divided into four groups: Control + Control diet(CD),epilepsy +CD,Control + Ketogenic diet(KD)and epilepsy + KD.Epileptic mice were induced epilepsy by the above method.For the first 6 weeks after pilocarpine injection,all mice were fed with CD,and from 7 weeks onwards,they were fed with either CD or KD in groups.After 21 weeks of pilocarpine injection,all mice were killed by pentobarbital overdose.Then the hippocampal tissues of each group were takenfor testing;11.To verify the effects of ketogenic diet on miR-330-5p /TLR4 axis in epileptic mice: Real-time PCR was used to detect the expression levels of miR-330-5p and TLR4 in hippocampus of each group;TLR4 protein levels in hippocampus were detected by Western blot.Results: 1.Racine score and EEG results showed that epileptic mice were successfully modeled.2.Double luciferase assay verified the targeting relationship between miR-330-5p and TLR4,and there were specific binding sites between the two.Meanwhile,q PCR and Western blot experiments showed that TLR4 level in miR-330-5p mimics group was lower than that in control group,while TLR4 level in miR-330-5p inhibitor group was increased.The targeting relationship between miR-330-5p and TLR4 was verified,that is,overexpression of miR-330-5p can reduce the expression of TLR4.3.TLR4 and miR-330-5p in hippocampal tissues of epileptic group and control group were detected by q PCR and Western Blot,and the levels of TLR4 in epileptic group were higher than those in control group.But the levels of miR-330-5p in epileptic group were lower than those in control group 4.HE staining was used to observe the morphology of hippocampal CA1 region of mice in each group.The results showed that neurons in the hippocampal CA1 region of mice in the control group were clearly arranged with normal morphology and no neuron damage was observed,while neurons in the hippocampal CA1 region of mice in the epilepsy group were obviously lost.Compared with the model group,neurons in the hippocampal CA1 region of mice injected with miRNA-330-5p mimics were significantly less damaged.However,the injection of miR-330-5p NC had no significant effect on neuron injury in hippocampal CA1 region.5.The results of q PCR、Western Blot and immunohistochemistry showed that the level of TLR4 in the epilepsy group was higher than that in the control group,while the level of TLR4 in the epilepsy + miR-330-5p mimics group was lower than that in the mimics NC group.It indicates that overexpression of miR-330-5p can reduce the expression of TLR4.6.ELISA showed that the expression levels of IL-1β,IL-6 and IL-18 were increased in the epilepsy group compared with the control group,while the expression levels of IL-1β,IL-6 and IL-18 were decreased in the epilepsy + miR-330-5p mimics group compared with the mimics NC group.These results suggest that overexpression of miR-330-5p may reduce the inflammatory response after epilepsy by decreasing the expression levels of IL-1β,IL-6 and IL-18.7.Western blot showed that the levels of cleaved caspase-3,BAX and p53 protein in hippocampal tissue of epileptic group were higher than those in the control group,while the levels of Bcl-2 were lower than those in the control group.Meanwhile,cleaved caspase-3,Bax and P53 protein levels were decreased in the epilepsy + miR-330-5p group compared with the mimics NC group,while Bcl-2 protein levels were increased compared with the NC group.These changes in apoptosis-related proteins suggest that overexpression of miR-330-5p can reduce Bcl-2 in the hippocampal region of epileptic mice.8.Compared with the control group,the expression of P-IκBα(phosphorylated IκBα)and NF-κB P65 in hippocampal tissues of epileptic group were increased,while the expression of IκBα(inhibitory protein A of NF-κB)was decreased.At the same time,the expression of NF-κB p65 and NF-κB was decreased in the epilepsy + miR-330-5p mimics group compared with the mimics NC group,while the expression of IκBα(NF-κB inhibitory protein A)was increased compared with the NC group.This suggests that overexpression of miR-330-5p inhibits the activation of NF-κB pathway in epileptic mice.9.Finally,western blot and q PCR experiments showed that TLR4 level and miR-330-5p level in Model+CD group were significantly increased compared with Control+CD group,and TLR4 level in Model+KD group was significantly decreased compared with Model +CD group.However,Through q PCR experiments,we found that miR-330-5p level in Model+KD group was slightly higher than that in Model +CD group,but the difference was not statistically significant.This suggests that ketogenic diet may reduce the level of TLR4 in epileptic mice,but this study did not prove that ketogenic diet can significantly increase the level of miR-330-5p.Conclusion: 1.There are specific binding sites between miR-330-5p and TLR4,and overexpression of miR-330-5p can reduce the expression of TLR4.2.Overexpression of miR-330-5p can down-regulate the expression of TLR4 in the hippocampus of epileptic mice,and reduce the levels of inflammatory cytokines IL-1β,IL-6 and IL-18.Overexpression of miR-330-5p can inhibit the activation of NF-κB signaling pathway,thus reducing the neuroinflammatory response of epileptic mice.3.Overexpression of miR-330-5p can alleviate nerve cell apoptosis and neuronal damage in hippocampus of epileptic mice.4.This study found that ketogenic diet could reduce the level of TLR4 in the hippocampus of epileptic mice,but ketogenic diet had no significant effect on the expression of miR-330-5p in the hippocampus of epileptic mice. |
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