| Objective Epilepsy(EP)is one of the most common diseases of the nervous system.Although most antidepressant drugs(ADEs)can alleviate the disease,about 30% of patients still show no response to ADEs and become intractable epilepsy(IE),of which temporal lobe epilepsy(TLE)is the most common type in adults.The pathogenesis of epilepsy is unclear,but it may be related to abnormal changes of ion channels,neurotransmitters and glial cells.Therefore,to find effective targets for epilepsy therapy has always been the research focus in epilepsy field.ATP,as a key upstream trigger factor of neuroinflammation,presents a dramatic increase in its concentration under pathological conditions,such as cerebral hypoxia,cerebral ischemia,brain trauma or seizures.High concentrations of ATP can activate P2 X receptors to participate in the generation and conduction of abnormal nerve signals,the regulation of synaptic plasticity and the secretion of neurotransmitters.These abnormal physiological activities are closely related with the onset of epilepsy.It has been reported that all P2 X receptor subtypes show changed expression in epileptic seizures,which suggests that they may play important roles on the onset and progression of epilepsy.In recent years,study on the diagnosis and treatment of epilepsy has gradually shifted from the level of protein to nucleic acid.Revealing the mechanisms governing inflammatory response from the molecular level of nucleic acid will help to achieve the purpose of targeted therapy.As a kind of non-coding RNA,micro RNAs play an important role in regulating the occurrence and development of epilepsy.Micro RNAs are reported to participate in the regulation of neural cell apoptosis,synaptic remodeling,glial fibroblast proliferation,inflammatory response,etc,which have been widely used in tumors and other fields.In this study,we injected the mi RNA-22 analog mi RNA-22 agomir into the brain of epileptic rats to achieve the over-expression of mi RNA-22 in the epileptogenic zone(EZ).This study was designed according to the current international research focus to further explore novel therapeutic strategies for intractable epilepsy from the perspective of mi RNAs and find the optimal concentration of mi RNA-22 agomir using single-injection for temporal lobe epilepsy.Method Lithium chloride-pilocarpine-induced rat epilepsy model is used in this study.And the study can be divided into two parts: One,3 days after successful modeling,twenty acute temporal epileptic rats were selected and the same number of randomly selected normal rats was used as a control group.The expression of P2X7,NF-?B and IL-1? was detected by Western-blot and q RT-PCR.Immunofluorescence was used to observe the distribution of P2X7 in epileptic foci and the proliferation of astrocytes.In addition,Nissl staining was used to observe the damage of hippocampal neurons.After that,the temporal lobe epilepsy rats in acute phase 3 days after modeling were selected and randomly divided into two groups,which were given equal amounts of mi RNA-22 agomir and mi RNA-22 agomir control respectively via the lateral ventricle.The expression of mi RNA-22,P2X7,NF-?B and IL-1? was detected by Western-blot and q RT-PCR.Immunofluorescence was used to observe the distribution of P2X7 in epileptic foci and the proliferation of astrocytes.Similarly,Nissl staining was used to observe the damage of hippocampal neurons.The purpose of this part is to investigate whether mi RNA-22 exerts its potential therapeutic value by down-regulating P2X7 in rats with acute temporal lobe epilepsy.Two,mi RNA-22 agomir with increasing doses(0.1 m M,2.5 m M,5 m M,10 m M,and 20 m M)was injected intracerebroventricularly and the brains were decapitated 3 days after the injection.The expression of P2X7 was detected by Western-blot and q RT-PCR.The optimal concentration of mi RNA-22 agomir used to treat temporal lobe epilepsy with unilateral lateral ventricle injection was investigated.1.Typical status epilepticus was induced by intraperitoneal injection of lithium-pilocarpine using SD rats.The successful modeling rats were characterized by initial salivation,congested eyes,and followed by stereotyped action,facial twitching,and repeated nodding until the eventual full-body rigidity-clonic epileptic seizure.The first part: the success rate of epilepsy model was 88%(A total of 75 rats,of which 69 acute temporal lobe epilepsy were successfully established,followed by 3 deaths,and a total of 66 survived).Sixty epilepsy rats were randomly selected and divided into 3 groups of 20 per group,one of which was an epilepsy group for comparison with normal rats.The other two groups were given equal doses of mi RNA-22 agomir and mi RNA-22 agomir control respectively.The second part: the success rate of epilepsy model was 89%(A total of 75 rats,of which 67 acute temporal lobe epilepsy were successfully established,followed by 4 deaths,and a total of 63 survived).60 epileptic rats were randomly selected and divided into 6 groups for exploring the optimal concentration of mi RNA-22 agomir used for the treatment of temporal lobe epilepsy.2.Western-blot results show:The first part: Compared with the normal group,the P2X7 protein exhibited increased expression in the epilepsy group.The expression of P2X7 in mi RNA-22 agomir injected group was significantly lower than that of mi RNA-22 agomir control group(P<0.05).The second part: The expression level of P2X7 protein in mi RNA-22 agomir injected groups decreased to varying degrees compared with the control group,and the difference was statistically significant(P<0.05).Rats injected with 2.5 m M,5 m M and 10 m M concentrations were observed in each experimental subgroup.The expression of P2X7 protein in hippocampus of the three subgroups was significantly decreased.P2X7 protein exhibited lowest expression of in 2.5 m M and 10 m M mi RNA-22 agomir injected groups,but no significant difference was observed between the two subgroups(P > 0.05).3.q RT-PCR results indicate:The first part:Compared with the normal group,the levels of mi RNA-22,P2X7 m RNA,NF-?b m RNA,and IL-1? m RNA in the epileptic group were increased,and the difference was statistically significant(P<0.05).After injecting mi RNA-22 agomir into the lateral ventricle,mi RNA-22 exhibited increased expression,which resulted in the decreased expression of P2X7,NF-?b and IL-1? m RNA compared with the mi RNA-22 agomir control group.The differences were significant between the two groups(P<0.05).The second part: The expression of P2X7 m RNA in each mi RNA-22 agomir injected group was significantly decreased compared with the control group,and the difference was statistically significant(P<0.05).The expression of P2X7 m RNA in the hippocampus of 2.5m M,5m M,and 10 m M mi RNA-22 agomir injected groups was significantly reduced with lowest expression in 2.5 m M and 10 m M mi RNA-22 agomir injected subgroups,and there was no significant difference between the two subgroups(P > 0.05).4.Nissl staining results suggest: A large number of abnormal neurons were observed in the CA3 region of the hippocampus in the epileptic group.The size of the neurons was smaller,the morphology was irregular,the chromatin exhibited pyknosis,the boundary between the nucleus and the cytoplasm was blurred,and the Nissl body was disintegrated.The differences were statistically significant between the normal and epileptic groups(P<0.05).The abnormality of neuron morphology and the disintegration of Nissl's bodies were significantly improved by mi RNA-22 agomir injection into the lateral ventricle compared with mi RNA-22 agomir control injection.5.Immunofluorescence results indicate:P2X7 displayed increased expression and a large number of proliferating astrocytes were observed in the hippocampus of epilepsy rats,which was significantly different from that of normal rats(P<0.05).Mi RNA-22 agomir injection decreased P2X7 expression and attenuated the astroglial proliferation and activation compared with mi RNA-22 agomir control injection,and the difference was statistically significant(P<0.05).Conclusion 1.The level of P2X7 in epileptic rats is higher than that in normal rats,indicating its potential value in epilepsy;at the same time,seizures lead to the activation and proliferation of astrocytes in rat hippocampal tissue.Expression of inflammatory factors such as NF-?b and IL-1? was up-regulated.2.After injecting mi RNA-22 agomir into the lateral ventricle,it was observed that the expression of P2X7 was down-regulated in epileptic rats,and the activation and proliferation of astrocytes were attenuated,and the expression of inflammatory factors such as NF-?b and IL-1? was down-regulated,indicating that mi RNA-22 agomir can ameliorate seizures by down-regulating its downstream target proteins and reducing the expression of inflammatory factors.3.Mi RNA-22 agomir had an optimal concentration to exert its anti-epileptic role by a single injection into the epileptic rat hippocampus through the lateral ventricle.4.Mi RNA-22 can be used as an intervention target to provide a new direction for the treatment of epilepsy in the future. |
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