| Research BackgroundEpilepsy is a chronic disease of the nervous system,which is characterize d by the corresponding sudden and transient brain dysfunction caused by recur rent abnormal discharges of brain neurons.Repeated seizures and the persistent state of epilepsy can damage the brain tissue,which often leaves some cogniti ve and motor dysfunction,which affects the physical and mental health of th e children.At present,the pathogenesis of epilepsy is not clear.The study sugg ests that it may be related to the neural-immune-endocrine network,which interact with each other.Inflammatory corpuscles have become a new research hotspot in central nervous system diseases in recent years.Inflammasome are composed of 14 subfamilies of NLRPl-NLRPl4.NLRP1、NLRP3 、 NLRC4 and AIM2 are the main inflammasome,At present,NLRP3 inflammasome which is composed of NOD like receptor,junction protein ASC and effector protein caspase-l are one of the most deeply studied inflammasome in the central nervous system.NLRP3 inflammasome was activated when the cells were stimulated by external signals,and activated NLRP3 further activated caspase-1.The activated caspase-1 cut pro-IL-1β into IL-1β.IL-1β is an important pro-inflammatory factor.It plays an important role in inducing cell activation and cytokine production.IL-1β can produce a series of amplifying effects,promote inflammatory reaction and cause brain injury.Melatonin is an indole hormone secreted by the pineal gland.Its secretion has the characteristic of circadian rhythm with photoperiod,It has scavenging free radical,antioxidation,anti-inflammation and immunomodulation.Melatonin has the effect of regulating sleep,and has been used in clinical practice abroad.In recent years,melatonin has been found to have a protective effect on brain damage caused by many diseases.For example: hypoxic ischemic encephalopathy,epilepsy,intracranial infection,stroke,bilirubin encephalopathy.In epilepsy models,melatonin can play a role in brain protection by influencing the release of neurotransmitters,the expression of receptors,and the expression of apoptosis factors.Whether melatonin,as a brain protective drug,can play a protective role in epilepsy through the effect of inflammatory factors has not been reported at home and abroad.Research purposesIn this study,through the establishment of status epilepticus model induced lithium-pilocarpine,using melatonin intervention,observation the expression changes of NLRP3、caspase-1、IL-1 protein and mRNA in the hippocampus of rats with time,the possible mechanismof melatonin brain protection,in order to provide a possible treatment for children with epilepsy.Research methods 1 Laboratory animals and groupingOne hundred and forty-four 21-30 day neonatal healthy,clear-class Sprague--Dawley rats(equivalent to human infancy),male or female,weighing 50g-100 g,they were randomly divided into control group,lithium-pilocarpine epilepsy group and melatonin treated group,n=48 in each group and by 24 h,48h,72 h,7day is divided into four subgroups,each subgroup 12(n=12)2 Model establishment and drug interventionThe model was established with reference.The rats in the melatonin group were given a single intraperitoneal injection of melatonin 25mg/kg 30 minutes after the onset of epilepsy,and the control group were injected with the same dose of 9g/L saline,The rats were observed immediately after drug administration.Continuous seizures of grade IV and above and lasting for more than 30 minutes were considered to be status epilepticus.Epileptic seizure symptoms in rats was assessed by Racine6 grade level(O-V)classification method.3 Specimen collection and preparationThe rats in each group were anesthetized by intraperitoneal injection of 100g/L chloride hydrate(3ml/kg)at 24 h,48h,72 h and 7d,respectively.Take supi ne position after anesthesia,The limbs were fixed slightly,thoracotomy expose d the heart after local disinfection,The puncture needle was punctured into the aorta,cut the right atrial appendae.Six rats in each group were perfused with 9g/L saline solution,The brain tissue was taken by craniotomy,and the hippoca mpus was immediately separated from the ice.The hippocampus was rapidly s tored in human liquid nitrogen and used for fluorescence quantitative PCR det ection.The remaining rats were fixed with 40g/L paraformaldehyde after 0.9%normal saline infusion,and the brain tissue was taken by craniotomy and pru ned.It was fixed in 40g/L paraformaldehyde for 48 hours,then was embedded in conventional dehydration section and coronal section.Immunohistochemistry staining was performed.4 immunohistochemicalParaffin sections were dewaxed and rehydrated,antigenic repair,dropping of hydrogen peroxide deionized water to inhibit endogenous peroxidase,normal goat serum working liquid seal tablets,drip first antibody,The second antibody and horseradish peroxidase labeled working solution of Streptomyces ovalbumin,Two amino diphenyl amine(DAB)chromogenic,hematoxylin restaining,dehydration seal tablet.In the cytoplasm,brown or brown cells were positive.5 Fluorescence quantitative PCRExtraction of total RNA in hippocampus of rats with Trizol reagent and reverse transcription into cDNA,cDNA was used as template for PCR amplification.SYBR Green I was used in the fluorescence quantitative PCR sample reaction system,and the final reaction system was 20 microliters.The reaction conditions are as follows: predenaturation at 95 ℃ for 30 s,denaturation at 95 ℃ for 5 s,annealing at 55 ℃ for 10 s,extension at 72 ℃ for 30 s,and a total of 40 cycles.6 Statistical analysisStatistical analysis was performed using SPSS 21.0.Measurement data are shown as mean ± SD.One-way analysis of variance(ANOVA)for multigroup data comparison,Comparison between two groups using LSD-t Test.The statistically significant level was P < 0.05 Research results 1 Immunohistochemical resultsThe number of NLRP3、caspase-1、IL-1β positive cells in epileptic group and melatonin group was higher than that in control group at each time point,The difference was significant(P < 0.05),and reached the peak at 72 h.The number of NLRP3、caspase-1、IL-1β positive cells in the melatonin group was lower than that in the epilepsy group at the same time point,The difference was significant(P < 0.05).2 Fluorescence quantitative PCR resultsThe relative expression of RNA of NLRP3、caspase-1 and IL-1β in epileptic group and melatonin group was higher than that in control group,The difference was significant(P < 0.05),and reached its peak at 72 h.The RNA relative expression level of NLRP3、caspase-1and IL-1 β in melatonin group was lower than that in epileptic group,The difference was significant(P < 0.05).ConclusionNLRP3 inflammasome is activated after seizure and play a certain role in epileptic brain injury.Melatonin may inhibit the expression of NLRP3 inflammasome through anti-inflammatory action and play a protective role in the brain. |
