RvD1-FPR2 Mediated Inflammatory Regression Disorder Participates In The Mechanism Of MCD Induced Epilepsy

BackgroundCortical malformations（MCD）is the main cause of refractory epilepsy.MCD is a group of severe brain malformations associated with intractable epilepsy,intellectual disability,cognitive impairment,and autism.Focal cortical dysplasia（FCDIIb）and tuberous sclerosis（TSC）are two classic MCD.They have the similar pathological characteristic,such as:dysmorphic neurons（DNs）,giant cell（GCs）and the balloon sample cell（BCs）.In MCD,there are not only electrophysiological abnormalities caused by abnormal cell and tissue structure,but also persistent chronic inflammatory responses and inflammatory mechanisms.In patients with FCDIIb and TSC,increased neuronal excitability and inflammatory activation ultimately lead to epilepsy.However,the detailed molecular mechanism of epilepsy in patients with FCDIIb and TSC remains unclear.Inflammation resolution is an active programmed process involving multiple cells and mediators.Among them,the synthesis of specialized proresolving mediators（SPMs）is the key link of inflammation resolution,such as the docosahexanoic acid（DHA）-derived resolvin D（RvD）series,neuroprotectin D（NPD1）,and maresins in peripheral tissues and the CNS.Resolvin D1（RvD1）is a specialized SPM that is synthesized from DHA by sequential oxygenation mediated by the enzymes 5-lipoxygenase（5-LOX）and 15-LOX and plays anti-inflammatory roles in vitro and in vivo.RvD1 affects the NF-κB pathway by activating ERK,MAPK,PI3K and other downstream signaling pathways through formyl peptide receptor 2（FPR2）,reducing the release of interleukin,tumor necrosis factor and other inflammatory factors,and promoting the inflammatory repair of M2 macrophages to mediate the resolution of inflammation.MethodTo investigate the role of RvD1 in patients with FCDIIb and TSC epilepsy,we detected the expression of RvD1 via ELISA,analyzed the correlation between the expression of RvD1and clinical variables,and explored the expression of key upstream enzymes 15-LOX and 5-LOX via western blots.In addition,we also investigated the expression and distribution of the RvD1 specific receptor FPR2 receptor using western blots and immunostainin,and the downstream NF-κB signaling pathway mediated by FPR2.In order to detect the effect of RvD1-FPR2 on neuronal excitability,The calcium imaging was used to measure intracellular calcium concentrations in FPR2-affected rat cortical neurons,and its effect on the expression of excitatory receptors（NR₂A and NR₂B）.Results1.Expression of RvD1 and its synthases 5-LOX and 15-LOX in surgical specimens of FCDIIb and TSC1.1 The expression levels of 15-LOX and 5-LOX in brain tissue samples resected from patients with FCDIIb and TSC.The RNA and protein expression levels of 15-LOX and 5-LOX in the epileptic lesions of patients with FCDIIb and TSC were significantly decreased compared with the control group.1.2 The expression level of RvD1 in epileptogenic foci resected from patients with FCDIIb and TSC.ELISA showed a significant decrease in RvD1 levels in epileptogenic foci from patients with FCDIIb and TSC compared with the control group.Associations between RvD1expression levels and different clinical variables（age at surgery,frequency of seizures,duration of seizures）were assessed in FCDIIb and TSC.RvD1 expression was negatively correlated with seizure frequency.2.Expression and distribution of FPR2 in surgical specimens of FCDIIb and TSC patients2.1 The expression levels of FPR2 in epileptogenic foci resected from FCDIIb and TSC patients.FPR2 m RNA and protein expression levels of 15-LOX and 5-LOX in the epileptogenic foci of FCDIIb and TSC patients were significantly decreased compared with the control group,and FPR2 protein expression level was significantly negatively correlated with seizure frequency.2.2 Immunohistochemistry（IHC）and immunofluorescence（IF）were used to detect the distribution of FPR2 in epileptogenic foci from FCDIIb and TSC patients.FPR2 was expressed in both dysmorphic neurons（DNs）and balloon-like cells（BCs）,and the mean optical density（MOD）of FPR2 in IHC was significantly lower than that in control group.IF showed that FPR2 was widely distributed in neurons and microglia of FCDIIb and TSC patients and controls.3.Inflammatory responses in epileptogenic foci of patients with FCDIIb and TSCWe investigated the expression of NF-κB mediated inflammatory pathways in epileptogenic foci in patients with FCDIIb and TSC.Western blots showed that NF-κB protein expression was significantly increased in FCDIIb and TSC patients compared with the control group,and q PCR showed that m RNA levels of downstream molecules IL-1β,IL-6 and TNF-αwere significantly increased compared with the control group.The expression of NF-κB protein was positively correlated with seizure frequency in FCDIIb and TSC patients.4.Effects of RvD1 and WRW4 on FPR2 signaling pathway.The cultured rat primary neurons were divided into 4 groups and treated with PBS,KA,RvD1+KA and FPR2 inhibitor（WRW4）+KA,respectively.Western blots and q PCR results showed that activation of the NF-κB pathway in an vitro epilepsy model was inhibited by FPR2 activation using RvD1,but the FPR2 antagonist WRW4 exerted the opposite effect.5.Effect of RvD1-FPR2 on neuronal excitability.Fluo-4 AM（Beyotime,China）was used to measure the intracellular calcium concentration.Primary cortical neurons were incubated with Fluo-4 AM.Then they were divided into 4 groups,PBS,KA,RvD1,and WRW4 were added.Fluo-4 AM fluorescence was used to monitor the changes in the intracellular Ca²⁺concentration.The fluorescence intensity was recorded with a confocal laser scanning microscope.RvD1-FPR2 reduced Ca²⁺influx in cortical neurons,whereas WRW4 increased intracellular Ca²⁺influx.ConclusionIn summary,these results suggest that the RvD1-FPR2 was involved in epilepsy caused by FCDIIb and TSC and FPR2 activation may contribute to control the epilepsy in FCDIIb and TSC.