GPR120 Regulates Neuroinflammation And Seizures In Epilepsy Through NLRP3/Caspase-1/IL-1Β Signaling Pathway

Part one:Expression and distribution of GPR120 in epileptic brainObjective:To detect the expression,distribution and localization of GPR120 in the brain tissues of epileptic mice and epileptic patients.Method:1.Male C57BL/6 adult mice were injected with kainic acid（KA）to establish temporal lobe epilepsy（TLE）model.The hippocampus and cortex of mice were taken at 1,3,7,14,21 and 28 days after the establishment of TLE model,and the expression changes were detected by Western blot.2.The brain tissues of 10 patients with TLE and 10 normal controls were randomly selected from the brain bank for immunohistochemical staining to verify the expression of GPR120 in human brain.3.At the time point of the highest expression of GPR120,the whole brain of mice was taken for immunofluorescence test to detect the distribution and localization of GPR120 in the hippocampus of epileptic mice.Result:1.Western blot showed that GPR120 was up-regulated in hippocampus and cortex of epileptic mice,and the expression of GPR120was the highest on the 7th day after KA injection.2.Immunohistochemical staining showed that the results of human brain tissue were consistent with those of animal model.Compared with the normal control group,the expression of GPR120 in brain tissue of epileptic patients was increased.3.Immunofluorescence staining showed that GPR120 was mainly expressed in hippocampal CA1 and CA3 regions associated with epilepsy,and co localized with neurons,but not with small glue and stellate glue.Conclusion:GPR120 was overexpressed in epileptic mice and epileptic brain tissues,and co localized with neurons in CA1 and CA3 regions of hippocampus,which suggested that GPR120 might be involved in epileptic activity.Part two:Effects of GPR120 on neuronal death and neuroinflammation in epilepsy after seizures and status epilepticusObjective:To investigate the role of GPR120 in epileptic seizures.Next,we used adeno-associated virus to overexpress and knock down GPR120 to explore the effects of GPR120 on neuronal damage and neuroinflammation after epileptic seizures,status epilepticus（SE）in KA induced tle epilepsy model.Methods:1.Male adult C57BL/6 mice were injected with GPR120-AAV-OE,Con-AAV-OE,GPR120-AAV-KD and Con-AAV-KD.Three weeks later,KA was injected into the brain to establish epilepsy model.The seizure was monitored by video.Local field potential was recorded one month later.2.Mice injected with adeno-associated virus were taken to prepare brain slices in vitro.In the absence of Mg²⁺,micro excitatory postsynaptic currents（m EPSCs）and micro inhibitory postsynaptic currents（m IPSCs）of CA1 vertebral neurons were recorded by whole cell patch clamp after GPR120-AAV-OE and GPR120-AAV-KD intervention.3.One month after SE induction,the survival number of neurons was detected by Nissl staining.4.On the 7th day after KA injection,the levels of IL-1β,IL-18 and IL-6 were detected by Western blot.Result:1.Frequent recurrent epileptic seizure like events（SLEs）were observed in all mice by LFP and behavioral monitoring.LFP analysis of 30minutes of SLE showed that there was no difference in the average attack time among groups.However,compared with con KD group,the number of SLE in KD group increased within 30 minutes,and the total time in SLE increased.Compared with con OE group,the number of SLE in OE group increased within 30 minutes,and the total time in SLE decreased.2.Whole cell patch clamp recording showed that the frequency and amplitude of m EPSC were increased in KD group and decreased in OE group,but GPR120-AAV-KD and GPR120-AAV-OE did not affect the frequency and amplitude of m IPSC.3.Nissl staining results showed that compared with normal control group and epilepsy group,the number of neurons in CA1,CA3 and DG areas of hippocampus in KD group was significantly reduced,while the result in OE group was just the opposite.Compared with Con-OE group,the number of neurons in CA1,CA3 and DG areas of hippocampus in OE group was significantly reduced,and there was no significant difference between OE group and normal control group.4.Western blot results showed that compared with the control group,the levels of IL-1β,IL-18 and IL-6 in hippocampus of mice with TLE induced by intracerebral injection of Con-AAV-KD and Con-AAV-KD were significantly increased.Compared with control group and Con-KD group,the levels of IL-1β,IL-18 and IL-6 in hippocampus of TLE epilepsy model group（KD group）were significantly increased.On the contrary,compared with Con-OE group,the levels of IL-1β,IL-18 and IL-6 in hippocampus of TLE epilepsy model group（OE group）were significantly decreased,and there was no statistical significance compared with control group.Conclusion:GPR120 can affect seizure activity,neuron damage and neuroinflammation after SE.Knock down of GPR120 can aggravate seizure activity,and overexpression of GPR120 can reduce seizure activity.In terms of neurotransmission,GPR120 mainly affects excitatory synaptic transmission,but has no significant effect on inhibitory synaptic transmission.Part three:GPR120 promotes neuroinflammation of IL-1β and IL-18 in epilepsy by activating NLRP3 inflammasomeObjective:In order to further explore the mechanism of GPR120 on epileptic seizures,based on the important role of NLRP3 inflammasome in neuroinflammation,we explored the effect of GPR120 on NLRP3inflammasome and its downstream signaling pathway in this part.Method:1.Male adult C57BL/6 mice were injected with GPR120-AAV-OE,Con-AAV-OE,GPR120-AAV-KD and Con-AAV-KD respectively.Three weeks later,KA was injected into the brain to establish epilepsy model.On the 7th day after KA injection,the expression of NLRP3,Caspase-1 and ASC in hippocampus of mice were detected by Western blot.2.Adult male C57BL/6 mice were injected with GPR120-AAV-KD and Con-AAV-KD,respectively.Three weeks later,KA was injected into the brain to establish epilepsy model.VX765 was administered by gavage to some mice injected with GPR120-AAV-KD（KA+KD+VX765 group）,and same dose corn oil was administered by gavage to the other mice injected with GPR120-AAV-KD（KA+KD group）.And the epileptic mice injected with Con-AAV-KD（KA group）were given the same amount of corn oil by gavage.On the 7th day after KA injection,the expression of IL-1β,IL-18 and IL-6 in hippocampus was detected by Western blot,the epileptic seizure activity was observed by behavioral monitoring and the survival of neurons was detected by Nissl staining.Result:1.The results of Western blot showed that the levels of NLRP3,ASC and Caspase-1 in hippocampus of mice in con KD and con OE groups were significantly higher than those in control group.Compared with control group and Con-KD group,the levels of NLRP3,ASC and Caspase-1 in hippocampus of KD group were significantly increased.However,compared with Con-OE group,the levels of NLRP3,ASC and Caspase-1 in hippocampus of mice in OE group were significantly decreased,and there was no statistical significance compared with control group.2.The Western blot results of VX765 after intragastric administration showed that compared with KD group,the levels of IL-1βand IL-18 in the brain of KA+KD+VX765 group were significantly decreased,and there was no statistical significance compared with KA group.However,VX765had little effect on the level of IL-6 in hippocampus of mice injected with GPR120-AAV-KD.3.Nissl staining showed that compared with KD group,the survival number of neurons in CA1,CA3 and DG of hippocampus in KA+KD+VX765 group was significantly increased,and there was no significant difference between KA+KD+VX765 group and KA group.4.Behavioral monitoring showed that compared with KA+KD group,KA+KD+VX765 group had longer latency and lower seizure frequency.Conclusion:GPR120 can regulate the activation of NLRP3 inflammasome,and VX765 can rescue the neuroinflammation and neuron death induced by GPR120-AAV-KD in the hippocampus of epileptic mice.