| Objective: Epilepsy is one of the most common neurological diseases in children,and its pathogenesis remain incompletely understood.At present,many factors are believed to contribute to the occurrence of epilepsy,including heredity factors,metabolism,immunity,inflammation,and so on.Microglia,as the innate immune cells in the central nervous system and main producer of proinflammatory cytokines,play an important role in epilepsy.Previous studies have revealed the presence of a large number of activated microglia in the hippocampus of patients with temporal lobe epilepsy as well as in animal models of epilepsy.Secretion of proinflammatory cytokines by activated microglia can increase neuronal excitability and cause direct damage to neurons,which is considered a major driving factor of epileptogenesis.Non-inflammatory activities of microglia,such as phagocytosis,synaptic pruning,and remodeling,also contribute to the process of epileptogenesis.Additionally,micro RNAs(miRNAs)can regulate whole gene networks at the post-transcriptional level.Previous research demonstrated significantly increased expression of miR-106b-5p in peripheral blood of epilepsy patients and a positive correlation between miR-106b-5p expression and the severity of epilepsy.This increased miR-106b-5p expression is speculated to be a response to chronic inflammation in the brain of patients with epilepsy.The purpose of our study was to study the dynamic expression of miR-106b-5p and its target gene RGMa as well as microglia phenotype markers and related cytokines in the hippocampus of the pilocarpine-induced status epilepticus(SE)mouse model;to analyze possible relationships among these factors;and to use lentivirus-mediated knockdown of miR-106b-5p in animal and cell experiments to confirm its effect on microglia phenotype and related cytokine expression and to explore the mechanism of its potential effect on epileptogenesis.Methods: For the first animal experiment,C57BL/6 mice were randomly assigned to a control group and an epilepsy group,and the mice of the epilepsy were further divided into 6 groups for time points of 6 h,1 d,3 d,7 d,14 d and 21 d.The SE model was established by intraperitoneal injection of pilocarpine.Hematoxylin and eosin(HE)and terminal deoxynucleotidyl transferase d UTP nick-end labeling(TUNEL)staining were used to observe damage to hippocampal neurons,and in situ hybridization immunofluorescence staining and immunofluorescence double staining were performed to miR-106b-5p and RGMa expression in microglia.The gene or protein expression levels of miR-106b-5p,RGMa,M1 and M2 microglia markers inducible nitric oxide synthase(i NOS)and arginase-1(Arg-1),and related cytokines interleukin(IL)-1β,IL-6,IL-4,IL-10,and myeloid cell trigger receptor 2(TREM2)in the mouse hippocampus were detected by RT-q PCR or western blotting.A double luciferase assay was performed to verify whether miR-106b-5p could target and inhibit RGMa expression.For the second animal experiment,C57BL/6 mice were randomly assigned to a control group and an epilepsy group,and the mice in the epilepsy group were further divided into epilepsy,epilepsy+negative control(NC),epilepsy+inhibitor,epilepsy+inhibitor+short hairpin(sh)control,and epilepsy+inhibitor+sh RGMa groups,according to different pretreatments.Mice were injected with serum-free medium or corresponding lentivirus into the bilateral hippocampus 1 week before the model established,according to groups.Seizure latency was observed,and in model mice,HE and TUNEL staining were used to observe damage among hippocampal neurons.RT-q PCR or western blotting were performed to detect the gene or protein expression of miR-106b-5p,RGMa,M1 and M2 microglia markers,and related cytokines in the mouse hippocampus.The proportion of M1 and M2 microglia was determined from flow cytometry data.The expression levels of Rac1,p-JNK and p-p38 proteins were detected by western blotting.Proliferation of neural stem cells in the hippocampal dentate gyrus was detected by Brd U staining.Behavioral changes of mice in open field test were observed.In the in vitro experiment,HT22 cells treated with magnesium(Mg)-free solution were co-cultured with BV2 cells,and lentiviral infection was used to inhibit the expression of miR-106b-5p and RGMa in BV2 cells.According to different pretreatments,the following co-culture groups were established: Control,Mg-free,Mg-free+NC,Mg-free+inhibitor,Mg-free+inhibitor+shcontrol,and Mg-free+inhibitor+sh RGMa.The expression levels of miR-106b-5p,RGMa,M1 and M2 microglia markers,and related cytokines in BV2 cells were detected by RT-q PCR or western blotting.RGMa,i NOS and Arg-1 expression in BV2 cells was observed by immunofluorescence double staining.The proportion of M1 and M2 BV2 cells was determined by flow cytometry.Rac1,p-JNK and p-p38 protein expression levels in BV2 cells were detected by western blotting.Damage among HT22 cells was detected by MTT and lactate dehydrogenase(LDH)release assays.Results: 1.In the SE mouse model,HE and TUNEL staining revealed neuronal damage in the mouse hippocampus within 6 hours after SE induction,which was most obvious at3 days and then recovered.2.miR-106b-5p expression in the hippocampus of SE model mice was greater than that in control mice,peaking at 3 days(P<0.05)before decreasing without returning to normal at 21 days(P<0.05).RGMa expression decreased and did not recover until 21 days(P<0.05).Expression of M1 microglia marker i NOS and related cytokines IL-1β and IL-6 increased,peaked at 3 days(P<0.05),then decreased,and returned to normal at 21 days(P>0.05).The expression of M2 microglia marker Arg-1 and cytokine IL-10 also increased,peaking at 3 days(P<0.05)before decreasing without returning to normal at 21 days(P<0.05).Although the IL-4expression also increased,it peaked at 7 days(P<0.05)before decreasing to the normal level by 21 days(P>0.05).TREM2 expression decreased and did not recover by 21days(P < 0.05).3.In situ hybridization immunofluorescence staining revealed co-expression of miR-106b-5p and Iba-1,and immunofluorescence double staining revealed co-expression of RGMa and Iba-1.4.Double luciferase assay confirmed that miR-106b-5p could target and inhibit RGMa expression.5.Lentivirus LV-miR-106b-5p inhibitor injection resulted in downregulation of miR-106b-5p expression in the mouse hippocampus after SE induction(P < 0.05).With the decrease in miR-106b-5p expression,RGMa expression increased(P<0.05).Additional injection of the lentivirus LV-sh RGMa resulted in reduced expression of RGMa again.6.Lentiviral knockdown of miR-106b-5p expression prolonged the latency of seizures(P<0.05),reduced the damage to hippocampal neurons(P<0.05),inhibited the proliferation of neural stem cells in the dentate gyrus(P<0.05),and increased the total moving distance(P<0.05),time in central area(P<0.05),and number of climbing attempts(P<0.05)in the open field test among mice.Lentiviral knockdown of RGMa expression could reverse the effects of miR-106b-5p knockdown.7.Lentiviral knockdown of miR-106b-5p decreased the expression of M1 microglia marker i NOS and cytokines IL-1β and IL-6(P<0.05)as well as the M1 cell proportion(P<0.05)while increasing the expression of M2 microglia marker Arg-1 and cytokines IL-4 and IL-10(P<0.05)along with the M2 cell proportion(P<0.05)in the mouse hippocampus after SE.Again,lentiviral knockdown of RGMa expression reversed the effects of miR-106b-5p knockdown.8.Lentiviral knockdown of miR-106b-5p also resulted in reduced expression of Rac1,p-JNK and p-p38 proteins(P<0.05)and increased expression of TREM2(P<0.05)in the mouse hippocampus after SE induction.Again,lentiviral knockdown of RGMa expression reversed these effects.9.In the in vitro experiment,miR-106b-5p expression was increased(P<0.05)and RGMa expression was decreased(P<0.05)in HT22 cells treated with Mg-free solution and in BV2 cells stimulated with corresponding culture medium,consistent with the expression changes observed in the SE mouse model induced by pilocarpine.10.In the co-culture system of HT22 cells treated with Mg-free solution and BV2 cells,lentiviral knockdown of miR-106b-5p in BV2 cells decreased the expression of M1 microglia marker i NOS and cytokines IL-1β and IL-6(P<0.05)as well as the M1 cell proportion(P<0.05),while increasing the expression of M2 microglia marker Arg-1 and cytokines IL-4 and IL-10(P<0.05)as well as the M2 cell proportion(P<0.05)among BV2 cells.Lentiviral knockdown of RGMa reversed these effects of miR-106b-5p knockdown.11.After lentiviral knockdown of miR-106b-5p,the expression levels of Rac1,p-JNK and p-p38 in BV2 cells were decreased(P<0.05)and that TREM2 was increased(P<0.05),and again,lentiviral knockdown of RGMa had the opposite effects.12.After knockdown of miR-106b-5p in BV2 cells,the activity of HT22 cells increased(P<0.05)and LDH release decreased(P<0.05).Again,lentiviral knockdown of RGMa expression in BV2 cells reversed these effects.Conclusion: After SE induction in mice,the trend in miR-106b-5p expression in the mouse hippocampus followed those of M1 and M2 type microglia markers and related cytokines and was opposite to those of RGMa and TREM2,suggesting that miR-106b-5p may affect the activation and polarization of microglia by targeting RGMa.Lentivirus-mediated knockdown of miR-106b-5p could switch the phenotypic ratio of M1/M2 microglia to M2 in the hippocampus of mice after SE induction,reducing the susceptibility to epilepsy,preventing hippocampal neuron damage and neurogenesis,and increasing the spontaneous movement of mice.The beneficial effects of reduced miR-106b-5p expression may be achieved by targeting the RGMa-Rac1-JNK/p38 MAPK axis.In the co-culture system of HT22 cells treated with Mg-free solution and BV2 cells,lentivirus-mediated downregulation of miR-106b-5p in BV2 cells could inhibit the transformation of BV2 cells to M1 type and promote to M2 type,while reducing production of proinflammatory cytokines IL-1β and IL-6,increasing the release of anti-inflammatory cytokines IL-4 and IL-10,and protecting HT22 cells from damage caused by Mg-free solution.These results caused by the reduced of miR-106b-5p expression may be achieved via the RGMa-Rac1-JNK/p38 MAPK axis. |
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