The Study On The Rhythmic Profiles Of Retina And The Regulation Of Clock Gene Nr1d1 In Retinal Inflammation

Objective:1.To explore the rhythmic transcriptome profiles of mouse retina and identify the physiological functions of retina which are under control of daily rhythm.To provide the theoretical evidence for establishing new treatment strategies of retinal diseases based on circadian clock.2.To investigate the relation between retinal inflammation and clock gene and the impact or influence of clock gene Nr1d1 on retinal inflammation and inflammatory microglial activation.3.To identify the pathway and molecular mechanisms of antiinflammation effects of clock gene Nr1d1 in microglia.Methods:1.6-week-old C57BL/6 mice were housed under 12:12 LD conditions with lights on at 6 a.m.and off at 6 p.m.for four weeks.From ZT0(Zeitgeber time)to ZT20,retina tissues were sampled with 4-h intervals,at ZT0,ZT4,ZT8,ZT12,ZT16,and ZT20.Three retina samples at each time point were collected.Each sample was then processed for RNA sequencing(RNA-seq).The Metacycle R package was used to determine the rhythmicity of gene expression.The Mfuzz R package was used to cluster rhythmic genes according to their expression patterns.The Cluster Profile R package was used to perform functional enrichment analysis.2.BALB/c mice were used to generate retinal inflammation animal model by intravitreal injection of lipopolysaccharide(LPS).We performed q PCR assays to evaluate the expression level of clock genes.Mice were randomized into three groups:(1)normal control group;(2)LPS + SR9009(an agonist of Nr1d1)group: mice were pretreated with intraperitoneal injection of SR9009(100mg/kg/day)for five days,and received intravitreal injection of LPS to induce retinal inflammation at day 5.(3)LPS + vehicle group: mice were injected with the vehicle at same dose.And the effect of SR9009 was evaluated according to the expression level of cytokines,the number of infiltrated immune cells,the morphology change of microglia in retina and the staining of Müller cell gliosis marker GFAP in retina sections.3.The microglia cell line BV2 and primary retinal microglia were used to perform in vitro experiments.The inflammatory action of microglia was induced by LPS,and SR9009 was administrated for treatment.The expression level of cytokines,the morphology change of microglia and the expression level of M2 polarization marker CD206 were measured in microglia.4.The western blotting assay was performed to evaluate the impact of SR9009 on NF-κB pathway and MAPK pathway.Besides,RNA-seq was conducted in Nr1d1 silenced BV2 cells,and Ch IP-seq of Nr1d1 in BV2 cell was performed.And we used BETA tool to integrate the Ch IP-seq and RNA-seq data to predict that Hmga2 gene might be the direct target gene of Nr1d1.In addition,we performed dual-luciferase assay and electrophoretic mobility shift assay(EMSA)to verify whether Nr1d1 regulate Hmga2.5.The lentivirus was applied to overexpress Hmga2 in BV2 cell,and the expression level of cytokines,the activation of NF-κB were measured after Hmga2 overexpression.SR9009 was administrated after Hmga2 overexpression to evaluate the influence of Hmga2 overexpression on antiinflammation effect of SR9009,by measuring expression level of cytokines and activation of NF-κB pathway.6.Each experiment was performed in three or more biological replicates,and the data were shown as mean ± SEM.The student t test was used to compare the difference between two groups,and one-way ANOVA was used to compare difference between multiple groups.p ≤ 0.05 was considered statistically significant.Results:1.We found that 1741 genes(10.26%)in the mouse retina were rhythmically expressed.According to their expression patterns,the rhythmically expressed genes were further grouped into four clusters,containing 396,492,492,and 361 genes respectively.The functional enrichment analysis showed that the activity of glycolysis and energy production processes were higher at night.Meanwhile,the genes were involved in Hif-1α signaling pathway and hypoxia associated cellular processes were highly expressed during night period.While the expression level of genes in assembly and organization of the photoreceptor cilia peak during day time.2.Comparing with normal control,the clock gene Nr1d1 was upregulated,while another clock gene Bmal1 was downregulated in retinal inflammation animal model.And SR9009 administration could inhibit the expression of cytokines Il-1β,Il-6,Ccl2,Tnfα in retina,decrease the infiltrated immune cells in vitreous and around the ciliary body,restore the ramified morphology of microglia,and suppress the staining of Müller gliosis marker GFAP in retina section.3.In vitro experiments,SR9009 treatment could also inhibited LPS induced expression of Il-1β,Il-6,Ccl2,Tnfα in BV2 cells and primary retinal microglia.Under LPS stimulation,the microglia transformed from a resting ramified phenotype to an activated amoeboid phenotype and expressed low level of M2 polarization marker CD206.Comparing with LPS group,SR9009 treatment group increased the proportion of ramified microglia and CD206 staining.Nr1d1 knockdown elevated Il-6,Il-1β and Tnfα under LPS stimulation,and attenuated the anti-inflammatory effect of SR9009 in BV2 cells.Alternatively,overexpression Nr1d1 inhibited LPSinduced expression of Il-6,Il-1β and Ccl2.4.Under LPS stimulation,the ratios of NF-κB pathway associated pp65/p65 and p-IKK/IKK were increased,and the ratios of MAPK pathway associated p-p38/p38 and p-ERK/ERK were also increased,and promoted p65 nuclear translocation.SR9009 treatment could inhibited the ratios of p-p65/p65 and p-IKK/IKK and p65 nuclear translocation,while no significant influence on ratios of p-p38/p38 and p-ERK/ERK.After Nr1d1 knockdown,RNA-seq results showed that upregulated genes were enriched in inflammation associated pathways,such as TNF signaling pathway and IL17 signaling pathway.Subsequently,we integrated Nr1d1 Ch IP-seq data and RNA-seq data by BETA tool,and found that Hmga2 might be downstream target of Nr1d1.Then dual-luciferase assay and EMSA assay found that Nr1d1 could bind to TGACCCTGAA sequence in Hmga2 promoter and transcriptional repress the expression of Hmga2.5.With overexpression of Hmga2,both the expression level of cytokines as well as ratios of p-p65/p65 and p-IKK/IKK in BV2 cell were further elevated under LPS stimulation.To establish whether the antiinflammatory effects of SR9009 are Hmga2-dependent,we used Hmga2 overexpressing SR9009-treated BV2 cells.Comparing with LPS + SR9009 group,the expression level of cytokines and phosphorylation of p65 and IKK were significantly increased in the Hmga2 overexpression + LPS +SR9009 group.Conclusions:1.In mouse retina,approximately 10.26% of genes are rhythmically expressed.The daily rhythm plays an important role in maintaining the retinal physiology and cellular metabolism.This provides crucial evidences for developing or establishing new prevent and even treat strategy of retinal diseases based on biological rhythm.2.Clock gene Nr1d1 is associated with retinal inflammation.And pharmacological activation of Nr1d1 with SR9009 could inhibited LPS induced retinal inflammation phenotypes.3.SR9009 could alleviate LPS induced microglia inflammatory activation.And Nr1d1 mainly suppresses the activation of NF-κB pathway to act anti-inflammation effects.4.In microglia,Hmga2 is a pro-inflammation gene.Nr1d1 could bind to the Hmga2 promoter and transcriptionally suppress the expression of Hmga2,thus alleviate the activation of NF-κB pathway.This means that Nr1d1/Hmga2/NF-κB might be a new molecular mechanism through which Nr1d1 exerts its anti-inflammation effects in the microglia.Targeting at this molecular mechanism might be a new strategy for treating retinal inflammation.