The Effect Of Exogenerous And Endogenerous Liver X Receptor Agonists And Dexamethasone On LPS-induced Retinal Inflammation

BackgroundInflammation is a response against the foreign invaders.In ophthalmology,LPS-induced inflammation in retinal pigment epithelium cells is involved in the pathological process of many retinal disease,including uveitis,age-related macular degeneration(AMD)and diabetic retinopathy(DR).Dexamethasone(DEX),a type of glucocorticoid,is widely used for the treatment of ocular inflammation disease,due to its strong anti-inflammatory and immunosuppressive effects.Whereas,long-term application of DEX may lead to a series of systemic and topical side-effects.Therefore,finding novel drug targets has become a research hotspot.In recent years,the relationship between the lipid homeostasis and inflammation is becoming the front line of medical research.Our lab and others have found that activation of liver X receptor(LXRs),which is a major regulator of cholesterol metabolism,with a synthesized ligand(TO90)inhibits retinal inflammation and is promising to become a novel class of drug for treating retinal inflammation.Nevertheless,limited reports suggest endogenerous LXRs agonist(7-KC)exaggerate retinal inflammation.Therefore,we raised a question:both 7-KC and TO90 activate LXRs and it's target genes,why do they play opposite roles in the regulation of inflammation?Based on the above background,this study was divided into two parts:(1)Investigate the opposite effects of 7-KC and TO90 on LPS-induced inflammation in RPE cells,and research the mechanisms with RNA-seq and TMT analysis.(2)Investigate the effects of DEX on EIU mouse model and explore the underlying mechanisms.PART I THE EFFECTS AND MECHANISM OF 7-KETOCHOLESTEROL AND TO901317 ON LPS-INDUCED INFLAMMATION IN HUMAN RETINAL PIGMENT EPITHELIUM CELLSPurpose:Liver X receptors are key regulators of cholesterol homeostasis,as well as important modulators of inflammation.Synthetic LXRs agonists(TO901317)exert anti-inflammatory effect,while the oxysterol LXRs agonists(7-KC)exert pro-inflammatory effect.We studied the paradoxical effects of 7-KC and TO901317 on inflammation and explored the underlying mechanisms with transcriptome and proteome analysis.Methods:ARPE-19 cells were incubated with different concentrations of 7-KC for 24 and 36 hours.CCK-8 assay and Annexin-V FITC/PI assay were used to detect the cell viability and apoptosis.EdU was used to detect cell proliferation.ARPE-19 cells were pretreated with 7-KC and TO90 for 12 hours,followed by stimulation with 5 ?g/mL of LPS for 24 hours.The mRNA levels of IL6,IL-8,LXRs and it's target genes ABCA1,ABCG1 were measured.Illumina Hiseq 2500 platform and Q Exactive mass spectrometer were applied to characterize the transcriptome and proteome profiles.Results:Incubation with high concentration of 7-KC markedly decreased the cell viability and increased cell apoptosis.There was no cell proliferation when stimulated with various concentrations of 7-KC for 24 and 36 hours.Thus,7.5 ?M of 7-KC was used as the optimum concentration.Both 7-KC and TO90 activated LXRs target genes ABCA1 and ABCG1,while they exhibited opposite effects on LPS-induced inflammatory response.7-KC increased the mRNA levels of IL-6,IL-8,LXRa,LXR? and ABCA1,ABCG1 in a dose dependent manner,while TO90 suppressed the inflammation.Transcriptome profile indicated that 7-KC activates the toll-like receptor and MAPK signal pathways,while TO90 suppressed those pathways.Combined analysis of transcriptome and proteome revealed that 7-KC not only regulated lipid and cholesterol metabolism,but also increased the expression of inflammation signal pathways,including Toll-like receptor,MAPK,mTOR,HIF-1,and VEGF signal pathwas and some inflammation related genes.Whereas,TO90 did not directly affect those inflammation signal pathways and genes.Conclusions:Both 7-KC and TO90 activated their receptors,whereas 7-KC exaggerated the LPS-induced inflammation and TO90 inhibited the inflammatory response.This paradox effects may be associated with the different regulation mechanisms of 7-KC and TO90 on Toll-like receptors,HIF-1,VEGF,mTOR and MAPK signal pathway and some lipid metabolism related genes.PART II RETINAL TRANSCRIPTOME PROFILE OF DEXAMETHASONE TREATED ENDOTOXIN-INDUCED UVEITIS IN MICEPurpose:To investigate the retinal transcriptome profile of dexamethasone(DEX)treated endotoxin-induced uveitis(EIU)in mice and to explore the underlying mechanisms of the protective effect of DEX.Method:EIU was induced in BALB/c mice by intravitreal injection of 125 ng lipopolysaccharide(LPS).DEX eyedrop(0.1%)was locally administrated every four hours for 24 hours.The anterior segment was examined with a slit lamp and clinical scores were assessed simultaneously.The morphology changes were assessed at the 24th hour after LPS injection.The transcriptome profile was conducted with next-generation sequencing(NGS)-based RNA sequencing(RNA-seq).The expression of inflammatory cytokines and selective differentially expressed genes(DEGs)were verified by real-time PCR.Results:DEX alleviated the inflammatory response and reduced the mRNA expression of IL-6,TNF-?,MCP-1 and ICAM-1 at the 24th hour after LPS injection.A total of 52 DEGs were identified by RNA-seq.Within these DEGs,37 genes were up-regulated and 15 genes were down-regulated in the LPS group when compared with D+LPS group.No significantly enriched Gene Ontology(GO)terms was noted.Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment showed 6 up-regulated and 2 down-regulated KEGG pathways.Interestingly,RIG-I like receptor signal pathway and several immune and inflammation related genes including Ifit1,H2-T24,Mx2 and Eif2ak2 were significantly down regulated by DEX.The gene expression was further validated with RT-PCR.Conlusion:DEX alleviated the inflammatory response induced by LPS and transcriptome profile indicated that the protective effect may be associated with RIG-I like receptor signal pathway and several other immune and inflammation related genes.