| Objective:This paper intends to investigate whether retinoic acid receptor blockade affects IgE-mediated mast cell activation and the main effector mechanisms during its occurrence,providing a scientific basis and a theoretical foundation for the treatment and intervention of allergic diseases and for the prevention of vitamin A deficiency and early intervention treatment of its patients.Method:Bone marrow-derived mast cells(BMMCs)were obtained by isolating and extracting 8-week-old female C57BL/6J mice bone marrow haematopoietic stem cells and cultured in RPMI-1640 medium containing 10% fetal bovine serum,1% double antibody and 10 ng/m L IL-3 for 4 weeks in vitro,and the cells were identified by flow cytometry and toluidine blue staining.Retinal at concentrations of 0.03 μM,0.3 μM and 3 μM was administered for 4 h.The cell activity assay was performed by CCK-8method,combined with toluidine blue staining and q RT-PCR to determine the effect of retinal on the activation of mast cells thereby determining the effect dose of retinal.The experimental groups were: Control group,IgE group,Re/IgE group,Rb/Re/IgE group.The release of histamine was measured by ELISA according to the different groups of drug interventions;gene and protein expression of signalling pathways and cytokines were measured by high-throughput transcriptome gene sequencing,q RT-PCR and western blot.Statistical analysis was performed using IBM SPSS 24.0software,and the measurement data were expressed as mean ± standard deviation.The One Way ANOVA method was used to compare between groups,and the LSD method was used to compare significant differences between groups.Differences were considered statistically significant when P < 0.05.Relevant images were created using Graph Pad Prism software(version 8.0.1).Result:1.Isolation of bone marrow-derived haematopoietic stem cells and identification of mast cells: Flow cytometry was used to obtain BMMCs with 99.2% purity of CD117 and FCεRIα.Toluidine blue staining showed blue nuclei and purple-red cytoplasm,consistent with mast cell characteristics.2.Effect of blocking retinoic acid receptors on mast cell activation: The effect of retinal on mast cell activity was measured by CCK-8 method,and the results showed a statistically significant decrease in cell activity at a retinoid concentration of 3 μM,and all three concentrations inhibited the activation of mast cells(level of IL-6production and mast cell volume and degranulation),with the most significant phenomenon at 3 μM.The most pronounced effect was observed at 3 μM.The activation of mast cells after retinoic acid receptor blockade was measured by ELISA,q RT-PCR and toluidine blue staining: compared to the Control group,the IgE group caused an activation of mast cells,as evidenced by increased cell volume,increased degranulation,increased expression of cytokines such as histamine and IL-4,IL-6,TNF-α,GM-CSF,IL-1β;after retinal intervention(i.e.Re/IgE group),toluidine blue staining results showed that retinal attenuated the activation state of mast cells,as evidenced by reduced cell morphology and degranulation.The ELISA and q RT-PCR results also showed that retinal reduced the release of histamine and cytokines such as IL-4,IL-6,IL-13,TNF-α,GM-CSF,IL-1β by mast cells,while in Rb/Re/IgE group the effect of retinal was blocked and this series of phenomena was counteracted in the Re/IgE group.3.Effect of blocking retinoic acid receptors on related pathways by sequencing analysis: Heat map and volcano map revealed 73 differential genes in the Re/IgE and Rb/Re/IgE groups,of which 25 were up-regulated and 48 were down-regulated,and 5genes were enriched in the My D88/NF-k B signalling pathway.In addition,we used Gene Set Enrichment Analysis(GSEA)plots to present the gene expression of multiple related signalling pathways.The results showed that the PPAR signalling pathway was inhibited.In addition,signaling pathways involved in inflammatory pathogenesis,such as phosphatidylinositol-4,5-bisphosphate 3-kinase(PI3K)/protein kinase B(AKT),toll-like receptors,tumor necrosis factor(TNF),IL-17,the Mitogen-activated protein kinase(MAPK)and epidermal growth factor receptor(Erb B)signaling pathways,as well as cytokine-cytokine receptor interactions,were upregulated(P < 0.05).These signalling pathways are associated with the My D88/NF-k B,mTOR signalling pathway4.The effect of blocking retinoic acid receptors on the expression levels of mast cell-related signalling pathways: The q RT-PCR method was used to detect the expression of My D88/NF-k B and its mTOR signalling pathway genes.The results showed that the IgE group exhibited increased gene expression of NF-κB,My D88,PI3 K,AKT,mTOR and S6K1 compared to the Control group;whereas the gene expression of the above indicators were all reduced in the Re/IgE group after the retinal intervention;in addition,the reduction in the Re/IgE group was partially offset when the retinal effect was blocked.Protein immunoblotting was used to detect the expression of My D88/NF-k B and its mTOR signalling pathway proteins,and the results showed that the IgE group exhibited increased protein expression of p-PI3 K,p-mTOR,p-AKT,p-P65,IKKβ,My D88,IκB-α and p-ERK compared to the Control group.After retinal intervention,the protein expression of the above indexes in the Re/IgE group was reduced.In addition,after the retinal effect was blocked,the decrease in the Re/IgE group was partially canceled.Conclusions.1.Retinoic acid receptor blockade can exacerbate mast cell activation by increasing the size of IgE-mediated mast cells and increasing the release of inflammatory mediators such as histamine and cytokines.2.Retinoic acid receptor blockade can exacerbate mast cell activation by enhancing gene or protein expression levels of My D88,IKKβ,IκB-α and NF-κB indicators leading to upregulation of the My D88-IKK-NF-κB signalling pathway.3.Retinoic acid receptor blockade can exacerbate mast cell activation by enhancing gene or protein expression levels of PI3 K,AKT,mTOR and S6K1 indicators leading to upregulation of the PI3K-AKT-mTOR signaling pathway. |
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