Fig 1: The proposed interplay between miR-15b-5p/miR-92b-3p and their target genes in the intermittent hypoxia with re-oxygenation (IHR) model of obstructive sleep apnea (OSA). (A) These interactions contribute to the development of depression by IHR-induced MAOA hyperactivity and IHR-induced oxidative stress/apoptosis/inflammation in OSA patients. (B) A schematic diaphragm depicts the protective effect of miR-15b-5p/miR-92b-3p mimics on IHR-induced oxidative stress and cell apoptosis through inhibiting MAOA via targeting PTGS1-NF-?B-SP1 signaling in OSA-related depression. IHR augments PTGS1, which increases the NF-?B expression that in turn activates MAOA gene transcription through augmenting the binding of SP1 to the promoter region of the MAOA gene. Down-regulation of miR-15b-5p/miR92b-3p in response to chronic IHR in OSA leads to MAOA hyperactivity, oxidative stress, and cell injury through targeting PTGS1-NF-?B1-SP1 signaling. MAOA = monoamine oxidase A; PTGS1 = prostaglandin-endoperoxide synthase 1; NF-?B1 = nuclear factor kappa B subunit 1; SP1 = Sp1 transcription factor.
Fig 2: Over-expression of miR-92b-3p or knockdown of PTGS1 reversed intermittent hypoxia with re-oxygenation (IHR)-induced cell injury and MAOA hyperactivity via targeting NF-?B1/SP1 signaling. Transfection with either PTGS1 SiRNA or miR-92b-3p mimic in SH-SY5Y cells reversed IHR-induced (A) down-regulation of miR-92b-3p, (B) cell injury, (C) MAOA hyperactivity, and up-regulations of (D) PTGS1, (E) NF-?B1, and (F) SP1. * p < 0.05 compared with normoxic (NOX) condition. # p < 0.05 compared with IHR condition.
Fig 3: MiR-15b-5p over-expression reversed intermittent hypoxia with re-oxygenation (IHR)-induced apoptosis, oxidative stress, and up-regulation of its target genes. Transfection with a miR-15b-5p mimic in THP-1 cells reversed IHR-induced (A) early apoptosis and reversed IHR-induced up-regulation of its predicted target mRNA, (B) PTGS1. Transfection with miR-15b-5p in HUVEC reversed IHR-induced (C) reactive oxygen species production, (D) early apoptosis, and up-regulation of its predicted target genes, including (E) NF-?B1, (F) TNF-a, and (G) TGF-ß. Transfection with a miR-15b-5p mimic in SH-SY5Y cells reversed IHR-induced (H) ROS production and (I) late apoptosis. * p < 0.05, compared with normoxic (NOX) condition. # p < 0.05, compared with IHR condition.
Fig 4: PGE2 Is Sufficient to Initiate Mesoderm Differentiation of hESCs(A) qPCR analysis of mesoderm marker gene expression levels in BMP4-induced hESCs transfected with two independent siRNAs against COX-1 or a scrambled control siRNA. *p < 0.05, **p < 0.01 compared with the scrambled control group. n = 3 independent experiments.(B) Immunostaining for BRA expression in BMP4-induced hESCs transfected with two independent siRNAs against COX-1 or a scramble control siRNA. Scale bars represent 50 µm.(C) qPCR analysis of mesoderm marker gene expression levels in hESCs with BMP4 induction plus DMSO (control), SC-560, or NS-398 for 48 hr. *p < 0.05, **p < 0.01 compared with control group. n = 3 independent experiments.(D and E) Immunostaining (D) and western blotting (E) analysis for BRA expression in hESCs with BMP4 induction plus DMSO (Control), SC-560, or NS398 for 48 hr. Scale bars represent 50 µm.(F) Mesoderm gene expression levels in pEGFP-hESCs and pEGFP-COX-1 hESCs during differentiation were determined by qPCR. *p < 0.05, **p < 0.01 compared with pEGFP-transfected cells. n = 3 independent experiments.(G) Western blotting analysis for COX-1 and BRA in pEGFP-hESCs and pEGFP-COX-1 transfected hESCs 2 days after transfection.(H) qPCR analysis of the BRA expression levels in hESCs with PGE2 treatment at different concentration for 2 days. *p < 0.05 compared with -PGE2 cells. n = 3 independent experiments.(I) qPCR analysis of mesodermal gene expression levels in hESCs treated with 1 µM PGE2. *p < 0.05 compared with -PGE2 2d. n = 3 independent experiments.(J and K) Western blotting (J) and immunostaining (K) analysis for BRA in hESCs treated with 1 µM PGE2 for 2 days. Scale bars represent 50 µm.(L) qPCR analysis of mesodermal gene expression levels in EpiSCs treated with 1 µM PGE2. *p < 0.05, **p < 0.01 compared with -PGE2 2d. n = 3 independent experiments.(M) Western blotting analysis for Bra in EpiSCs treated with 1 µM PGE2 for 2 days.Error bars indicate SD. See also Figure S3.
Fig 5: Western blot analysis of COX-1 and COX-2 in gastric mucosal tissues after 6 days of treatment. CON: vehicle control; AOE-H: 0.18 g/kg A. officinarum extract; AOE-M: 0.09 g/kg A. officinarum extract; AOE-L: 0.03 g/kg A. officinarum extract; GAL: 0.2 g/kg galangin; POS: 0.08 g/kg bismuth potassium citrate; MOD: 0.3 g/kg indomethacin. #p < 0.05 and ##p < 0.01 compared with the CON group; *p < 0.05 and ***p < 0.001 compared with the MOD group.
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