Journal: Materials Today Bio

Article Title: Exosomal lncRNA OTUD6B-AS1 as a pathogenic nanocarrier promotes inflammatory macrophage polarization in endometritis via a targetable ceRNA circuit

doi: 10.1016/j.mtbio.2026.103027

Figure Lengend Snippet: Exosomes from LPS-stimulated EECs induce pro-inflammatory macrophage activation. (A) Schematic diagram of the experimental setup for exosome uptake. (B) Fluorescence microscopy images showing the uptake of PKH67-labeled exosomes (green) by macrophages. Cytoskeleton was stained with Phalloidin (red), and nuclei were stained with DAPI (blue). (C) Western blotting analysis of phosphorylated NF-κB p65 (p-p65) in macrophages treated with Control-exo or LPS-exo. (D, E) Representative immunofluorescence (IF) staining images (D) and quantitative analysis (E) of iNOS (greed) in macrophages. (F, G) Representative IF staining images (F) and quantitative analysis (G) of Arg1 (red) in macrophages. (H) Schematic diagram of co-culture experiments. (I, J) Relative mRNA expression levels of iNOS (I) and Arg1 (J) in macrophages after co-culture with EECs. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The antibodies used include TSG101 (Genuin Biotech, Cat # 51942), CD81 (Santa Cruz Biotechnology, Cat # sc-18877), AKT (abcam, Cat # AB81283), Phospho-AKT (abcam, Cat # AB179463 ), NF-κB p65 (abcam, Cat # AB32536), Phospho-NF-κB p65 (Cell Signaling Technology, Cat # 3033S), Notch2 (Santa Cruz Biotechnology, Cat # sc-51869), RBP-Jκ (Santa Cruz Biotechnology, Cat # sc-271128), and β-actin (Bioss, Cat # bs-0061R).

Techniques: Activation Assay, Fluorescence, Microscopy, Labeling, Staining, Western Blot, Control, Immunofluorescence, Co-Culture Assay, Expressing

Journal: Materials Today Bio

Article Title: Exosomal lncRNA OTUD6B-AS1 as a pathogenic nanocarrier promotes inflammatory macrophage polarization in endometritis via a targetable ceRNA circuit

doi: 10.1016/j.mtbio.2026.103027

Figure Lengend Snippet: Transcriptomic profiling reveals significant enrichment of lncRNA OTUD6B-AS1 in exosomes derived from LPS-stimulated EECs. (A, B) LPS-exo was treated with RNase A alone or in combination with Triton X-100 for 4 h, and then co-incubated with macrophages. Relative expression levels of iNOS (A) and Arg1 (B) in macrophages. (C) Schematic overview of the RNA sequencing and analysis workflow. (D) Volcano plot showing differentially expressed lncRNAs in LPS-exo compared to Control-exo. (E, F) Gene Ontology (GO) biological process enrichment analysis (E) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis (F) of the differentially expressed lncRNAs. (G) qPCR validation of the 6 upregulated lncRNAs in Control-exo and LPS-exo. (H) LPS-exo was treated with RNase A alone or in combination with Triton X-100 for 4 h, and then co-incubated with macrophages. Relative mRNA expression level of lncRNA OTUD6B-AS1 in macrophages. (I) A proposed competing endogenous RNA (ceRNA) network involving lncRNA OTUD6B-AS1, miR-128, and Notch2. (J) Relative mRNA expression level of lncRNA OTUD6B-AS1 in control and LPS-stimulated EECs. (K, L) RNA fluorescence in situ hybridization (RNA-FISH) showing the subcellular localization of lncRNA OTUD6B-AS1 (red) in EECs (K) and its quantitative cytoplasmic/nuclear distribution (L). Nuclei were stained with DAPI (blue). (M – P) Relative mRNA expression levels of lncRNA OTUD6B-AS1 (M − O) and miR-128 (P) in endometrial tissues from healthy cows and cows with endometritis, as determined by qPCR (O, P) and RNA-FISH (M) with quantification (N). (Q) Western blotting analysis of Notch2, RBP-Jκ, and p-p65 protein levels in endometrial tissues. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The antibodies used include TSG101 (Genuin Biotech, Cat # 51942), CD81 (Santa Cruz Biotechnology, Cat # sc-18877), AKT (abcam, Cat # AB81283), Phospho-AKT (abcam, Cat # AB179463 ), NF-κB p65 (abcam, Cat # AB32536), Phospho-NF-κB p65 (Cell Signaling Technology, Cat # 3033S), Notch2 (Santa Cruz Biotechnology, Cat # sc-51869), RBP-Jκ (Santa Cruz Biotechnology, Cat # sc-271128), and β-actin (Bioss, Cat # bs-0061R).

Techniques: Derivative Assay, Incubation, Expressing, RNA Sequencing, Control, Biomarker Discovery, Fluorescence, In Situ Hybridization, Staining, Western Blot

Journal: Materials Today Bio

Article Title: Exosomal lncRNA OTUD6B-AS1 as a pathogenic nanocarrier promotes inflammatory macrophage polarization in endometritis via a targetable ceRNA circuit

doi: 10.1016/j.mtbio.2026.103027

Figure Lengend Snippet: EECs-derived exosomes induce pro-inflammatory macrophage activation via delivery of lncRNA OTUD6B-AS1. (A) Relative mRNA expression level of lncRNA OTUD6B-AS1 in macrophages treated with Control-exo or LPS-exo. (B, C) RNA-FISH images (B) and quantitative analysis (C) showing lncRNA OTUD6B-AS1 (red) transfer to macrophages after co-culture with Control-exo or LPS-exo. Nuclei were stained with DAPI (blue). (D) Relative mRNA expression level of lncRNA OTUD6B-AS1 in macrophages after transfection with lncRNA OTUD6B-AS1 overexpression plasmids (OE-lncRNA) or control plasmids (OE-NC). ( E – I) Western blotting analysis of Notch2, RBP-Jκ, and p-p65 (E), along with immunofluorescence (IF) quantitative analysis of iNOS (F, G) and Arg1 (H, I) protein levels in macrophages after transfection with OE-lncRNA or OE-NC. (J – N) Western blotting analysis of Notch2, RBP-Jκ, and p-p65 (J), along with IF quantitative analysis of iNOS (K, L) and Arg1 (M, N) protein levels in macrophages after lncRNA OTUD6B-AS1 knockdown (si-lncRNA) or control treatment (si-NC). (O) Relative mRNA expression level of lncRNA OTUD6B-AS1 in exosomes isolated from lncRNA OTUD6B-AS1-knockdown LPS-stimulated EECs (si-lncRNA-LPS-exo) or exosomes from siRNA NC-transfected LPS-stimulated EECs (si-NC-LPS-exo). (P – S) IF quantitative analysis of iNOS (P, Q) and Arg1 (R, S) protein levels in macrophages treated with si-lncRNA-LPS-exo or si-NC-LPS-exo. (T) Relative mRNA expression levels of iNOS and Arg1 in macrophages treated with exosomes isolated from control EECs overexpressing lncRNA OTUD6B-AS1 (OE-lncRNA-Control-exo) or exosomes from control plasmids-transfected EECs (OE-NC-Control-exo). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The antibodies used include TSG101 (Genuin Biotech, Cat # 51942), CD81 (Santa Cruz Biotechnology, Cat # sc-18877), AKT (abcam, Cat # AB81283), Phospho-AKT (abcam, Cat # AB179463 ), NF-κB p65 (abcam, Cat # AB32536), Phospho-NF-κB p65 (Cell Signaling Technology, Cat # 3033S), Notch2 (Santa Cruz Biotechnology, Cat # sc-51869), RBP-Jκ (Santa Cruz Biotechnology, Cat # sc-271128), and β-actin (Bioss, Cat # bs-0061R).

Techniques: Derivative Assay, Activation Assay, Expressing, Control, Co-Culture Assay, Staining, Transfection, Over Expression, Western Blot, Immunofluorescence, Knockdown, Isolation

Journal: Materials Today Bio

Article Title: Exosomal lncRNA OTUD6B-AS1 as a pathogenic nanocarrier promotes inflammatory macrophage polarization in endometritis via a targetable ceRNA circuit

doi: 10.1016/j.mtbio.2026.103027

Figure Lengend Snippet: lncRNA OTUD6B-AS1 acts as a ceRNA by sponging miR-128 to facilitate pro-inflammatory macrophage activation. (A) Relative mRNA expression level of miR-128 in macrophages treated with Control-exo or LPS-exo. (B) Luciferase reporter assay in HEK293T cells co-transfected with wild-type (WT) or mutant (MUT) lncRNA OTUD6B-AS1 reporter plasmids and miR-128 mimic or mimic NC. (C) RNA pull-down detection of the enrichment of miR-128 to lncRNA OTUD6B-AS1. (D) Ago2 RIP assay analysis of the enrichment of lncRNA OTUD6B-AS1 pulled-down from the Ago2 protein. (E) Relative mRNA expression level of miR-128 in macrophages transfected with OE-NC or OE-lncRNA. (F – J) Western blotting analysis of Notch2, RBP-Jκ, and p-p65 (F), along with immunofluorescence (IF) quantitative analysis of iNOS (G, H) and Arg1 (I, J) protein levels in macrophages co-transfected with OE-lncRNA and miR-128 mimic or mimic NC. (K, L) Relative mRNA expression levels of IL-1β (K) and IL-6 (L) in macrophages co-transfected with OE-lncRNA and miR-128 mimic or mimic NC. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Article Snippet: The antibodies used include TSG101 (Genuin Biotech, Cat # 51942), CD81 (Santa Cruz Biotechnology, Cat # sc-18877), AKT (abcam, Cat # AB81283), Phospho-AKT (abcam, Cat # AB179463 ), NF-κB p65 (abcam, Cat # AB32536), Phospho-NF-κB p65 (Cell Signaling Technology, Cat # 3033S), Notch2 (Santa Cruz Biotechnology, Cat # sc-51869), RBP-Jκ (Santa Cruz Biotechnology, Cat # sc-271128), and β-actin (Bioss, Cat # bs-0061R).

Techniques: Activation Assay, Expressing, Control, Luciferase, Reporter Assay, Transfection, Mutagenesis, Western Blot, Immunofluorescence

Journal: Materials Today Bio

Article Title: Exosomal lncRNA OTUD6B-AS1 as a pathogenic nanocarrier promotes inflammatory macrophage polarization in endometritis via a targetable ceRNA circuit

doi: 10.1016/j.mtbio.2026.103027

Figure Lengend Snippet: Notch2 mediates the regulatory effect of the lncRNA OTUD6B-AS1/miR-128 axis on macrophage activation. (A) Predictive analysis of miR-128 targets using multiple databases. (B) Western blotting analysis of Notch2 protein levels in macrophages treated with Control-exo or LPS-exo. (C) Western blotting analysis of Notch2 protein levels in macrophages transfected with OE-NC or OE-lncRNA. (D – H) Western blotting analysis of Notch2, RBP-Jκ, and p-p65 (D), along with immunofluorescence (IF) quantitative analysis of iNOS (E, F) and Arg1 (G, H) protein levels in macrophages treated with OE-NC or OE-lncRNA and the Notch2 inhibitor DAPT. (I) Luciferase reporter assay in HEK293T cells co-transfected with WT or MUT Notch2 3′UTR reporter plasmids and miR-128 mimic or mimic NC. (J, K) Relative protein (J) and mRNA (K) expression levels of Notch2 in macrophages transfected with miR-128 mimic or mimic NC. (L – P) Western blotting analysis of Notch2, RBP-Jκ, and p-p65 (L), along with IF quantitative analysis of iNOS (M, N) and Arg1 (O, P) protein levels in macrophages co-treated with miR-128 inhibitor or inhibitor NC and DAPT. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Article Snippet: The antibodies used include TSG101 (Genuin Biotech, Cat # 51942), CD81 (Santa Cruz Biotechnology, Cat # sc-18877), AKT (abcam, Cat # AB81283), Phospho-AKT (abcam, Cat # AB179463 ), NF-κB p65 (abcam, Cat # AB32536), Phospho-NF-κB p65 (Cell Signaling Technology, Cat # 3033S), Notch2 (Santa Cruz Biotechnology, Cat # sc-51869), RBP-Jκ (Santa Cruz Biotechnology, Cat # sc-271128), and β-actin (Bioss, Cat # bs-0061R).

Techniques: Activation Assay, Western Blot, Control, Transfection, Immunofluorescence, Luciferase, Reporter Assay, Expressing

Journal: Materials Today Bio

Article Title: Exosomal lncRNA OTUD6B-AS1 as a pathogenic nanocarrier promotes inflammatory macrophage polarization in endometritis via a targetable ceRNA circuit

doi: 10.1016/j.mtbio.2026.103027

Figure Lengend Snippet: A proposed model illustrating the exosome-mediated lncRNA OTUD6B-AS1/miR-128/Notch2 axis in aggravating endometritis. Upon LPS-induced damage, endometrial epithelial cells (EECs) release increased exosomes carrying elevated levels of lncRNA OTUD6B-AS1. These exosomes are taken up by endometrial macrophages. The transferred lncRNA OTUD6B-AS1 acts as a molecular sponge to sequester miR-128, leading to the derepression and upregulation of its target gene, Notch2. The enhanced Notch2 signaling subsequently promotes macrophage polarization towards a pro-inflammatory M1 phenotype, characterized by increased NF-κB activation and iNOS expression, thereby exacerbating endometrial inflammation and tissue damage.

Article Snippet: The antibodies used include TSG101 (Genuin Biotech, Cat # 51942), CD81 (Santa Cruz Biotechnology, Cat # sc-18877), AKT (abcam, Cat # AB81283), Phospho-AKT (abcam, Cat # AB179463 ), NF-κB p65 (abcam, Cat # AB32536), Phospho-NF-κB p65 (Cell Signaling Technology, Cat # 3033S), Notch2 (Santa Cruz Biotechnology, Cat # sc-51869), RBP-Jκ (Santa Cruz Biotechnology, Cat # sc-271128), and β-actin (Bioss, Cat # bs-0061R).

Techniques: Activation Assay, Expressing