Oleic acid induces lipogenesis and NLRP3 inflammasome activation in organotypic mouse meibomian gland and human meibomian gland epithelial cells.

The accumulation of oleic acid (OA) in the meibum from patients with meibomian gland dysfunction (MGD) suggests that it may contribute to meibomian gland (MG) functional disorder, as it is a potent stimulator of acne-related lipogenesis and inflammation in sebaceous gland. Therefore, we investigate whether OA induces lipogenesis and inflammasome activation in organotypic cultured mouse MG and human meibomian gland epithelial cells (HMGECs). Organotypic cultured mouse MG and HMGECs were exposed to OA or combinations with specific AMPK agonists 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR). Lipogenic status, ductal keratinization, squamous metaplasia, NLRP3/ASC/Caspase-1 inflammasome activation, proinflammatory cytokine IL-1ß production, and AMPK pathway phosphorylation in MG were subsequently examined by lipid staining, immunofluorescence staining, immunohistochemical staining, ELISA assay, and Western blot analyses. We found that OA significantly induced lipid accumulation, ductal keratinization, and squamous metaplasia in organotypic cultured MG, as evidenced by increased lipids deposition within acini and duct, upregulated expression of lipogenic proteins (SREBP-1 and HMGCR), and elevation of K10/Sprr1b. Additionally, OA induced NLRP3/ASC/Caspase-1 inflammasome activation, cleavage of Caspase-1, and production of downstream proinflammatory cytokine IL-1ß. The findings of lipogenesis and NLRP3-related proinflammatory response in OA-stimulated HMGECs were consistent with those in organotypic cultured MG. OA exposure downregulated phospho-AMPK in two models, while AICAR treatment alleviated lipogenesis by improving AMPK/ACC phosphorylation and SREBP-1/HMGCR expression. Furthermore, AMPK amelioration inhibited activation of the NLRP3/ASC/Caspase-1 axis and secretion of IL-1ß, thereby relieving the OA-induced proinflammatory response. These results demonstrated that OA induced lipogenic disorder and NLRP3 inflammasome activation in organotypic cultured mouse MG and HMGECs by suppressing the AMPK signaling pathway, indicating OA may play an etiological role in MGD.

Urban Particulate Matter Triggers Meibomian Gland Dysfunction.

Purpose: The meibomian gland (MG), as the largest modified sebaceous gland, is potentially damaged by urban particulate matter (UPM) based on epidemiological evidence, but the specific experimental mechanisms remain unknown. This study investigated the effects of UPM on MG dysfunction (MGD) in rodent models. Methods: Female C57BL/6J mice received eye drops containing UPM suspension or PBS for 14 days. The proliferative capacity and progenitor of MG were evaluated by immunofluorescence. Cell apoptosis was confirmed by TUNEL assay, along with the analysis of caspase family expression. Lipid accumulation was visualized by Oil Red O staining and LipidTox staining. Ductal hyperkeratinization, neutrophil infiltration, and pyroptosis activation were detected through immunostaining. The relative gene expression and signaling pathway activation were determined by Western blot analysis. Results: Administration of UPM caused MGD-like clinical signs, manifested as distinct corneal epithelial erosion, increased MG orifice occlusion, and glandular dropout. UPM exposure significantly induced progenitor loss, cellular apoptosis, and lipogenic disorder in MG, by reducing P63/Lrig1 expression and increasing cleaved caspase-8, -9, and -3 and meibum lipogenic protein (HMGCR/SREBP-1) expression. UPM-treated mice exhibited ductal hyperkeratinization and neutrophil recruitment. Simultaneously, pyroptosis was motivated, as indicated by the heightened expression of NLRP3 and the cleavage of caspase-1 and -4 and gasdermin D, as well as the increase in IL-1ß and IL-18 downstream. The underlying pathological mechanisms of UPM involve the phosphorylation of mitogen-activated protein kinase and nuclear factor-κB. Conclusions: These results provided direct evidence for the toxicity of UPM in MG. UPM-induced activation of pyroptosis and mitogen-activated protein kinase/nuclear factor-κB signaling pathway might account for the inflammatory MGD.

The dietary flavonoid isoliquiritigenin induced apoptosis and suppressed metastasis in melanoma cells: An in vitro and in vivo study.

AIMS: This study aimed to explore the role of Isoliquiritigenin (ISL) in the proliferation and invasion of melanoma cells and investigate the mechanism of action of this compound. MAIN METHODS: The functional roles of ISL in melanoma cells were determined by CCK8 assay, colony formation assay, flow cytometry and wound healing assay. The antitumor activity of ISL was assessed in vivo in a mouse xenograft model using A2058 cells. Quantitative real-time PCR analysis (RT-qPCR) and western blot assays were used to evaluate the gene and protein expression in cell lines or tumor tissue samples. Bioinformatic analysis, luciferase reporter assay, and gene set enrichment analysis (GSEA) were performed to confirm the mechanism of ISL effect on cell growth and metastasis of melanoma. KEY FINDINGS: ISL suppressed proliferation and migration of melanoma cells via downregulation of miR-27a expression. The inhibitory effect of ISL on growth and metastasis of melanoma cells was reversed by ectopic expression of miR-27a. Bioinformatic analysis showed that miR-27a targets POU class 2 homeobox 3 (POU2F3); this result was verified by the luciferase reporter assay and by a decrease in the expression of POU2F3 by miR-27a intervention. GSEA demonstrated that POU2F3 is associated with the c-MYC/p53 signaling pathway and metastasis. POU2F3 knockdown reversed the inhibitory effect of ISL on the growth and metastasis of melanoma. Additionally, POU2F3 was found to be downregulated in melanoma tissue samples and was negatively correlated with miR-27a. SIGNIFICANCE: ISL inhibits proliferation and metastasis of melanoma via the miR-27a/POU2F3/c-MYC/p53 axis; these results may provide a new thought for the treatment of melanoma.