Meloxicam Inhibited the Proliferation of LPS-Stimulated Bovine Endometrial Epithelial Cells Through Wnt/β-Catenin and PI3K/AKT Pathways

Abstract

Meloxicam is a non-steroidal anti-inflammatory drug and has been used to relieve pain and control inflammation in cows with metritis and endometritis. Meloxicam has been found to be effective in inhibiting tissue or cell growth when it is used as an anti-inflammatory therapy. However, the influence of meloxicam on bovine endometrial regeneration has not been reported. This study was to research the effect of meloxicam (0.5 and 5 μM) on the proliferation of primary bovine endometrial epithelial cells (BEECs) stimulated by Escherichia coli lipopolysaccharide. The cell viability, cell cycle, and cell proliferation were evaluated by Cell Counting Kit-8, flow cytometry, and cell scratch test, respectively. The mRNA transcriptions of prostaglandin-endoperoxide synthase 1 (PTGS1) and PTGS2, Toll-like receptor 4, and proliferation factors were detected using quantitative reverse-transcription polymerase chain reaction. The activations of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) and Wnt/β-catenin pathways were determined using western blot and immunofluorescence. As a result, co-treatment of meloxicam and lipopolysaccharide inhibited (P < 0.05) the cell cycle progression and reduced (P < 0.05) the cell healing rate and the mRNA level of proliferation factors as compared with the cells treated with lipopolysaccharide alone. Meloxicam decreased (P < 0.05) the lipopolysaccharide-induced PTGS2 gene expression. Neither lipopolysaccharide nor meloxicam changed PTGS1 mRNA abundance (P > 0.05). Meloxicam inhibited (P < 0.05) the lipopolysaccharide-activated Wnt/β-catenin pathway by reducing (P < 0.05) the protein levels of β-catenin, c-Myc, cyclin D1, and glycogen synthase kinase-3β and prevented the lipopolysaccharide-induced β-catenin from entering the nucleus. Meloxicam suppressed (P < 0.05) the phosphorylation of PI3K and AKT. In conclusion, meloxicam alone did not influence the cell cycle progression or the cell proliferation in BEEC but caused cell cycle arrest and inhibited cell proliferation in lipopolysaccharide-stimulated BEEC. This inhibitory effect of meloxicam was probably mediated by Wnt/β-catenin and PI3K/AKT pathways.

The effect of selenium on the proliferation of bovine endometrial epithelial cells in a lipopolysaccharide-induced damage model

“…The migration of cells was determined by scratch wound healing assay [ 13 ]. Briefly, BEECs grew to 90% confluence in a six-well plate.…”

“…The cDNA was synthesized from 900 ng extracted RNA from each sample using a HiScript II 1st Strand cDNA Synthesis Kit (R211-1, Vazyme, China) according to the manufacturer’s instructions. Primer sequences are listed in Table 1 according to a previous paper [ 13 ] and were purified and sequenced by TsingKe Biotech, China. The sequence results were analyzed using BLAST and compared to the GenBank database.…”

“…Gram-negative bacteria such as Escherichia coli ( E. coli ) have been identified as the main pathogenic microorganism causing bovine endometritis. Lipopolysaccharide (LPS), as its main virulence factor, has been widely used to establish endometritis models in vitro [ 12 , 13 ].…”

Anti-inflammatory effects of progesterone through NF-κB and MAPK pathway in lipopolysaccharide- or Escherichia coli-stimulated bovine endometrial stromal cells

“…The RNA was reverse transcribed into cDNA using the PrimerScript RT regent Kit gDNA Eraser (DRR047A, TaKaRa, Japan). The quantitative PCR was carried out using a CFX 96 Real-Time PCR Detection System (Bio-Rad, USA) as previously described [ 20 ]. The 2 -△△Ct method was used to analyze the relative mRNA abundance.…”

The effect and mechanism of selenium supplementation on the proliferation capacity of bovine endometrial epithelial cells exposed to lipopolysaccharide in vitro under high cortisol background

“…Therefore, preventing E. coli infection will prevent the adverse effects associated with lipopolysaccharide ( LPS ) and other bacterial infections during the later postpartum period. For these reasons, E. coli was chosen for the construction of a cell model, and LPS was widely used in vitro endometritis models ( Zhao et al, 2018 ; Cui et al, 2021 ).…”