Effect of VD3 on cell proliferation and the Wnt signaling pathway in bovine endometrial epithelial cells treated with lipopolysaccharide.

ABSTRACT

Vitamin D (VD) deficiency plays an important role in the occurrence and development of various uterine diseases. At present, most studies on the mechanism of VD in the Wnt signaling pathway focus on cancer, while there are no relevant reports on its mechanism in endometritis. This study investigated the effect of vitamin D3 (VD3) on the Wnt signaling pathway in endometrial epithelial cells (BEECs) induced by lipopolysaccharide (LPS). BEECs obtained from bovine uteri were treated with VD3 (0, 50 ng/mL) and LPS (0, 10, 100 ng/mL) separately or in combination, and treated with the Wnt signaling pathway inhibitor IWR-1 to study the mechanism of action. The proliferation of BEECs was evaluated by a CCK-8 assay. qRT-PCR was used to assess the gene expression of Wnt pathway-related factors, including MYC, PCNA, LGR5, GREM1, ß-catenin, FZD7, FZD2, Wnt4 and VDR. The results showed that VD3 had no significant effect on cell proliferation (P > 0.05); LPS inhibited BEEC proliferation in a time- and dose-dependent manner, and cells treated with LPS at different concentrations for 24-48 h in combination with VD3 promoted cell proliferation to varying degrees. IWR-1 inhibited cell proliferation in a time- and concentration-dependent manner, while LPS + IWR-1 treatment also significantly promoted cell proliferation after VD3 treatment (P < 0.01). The qRT-PCR results showed that the expression of Wnt4 and PCNA genes showed different trends with different LPS concentrations for stimulation, and the expression of the MYC and GREM1 genes was only stimulated by high-dose (100 ng/mL) LPS stimulation. The expression of FZD7, LGR5, FZD2 and ß-catenin was upregulated by LPS at both concentrations. LPS + VD3 significantly downregulated the expression of the Wnt pathway-related genes MYC, PCNA, LGR5, GREM1 and ß-catenin (P < 0.001), Wnt4 and FZD2 (P < 0.01), and significantly upregulated the expression of VDR (P < 0.05). After LPS + IWR-1 treatment, the expression of the ß-catenin (P < 0.01) and LGR5 (P < 0.05) genes was significantly downregulated, while the Wnt4 (P < 0.01) and VDR (P < 0.001) genes were significantly upregulated, MYC was downregulated but without a significant difference (P > 0.05). In conclusion, VD3 treatment can mitigate the LPS-induced abnormal expression of Wnt signaling pathway genes in BEECs, showing that the Wnt pathway may be a protective pathway of VD3 against LPS-induced gene overexpression in BEECs. The results suggest that VD3 may play a regulatory role in pathways other than the Wnt signaling pathway. Whether VD3 affects the Wnt signaling pathway by affecting Wnt4 gene expression requires further study.