MicroRNA-146 attenuates lipopolysaccharide induced ovarian dysfunction by inhibiting the TLR4/NF- κB signaling pathway

Abstract

Premature ovarian insufficiency (POI) is a disease that seriously affects women's reproductive function and even leads to lifelong infertility. Little is known about the mechanism of lipopolysaccharide (LPS)-induced ovarian dysfunction. Thus, we aimed to identify the role of the up-regulation of microRNA (miRNA)-146 expression offered protection against ovarian dysfunction by inhibiting the toll-like receptor (TLR) 4, TLR4/phosphorylated (p)-nuclear factor (NF)-κB signaling pathway and inflammatory cytokine tumor necrosis factor (TNF)-a and Interleukin (IL)-6. In an in vivo study, we established an LPS-induced ovarian dysfunction mouse model. The mouse ovarian granulosa cells were transfected with miR-146 mimic or negative controls or inhibitor and then treated with LPS. Therefore, cell viability, cells apoptosis, IL-6 and TNF-a, TLR4, NF- κB were assessed, respectively. These results demonstrated that the up-regulation of miRNA-146 expression may protect against LPS-induced ovarian dysfunction and markedly increased the cell viability, and significantly reduced the ovarian granulosa cells apoptotic rate, and down-regulated IL-6 and TNF-a expression. In addition, miRNA-146 exerted protective ovarian functions might be via inhibiting TLR4/NF-κB signaling pathway. In summary, we reveal the up-regulation of miRNA-146 expression mitigated ovarian dysfunction by negatively regulating expression of the IL-6 and TNF-a, which may shed light on the potential molecular mechanisms of overexpression of miRNA-146 may reversed the ovarian dysfunction by inhibiting the TLR4/ NF-κB signaling pathway.

Keywords: apoptosis; lipopolysaccharide (LPS); miRNA-146; ovarian dysfunction; ovarian insufficiency (POI).

Graphical abstract

Figure 1.

LPS suppresses viability of granulosa cells.

Figure 2.

Transfection efficiency of miRNA-146 in granulosa cells. a: miNA-146 mimics, inhibitors and transfection efficiency. The expression levels of miRNA-146 in granulosa cells and negative control group were detected by PCR. GAPDH is quality control, *** P < 0.001.

Figure 3.

The levels of miRNA-146 expression and IL-6, and TNF-a in granulosa cells. C: Granulosa cells were transfected using miR-146 mimics, miRNA-146 inhibitors or negative control (NC). The effect of miRA-146 on siTLR4 expression in granulosa cells was measured by qRT PCR and Western blot. *P < 0.01.

Figure 4.

The levels of Bcl-2 and Bax protein were analyzed by western blotting. A) Overexpression of miR-146 repress apoptosis of OGCs by increasing Bcl-2, ** P < 0.01, * P < 0.05, B) versus the control. C) Overexpression of miR-146 inhibited apoptosis of OGCs by reducing Bax, ***P < 0.001, ** P < 0.01, * P < 0.05, versus the control.

Figure 5.

The levels of miR-146 expression and TLR4 and NF-ĸB expression. a: TLR4 expression. These granulosa cells were transfected with miR-146 mimics, miR-146 inhibitors, control, or TLR4 siRNA (si-TLR4), and were treated using concentration of 10 μg/mL LPS for 12 h by qPCR. * P < 0.05; ** P < 0.01; *** P < 0.001. B: NF-ĸB expression. These granulosa cells were transfected with miR-146 mimics, miR-146 inhibitors, control, or TLR4 siRNA (si-TLR4), and were treated using concentration of 10 μg/mL LPS for 12 h by qPCR. * P < 0.05; ** P < 0.01; *** P < 0.001. C: The levels of TLR4 and NF-ĸB expression in granulosa cells were detected by western blot analysis.