LPS/TLR4-mediated stromal cells acquire an invasive phenotype and are implicated in the pathogenesis of adenomyosis

Abstract

The present study tested whether the LPS/TLR4 signal pathway in endometrial stromal cells is essential for the pathogenesis of adenomyosis. We tested the expression of TLR4, MD2 in the endometrium without adenomyosis (CE), the eutopic endometrium with adenomyosis (EuE) and the ectopic endometrium with adenomyosis (EE). We isolated the stromal cells from CE, EuE and EE (CESC, EuESC, EESC), treated with lipopolysaccharide (LPS) and TLR4 antagonist and detected the cell viability. And we also measured the key protein of the TLR4 signal pathway and inflammatory proliferation and invasive growth of experimental cells. We found that the viability of experimental cells treated with LPS was significantly greater than that of the non-treated cells, blocked by the TLR4 antagonist VIPER. TLR4 signal pathway and inflammatory proliferation and invasive growth of experimental cells stimulated by LPS, and it was inhibited by VIPER. This study suggested that stromal cells were activated by the TLR4 signalling pathway, which processed the cellular inflammatory proliferation and invasive growth involved in the pathogenesis of adenomyosis.

Figure 1

The expression of TLR4 in CE, EuE, and EE by IHC (A), WB (B,C), and RT-PCR (D). The gels had been run under the same experimental conditions. CE = endometrium without adenomyosis, EuE = eutopic endometrium with adenomyosis, EE = ectopic endometrium with adenomyosis. 1–10 means the sample label. (Ns p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.005).

Figure 2. CESC, EuESC, and EESC were isolated.

The immunoexpression of TLR4 was found in CK19-negative and vimentin-positive stromal cells. The expression of MD2, MyD88, NF-κB, VEGF, EGF, MMP2, IL-6 and TGF-β in CESC ranged 0–100, in EuESC range 200–210, and in EESC ranged 240–270 (×200).

Figure 3. Immunofluorescence showed the expression of TLR4 in vimentin-positive stromal cells (×200).

Figure 4. The cell viability was assessed by CCK-8 assay (J).

The expression of TLR4, MD2, MyD88 and NF-κB in CESC, EuESC and EESC was measured by WB (A–E) and RT-PCR (F–I) after treated with LPS and VIPER. CESC: stromal cells from endometrium without adenomyosis, EuESC: stromal cells from eutopic endometrium with adenomyosis, EESC: stromal cells from ectopic endometrium with adenomyosis (Ns p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.005).

Figure 5

The expression of VEGF, EGF, MMP2, IL-6, TGF-β in CESC, EuESC and EESC was measured by WB/ELISA (A–F) and RT-PCR (G–K) after treated with LPS and VIPER. (Ns p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.005).