Fig 1.
Immunohistochemistry analysis of HMGB-1 expression in the human endometrium.
In control endometrium, HMGB-1 was expressed in the glandular cells of the epithelium and in stromal cells (N = 27). No significant differences were observed according to the menstrual cycle (A). In EM from a patient with endometriosis, significantly increased HMGB-1 expression was detected in the early and mid-secretory phase as compared to the control EM (N = 33, B). There were no significant differences in HMGB-1 expression between the two groups during the proliferative phase. *, P < 0.05 versus the control.
Fig 2.
Induction of necrosis in HESCs altered the secretion of HMGB-1.
Cell viability was detected by MTT assays after treatment of HESCs with H2O2 (A). Extracellular release of HMGB-1 was measured following H2O2-induced necrosis. Relative mRNA (B) and protein levels (C, D) are shown. *, P < 0.05 versus 0 μM H2O2; **, P < 0.05 versus 25 μM H2O2. a, fresh medium was used as a control; b, 0 μM H2O2; c, 25 μM H2O2; d, 50 μM H2O2.
Fig 3.
HESC proliferation and TLR4 expression were altered in a concentration-dependent manner after treatment with rHMGB-1.
At 48 h after treatment with different concentrations of rHMGB-1, cell proliferation was measured (A). Cell proliferation after 24 or 48 h of treatment with rHMGB-1 (B). Relative mRNA and protein levels of TLR4 are shown following treatment with rHMGB-1 (C, D). *, P < 0.05 versus the control; **, P < 0.05 versus 5 ng/mL rHMGB-1; ***, P < 0.05 versus 10 ng/mL rHMGB-1.
Fig 4.
HESC proliferation and TLR4 expression were altered in a time-dependent manner after treatment with rHMGB-1.
Changes in cell proliferation were measured at 0, 12, 24, and 48 h after treatment with 15 ng/mL rHMGB-1 (A). Relative mRNA (B) and protein levels (C, D) of TLR4 are shown. #, P < 0.05 versus 12 h; ##, P < 0.05 versus 24 h.
Fig 5.
Effects of TLR4 inhibition on HESC proliferation and TLR4 expression following treatment with rHMGB-1.
Cells were treated with the TLR4 antagonist LPS-RS, rHMGB-1-induced cell proliferation was measured (A). TLR4 mRNA and protein levels were measured (B–D). *, P < 0.05 versus the control.
Fig 6.
Effects of NF-κB inhibition on rHMGB-1-induced HESC proliferation and TLR4 expression.
The NF-κB pathways was blocked using TPCA-1, and rHMGB-1-dependent cell proliferation was measured (A). TLR4 expression was analyzed using qRT-PCR (B) and western blotting (C, D) following treatment with different concentrations of rHMGB-1. Changes in IL-6 secretion were measured following inhibition of the NF-κB pathway by TPCA-1 (E). *, P < 0.05 versus the control.