Oxycodone Alleviates Endometrial Injury via the TLR4/NF- κ B Pathway

Abstract

Endometrial injury is a common female disease. This study was designed to illustrate the effects of oxycodone on mifepristone-induced human endometrial stromal cells (hEndoSCs) injury and delineate the underlying molecular mechanism. hEndoSCs were stimulated with mifepristone to generate the endometrial injury in vitro model. hEndoSCs viability, cytotoxicity, and apoptosis were measured by methyl thiazolyl tetrazolium (MTT) assay, lactate dehydrogenase assay (LDH), and flow cytometry (FCM) analysis, respectively. Meanwhile, quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Western blot assay were conducted to evaluate gene and protein expressions. The secretions of inflammatory cytokines (TNF-α, IL-1β, and IL-6) were measured using enzyme-linked immunosorbent assay (ELISA). The data revealed that mifepristone exposure memorably inhibited hEndoSCs viability and promoted cell apoptosis and inflammatory cytokines secretion, and oxycodone had no cytotoxicity on hEndoSCs. Oxycodone increased hEndoSCs growth, blocked cell apoptosis, enhanced Bcl-2 expression, reduced Bax levels, and decreased the secretion of inflammatory cytokines in mifepristone-induced hEndoSCs, exhibiting the protective effects in endometrial injury. In addition, the TLR4/NF-κB pathway-related protein levels (TLR4 and p-p65) in mifepristone-treated hEndoSCs were enhanced, while these enhancements were inhibited by oxycodone treatment. In conclusion, oxycodone exhibited the protective role in mifepristone-triggered endometrial injury via inhibiting the TLR4/NF-κB signal pathway.

Figure 1

Establishment of endometrial injury model. hEndoSCs were treated with 60 μmol/l mifepristone to generate endometrial injury model in vitro. (a) MTT assay applied to determine cell viability. (b) Apoptotic cells calculated using flow cytometry assay. (c) Quantitative analysis of apoptotic cells. (d–f) Western blot analysis of Bax and Bcl-2 expressions. (h-i) The secretion of inflammatory cytokines (TNF-α, IL-1β, and IL-6) evaluated using ELISA. All experiments were repeated for three times. ^∗∗P < 0.01 vs. control.

Figure 2

Effects of oxycodone on the viability and LDH release of hEndoSCs. hEndoSCs were induced by 1, 5, 10, 15, or 20 μg/ml oxycodone for 24 h. (a) Cell viability evaluated using MTT assay. (b) LDH activity measured and presented as fold of control. All experiments were repeated for three times.

Figure 3

Effects of oxycodone on the mifepristone-induced hEndoSCs injury. hEndoSCs were subjected to 60 μmol/l mifepristone for 48 h and then incubated in 1, 5, or 10 μg/mL oxycodone for 24 h. Cells were divided into five groups: control group, mifepristone group, mifepristone + oxycodone-1 group, mifepristone + oxycodone-5 group, and mifepristone + oxycodone-10 group. (a) Cell viability evaluated using MTT assay. (b) The level of apoptotic hEndoSCs measured using flow cytometry. (c) Quantification of apoptotic cells in different groups. (d–f) Protein and mRNA levels of Bax and Bcl-2 checked using Western blot assay and RT-qPCR. All experiments were repeated for three times. ^∗∗P < 0.01 vs. control. ^#,##P < 0.05, 0.01 vs. the mifepristone treatment group.

Figure 4

Effects of oxycodone on the mifepristone-induced inflammatory response in hEndoSCs. hEndoSCs were subjected to 60 μmol/l mifepristone for 48 h and then incubated in 1, 5, or 10 μg/mL oxycodone for 24 h. Cells were divided into five groups: control group, mifepristone group, mifepristone + oxycodone-1 group, mifepristone + oxycodone-5 group, and mifepristone + oxycodone-10 group. ELISA assay was conducted to evaluate TNF-α, IL-1β, and IL-6. (a–c) Expression in different groups. All experiments were repeated for three times. ^∗∗P < 0.01 vs. control. ^#,##P < 0.05, 0.01 vs. the mifepristone treatment group.

Figure 5

Effects of oxycodone on the TLR4/NF-κB signal pathway in mifepristone-induced hEndoSCs. After treated with mifepristone, hEndoSCs were incubated in different concentrations of oxycodone for 24 h. (a) Protein expression of TLR4 and abundance of p-p65 determined by Western blot assay. (b) mRNA levels of TLR4 determined by RT-qPCR analysis. (c) Quantification of p-p65/p65 ratio expressed. (d) mRNA levels of p65 in different groups measured using RT-qPCR analysis. All experiments were repeated for three times. ^∗∗P < 0.01 vs. control. ^#,##P < 0.05, 0.01 vs. the mifepristone treatment group.