Department of Laboratory Medicine, Putuo Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, China.
Department of Obstetrics and Gynecology, Putuo Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, China.
Front Endocrinol (Lausanne). 2023 Oct 5;14:1255925. doi: 10.3389/fendo.2023.1255925. eCollection 2023.
Increased inflammation and cytokine levels are considered risk factors and promoters of preterm birth (PTB). However, the regulatory mechanism of pregnancy-related inflammation remains unclear. Toll-like receptor 4 (TLR4) plays a critical role in inflammatory responses in various diseases. Therefore, our study aimed to investigate whether TLR4 is involved in the inflammatory responses during uterine activation for labor, with the goal of identifying potential biomarkers for uterine activation at term.
We used flow cytometry to detect TLR4 expression on CD14+ maternal blood monocytes in the first, second, and third trimesters. ELISA was employed to measure TLR4 and cytokines levels in the maternal serum of term non-labor (TNL), term labor (TL) women and LPS induced preterm labor and PBS injected controls. TLR4siRNA was transfected into the human myometrial smooth muscle cells (HMSMCs), which were subsequently treated with IL-1β. The mRNA and protein levels of TLR4, uterine contraction-related protein connexin 43 (CX43), oxytocin receptor (OTR), MAPK/NF-κB signaling pathway, and cytokines were analyzed using qRT-PCR, western blotting, and immunohistochemistry.
The study revealed TLR4 expression on CD14+ maternal blood monocytes was higher in the third trimester group compared to the first and second trimester groups (p<0.001). Maternal serum concentrations of TLR4 and cytokines were significantly higher in the TL group than the TNL group (p<0.001). TLR4, OTR, CX43, activated MAPK/NF-κB expression, and cytokines levels were upregulated in TL group, and similarly significantly higher in the LPS-induced preterm group than in the control group. Using the HMSMCs we demonstrated that TLR4siRNA transfection suppressed contractility. Interfering with TLR4 expression reduced the expression of OTR, CX43, cytokines, and MAPK/NF-κB activation. There was a significant positive relationship between TLR4 expression and the inflammatory status in the myometrium. ROC analysis indicated that TLR4 and cytokines may serve as potential biomarkers for predicting uterine activation for labor.
Our data suggest that TLR4 and cytokines can act as stimulators of uterine activation for labor at term. Furthermore, the MAPK/NF-κB pathway appears to be one of the potential signaling pathways mediating TLR4's regulation of parturition initiation.
炎症和细胞因子水平的增加被认为是早产（PTB）的风险因素和促进因素。然而，与妊娠相关的炎症的调节机制尚不清楚。Toll 样受体 4（TLR4）在各种疾病的炎症反应中起着关键作用。因此，我们的研究旨在探讨 TLR4 是否参与子宫激活分娩的炎症反应，以确定足月子宫激活的潜在生物标志物。
我们使用流式细胞术检测第一、二、三孕期 CD14+母体血单核细胞上 TLR4 的表达。采用 ELISA 法检测足月未临产（TNL）、足月临产（TL）妇女及 LPS 诱导早产和 PBS 注射对照组母血清中 TLR4 和细胞因子水平。将 TLR4siRNA 转染入人子宫平滑肌细胞（HMSMCs），用 IL-1β 处理。采用 qRT-PCR、western blot 和免疫组化分析 TLR4、与子宫收缩相关的蛋白连接蛋白 43（CX43）、催产素受体（OTR）、MAPK/NF-κB 信号通路和细胞因子的 mRNA 和蛋白水平。
研究显示，第三孕期组 CD14+母体血单核细胞上 TLR4 的表达高于第一和第二孕期组（p<0.001）。TL 组母血清 TLR4 和细胞因子浓度明显高于 TNL 组（p<0.001）。TL 组 TLR4、OTR、CX43、激活的 MAPK/NF-κB 表达和细胞因子水平上调，LPS 诱导的早产组明显高于对照组。我们用 HMSMCs 证明 TLR4siRNA 转染抑制了收缩性。干扰 TLR4 表达降低了 OTR、CX43、细胞因子和 MAPK/NF-κB 激活的表达。TL 组 TLR4 表达与子宫肌层炎症状态呈显著正相关。ROC 分析表明，TLR4 和细胞因子可能是预测子宫激活分娩的潜在生物标志物。
我们的数据表明，TLR4 和细胞因子可作为足月子宫激活分娩的刺激物。此外，MAPK/NF-κB 通路可能是 TLR4 调节分娩启动的潜在信号通路之一。
