Gynecology, Fuzhou Second Hospital, 350007 Fuzhou, Fujian, China.
Experimental Animal Center, Fujian University of Traditional Chinese Medicine, 350122 Fuzhou, Fujian, China.
Discov Med. 2024 Jan;36(180):140-149. doi: 10.24976/Discov.Med.202436180.13.
Endometritis is a condition usually resulted from the bacterial infection of uterus, causing pelvic disease, sepsis, shock, uterine necrosis and even death if it is inappropriately treated. The aim of this study is to explore the pathogenesis of endometritis, and investigate whether the combination of doxycycline and metronidazole offers stronger protection against lipopolysaccharide (LPS)-induced endometritis, and decipher more about the mechanisms underlying endometritis-related pyroptosis.
Sprague-Dawley (SD) rats were divided into five groups (n = 8 per group): control, model, metronidazole, doxycycline, and combination groups. In control group, the rats were injected with saline, while in other groups, lipopolysaccharide was injected into uterus of the rats to establish endometritis. Hematoxylin-eosin (H&E) staining was performed as part of the histopathological examination of endometrium. The integrity of chromatin and pyroptosis were evaluated by terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay. Western blot and quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) were performed to ascertain the activation of toll-like receptors ()/nuclear factor-kappa B () pathway by detecting protein levels of phosphorylated p50 (p-p50)/p50, phosphorylated nuclear factor-kappa B (p-NF-κB)/NF-κB, phosphorylated IkappaB (p-IκB), and TLR4 protein and mRNA. Development of pyroptosis was also detected by determining the levels of caspase-1 and caspase-5 through Western blot and qRT-PCR. Enzyme-linked immunosorbent assay (ELISA) was used to detect levels of interleukin (IL)-1β, IL-18, IL-2, IL-4, IL-6 and tumor necrosis factor alpha (TNF-α), and flow cytometry was adopted to determine T-helper (Th)1 and Th2 cell percentage to assess the extent of pyroptosis and Th1/Th2 imbalance.
The uterine of the model group exhibited pathological alterations and higher degree of cell apoptosis. Compared with the control rats, model group showed lower protein levels of p-p50/p50 ( < 0.001), p-NF-κB/NF-κB ( < 0.001), p-IκB ( < 0.001), and TLR4 protein ( < 0.001) and mRNA ( < 0.001). Elevated levels of caspase-1 ( < 0.001), caspase-5 ( < 0.001), IL-1β ( < 0.001), IL-18 ( < 0.001), IL-2 ( < 0.01), TNF-α ( < 0.05) and Th1/Th2 ( < 0.001) as well as reduced levels of IL-4 ( < 0.05) and IL-6 ( < 0.01) were observed in the model group, which could however be reversed by metronidazole ( < 0.01) or doxycycline ( < 0.01), with a more significant effect detected if a combination of the two drugs was administered ( < 0.01).
The combination of doxycycline and metronidazole protects against rat endometritis by inhibiting / pathway-mediated inflammation and suppressing pyroptosis.
子宫内膜炎通常是由细菌感染子宫引起的一种疾病，若不及时治疗，可导致盆腔疾病、败血症、休克、子宫坏死甚至死亡。本研究旨在探讨子宫内膜炎的发病机制，研究多西环素和甲硝唑联合应用是否能更有效地预防脂多糖（LPS）诱导的子宫内膜炎，并进一步探讨子宫内膜炎相关细胞焦亡的机制。
将 SD 大鼠分为五组（每组 8 只）：对照组、模型组、甲硝唑组、多西环素组和联合组。对照组大鼠注射生理盐水，其余组大鼠注射 LPS 建立子宫内膜炎模型。采用苏木精-伊红（H&E）染色进行子宫内膜组织病理学检查。末端脱氧核苷酸转移酶（TdT）dUTP 缺口末端标记（TUNEL）检测法评估染色质完整性和细胞焦亡。Western blot 和实时定量逆转录聚合酶链反应（qRT-PCR）检测 toll 样受体（TLR）/核因子-κB（NF-κB）通路的激活，通过检测磷酸化 p50（p-p50）/p50、磷酸化核因子-κB（p-NF-κB）/NF-κB、磷酸化 IκB（p-IκB）和 TLR4 蛋白和 mRNA 的水平来确定。通过 Western blot 和 qRT-PCR 检测半胱氨酸蛋白酶-1（caspase-1）和半胱氨酸蛋白酶-5（caspase-5）的水平来检测细胞焦亡的发生。酶联免疫吸附试验（ELISA）检测白细胞介素（IL）-1β、IL-18、IL-2、IL-4、IL-6 和肿瘤坏死因子-α（TNF-α）的水平，采用流式细胞术检测辅助性 T 细胞（Th）1 和 Th2 细胞的比例，以评估细胞焦亡和 Th1/Th2 失衡的程度。
模型组子宫出现病理改变，细胞凋亡程度较高。与对照组大鼠相比，模型组 p-p50/p50（<0.001）、p-NF-κB/NF-κB（<0.001）、p-IκB（<0.001）和 TLR4 蛋白（<0.001）和 mRNA（<0.001）水平降低。caspase-1（<0.001）、caspase-5（<0.001）、IL-1β（<0.001）、IL-18（<0.001）、IL-2（<0.01）、TNF-α（<0.05）和 Th1/Th2（<0.001）水平升高，IL-4（<0.05）和 IL-6（<0.01）水平降低，但甲硝唑（<0.01）或多西环素（<0.01）治疗后均可逆转，两药联合应用时效果更为显著（<0.01）。
多西环素和甲硝唑联合应用通过抑制 TLR/NF-κB 通路介导的炎症反应和抑制细胞焦亡来保护大鼠子宫内膜炎。
