College of Pharmacy, Anhui University of Chinese Medicine, Hefei, 230012, PR China; Institute of Traditional Chinese Medicine Resources Protection and Development, Anhui Academy of Chinese Medicine, Hefei, 230012, PR China.
College of Pharmacy, Anhui University of Chinese Medicine, Hefei, 230012, PR China.
J Ethnopharmacol. 2023 Aug 10;312:116489. doi: 10.1016/j.jep.2023.116489. Epub 2023 Apr 11.
Clinopodium chinense (Benth.) O. Kuntze (C. chinense) is a Chinese herbal medicine used in treating gynecological hemorrhagic diseases for hundreds of years. Flavonoids are one kind of the major components in C. chinense. The flavonoids of C. chinense (TFC) have a vital role in treating endometritis but the underlying therapeutic mechanisms of TFC against endometritis have been rarely reported.
To elucidate the therapeutic effect and possible mechanisms of TFC against lipopolysaccharide (LPS)-induced endometritis in vivo and LPS-induced primary mouse endometrial epithelial cells (MEECs) injury in vitro.
The holistic phytochemicals of the TFC and TFC-contained serum were screened and identified using UPLC-Q-TOF-MS. The model of endometritis was established by intrauterine injection of LPS (5 mg/mL) into female BALB/c mice, and the model mice were treated with TFC for 7 days. The value of MPO was measured by Myeloperoxidase assay kit, the pathological changes in the endometrium were evaluated using H&E staining and transmission electron microscope (TEM), the secretions of IL-18, IL-1β and TNF-α were determined by ELISA kits, the mRNA expressions of IL-18, IL-1β and TNF-α were determined by RT-PCR assay, and the protein levels of TLR4, IKBα, p-IKBα, p65, p-p65, caspase-1, ASC, NLRP3 and GSDMD were measured by Western blot. Subsequently, MEECs were isolated from the uterus of pregnant female mice, injured by LPS for 24 h and incubated with the TFC-contained serum. Finally, cell viability, LDH release, hoechst 33342/PI staining, immunofluorescence staining, scanning electron microscope observation, ELISA assay, RT-PCR detection and Western blot analysis were carried out to further validate the therapeutic effect and the underlying mechanisms of TFC.
A total of 6 compounds in the plasma of mice after being intragastric administrated of TFC were identified. The results in vivo showed that TFC significantly reduced MPO value and alleviated pathological injury of the endometrium. Furthermore, TFC significantly decreased the serum IL-18, IL-1β and TNF-α levels, and the mRNA levels of IL-18, IL-1β and TNF-α. TFC also inhibited the expressions of TLR4, p-IKBα, p-p65, caspase-1, ASC, NLRP3 and GSDMD. Besides, compared with the model group in MEECs cells, TFC-contained serum prevented pyroptosis, decreased the levels of IL-18 and IL-1β, and inhibited the mRNA expressions of IL-18, IL-1β and GSDMD. TFC-contained serum also reversed the activation of NLRP3 inflammasome caused by nigericin, and restrainted the translocation of NF-κB into nuclear.
TFC protects mice endometritis from the injury of LPS via suppressing the activation of NLRP3 inflammasome and pyroptosis, the underlying mechanisms of which were related to restraining the TLR4/NF-κB/NLRP3 pathway activation.
荆芥（Benth.）O. Kuntze（C. chinense）是一种中药，用于治疗妇科出血性疾病已有数百年历史。类黄酮是 C. chinense 的主要成分之一。荆芥中的类黄酮（TFC）在治疗子宫内膜炎方面起着重要作用，但 TFC 治疗子宫内膜炎的潜在治疗机制却鲜有报道。
阐明 TFC 对体内脂多糖（LPS）诱导的子宫内膜炎和体外 LPS 诱导的原代小鼠子宫内膜上皮细胞（MEEC）损伤的治疗作用及可能机制。
采用 UPLC-Q-TOF-MS 对 TFC 及其含药血清中的全植物化学成分进行筛选和鉴定。通过向雌性 BALB/c 小鼠宫腔内注射 LPS（5mg/mL）建立子宫内膜炎模型，并用 TFC 处理 7 天。用髓过氧化物酶（MPO）测定试剂盒测定 MPO 值，用 H&E 染色和透射电镜（TEM）评估子宫内膜的病理变化，用 ELISA 试剂盒测定 IL-18、IL-1β 和 TNF-α 的分泌量，用 RT-PCR 检测 IL-18、IL-1β 和 TNF-α 的 mRNA 表达水平，用 Western blot 测定 TLR4、IKBα、p-IKBα、p65、p-p65、caspase-1、ASC、NLRP3 和 GSDMD 的蛋白水平。随后，从怀孕雌鼠子宫中分离 MEECs，用 LPS 损伤 24 小时，并用含 TFC 的血清孵育。最后，进行细胞活力、LDH 释放、hoechst 33342/PI 染色、免疫荧光染色、扫描电子显微镜观察、ELISA 检测、RT-PCR 检测和 Western blot 分析，进一步验证 TFC 的治疗效果和潜在机制。
共鉴定出 TFC 灌胃后小鼠血浆中的 6 种化合物。体内研究结果表明，TFC 能显著降低 MPO 值，减轻子宫内膜的病理损伤。此外，TFC 还能显著降低血清中 IL-18、IL-1β 和 TNF-α水平，以及 IL-18、IL-1β 和 TNF-α 的 mRNA 水平。TFC 还抑制了 TLR4、p-IKBα、p-p65、caspase-1、ASC、NLRP3 和 GSDMD 的表达。此外，与模型组 MEECs 细胞相比，TFC 含药血清可预防细胞焦亡，降低 IL-18 和 IL-1β 水平，并抑制 IL-18、IL-1β 和 GSDMD 的 mRNA 表达。TFC 含药血清还可逆转 Nigericin 诱导的 NLRP3 炎性小体的激活，并抑制 NF-κB 向核内转移。
TFC 通过抑制 NLRP3 炎性小体和细胞焦亡的激活，对 LPS 诱导的小鼠子宫内膜炎起到保护作用，其潜在机制与抑制 TLR4/NF-κB/NLRP3 通路的激活有关。
