Clinical Medical School, Chengdu University of Traditional Chinese Medicine, Chengdu, China.
Department of Gynecology, Chongqing Hospital of Traditional Chinese Medicine, Chongqing 400021, China.
Mol Immunol. 2024 Jul;171:105-114. doi: 10.1016/j.molimm.2024.05.007. Epub 2024 May 30.
Chlamydia trachomatis (CT) is the leading cause of bacterial sexually transmitted diseases worldwide, which can cause diseases such as pelvic inflammatory disease, and cervical and fallopian tube inflammation, and poses a threat to human health. Rosmarinic acid (RosA) is an active ingredient of natural products with anti-inflammatory and immunomodulatory effects. This study aimed to investigate the role of RosA in inhibiting autophagy-regulated immune cells-CD8+ T cells via the Ras/Raf/MEK/ERK signaling pathway in a CT-infected mouse model. Mice were inoculated with CT infection solution vaginally, and the mechanistic basis of RosA treatment was established using H&E staining, flow cytometry, immunofluorescence, transmission electron microscopy, and western blot. The key factors involved in RosA treatment were further validated using the MEK inhibitor cobimetinib. Experimental results showed that both RosA and the reference drug azithromycin could attenuate the pathological damage to the endometrium caused by CT infection; flow cytometry showed that peripheral blood CD8+ T cells increased after CT infection and decreased after treatment with RosA and the positive drug azithromycin (positive control); immunofluorescence showed that endometrial CD8 and LC3 increased after CT infection and decreased after RosA and positive drug treatment; the results of transmission electron microscopy showed that RosA and the positive drug azithromycin inhibited the accumulation of autophagosomes; western bolt experiments confirmed the activation of autophagy proteins LC3Ⅱ/Ⅰ, ATG5, Beclin-1, and p62 after CT infection, as well as the inhibition of Ras/Raf/MEK/ERK signaling. RosA and azithromycin inhibition of autophagy proteins activates Ras/Raf/MEK/ERK signaling. In addition, the MEK inhibitor cobimetinib attenuated RosA's protective effect on endometrium by further activating CD8+ T cells on a CT-induced basis, while transmission electron microscopy, immunofluorescence, and western blots showed that cobimetinib blocked ERK signals activation and further induced phagocytosis on a CT-induced basis. These data indicated that RosA can activate the Ras/Raf/MEK/ERK signaling pathway to inhibit autophagy, and RosA could also regulate the activation of immune cells-CD8+T cells to protect the reproductive tract of CT-infected mice.
沙眼衣原体（CT）是全球细菌性性传播疾病的主要原因，可引起盆腔炎、宫颈和输卵管炎症等疾病，对人类健康构成威胁。迷迭香酸（RosA）是具有抗炎和免疫调节作用的天然产物的活性成分。本研究旨在探讨 RosA 通过 Ras/Raf/MEK/ERK 信号通路抑制 CT 感染小鼠模型中自噬调节免疫细胞-CD8+T 细胞的作用。通过阴道接种 CT 感染溶液感染小鼠，使用 H&E 染色、流式细胞术、免疫荧光、透射电子显微镜和 Western blot 建立 RosA 治疗的机制基础。使用 MEK 抑制剂 cobimetinib 进一步验证 RosA 治疗的关键因素。实验结果表明，RosA 和参考药物阿奇霉素均可减轻 CT 感染引起的子宫内膜病理损伤；流式细胞术显示 CT 感染后外周血 CD8+T 细胞增加，RosA 和阳性药物阿奇霉素（阳性对照）治疗后减少；免疫荧光显示 CT 感染后子宫内膜 CD8 和 LC3 增加，RosA 和阳性药物治疗后减少；透射电镜结果表明 RosA 和阳性药物阿奇霉素抑制自噬体的积累；Western bolt 实验证实 CT 感染后自噬蛋白 LC3Ⅱ/Ⅰ、ATG5、Beclin-1 和 p62 激活，Ras/Raf/MEK/ERK 信号抑制。RosA 和阿奇霉素抑制自噬蛋白激活 Ras/Raf/MEK/ERK 信号。此外，MEK 抑制剂 cobimetinib 通过进一步激活 CT 诱导的 CD8+T 细胞，减弱 RosA 对子宫内膜的保护作用，而透射电子显微镜、免疫荧光和 Western blot 表明 cobimetinib 阻断 ERK 信号激活并进一步诱导 CT 诱导的吞噬作用。这些数据表明 RosA 可以激活 Ras/Raf/MEK/ERK 信号通路抑制自噬，并且 RosA 还可以调节免疫细胞-CD8+T 细胞的激活以保护 CT 感染小鼠的生殖道。
