Huang Xiaolan, Luo Xiangmin, Huang Suzhen, Chen Xiaoqing, Qiu Lingling
Department of Reproductive Medicine, The Second Affiliated Hospital of Fujian Medical University, 34 North Zhongshan Road, Licheng District, Quanzhou, Fujian, China.
Department of Rheumatology, The Second Affiliated Hospital of Fujian Medical University, 34 North Zhongshan Road, Licheng District, Quanzhou, Fujian, China.
Funct Integr Genomics. 2024 Jan 8;24(1):6. doi: 10.1007/s10142-024-01284-4.
The aim of this study was to explore the role of forkhead box transcription Factor O1 (FoxO1) in chronic inflammation in polycystic ovary syndrome (PCOS). A PCOS rat model was constructed as an in vivo model by letrozole induction, and granulosa cells (GCs) from PCOS rats were isolated and cultured as an in vitro cellular model. FoxO1 was knocked down by shRNA and siRNA in the PCOS rat model and GCs model, respectively. H&E staining was conducted to evaluate the effect of FoxO1 inhibition on ovarian pathology and dysfunction in PCOS rats. The levels of inflammatory cytokines in the ovaries and uterus of PCOS rats and in GCs were assessed by ELISA. Flow cytometry was used to evaluate the changes in the contents of neutrophils and macrophages in the peripheral blood and spleen of PCOS rats. CCK-8 assays and Annexin V-FITC/PI staining were performed to evaluate the proliferation and apoptosis of GCs. The expression of genes and proteins related to the TLR4/NF-κB/NLRP3 pathway in GCs was determined by RT-qPCR and Western blotting. The results indicated that FoxO1 was highly expressed in PCOS rat model. Inhibition of FoxO1 significantly mitigated the pathological changes and dysfunction in the ovaries of PCOS rats while also suppressing inflammation and fibrosis in the ovaries and uterus. Moreover, knocking down FoxO1 facilitated the restoration of the normal ratio of neutrophils and macrophages in the peripheral blood and spleen of PCOS rats and promoted M2 polarization of macrophages. Additionally, inhibition of FoxO1 promoted the proliferation of GCs and inhibited the inflammatory response in GCs. Furthermore, FoxO1 knockdown inhibited the activation of the NF-κB pathway and the formation of the NLRP3 inflammasome in GCs. In conclusion, inhibition of FoxO1 can alleviate PCOS by inhibiting the TLR4/NF-κB/NLRP3 pathway to reduce inflammation and the immune response.
本研究旨在探讨叉头框转录因子O1（FoxO1）在多囊卵巢综合征（PCOS）慢性炎症中的作用。通过来曲唑诱导构建PCOS大鼠模型作为体内模型，并分离培养PCOS大鼠的颗粒细胞（GCs）作为体外细胞模型。分别在PCOS大鼠模型和GCs模型中，通过短发夹RNA（shRNA）和小干扰RNA（siRNA）敲低FoxO1。进行苏木精-伊红（H&E）染色以评估FoxO1抑制对PCOS大鼠卵巢病理和功能障碍的影响。通过酶联免疫吸附测定（ELISA）评估PCOS大鼠卵巢和子宫以及GCs中炎性细胞因子的水平。采用流式细胞术评估PCOS大鼠外周血和脾脏中中性粒细胞和巨噬细胞含量的变化。进行细胞计数试剂盒-8（CCK-8）检测和膜联蛋白V-异硫氰酸荧光素/碘化丙啶（Annexin V-FITC/PI）染色以评估GCs的增殖和凋亡。通过逆转录定量聚合酶链反应（RT-qPCR）和蛋白质免疫印迹法检测GCs中与Toll样受体4（TLR4）/核因子κB（NF-κB）/NOD样受体蛋白3（NLRP3）通路相关的基因和蛋白表达。结果表明，FoxO1在PCOS大鼠模型中高表达。抑制FoxO1可显著减轻PCOS大鼠卵巢中的病理变化和功能障碍，同时还可抑制卵巢和子宫中的炎症和纤维化。此外，敲低FoxO1有助于恢复PCOS大鼠外周血和脾脏中中性粒细胞和巨噬细胞的正常比例，并促进巨噬细胞的M2极化。此外，抑制FoxO1可促进GCs的增殖并抑制GCs中的炎症反应。此外，敲低FoxO1可抑制GCs中NF-κB通路的激活和NLRP3炎性小体的形成。总之，抑制FoxO1可通过抑制TLR4/NF-κB/NLRP3通路减轻炎症和免疫反应，从而缓解PCOS。
