Zhang Yuwei, Zhou Haili, Ding Caifei
Second Clinical Medical College of Zhejiang Chinese Medical University, Hangzhou, Zhejiang, China.
Department of Reproductive Medicine, Zhejiang Hospital of Integrated Traditional Chinese and Western Medicine, Hangzhou, Zhejiang, China.
Gynecol Endocrinol. 2023 Dec;39(1):2181637. doi: 10.1080/09513590.2023.2181637. Epub 2023 Feb 23.
This study investigates the effects of CangFu Daotan Decoction (CDD) on m6A methylation and Wnt/β-catenin pathway in rats with polycystic ovary syndrome (PCOS). The PCOS rat model was established by letrozole gavage. The rats were fed high-fat chow, and their body weight and blood glucose were recorded. The expressions of follicle-stimulating hormone(FSH), luteinizing hormone(LH), and testosterone(T) were quantified by ELISA. Chemical components in CDD were analyzed using UPLC-Q/TOF-MS. Based on network pharmacology methods, related targets of CDD on PCOS were screened. An enrichment analysis according to Tokyo Encyclopedia of Genes and Genomes (KEGG) was conducted to predict the potential signaling pathway of CDD in PCOS. The expressions of Wnt-1, β-Catenin, GSK-3β, C-MYC, Beclin1, LC3II, Bax, and PCNA were detected by western blotting. The expressions of Mettl3, Mettl14, Fto, Alkbh5, Ythdf1, and Ythdf2 were monitored by RT-PCR. The expressions of Mettl3, Fto, and Ythdf1 were detected by western blotting. Letrozole and a high-fat diet induced ovarian dysfunction in rats, which was attenuated by CDD. CDD decreased blood glucose, LH, and T concentrations and increased FSH expression in PCOS. After removing duplicates, a total of 71 compounds were identified by UHPLC-Q/TOF-MS, among which terpenoids and flavonoids account for the main proportion. The clustering analysis showed that the active site of CDD might be in the Wnt/β-catenin pathway. CDD decreased the expressions of Wnt-1, β-Catenin, GSK-3β, C-MYC, Beclin1, LC3II, and Bax and increased PCNA expression in the ovarian tissue of PCOS rats. CDD decreased the m6A gene expressions of Mettl3, Mettl14, Fto, Alkbh5, Ythdf1, and Ythdf2 in peripheral blood and ovarian tissue of PCOS rats. CDD reduced the m6A proteins expressions of Mettl3, Fto, and Ythdf1 in the ovarian tissue of PCOS rats. CDD can regulate m6A modification and inhibit the Wnt/β-catenin signaling pathway in PCOS rats, thereby reducing body weight, lowering blood glucose levels, improving sex hormone disorders, and decreasing autophagy and apoptosis in ovarian tissue to promote the recovery of ovarian morphology.
本研究探讨苍附导痰汤（CDD）对多囊卵巢综合征（PCOS）大鼠m6A甲基化及Wnt/β-连环蛋白通路的影响。通过来曲唑灌胃建立PCOS大鼠模型。给大鼠喂食高脂饲料，并记录其体重和血糖。采用酶联免疫吸附测定（ELISA）法测定促卵泡生成素（FSH）、促黄体生成素（LH）和睾酮（T）的表达。运用超高效液相色谱-四极杆飞行时间质谱联用仪（UPLC-Q/TOF-MS）分析CDD中的化学成分。基于网络药理学方法，筛选CDD对PCOS的相关靶点。依据京都基因与基因组百科全书（KEGG）进行富集分析，以预测CDD在PCOS中的潜在信号通路。通过蛋白质免疫印迹法检测Wnt-1、β-连环蛋白、糖原合成酶激酶-3β（GSK-3β）、原癌基因C-MYC、自噬相关蛋白1（Beclin1）、微管相关蛋白1轻链3Ⅱ（LC3II）、凋亡相关蛋白Bax和增殖细胞核抗原（PCNA）的表达。采用逆转录聚合酶链反应（RT-PCR）监测甲基转移酶样蛋白3（Mettl3）、甲基转移酶样蛋白14（Mettl14）、脂肪量和肥胖相关蛋白（Fto）、alkB同源蛋白5（Alkbh5）、YTH结构域家族蛋白1（Ythdf1）和YTH结构域家族蛋白2（Ythdf2）的表达。通过蛋白质免疫印迹法检测Mettl3、Fto和Ythdf1的表达。来曲唑和高脂饮食诱导大鼠卵巢功能障碍，而CDD可使其减轻。CDD可降低PCOS大鼠的血糖、LH和T浓度，并增加FSH表达。去除重复项后，通过UHPLC-Q/TOF-MS共鉴定出71种化合物，其中萜类和黄酮类占主要比例。聚类分析表明，CDD的活性位点可能位于Wnt/β-连环蛋白通路。CDD可降低PCOS大鼠卵巢组织中Wnt-1、β-连环蛋白、GSK-3β、C-MYC、Beclin1、LC3II和Bax的表达，并增加PCNA表达。CDD可降低PCOS大鼠外周血和卵巢组织中Mettl3、Mettl14、Fto、Alkbh5、Ythdf1和Ythdf2的m6A基因表达。CDD可降低PCOS大鼠卵巢组织中Mettl3、Fto和Ythdf1的m6A蛋白表达。CDD可调节PCOS大鼠的m6A修饰并抑制Wnt/β-连环蛋白信号通路，从而减轻体重、降低血糖水平、改善性激素紊乱，并减少卵巢组织中的自噬和凋亡，以促进卵巢形态的恢复。
