Zhang Kang, Zhao Ting, Xia Dan, Zhang Meng-Xiang, Wu Da-Qiang, Wang Tian-Ming, Shao Jing, Wang Chang-Zhong
School of Integrated Traditional Chinese and Western Medicine, Anhui University of Chinese Medicine Hefei 230012, China.
School of Integrated Traditional Chinese and Western Medicine, Anhui University of Chinese Medicine Hefei 230012, China Institute of Integrated Traditional Chinese and Western Medicine, Anhui Academy of Chinese Medicine Hefei 230012, China Anhui Province Key Laboratory of Chinese Medicinal Formula, Anhui University of Chinese Medicine Hefei 230012, China.
Zhongguo Zhong Yao Za Zhi. 2022 May;47(9):2516-2524. doi: 10.19540/j.cnki.cjcmm.20211223.701.
This study aims to explore the effect of butyl alcohol extract of Baitouweng Decoction(BAEB) on vulvovaginal candidiasis(VVC) in mice and to clarify the mechanism from Toll-like receptors(TLRs)/MyD88 and Dectin-1/Syk signal pathways and NLRP3 inflammasome. To be specific, female KM mice were randomized into control group(i.g., normal saline), model group, fluco-nazole group(i.g., 20 mg·kg(-1)), and low-dose, medium-dose, and high-dose BAEB groups(i.g., 20, 40, and 80 mg·kg(-1), respectively). VVC was induced in mice except the control group. After the modeling, administration began and lasted 7 days. The ge-neral conditions and body weight of mice were recorded every day. On the 1 st, 3 rd, 7 th, and 14 th after vaginal infection by Candida albicans, the fungal load in the vaginal lavage fluid of the mice was measured with the plate method, and the morphology of C. albicans in vaginal lavage fluid was observed based on Gram staining. After the mice were killed, vaginal tissues were subjected to hematoxylin-eosin(HE) staining and periodic acid-Schiff(PAS) staining for vaginal histopathological analysis. The content of cytokines in vaginal lavage fluid, such as interleukin(IL)-1β, IL-18, tumor necrosis factor-α(TNF-α), IL-6, and S100 a8, was determined by enzyme-linked immunosorbent assay(ELISA), and content of reactive oxygen species(ROS) in vaginal tissues by tissue ROS detection kit. The protein expression of NLRP3, ASC, caspase-1, Dectin-1, Syk, MyD88, TLR2, TLR4, and nuclear factor-κB(NF-κB) in vaginal tissues was detected by Western blot, and the levels and distribution of NLRP3, Dectin-1, Syk, MyD88, TLR2, and TLR4 in vaginal tissues were determined with the immunohistochemical method. The results show that BAEB can improve the general conditions of VVC mice, reduce the fungal load and C. albicans hyphae in vaginal secretion, decrease ROS content in vaginal tissues and content of cytokines in vaginal lavage fluid, and down-regulate the expression of NLRP3, ASC, caspase-1, Dectin-1, Syk, MyD88, TLR2, TLR4, and NF-κB in vaginal tissues. The above results indicate that BAEB exerts therapeutic effect on VVC mice by down-regulating the key proteins in the TLRs/MyD88 and Dectin-1/Syk signal pathways and NLRP3 inflammasome.
本研究旨在探讨白头翁汤丁醇提取物(BAEB)对小鼠外阴阴道假丝酵母菌病(VVC)的影响,并从Toll样受体(TLRs)/髓样分化因子88(MyD88)和C型凝集素受体1(Dectin-1)/脾酪氨酸激酶(Syk)信号通路及NLRP3炎性小体方面阐明其作用机制。具体而言,将雌性KM小鼠随机分为对照组(即生理盐水)、模型组、氟康唑组(即20 mg·kg⁻¹)以及低剂量、中剂量和高剂量BAEB组(分别为20、40和80 mg·kg⁻¹)。除对照组外,其余小鼠均诱导建立VVC模型。建模后开始给药,持续7天。每天记录小鼠的一般情况和体重。在白色念珠菌阴道感染后的第1、3、7和14天,采用平板法检测小鼠阴道灌洗液中的真菌载量,并基于革兰氏染色观察阴道灌洗液中白色念珠菌的形态。处死小鼠后,取阴道组织进行苏木精-伊红(HE)染色和过碘酸-希夫(PAS)染色,用于阴道组织病理学分析。采用酶联免疫吸附测定(ELISA)法测定阴道灌洗液中细胞因子如白细胞介素(IL)-1β、IL-18、肿瘤坏死因子-α(TNF-α)、IL-6和S100 a8的含量,并用组织活性氧(ROS)检测试剂盒检测阴道组织中ROS的含量。通过蛋白质免疫印迹法检测阴道组织中NLRP3、凋亡相关斑点样蛋白(ASC)、半胱天冬酶-1、Dectin-1、Syk、MyD88、TLR2、TLR4和核因子κB(NF-κB)的蛋白表达,并用免疫组织化学方法测定阴道组织中NLRP3、Dectin-1、Syk、MyD88、TLR2和TLR4的水平及分布。结果表明,BAEB可改善VVC小鼠的一般情况,降低阴道分泌物中的真菌载量和白色念珠菌菌丝,降低阴道组织中ROS含量及阴道灌洗液中细胞因子含量,并下调阴道组织中NLRP3、ASC、半胱天冬酶-1、Dectin-1、Syk、MyD88、TLR2、TLR4和NF-κB的表达。上述结果表明,BAEB通过下调TLRs/MyD88和Dectin-1/Syk信号通路及NLRP3炎性小体中的关键蛋白,对VVC小鼠发挥治疗作用。