白头翁汤调控PI3K/AKT-HIF-1α通路治疗VVC小鼠的研究

Study on the Effect of Baitouweng Decoction on VVC Mice by Regulating PI3K/AKT-HIF-1α Pathway

    摘要
  • 摘要:
    目的 基于磷脂酰肌醇-3激酶(Phosphoinositide 3-kinase, PI3K)/蛋白激酶B (Protein kinase B, AKT)-缺氧诱导因子-1α(Hypoxia-inducible factor-1α, HIF-1α)通路探究白头翁汤对外阴阴道假丝酵母菌病(Vulvovaginal candidiasis, VVC)小鼠的作用机制。
    方法 雌性SPF级昆明种小鼠采用苯甲酸雌二醇皮下注射联合白色念珠菌混悬液阴道接种建立VVC小鼠模型。造模成功小鼠随机分为模型组, 白头翁汤高、中、低剂量组(23.4、11.7、5.85 g·kg-1), 氟康唑组(0.02 g·kg-1), 每组20只。另取20只小鼠只注射苯甲酸雌二醇作为空白组。模型成功后次日给药, 连续14 d。末次给药2 h后, 巴氏染色法观测阴道灌洗液内中性粒细胞数量; HE染色法观察阴道组织形态; ELISA法测定阴道组织中烟酰胺腺嘌呤二核苷酸磷酸氧化酶2(Nicotinamide adenine dinucleo-tide phosphate oxidase 2, NOX2)、超氧化物歧化酶(Superoxide dismutase, SOD)活性及活性氧(Reactive oxygen species, ROS)、丙二醛(Malondialdehyde, MDA)水平; qPCR检测阴道组织PI3K、AKT、HIF-1α的mRNA表达; Western blot检测阴道组织p-PI3K、PI3K、p-AKT、AKT、HIF-1α蛋白表达。
    结果 与空白组小鼠相比, 模型组小鼠阴道灌洗液含有大量中性粒细胞(P < 0.001);阴道黏膜层脱落严重, 大量炎细胞浸润; 阴道组织ROS、MDA水平升高(P < 0.001), NOX2活性提高(P < 0.001), SOD活性降低(P < 0.001), PI3K、AKT、HIF-1α mRNA表达降低(P < 0.001), p-PI3K/PI3K和p-AKT/AKT的比值、HIF-1α蛋白表达降低(P < 0.001)。与模型组小鼠相比, 白头翁汤高、中剂量组阴道灌洗液内中性粒细胞数量降低明显(P < 0.01, P < 0.001);阴道组织黏膜状况呈现不同程度的改善; 阴道组织ROS、MDA水平有所降低(P < 0.05, P < 0.01, P < 0.001), NOX2活性降低(P < 0.01, P < 0.001), SOD活性升高(P < 0.01, P < 0.001);白头翁汤高剂量组阴道组织PI3K、AKT、HIF-1α mRNA表达升高(P < 0.001), p-PI3K/PI3K和p-AKT/AKT的比值、HIF-1α蛋白表达升高(P < 0.001)。
    结论 白头翁汤可通过调控PI3K/AKT-HIF-1α信号通路发挥对VVC的治疗作用。

     

    Abstract:
    OBJECTIVE To explore the mechanism Baitouweng Decoction on mice with vulvovaginal candidiasis (VVC) based on phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)-hypoxia-inducible factor-1α(HIF-1α) pathway.
    METHODS Female SPF-grade Kunming mice were injected subcutaneously with estradiol benzoate and vaginally inoculated with Candida albicans suspension to establish a VVC mouse model.The mice with successful modeling were randomly divided into model group, high-, medium-, and low-dose Baitouweng Decoction groups (23.4, 11.7, and 5.85 g·kg-1), and fluconazole group (0.02 g·kg-1), with 20 mice in each group.Another 20 mice were injected with estradiol benzoate as control group.Treatment started the day after successful model establishment and continued for 14 days.Two hours after the last administration, the number of neutrophils in vaginal lavage fluid was examined with Papanicolaou stain.The HE staining method was used to observe the morphology of vaginal tissue.ELISA was used to measure the levels of nicotinamide adenine dinucleo-tide phosphate oxidase 2(NOX2), reactive oxygen species (ROS), superoxide dismutase (SOD), malondialdehyde (MDA) in the vaginal tissues.The mRNA expression levels of PI3K, AKT and HIF-1α were detected using qPCR.Western blot was employed to evaluate the protein expression of p-PI3K, PI3K, p-AKT, AKT, and HIF-1α in vaginal tissue.
    RESULTS Compared to the control group, the model group exhibited a significant increase in neutrophils in vaginal lavage fluid (P < 0.001);the vaginal mucosal layer was severely desquamated and a large number of inflammatory cells infiltrated.Additionally, there was an elevation in NOX2, ROS, and MDA levels in vaginal tissue (P < 0.001), while SOD content decreased (P < 0.001).Furthermore, the mRNA expression of PI3K, AKT and HIF-1α was reduced along with decreased protein expression of p-PI3K/PI3K, p-AKT/AKT, and HIF-1α(P < 0.001).In comparison to the model group, both high-dose and medium-dose groups treated with Baitouweng Decoction demonstrated a significant decrease in neutrophil count in lavage fluid (P < 0.01, P < 0.001);the condition of the vaginal mucosa improved to varying degrees.Moreover, the levels of ROS and MDA in vaginal tissue were reduced (P < 0.05, P < 0.01, P < 0.001), the activity of NOX2 was decreased (P < 0.01, P < 0.001), and the activity of SOD was increased (P < 0.01, P < 0.001).The high-dose group showed that, the mRNA expression of PI3K, AKT, and HIF-1α in vaginal tissue was increased (P < 0.001), the ratios of p-PI3K/PI3K and p-AKT/AKT, and the protein expression of HIF-1α were increased (P < 0.001).
    CONCLUSION Baitouweng Decoction has a therapeutic effect on VVC, and its mechanism of action may be related to the PI3K/AKT-HIF-1α signal pathway.

     

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