背景:Graves病(GD)的病理生理学涉及滤泡辅助性T(Tfh)和滤泡调节性T(Tfr)细胞之间的失衡,以及氧化应激(OS)。夏枯草(夏酷曹,XKC)及其主要生物活性化合物,木犀草素,因其治疗GD的潜力而得到认可。然而,解释XKC的免疫调节和抗氧化作用的机制仍然难以捉摸。
目的:本研究旨在评估XKC和木犀草素在重组TSH受体A亚基(Ad-hTSHR-289)腺病毒诱导的GD小鼠模型中的药理作用并阐明其潜在机制。
方法:采用高效液相色谱-四极杆飞行时间质谱(HPLC-QTOFMS)检测XKC的成分。通过三次肌肉注射Ad-TSHR-289诱导雌性BALB/c小鼠建立GD模型。甲状腺功能,通过ELISA测量自身抗体和OS参数。流式细胞术检测Tfh细胞和Tfr细胞的变更。RT-qPCR,西方印迹,用免疫组织化学方法探讨相关的分子机制。
结果:通过HPLC-QTOFMS鉴定了XKC中的37种化学成分,以类黄酮为代表,类固醇,萜类化合物,和木犀草素.XKC和木犀草素降低了GD小鼠的T4、TRAb水平,促进了甲状腺损伤的恢复。同时,XKC和木犀草素通过降低MDA水平有效缓解OS,NOX2,4-HNE,8-OHdG,同时提高GSH水平。流式细胞仪检测显示,XKC和木犀草素恢复了Tfh/Tfr和Tfh/Treg的异常比例,GD小鼠IL-21、Bcl-6和Foxp3的mRNA水平。此外,XKC和木犀草素抑制PI3K,Akt,p-PI3K和p-Akt,但激活了Nrf2和HO-1。
结论:XKC和木犀草素可通过重新平衡Tfh/Tfr细胞和减轻OS来抑制GD的发展。这种治疗机制可能涉及Nrf2/HO-1和PI3K/Akt信号通路。木犀草素是XKC对抗GD的主要功效物质基础。第一次,我们从自身免疫和OS的角度揭示了XKC和木犀草素治疗GD的机制。
BACKGROUND: The pathophysiology of Graves\' disease (GD) involves imbalances between follicular helper T (Tfh) and follicular regulatory T (Tfr) cells, as well as oxidative stress (OS). Prunella vulgaris L. (Xia Ku Cao, XKC) and its primary bioactive compound, luteolin, are recognized for their potential in treating GD. Yet, the mechanism accounting for the immune-modulatory and antioxidant effects of XKC remains elusive.
OBJECTIVE: This study aims to evaluate the pharmacological effects and elucidate the underlying mechanism of XKC and luteolin in a GD mouse model induced by recombinant adenovirus of TSH receptor A subunit (Ad-hTSHR-289).
METHODS: High-Performance Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry (HPLC-QTOF MS) was used to detect the constituents of XKC. The GD model was established through inducing female BALB/c mice with three intramuscular injections of Ad-TSHR-289. Thyroid function, autoantibody and OS parameters were measured by ELISA. Changes of Tfh cells and Tfr cells were detected by flow cytometry. RT-qPCR, Western Blotting, immunohistochemistry were used to explore the related molecular mechanisms.
RESULTS: A total of 37 chemical components from XKC were identified by HPLC-QTOF MS, represented by flavonoids, steroids, terpenoids, and luteolin. XKC and luteolin reduced T4, TRAb levels and facilitated the recovery from thyroid damage in GD mice. Meanwhile, XKC and luteolin effectively alleviated OS by decreasing the levels of MDA, NOX2, 4-HNE, 8-OHdG, while increasing GSH level. Flow cytometry showed that XKC and luteolin restored the abnormal proportions of Tfh/Tfr and Tfh/Treg, and the mRNA levels of IL-21, Bcl-6 and Foxp3 in GD mice. In addition, XKC and luteolin inhibited PI3K, Akt, p-PI3K and p-Akt, but activated Nrf2 and HO-1.
CONCLUSIONS: XKC and luteolin could inhibit the development of GD in vivo by rebalancing Tfh/Tfr cells and alleviating OS. This therapeutic mechanism may involve the Nrf2/HO-1 and PI3K/Akt signaling pathways. Luteolin is the main efficacy material basis of XKC in countering GD. For the first time, we revealed the mechanism of XKC and luteolin in the treatment of GD from the perspective of
autoimmune and OS.