Institute of Integrated Traditional Chinese and Western Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China.
Department of Integrated Traditional Chinese and Western Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China.
J Ethnopharmacol. 2020 May 23;254:112611. doi: 10.1016/j.jep.2020.112611. Epub 2020 Feb 20.
The Chinese herbal prescription JieZe-1 (JZ-1) is based on the modification of Yihuang Tang, which was first described in Fu Qingzhu Nvke by the famous Qing Dynasty doctor Shan Fu as a treatment for leukorrheal diseases. As an in-hospital preparation, JZ-1 has been used in Tongji Hospital for many years to treat various infectious diseases of the lower female genital tract, including cervicitis, vaginitis, genital herpes and condyloma acuminatum. Our previous studies have shown that JZ-1 has curative effects on Candida albicans, Trichomonas vaginalis and Ureaplasma urealyticum infections.
Genital herpes is among the most common sexually transmitted diseases (STDs) worldwide and is mainly caused by herpes simplex virus type-2 (HSV-2). Current therapies can relieve symptoms in patients but do not cure or prevent the spread of the virus. This study was designed to investigate the effect of JZ-1 on HSV-2 infection and its mechanism, which is based on autophagy induction, to provide new ideas and a basis for the study of antiviral drugs.
Evaluation of the antiviral activity of JZ-1 was conducted by MTT assay and western blotting. Then, Western blot and immunofluorescence analyses, observations through transmission electron microscopy and experiments with the recombinant lentivirus vector mRFP-GFP-LC3B were used to monitor autophagic flux in VK2/E6E7 cells. To explore the mechanism by which JZ-1 regulates autophagy, western blotting and real-time quantitative PCR (qRT-PCR) were used to determine the expression of phosphoinositide 3-kinase (PI3K)/Akt/mTOR pathway proteins and to detect changes in critical molecules in the pathway after the application of a PI3K inhibitor. Additionally, the mRNA expression levels of inflammatory cytokines, namely, IL-6, IFN-α, IFN-β and TNF-α, were measured with qRT-PCR.
HSV-2 infection inhibited autophagy in the VK2/E6E7 cells. Further study revealed that the activation of the PI3K/Akt/mTOR pathway induced by HSV-2 infection may result in the blocked autophagic flux and inhibited autophagosome and autolysosome formation. JZ-1 exhibited significant antiviral activity in the VK2/E6E7 cells, which showed increased cell vitality and reduced viral protein expression, namely, earliest virus-specific infected cell polypeptides 5 (ICP5) and glycoprotein D (gD). We found that JZ-1 treatment inhibited the upregulation of the PI3K/Akt/mTOR pathway proteins and promoted autophagy to combat HSV-2 infection, while PI3K inhibitor pretreatment prevented the enhanced autophagy induced by JZ-1. Moreover, JZ-1 attenuated the increase in inflammatory cytokines that had been induced HSV-2 infection.
Our results showed that JZ-1 protects against HSV-2 infection, and this beneficial effect may be mediated by inducing autophagy via inhibition of the PI3K/Akt/mTOR signaling axis.
中药方剂 JieZe-1（JZ-1）是基于医圣傅青主的《傅青主女科》中易黄汤的改良，最初用于治疗带下病。作为院内制剂，JZ-1 多年来一直被用于同济医院治疗各种下生殖道感染性疾病，包括宫颈炎、阴道炎、生殖器疱疹和尖锐湿疣。我们之前的研究表明，JZ-1 对白色念珠菌、阴道毛滴虫和解脲支原体感染有疗效。
生殖器疱疹是全球最常见的性传播疾病（STD）之一，主要由单纯疱疹病毒 2 型（HSV-2）引起。目前的治疗方法可以缓解患者的症状，但不能治愈或预防病毒的传播。本研究旨在探讨 JZ-1 通过诱导自噬对抗 HSV-2 感染的作用及其机制，为抗病毒药物的研究提供新思路和依据。
通过 MTT 检测和 Western blot 评估 JZ-1 的抗病毒活性。然后，通过 Western blot 和免疫荧光分析、透射电镜观察以及使用重组慢病毒载体 mRFP-GFP-LC3B 进行实验，监测 VK2/E6E7 细胞中的自噬流。为了探讨 JZ-1 调节自噬的机制，通过 Western blot 和实时定量 PCR（qRT-PCR）检测磷酸肌醇 3-激酶（PI3K）/Akt/mTOR 通路蛋白的表达，并检测应用 PI3K 抑制剂后通路中关键分子的变化。此外，通过 qRT-PCR 检测炎症细胞因子白细胞介素 6（IL-6）、干扰素-α（IFN-α）、干扰素-β（IFN-β）和肿瘤坏死因子-α（TNF-α）的 mRNA 表达水平。
HSV-2 感染抑制了 VK2/E6E7 细胞中的自噬。进一步的研究表明，HSV-2 感染诱导的 PI3K/Akt/mTOR 通路的激活可能导致自噬流受阻，自噬体和自噬溶酶体的形成受到抑制。JZ-1 在 VK2/E6E7 细胞中表现出显著的抗病毒活性，表现为细胞活力增加和病毒蛋白表达减少，即早期病毒特异性感染细胞多肽 5（ICP5）和糖蛋白 D（gD）。我们发现 JZ-1 处理抑制了 PI3K/Akt/mTOR 通路蛋白的上调，并促进了自噬以对抗 HSV-2 感染，而 PI3K 抑制剂预处理可防止 JZ-1 诱导的自噬增强。此外，JZ-1 减轻了 HSV-2 感染诱导的炎症细胞因子的增加。
我们的研究结果表明，JZ-1 可预防 HSV-2 感染，这种有益作用可能是通过抑制 PI3K/Akt/mTOR 信号通路诱导自噬来介导的。
