Third Level Research Laboratory of State Administration of Traditional Chinese Medicine, School of Traditional Chinese Medicine, Southern Medical University, Guangzhou 510515, PR China; Guangdong Provincial Key Laboratory of Chinese Medicine Pharmaceutics, School of Traditional Chinese Medicine, Southern Medical University, Guangzhou 510515, PR China.
Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health, Southern Medical University, Guangzhou, 510515, PR China.
Phytomedicine. 2023 Oct;119:154977. doi: 10.1016/j.phymed.2023.154977. Epub 2023 Jul 18.
Dengue virus (DENV) is a major public health threat. However, there are no specific therapeutic drugs for DENV. Many Chinese heat-cleaning formulas, such as Liang-Ge-San (LGS), have been frequently used in the virus-induced diseases. The antiviral effect of LGS has not been reported yet.
In this study, the effect of LGS on the inhibition of dengue virus serotype 2 (DENV-2) was investigated and the relevant mechanism was explored.
High-performance liquid chromatography was applied to analyze the chemical characterization of LGS. The in vitro antiviral activities of LGS against DENV-2 were evaluated by time-of-drug-addition assay. The binding of heat shock protein 70 (Hsp70) and envelope (E) protein or caveolin1 (Cav1) were analyzed by immunofluorescence and immunoprecipitation assays. Then the role of Cav1 in the anti-DENV-2 effects of LGS was further examined. DENV-2 infected Institute of Cancer Research suckling mice (n = 10) and AG129 mice (n = 8) were used to examine the protective effects of LGS.
It was found that geniposide, liquiritin, forsythenside A, forsythin, baicalin, baicalein, rhein, and emodin maybe the characteristic components of LGS. LGS inhibited the early stage of DENV-2 infection, decreased the expression levels of viral E and non-structural protein 1 (NS1) proteins. LGS also reduced E protein and Hsp70 binding and attenuated the translocation of Hsp70 from cytoplasm to the cell membrane. Moreover, LGS decreased the binding of Hsp70 to Cav1. Further study showed that the overexpression of Cav1 reversed LGS-mediated E protein and Hsp70 inhibition in the plasma membrane. In the in vivo study, LGS was highly effective in prolonging the survival time, reducing viral loads.
This work demonstrates for the first time that LGS exerts anti-DENV-2 activity in vitro and in vivo. LGS decreases DENV-2-stimulated cytoplasmic Hsp70 translocation into the plasma membrane by Cav1 inhibition, thereby inhibiting the early stage of virus infection. These findings indicate that LGS may be a candidate for the treatment of DENV.
登革热病毒（DENV）是一种主要的公共卫生威胁。然而，目前还没有针对 DENV 的特定治疗药物。许多中国清热方剂，如凉膈散（LGS），在病毒诱导的疾病中经常使用。LGS 的抗病毒作用尚未报道。
本研究旨在探讨 LGS 对抑制登革热病毒血清型 2（DENV-2）的作用，并探讨其相关机制。
采用高效液相色谱法分析 LGS 的化学成分。通过时效加药法评价 LGS 对 DENV-2 的体外抗病毒活性。通过免疫荧光和免疫沉淀试验分析热休克蛋白 70（Hsp70）与包膜（E）蛋白或小窝蛋白 1（Cav1）的结合。然后进一步研究 Cav1 在 LGS 抗 DENV-2 作用中的作用。用登革热病毒感染的 ICR 乳鼠（n=10）和 AG129 小鼠（n=8）检测 LGS 的保护作用。
研究发现，龙胆苦苷、甘草苷、当药苦苷 A、当药苷、黄芩苷、黄芩素、大黄酸和大黄素可能是 LGS 的特征成分。LGS 抑制 DENV-2 感染的早期阶段，降低病毒 E 和非结构蛋白 1（NS1）蛋白的表达水平。LGS 还减少了 E 蛋白和 Hsp70 的结合，并减弱了 Hsp70 从细胞质向细胞膜的易位。此外，LGS 减少了 Hsp70 与 Cav1 的结合。进一步的研究表明，Cav1 的过表达逆转了 LGS 介导的质膜中 E 蛋白和 Hsp70 的抑制。在体内研究中，LGS 能显著延长生存时间，降低病毒载量。
本研究首次证明 LGS 在体外和体内均具有抗 DENV-2 活性。LGS 通过抑制 Cav1 减少 DENV-2 刺激的细胞质 Hsp70 易位到质膜，从而抑制病毒感染的早期阶段。这些发现表明，LGS 可能是治疗 DENV 的候选药物。
