Background

COVID-19 (Coronavirus Disease-2019) has spread widely around the world and impacted human health for millions. The lack of effective targeted drugs and vaccines forces scientific world to search for new effective antiviral therapeutic drugs. It has reported that flavonoids have potential inhibitory activity on SARS-CoV-2 M^pro and anti-inflammatory properties. Dihydromyricetin, as a flavonol, also has antiviral and anti-inflammatory potential. However, the inhibition of dihydromyricetin on SARS-CoV-2 M^pro and the protective effect of dihydromyricetin on pulmonary inflammation and fibrosis have not been proved and explained.

Purpose

The coronavirus main protease (M^pro) is essential for SARS-CoV-2 replication and to be recognized as an attractive drug target, we expect to find the inhibitor of M^pro. Novel coronavirus infection can cause severe inflammation and even sequelae of pulmonary fibrosis in critically ill patients. We hope to find a drug that can not only inhibit virus replication but also alleviate inflammation and pulmonary fibrosis in patients.

Methods

FRET-based enzymatic assay was used to evaluate the inhibit activity of dihydromyricetin on SARS-CoV-2 M^pro. Molecular docking was used to identify the binding pose of dihydromyricetin with SARS-CoV-2 M^pro. The protective effects of dihydromyricetin against BLM-induced pulmonary inflammation and fibrosis were investigated in C57BL6 mice. BALF and lung tissue were collected for inflammation cells count, ELISA, masson and HE staining, western blotting and immunohistochemistry to analyze the effects of dihydromyricetin on pulmonary inflammation and fibrosis. MTT, western blotting, reverse transcription-polymerase chain reaction (RT-PCR) and wound healing were used to analyze the effects of dihydromyricetin on lung fibrosis mechanisms in Mlg cells.

Results

In this study, we found that dihydromyricetin is a potent inhibitor targeting the SARS-CoV-2 M^pro with a half-maximum inhibitory concentration (IC₅₀) of 1.716 ± 0.419 μM, using molecular docking and the FRET-based enzymatic assay. The binding pose of dihydromyricetin with SARS-CoV-2 M^pro was identified using molecular docking method. In the binding pocket of SARS-CoV-2 M^pro, the dihydrochromone ring of dihydromyricetin interact with the imidazole side chain of His163 through π-π stacking. The 1-oxygen of dihydromyricetin forms a hydrogen bond with the backbone nitrogen of Glu166. The 3-, 7-, 3’- and 4’-hydroxyl of dihydromyricetin interact with Gln189, Leu141, Arg188 and Thr190 through hydrogen bonds. Moreover, our results showed that dihydromyricetin can significantly alleviate BLM-induced pulmonary inflammation by inhibiting the infiltration of inflammation cells and the secretion of inflammation factors in the early process and also ameliorate pulmonary fibrosis by improving pulmonary function and down-regulate the expression of α-SMA and fibronectin in vivo. Our results also showed that dihydromyricetin inhibits the migration and activation of myofibroblasts and extracellular matrix production via transforming growth factor (TGF)-β1/Smad signaling pathways.

Conclusion

Dihydromyricetin is an effective inhibitor for SARS-CoV-2 M^pro and it prevents BLM-induced pulmonary inflammation and fibrosis in mice. Dihydromyricetin will be a potential medicine for the treatment of COVID-19 and its sequelae.

中文翻译：

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二氢杨梅素对 SARS-CoV-2 病毒复制及肺部炎症和纤维化的影响

背景

COVID-19（2019 年冠状病毒病）已在世界范围内广泛传播，影响了数百万人的健康。缺乏有效的靶向药物和疫苗迫使科学界寻找新的有效的抗病毒治疗药物。据报道，类黄酮对 SARS-CoV-2 M ^pro和抗炎特性具有潜在的抑制活性。二氢杨梅素作为一种黄酮醇，还具有抗病毒和抗炎作用。然而，二氢杨梅素对SARS-CoV-2 M ^pro的抑制作用以及二氢杨梅素对肺部炎症和纤维化的保护作用尚未得到证实和解释。

目的

冠状病毒主蛋白酶（M ^pro）是SARS-CoV-2复制所必需的，被认为是一个有吸引力的药物靶点，我们期待找到M ^pro的抑制剂。新型冠状病毒感染可引起重症患者严重炎症甚至肺纤维化后遗症。我们希望找到一种既能抑制病毒复制又能减轻患者炎症和肺纤维化的药物。

方法

基于 FRET 的酶促测定法用于评估二氢杨梅素对 SARS-CoV-2 M ^pro的抑制活性。分子对接用于鉴定二氢杨梅素与 SARS-CoV-2 M ^pro的结合位点。在 C57BL6 小鼠中研究了二氢杨梅素对 BLM 诱导的肺部炎症和纤维化的保护作用。收集BALF和肺组织进行炎症细胞计数、ELISA、masson和HE染色、western blotting和免疫组织化学分析二氢杨梅素对肺部炎症和纤维化的影响。MTT、蛋白质印迹、逆转录-聚合酶链反应（RT-PCR）和伤口愈合用于分析二氢杨梅素对Mlg细胞肺纤维化机制的影响。

结果

在这项研究中，我们发现二氢杨梅素是一种针对 SARS-CoV-2 M ^pro的有效抑制剂，半数最大抑制浓度 (IC ₅₀ ) 为 1.716 ± 0.419 μM，使用分子对接和基于 FRET 的酶促测定。使用分子对接方法鉴定了二氢杨梅素与 SARS-CoV-2 M ^{pro的结合位点。}在 SARS-CoV-2 M ^{pro的结合口袋中}，二氢杨梅素的二氢色酮环通过π-π堆积与His163的咪唑侧链相互作用。二氢杨梅素的 1-氧与 Glu166 的主链氮形成氢键。二氢杨梅素的 3-、7-、3'- 和 4'-羟基通过氢键与 Gln189、Leu141、Arg188 和 Thr190 相互作用。此外，我们的研究结果表明，二氢杨梅素可通过早期抑制炎症细胞浸润和炎症因子的分泌，显着减轻 BLM 诱导的肺部炎症，并通过改善肺功能和下调 α-体内 SMA 和纤连蛋白。

结论

Dihydromyricetin 是 SARS-CoV-2 M ^pro的有效抑制剂，可预防 BLM 诱导的小鼠肺部炎症和纤维化。二氢杨梅素将成为治疗COVID-19及其后遗症的潜在药物。