Effect of dihydromyricetin on SARS-CoV-2 viral replication and pulmonary inflammation and fibrosis

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 Mᵖʳᵒ and anti-inflammatory properties. Dihydromyricetin, as a flavonol, also has antiviral and anti-inflammatory potential. However, the inhibition of dihydromyricetin on SARS-CoV-2 Mᵖʳᵒ and the protective effect of dihydromyricetin on pulmonary inflammation and fibrosis have not been proved and explained.The coronavirus main protease (Mᵖʳᵒ) is essential for SARS-CoV-2 replication and to be recognized as an attractive drug target, we expect to find the inhibitor of Mᵖʳᵒ. 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.FRET-based enzymatic assay was used to evaluate the inhibit activity of dihydromyricetin on SARS-CoV-2 Mᵖʳᵒ. Molecular docking was used to identify the binding pose of dihydromyricetin with SARS-CoV-2 Mᵖʳᵒ. 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.In this study, we found that dihydromyricetin is a potent inhibitor targeting the SARS-CoV-2 Mᵖʳᵒ 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 Mᵖʳᵒ was identified using molecular docking method. In the binding pocket of SARS-CoV-2 Mᵖʳᵒ, 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.Dihydromyricetin is an effective inhibitor for SARS-CoV-2 Mᵖʳᵒ 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.