Liang–Ge–San protects against viral infection-induced acute lung injury through inhibiting α7nAChR-mediated mitophagy

•Liang–Ge–San (LGS) attenuates SARS-CoV-2, H 1N1 and Poly(I:C)-induced acute lung injury.•LGS suppresses virus-induced inflammation by activating α7nAChR in RAW264.7 cells.•LGS inhibits mitophagy in MLE-12 cells which is mainly caused by TNF-α. Acute lung injury (ALI) is the main cause of death in clinical respiratory virus infection. Liang–Ge–San (LGS), a famous traditional Chinese formula, has been proved to be effective in treating ALI caused by lipopolysaccharide. However, the effects of LGS on ALI induced by viral infections remain uncertain. To investigate the effect and mechanism of action of LGS on viral infection-induced ALI. The inhibitory effects of LGS on virus-induced inflammation in vitro were evaluated by qRT-PCR and ELISA. The protein expression of α7nAChR was examined by Western blotting. α7nAChR was inhibited by the transfection of siRNA or methyllycaconitine citrate (MLA, an α7nAChR inhibitor) to investigate the role of α7nAChR in the anti-inflammatory effect of LGS. Adoptive culture and co-culture systems of macrophages RAW264.7 and alveolar epithelial cells MLE-12 were established to mimic their interaction. Western blotting, immunofluorescence, flow cytometry and transmission electron microscopy were used to examine the effects of LGS on mitophagy inhibition. In vivo, ALI mouse models induced by SARS-CoV-2, H1N1 or Poly(I:C) infection were established to explore the therapeutic effect and mechanism of LGS. LGS reduced the release of IL-6, TNF-α and IL-1β and increased the expression of α7nAChR in virus-infected RAW264.7 cells. The blockage of α7nAChR counteracted the anti-inflammatory effect of LGS. Moreover, LGS significantly inhibited autophagy in MLE-12 cells induced by Poly(I:C) in adoptive culture and co-culture systems of RAW264.7 and MLE-12 cells, which could be attenuated after the inhibition of α7nAChR in RAW264.7 cells by decreasing the secretion of IL-6, TNF-α and IL-1β. Further study showed that LGS suppressed TNF-α-induced mitochondrial damage and mitophagy by inhibiting the generation of ROS in MLE-12 cells. In vivo, LGS significantly prolonged the survival time, alleviated pathological injury and acute inflammation of ALI mice induced by SARS-CoV-2, H1N1 or Poly(I:C) infection which were associated with the inhibition of α7nAChR-mediated mitophagy. This study first demonstrates that LGS inhibits virus infection-induced inflammation in RAW246.7 cells by increasing the expression of α7nAChR, thereby inhibiting mitophagy i