Total tanshinones protect against acute lung injury through the PLCγ2/NLRP3 inflammasome signaling pathway

Salvia miltiorrhiza Bunge is a widely used traditional Chinese medicine with anticholinesterase, antitumor, and anti-inflammatory. Total Tanshinones (TTN), the most significant active ingredient of Salvia miltiorrhiza Bunge, exerts anti-inflammatory activity. However, the protective mechanism of total Tanshinones on acute lung injury (ALI) still needs to be explored.In this study, the underlying mechanisms of TTN to treat with ALI were investigated in vitro and in vivo.Cell experiments established an in vitro model of LPS-induced J774A.1 and MH-S macrophages to verify the mechanism. The levels of inflammatory cytokines (TNF-α, IL-6 and IL-1β) were estimated by ELISA. The changes of ROS, Ca2+ and NO were detected by flow cytometry. The expression levels of proteins related to the NLRP3 inflammasome were determined by Western blotting. The effect of TTN on NLRP3 inflammasome activation was examined by immunofluorescence analysis of caspase-1 p20. Male BALB/c mice were selected to establish the ALI model. The experiment was randomly divided into six groups: control, LPS, LPS + si-NC, LPA + si-Nek7, LPS + TTN, and DEX. Pathological alterations were explored by H&E staining. The expression levels of proteins related to the NLRP3 inflammasome were analyzed by Western blotting.TTN decreased pro-inflammatory cytokines levels like TNF-α, IL-6, IL-1β, NO, and ROS in alveolar macrophages. TTN bound to NIMA-related kinase 7 (NEK7), a new therapeutic protein to modulate NLRP3 inflammasome and PLCγ2-PIP2 signaling pathway. In ALI mice, LPS enhanced IL-1β levels in the serum, lung tissues, and bronchoalveolar lavage fluid (BALF),which were reversed by TTN. TTN decreased cleaved-caspase-1 and NLRP3 expressions in lung tissues. When Nek7 was knocked down in mice by siRNA, the syndrome of ALI in mice was significantly suppressed, of which the effect was similar to that of TTN.This research demonstrates that TTN alleviated ALI by binding to NEK7 in vitro and in vivo to modulate NLRP3 inflammasome activation and PLCγ2-PIP2 signaling pathways.