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.

Li X ，Qiu H ，Gan J ，Liu Z ，Yang S ，Yuan R ，Gao H ... -  《-》

Glycyrrhizin mitigates acute lung injury by inhibiting the NLRP3 inflammasome in vitro and in vivo.

Glycyrrhiza glabra L. is a widely used traditional Chinese medicine with antipyretic, detoxification, antibacterial and therapeutic effects against various diseases, including liver diseases. Glycyrrhizin (GL), the most significant active ingredient of Glycyrrhiza glabra L., exerts anti-inflammatory activity. However, the anti-inflammatory effect of GL remains to be determined. Consequently, this research was carried out to discover the effects and mechanism of action of GL on ALI. Cell experiments established an in vitro model of LPS-induced RAW 264.7 macrophages to verify the mechanism. The levels of NO, PEG2, and inflammatory cytokines were estimated by ELISA. The expression levels of proteins related to the NF-κB signalling pathway and NLRP3 inflammasome were determined by Western blotting. The nuclear translocation of NF-κB p65 and ASC was tested through immunofluorescence analysis. The inhibitory effect of NLRP3 inhibitor MCC950 on macrophage was evaluated. Male BALB/C mice were selected to establish the ALI model. The experiment was randomly divided into five groups: control, ALI, GLL, GLH, and DEX. Pathological alterations were explored by H&E staining. The weight ratios of lung W/D, MPO, and inflammatory cytokines were evaluated by ELISA. The expression levels of proteins related to the NF-κB signalling pathway or NLRP3 inflammasome were analysed by Western blotting. Here, we demonstrate that GL attenuates inflammation, nitric oxide, IL-18, IL-1β, TNF-α, IL-6, and PGE2 levels and alveolar epithelial barrier permeability in macrophages and mice challenged with LPS. In addition, GL inhibits NLRP3 inflammasome initiation and activation and NF-κB signalling pathway activation. This research demonstrates that GL may alleviate ALI inflammation by interfering with the NF-κB/NLRP3 inflammasome signalling pathway.

Wang J ，Ren C ，Bi W ，Batu W ... -  《-》

Intranasal Application of Budesonide Attenuates Lipopolysaccharide-Induced Acute Lung Injury by Suppressing Nucleotide-Binding Oligomerization Domain-Like Receptor Family, Pyrin Domain-Containing 3 Inflammasome Activation in Mice.

Dong L ，Zhu YH ，Liu DX ，Li J ，Zhao PC ，Zhong YP ，Chen YQ ，Xu W ，Zhu ZQ ... -  《-》

Pirfenidone ameliorates lipopolysaccharide-induced pulmonary inflammation and fibrosis by blocking NLRP3 inflammasome activation.

Acute respiratory distress syndrome(ARDS)is a severe clinical disorder characterized by its acute onset, diffuse alveolar damage, intractable hypoxemia, and non-cardiogenic pulmonary edema. Acute lung injury(ALI) can trigger persistent lung inflammation and fibrosis through activation of the NLRP3 inflammasome and subsequent secretion of mature IL-1β, suggesting that the NLRP3 inflammasome is a potential therapeutic target for ALI, for which new therapeutic approaches are needed. Our present study aims to assess whether pirfenidone,with anti-fibrotic and anti-inflammatory properties, can improve LPS-induced inflammation and fibrosis by inhibiting NLRP3 inflammasome activation. Male C57BL/6 J mice were intratracheally injected with LPS to induce ALI. Mice were administered pirfenidone by oral gavage throughout the entire experimental course. The mouse macrophage cell line (J774 A.1) was incubated with LPS and ATP, with or without PFD pre-treatment. We demonstrated that PFD remarkably ameliorated LPS-induced pulmonary inflammation and fibrosis and reduced IL-1β and TGF-β1 levels in bronchoalveolar lavage fluid(BALF). Pirfenidone substantially reduced NLRP3 and ASC expression and inhibited caspase-1 activation and IL-1β maturation in lung tissues. In vitro, the experiments revealed that PFD significantly suppressed LPS/ATP-induced production of reactive oxygen species (ROS) and decreased caspase-1 activation and the level of IL-1β in J774 A.1 cells. Taken together, the administration of PFD reduced LPS-induced lung inflammation and fibrosis by blocking NLRP3 inflammasome activation and subsequent IL-1β secretion. These findings indicated that PFD can down-regulate NLRP3 inflammasome activation and that it may offer a promising therapeutic approach for ARDS patients.

Li Y ，Li H ，Liu S ，Pan P ，Su X ，Tan H ，Wu D ，Zhang L ，Song C ，Dai M ，Li Q ，Mao Z ，Long Y ，Hu Y ，Hu C ... -  《-》

Xuanbai Chengqi Decoction alleviates acute lung injury by inhibiting NLRP3 inflammasome.

Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) is a prevalent critical respiratory disorder caused mostly by infection and other factors. However, effective drug therapies are currently lacking. Xuanbai Chengqi Decoction (XCD), a traditional Chinese medicine (TCM) prescription, is commonly employed to treat lung diseases. It has been recommended by Chinese health authorities as one of the TCM prescriptions for COVID-19. Nonetheless, its underlying mechanism for the treatment of ALI has not been fully understood. The study aims to investigate the therapeutic effect of XCD on lipopolysaccharide (LPS) -induced ALI in mice and explore its anti-inflammatory mechanism involving pyroptosis. Ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) was employed to identify the active compounds of XCD, and quantitative analysis of the main compounds was conducted. Male C57BL/6J mice were given different doses of XCD (4.5 and 9.0 g/kg/day) or dexamethasone (5 mg/kg/day) by oral gavage for 5 consecutive days. Subsequently, ALI was induced by injecting LPS (20 mg/kg) intraperitoneally 2 h after the last administration, and serum and lung tissues were collected 8 h later. J774A.1 cells were pretreated with different doses of XCD (100, 200, 400 μg/ml) for 12 h, then incubated with LPS (1 μg/ml) for 4 h and ATP (1 mM) for 2 h to induce pyroptosis. Supernatant and cells were collected. Moreover, J774A.1 cells were transfected with an NLRP3 overexpression plasmid for 24 h, followed by subsequent experiments with XCD (400 μg/ml). Lung histopathological changes were evaluated using hematoxylin and eosin (HE) staining. To assess the efficacy of XCD on ALI/ARDS, the levels of inflammatory factors, chemokines, and proteins associated with NLRP3 inflammasome signaling pathway were evaluated. XCD was found to ameliorate lung inflammation injury in ALI mice, and reduce the protein expression of TNF-α, IL-1β, and IL-6 in both mouse serum and J774A.1 cell supernatant. Meanwhile, XCD significantly decreased the mRNA levels of IL-1β, pro-IL-1β, CXCL1, CXCL10, TNF-α, NLRP3, NF-κB P65, and the protein expression of NLRP3, Cleaved-Caspase1, and GSDMD-N in the lung and J774A.1 cells. These effects were consistent with the NLRP3 inhibitor MCC950. Furthermore, overexpression of NLRP3 reversed the anti-inflammatory effect of XCD. The therapeutic mechanism of XCD in ALI treatment may involve alleviating inflammatory responses in lung tissues by inhibiting the activation of the NLRP3 inflammasome-mediated pyroptosis in macrophages.

Wang S ，Lin F ，Zhang C ，Gao D ，Qi Z ，Wu S ，Wang W ，Li X ，Pan L ，Xu Y ，Tan B ，Yang A ... -  《-》