Exocarpium Citri Grandis ameliorates LPS-induced acute lung injury by suppressing inflammation, NLRP3 inflammasome, and ferroptosis

Ethnopharmacological relevance: Exocarpium Citri Grandis (ECG), the epicarp of C. grandis 'Tomentosa ' which is also known as Hua-Ju-Hong in China, has been widely used for thousands of years to treat inflammatory lung disorders such as asthma, and cough as well as dispelling phlegm. However, its underlying pharmacological mechanisms in acute lung injury (ALI) remain unclear. Aim of the study: To explore the therapeutic effect of ECG on ALI and reveal the potential mechanisms based on experimental techniques in vivo and in vitro . Materials and methods: Lipopolysaccharides (LPS) induced ALI in mice and induced RAW 264.7 cell inflammatory model were established to investigate the pharmacodynamics of ECG. ELISA kits, commercial kits, Western Blot, qPCR, Hematoxylin and Eosin (H &E) staining, immunohistochemistry, and immunofluorescence technologies were used to evaluate the pharmacological mechanisms of ECG in ameliorating ALI. Results: ECG significantly attenuated pulmonary edema in LPS-stimulated mice and decreased the levels of IL1ll, IL6, and TNF- alpha in serum and BALF, reduced MDA and iron concentration as well as increased SOD and GSH levels in lung tissues, and also decreased the ROS level in BALF and Lung tissue. Further pharmacological mechanism studies showed that ECG significantly inhibited mRNA expression of inflammatory signaling factors and chemokines, and down -regulated the expression of TLR4, MyD88, NF- kappa B p65, NF- kappa B p -p65 (S536), COX2, iNOS, Txnip, NLRP3, ASC, Caspase-1, JAK1, p-JAK1 (Y1022), JAK2, STAT1, p-STAT1 (S727), STAT3, p-STAT3 (Y705), STAT4, p-STAT4 (Y693), and Keap1, and also up -regulated the expression of Trx-1, Nrf2, HO -1, NQO1, GPX4, PCBP1, and SLC40A1. In the LPS-induced RAW264.7 cell inflammatory model, ECG showed similar results to animal experiments. Conclusions: Our results showed that ECG alleviated ALI by inhibiting TLR4/MyD88/NF- kappa B p65 and JAK/STAT signaling pathway -mediated inflammatory response, Txnip/NLRP3 signaling pathway -mediated inflammasome activation, and regulating Nrf2/GPX4 axis -mediated ferroptosis. Our findings provide an experimental basis for the application of ECG.