Single-cell transcriptomics reveals macrophage-neutrophil crosstalk in Haematitum-induced lung injury associated with gut dysbiosis

Haematitum is a mineral-based traditional Chinese medicine widely used in clinical applications. Both the Chinese Materia Medica and modern research indicate that it exhibits lung toxicity and should not be administered over prolonged periods. The precise mechanism underlying this toxicity, however, remains incompletely understood. This study aims to systematically elucidate the toxic mechanism of lung injury induced by Haematitum administration, emphasizing the central role of the gut–lung axis in this process. The mouse model of lung injury induced by Haematitum was established. A combination of 16S rRNA sequencing, untargeted metabolomics, and single-cell RNA sequencing was utilized to comprehensively analyze alterations in the intestinal microbiota, lung tissue metabolism, and lung cell atlas. The pharmacological validation was conducted by administering PPARγ agonists and TNF-α inhibitors. Furthermore, the detoxification effect of its classical combination with Inula japonica Thunb. was also evaluated. High-dose Haematitum disrupted the intestinal barrier and induced dysbiosis. Metabolites from the gut microbiota entered the bloodstream and exerted a pathogenic effect. Correlation analysis showed that gut microbiota dysbiosis is strongly associated with the pulmonary glycerophospholipid metabolic pathway. Results from single-cell sequencing and macrophage/TNF-α colocalization experiments indicated that the macrophages’ PPAR signaling pathway was inhibited, inactivating their anti-inflammatory function and triggering the release of inflammatory factors such as TNF-α. Acting as a key messenger, TNF-α activated the TNF signaling pathway in neutrophils, driving them to acquire a highly destructive phenotype. The synergy between macrophages and neutrophils ultimately provoked inflammation and cell apoptosis in lung tissue. Pharmacological intervention of the PPARγ/TNF-α axis can effectively reverse cellular abnormalities and significantly alleviate lung injury. The combination with Inula japonica Thunb. also reduced lung toxicity, and components within this herb are predicted to act as natural PPARγ agonists. This study has revealed a potential toxic mechanism. The intestinal barrier in mice was compromised, with an increase in the relative abundance of Klebsiella , entry of microbial metabolites into the bloodstream, and disruption of glycerophospholipid metabolism in the lungs, which in turn resulted in dysfunction of pulmonary immune cells. It clarifies that the core mechanism of Haematitum -induced lung injury lies in inhibiting the PPAR signaling pathway in macrophages and activating the TNF signaling pathway in neutrophils. Administering Haematitum at a common dose or in combination with Inula japonica Thunb. mitigates these safety risks. These findings advance understanding of the toxicology of mineral-based medicines and provide a reference for the safe clinical application of these traditional Chinese medicines.