Single-Cell RNA Sequencing Revealing Yiqi Huayu Jiedu Formula Mediated Alleviation of Acute Respiratory Distress Syndrome via Macrophage Chemotaxis.

Acute Respiratory Distress Syndrome (ARDS) is a life-threatening form of respiratory failure with widespread inflammation in the lungs. Based on decades of clinical practice by Professor Du Huaitang, the Yiqi Huayu Jiedu Formula (YQHYJDF) has been used to alleviate symptoms in ARDS patients. Although formal clinical trials are lacking, preliminary observations indicate its therapeutic potential, justifying further investigation. To elucidate its protective mechanisms, network pharmacology, single-cell RNA sequencing, and validation experiments both in vivo and in vitro were integrated. An ARDS rat model was established by tail vein injection of LPS, after pre-prepared intervention with YQHYJDF. Histopathological analysis was conducted to evaluate the pathological alterations. Single-cell RNA sequencing was performed to explore differentially expressed genes between groups. Network pharmacology analysis was executed for the prediction of the potential protective mechanism of YQHYJDF against ARDS. Following this prediction, the mechanism was further validated through both in vivo and in vitro experiments via quantitative real-time PCR (qPCR), enzyme-linked immunosorbent assay (ELISA), and Western blot. Hematoxylin-eosin (HE) staining demonstrated that YQHYJDF significantly ameliorated lung histopathological alterations, reduced inflammatory cell infiltration, and lowered lung injury scores. Single-cell RNA sequencing revealed that YQHYJDF effectively regulated macrophage chemotaxis and significantly attenuated neutrophil recruitment in ARDS lung tissues. Network pharmacology analysis illustrated that the efficacy of YQHYJDF on ARDS may be realized by inhibiting PI3K/AKT, MAPK, and NF-κB signaling pathways. In vivo experiments demonstrated that YQHYJDF markedly suppressed the mRNA expression of IL-6, IL-1ß, and TNF-α in lung tissues, and reduced aberrant activation of the PI3K/AKT, MAPK, and NF-κB signaling pathways. In vitro experiments showed that YQHYJDF effectively inhibited the synthesis and secretion of Ccl2, Ccl7, and Ccl12 in LPS-induced RAW264.7 cells. The protective effect of YQHYJDF on ARDS is likely associated with the suppression of macrophage chemotaxis and inflammatory responses in lung tissues.