Integration of single-cell RNA sequencing and network pharmacology to elucidate the effect of Yantiao Formula on alleviating ALI by regulating the polarization of alveolar macrophages.

Ethnopharmacological Relevance: Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) has a high mortality rate and often occurs in sepsis. Yantiao Formula (YTF) is used effectively in clinic but its mechanism in the treatment of ALI induced by sepsis remains unelucidated.

Aim Of The Study: This study aims to explore the potential molecular mechanisms of YTF in the treatment of sepsis-induced ALI.

Materials And Methods: Using ACQUITY UPLC I-Class, the chemical components contained in YTF were characterized. The network pharmacology approach was used to predict the components and targets of YTF for treating sepsis-induce ALI. Single-cell RNA sequencing (scRNA-seq) was used to find changes in the lung microenvironment after CLP-induced sepsis. Experimental validation was also performed in vitro and in vivo. Using molecular docking, we speculated on the potential pharmacological substances of YTF.

Results: We detected 596 ingredients in YTF and identified 7 absorbed prototypes in serum. 1031 targets for 596 components were retrieved through TCMSP and SwissTargetPrediction databases. 365 potential targets for YTF and sepsis were identified. We observed that the targets of YTF for sepsis were significantly enriched in TNF and chemokine related pathway using GO and KEGG analysis. It was confirmed that at different time points, different doses of YTF increased the CLP-induced PaO, reduced PaCO levels and W/D ratio of lung tissue. CLP- decreased survival rates was also significantly improved by YTF. YTF reversed the increase of IL-6 and IL-1β caused by CLP. Using scRNA-seq analysis, we found that changes in the proportion of cell types and the polarization state of macrophages were evident. Furthermore, the altered levels of biomarkers (M1: IL-1β, iNOS and TNF- α; M2: CD206/Mrc1 and Arg-1) provided evidence of macrophages polarization. We found that CLP-challenged group presented enhanced iNOS and IL-1β expression and YTF increased CD206 and Arg-1 expression in CLP- induced sepsis using immunohistochemical analysis. Similarly, the same results were validated in LPS- induced ALI in NR8383 cells. The material basis and potential therapeutic targets of YTF were also demonstrated using molecular docking.

Conclusions: YTF reduced the release of inflammatory factors and attenuated sepsis-induced ALI. The combined application of scRNA-seq, network pharmacology and molecular docking was helpful for revealing the mechanism of YTF, which was related to altering levels of M1 and M2 biomarkers to regulate macrophage polarization. The role of YTF in exerting its effects was closely relevant to the potential binding targets of its absorbed prototypes.