Protective Effects of Wenqingyin on Sepsis-Induced Acute Lung Injury Through Regulating RAGE Pathway

Abstract

Background: Wenqingyin (WQY), an ancient Chinese medicinal formula known for its ability to eliminate heat and toxins, as well as improve blood circulation and alleviate blood stagnation, has been extensively utilized in the treatment of infectious ailments throughout history. However, its anti-sepsis mechanism remains ambiguous.

Purpose: The objective of this research was to investigate the diverse mechanisms of WQY in treating acute lung injury (ALI) caused by sepsis. This was achieved by employing ultrahigh-performance liquid chromatography quadrupole-Orbitrap high-resolution mass spectrometry (UHPLC-Orbitrap-HRMS) and a network pharmacology approach. Additionally, the effects of WQY were validated through biological experiments.

Methods: UHPLC-Orbitrap-HRMS and Network pharmacology were employed to forecast the potential mechanism of WQY in combating sepsis. Afterwards, an in vivo model of sepsis-induced acute lung injury (ALI) was created through the intraperitoneal administration of lipopolysaccharide (LPS), while an in vitro model was established by stimulating RAW264.7 macrophages with LPS. To assess the extent of lung damage and the levels of inflammatory cytokines, various techniques including Hematoxylin-eosin (HE) staining, Tunel, qPCR, and ELISA were employed. The infiltration of inflammatory cells was observed by immunohistochemistry staining. Western blot, immunohistochemistry, and immunofluorescence staining were utilized to identify the hub targets and signal pathway.

Results: A total of 75 active constituents and 237 associated targets of WQY were acquired, with 145 overlapping targets identified as sepsis-related. JUN, AKT1, TP53, IL-6, HSP90AA1, CASP3, VEGFA, IL-1β, RELA and EGFR may be targets of WQY for sepsis based on PPI network analysis. The analysis of KEGG revealed that WQY is implicated in the signaling pathway of advanced glycation end products-receptor of advanced glycation end products (AGE-RAGE). In an in vivo experiment, we verified that WQY reduced sepsis-induced lung damage by suppressing the production of proinflammatory cytokines like IL-6, TNF-α and IL1-β, and also by inhibiting the infiltration of macrophages and neutrophils in mice with LPS-induced ALI. The in vitro tests showed that WQY suppressed the production and release of IL-1β, IL-6, iNOS and TNF-α in LPS-induced RAW264.7 macrophages. We discovered that WQY effectively treated acute lung injury induced by LPS by targeting the RAGE pathway.

Conclusion: In conclusion, the primary way in which WQY combats sepsis-induced acute lung injury is by controlling the levels of RAGE and the PI3K/AKT pathway, thereby suppressing inflammation and mitigating damage to the lungs. The research we conducted establishes a scientific foundation for understanding the mechanism and clinical use of WQY in sepsis treatment.

Note:
Funding declaration: This work was supported by National Natural Science Foundation of China (Nos. 82205083, 82305110 and 82204959), Chinese Postdoctoral Science Foundation (2022M711538), Natural Science Foundation of Guangdong Province (No. 2022A1515012620), Guangdong Basic and Applied Basic Research Fund (Nos. 2021A1515110786), Guangdong Provincial Administration of Traditional Chinese Medicine Research Project (20221257), Science and Technology Project of Guangzhou (2023A04J1824 and 202201011196).

Conflict of Interests: The authors declare no conflict of interest.

Ethical Approval: Approval for the animal experiment was granted by the Ethical Committee on Animal Experimentation of Southern Medical University (Registration number L2019147, July 10, 2019).

Keywords: Wenqingyin, Network Pharmacology, Sepsis, Acute Lung Injury, RAGE