Renshen Baidu Powder mitigates ALI by inhibiting the RORγt/IL-17 pathway and restoring the Treg/Th17 balance.

ETHNOPHARMACOLOGICAL RELEVANCE: Acute lung injury (ALI), a life-threatening inflammatory respiratory condition often progressing to respiratory distress syndrome (ARDS), has high mortality due to dysregulated immunity and lack of targeted therapies. Renshen Baidu Powder (RSBD) is a Traditional Chinese formula from the Song Dynasty's Taiping Huimin Heji Jufang, historically used for wind-cold-dampness-induced respiratory disorders in patients with Qi deficiency. Although several RSBD herbs show anti-inflammatory and immune-modulating effects, its specific mechanisms and therapeutic impact on ALI remain unclear.

AIM OF THIS STUDY: To investigate the therapeutic effects of RSBD on mice with lipopolysaccharide (LPS)-induced ALI and to elucidate its underlying mechanism.

STUDY DESIGN AND METHODS: A model of LPS-induced ALI was developed, wherein the mice received treatment with RSBD or the positive control drug, dexamethasone. The extent of lung injury and inflammation was evaluated by determining the pulmonary wet-to-dry weight ratio, conducting histopathological staining, and measuring the levels of inflammatory cytokines. The proportions of T helper 17 (Th17) and regulatory T (Treg) cells in the spleen were quantified through flow cytometry. The activity of the retinoic acid-related orphan receptor gamma t (RORγt)/interleukin-17 (IL-17) pathway was assessed using quantitative real-time PCR, western blotting, enzyme-linked immunosorbent assay and immunofluorescence. Mechanistic validation was conducted using the RORγt inverse agonist 13 (RORγt13), an IL-17 neutralizing antibody (IL-17NAb) and Hexyl 4-hydroxybenzoate (HexP). Additionally, the chemical constituents, potential targets, and signaling pathways of RSBD were explored by integrating liquid chromatography-mass spectrometry (LC-MS), network pharmacology, and mRNA sequencing.

RESULTS: RSBD significantly alleviated LPS-induced lung injury by reducing pulmonary edema, inflammatory cell infiltration, and pro-inflammatory cytokine release. It restored immune balance by decreasing Th17 cells and increasing Treg cells, resulting in a higher Treg/Th17 ratio. RSBD suppressed RORγt and IL-17 expression while promoting IL-10 production. Network pharmacology and transcriptomics identified the IL-17 signaling pathway as a key target of RSBD, while LC-MS revealed abundant bioactive compounds-such as flavonoids and phenolics-that might contribute to its effects. Notably, RORγt13 and IL-17NAb replicated RSBD's protective effect by suppressing the differentiation into Th17 cells and preventing lung damage. In contrast, HexP, an RORγt agonist, nullified the protective effects of RSBD, which further validated the crucial role of the RORγt/IL-17 pathway in mediating its therapeutic action.

CONCLUSION: RSBD alleviated LPS-induced ALI by inhibiting the RORγt/IL-17 pathway, restoring Treg/Th17 balance, and reducing pulmonary inflammation and damage. These results provided mechanistic support for RSBD as a multi-component immunomodulatory agent for ALI treatment.