Shengjiang San limits lung injury alongside complement-linked neutrophil responses in mice

In a mouse model of lipopolysaccharide-induced acute lung injury, pretreatment with Shengjiang San reduced pulmonary inflammation, edema, vascular leakage and neutrophil activation while preserving the alveolar-capillary barrier. The traditional Chinese medicine formula also lowered circulating and lung concentrations of the complement protein C5a, reduced pulmonary expression of its receptor C5aR1, and suppressed C5a-triggered neutrophil migration, calcium mobilization, oxidative burst and neutrophil extracellular trap formation. These findings indicate that the formula’s protective effects in this experimental model are associated with restraint of pathological complement-linked neutrophil responses, although the study did not establish that this pathway explains all of its effects.

Researchers assigned male C57BL/6J mice to a control group, an injury group, three Shengjiang San dose groups and a dexamethasone comparison group, with six animals in each group. The animals received their assigned treatment orally for five consecutive days. Two hours after the final pretreatment, acute lung injury was induced by intratracheal administration of lipopolysaccharide, or LPS, and treatment continued once daily after the challenge. Samples were collected 48 hours after LPS instillation. Histological analysis showed that LPS caused inflammatory-cell infiltration, thickening of the alveolar walls and interstitial edema, while Shengjiang San attenuated these changes and lowered lung-injury scores. The formula also reduced neutrophils and total inflammatory cells in bronchoalveolar lavage fluid and lowered interleukin-1β, tumor necrosis factor-α and interleukin-6 concentrations in lavage fluid and serum.

The protective response extended to the lung’s blood-air barrier. After LPS exposure, the animals developed increased vascular leakage, measured by Evans blue extravasation, together with higher lung wet-to-dry ratios and greater protein concentration in bronchoalveolar lavage fluid. Shengjiang San reduced all three measures, consistent with less edema and lower pulmonary vascular permeability. Fluorescence imaging also showed that LPS disrupted the continuous distribution of the junctional proteins ZO-1, occludin and VE-cadherin. Treatment with the formula preserved both the intensity and continuity of these markers. The results therefore link reduced inflammatory injury with maintenance of epithelial and endothelial barrier integrity, although the experiments did not isolate neutrophil responses as the sole cause of improved barrier function.

Lung RNA sequencing provided a broader view of the processes altered by the formula. The researchers identified 2,175 genes that changed in opposite directions after LPS challenge and Shengjiang San treatment. Gene Ontology analysis associated this responsive set with neutrophil activation, complement activation and regulation of inflammatory responses, while pathway analysis highlighted complement and coagulation cascades and neutrophil extracellular trap formation. Among 27 representative genes, C5ar1 and Padi4 were elevated in injured lungs and reduced after treatment. These transcriptional associations directed attention toward C5a/C5aR1 signaling and neutrophil extracellular trap formation, but the sequencing data alone did not establish a causal mechanism.

Consistent with the transcriptional findings, Shengjiang San reduced the accumulation and activation of neutrophils in injured lungs. Flow cytometry showed that LPS increased the proportion of viable CD45-positive, Ly6G-positive, CD11b-positive cells, and the formula lowered that increase, with the largest reduction at the highest dose. Surface CD11b signals on pulmonary neutrophils and CD11b abundance in whole-lung homogenates also declined. Immunohistochemical measurements of the neutrophil granule proteins myeloperoxidase and neutrophil elastase showed the same pattern. At the same time, pulmonary myeloperoxidase and citrullinated histone H3 signals became less colocalized after treatment, indicating reduced extracellular trap formation. Myeloperoxidase-DNA complexes reached 70.98 nanograms per milliliter in untreated injured lungs but fell to 38.07 nanograms per milliliter with the high dose, alongside lower PAD4 and citrullinated H3 abundance.

The complement measurements supplied a mechanistic link between the formula and the neutrophil responses. LPS raised plasma C5a from 120.1 to 259.4 picograms per milliliter and lung-homogenate C5a from 78.9 to 175.7 picograms per milliliter. Shengjiang San lowered these concentrations in a dose-dependent pattern: plasma C5a reached 156.2, 153.1 and 122.8 picograms per milliliter at the low, medium and high doses, while corresponding lung concentrations were 141.3, 115.7 and 102.9 picograms per milliliter. The treatment also reduced lung C5ar1 messenger RNA and C5aR1 immunostaining. Reanalysis of a separate single-cell RNA-sequencing dataset detected C5ar1 transcripts most frequently in neutrophils, followed by macrophages and monocytes, providing cellular context for the pathway without establishing that the formula acts only on one cell type or directly antagonizes the receptor.

Functional tests with purified mouse neutrophils showed that the effect was not confined to tissue measurements. In Transwell assays, C5a increased neutrophil migration through a 3-micrometer membrane, while serum collected from Shengjiang San-treated rats reduced both migration and the number of cells entering the lower chamber. PMX205 produced a similar inhibitory pattern. Shengjiang San-containing serum at 10% did not reduce neutrophil viability or alter apoptosis under these conditions. It also reduced C5a-induced increases in intracellular calcium, reactive oxygen species, myeloperoxidase and citrullinated H3 signals, and the area occupied by extracellular traps. Together, the in vivo and cell experiments support involvement of C5a/C5aR1-associated neutrophil activation, but they do not show that the formula directly antagonizes the receptor.

The formula also restrained a different route to extracellular trap formation. When primary neutrophils were stimulated with phorbol 12-myristate 13-acetate, or PMA, they developed increased myeloperoxidase, neutrophil elastase, PAD4 and citrullinated H3 signals, along with extracellular DNA and web-like structures visible by microscopy. Serum containing Shengjiang San reduced these changes without altering basal marker levels in unstimulated cells, and its inhibitory pattern resembled that of GSK484. Because PMA activates intracellular protein kinase C and NADPH oxidase rather than depending on C5a receptor engagement, this result indicates that Shengjiang San-containing serum can affect intracellular and downstream processes involved in extracellular trap formation as well as complement-triggered signaling. The active material basis and precise intracellular targets remain unresolved.

Chemical analysis identified 95 constituents or tentatively characterized compounds in the aqueous Shengjiang San extract, including anthraquinones and anthrones, phenylpropanoids and tannins, and terpenoids. In serum from treated rats, the researchers retained 79 unique SJS-related entries after consolidating different adduct signals, comprising 11 prototype constituents and 68 putative metabolites. Ferulic acid, also detected in the circulating serum profile, was selected for further testing. Its plasma concentration after intragastric administration peaked at 10.3 micrograms per milliliter at 30 minutes and declined to approximately 1.6 micrograms per milliliter at four hours. Biolayer interferometry showed concentration-dependent binding between ferulic acid and recombinant human C5a, with an apparent dissociation constant of 21.65 micromolar, while molecular docking proposed contacts involving Arg27, Arg62 and Lys68 of C5a. Ferulic acid also reduced citrullinated H3 fluorescence and extracellular-trap area in C5a-stimulated primary neutrophils. The peak plasma concentration was approximately 53 micromolar, but this comparison does not establish target engagement in vivo.

These findings support Shengjiang San as a candidate for further investigation in acute lung injury, but they remain preclinical and should not be interpreted as evidence of clinical efficacy. The study used a single LPS-induced mouse model, a pretreatment regimen and male animals, and it did not test treatment begun after injury or reproduce the varied causes and stages of acute lung injury and acute respiratory distress syndrome. Ferulic acid was identified as a candidate circulating constituent, but its contribution to the formula’s overall effects in vivo was not established, and other serum constituents were not systematically evaluated. The experiments also did not show that direct C5a binding or reduced extracellular-trap formation alone explained the preserved barrier. Prospective clinical studies, additional disease models and post-injury treatment schedules would be needed to determine whether the formula’s effects translate beyond experimental acute lung injury.

Subject of Research: Preclinical effects of Shengjiang San on complement-linked neutrophil activation and lung injury

Article Title: Shengjiang San suppresses C5a/C5aR1-mediated neutrophil activation and NET formation to alleviate acute lung injury

Article References: Zhang, D., Li, X., Zeng, J., Cheng, D., Wang, Y., Liu, S., Shao, B., Liu, C., Chen, H., Liu, Y., Zhao, X., Huang, G., & Xu, A. (2026). Shengjiang San suppresses C5a/C5aR1-mediated neutrophil activation and NET formation to alleviate acute lung injury. Frontiers in Pharmacology, 17, Article 1955412. https://doi.org/10.3389/fphar.2026.1955412

Image Credits: AI Generated

DOI: 10.3389/fphar.2026.1955412

Keywords: acute lung injury, Shengjiang San, C5a, C5aR1, neutrophils, neutrophil extracellular traps, ferulic acid, complement signaling, lung inflammation, mouse model, Shengjiang, suppresses