Association between systemic immune-inflammation indexes and Nuodikang capsule's protection in LPS-induced acute lung injury in mice.

BACKGROUND

Acute lung injury (ALI) is a prevalent clinical syndrome characterized by respiratory distress and progressive hypoxemia, which arises from diverse intrapulmonary and extrapulmonary etiological factors. Currently, there is no definitive treatment for ALI. The Nuodikang capsule (NDK) is a traditional Tibetan medicine composed of Rhodiola crenulata (Hook. f. & Thomson) H. Ohba (R. crenulata), which is known for its remarkable therapeutic effects on lung diseases by regulating Chiba, benefiting Qi and clearing lung-heat. However, its efficacy in treating ALI and the underlying mechanisms remain unclear.

METHODS

This study initially employed UPLC-Q-Exactive Orbitrap/MS technology to analyze the chemical constituents of NDK. Subsequently, the protective effects and underlying mechanisms of NDK on an ALI model in C57BL/6 J mice were investigated. Following a 14-day intragastric administration of varying doses of NDK (0.14, 0.28 and 0.56 g/kg), an ALI model was constructed by noninvasive intratracheal instillation of lipopolysaccharide (LPS, 5 mg/kg). Respiratory function and the degree of lung injury in ALI mice were assessed using whole-body plethysmography (WBP). Additionally, complete blood counts in blood and bronchoalveolar lavage fluid (BALF) of mice were analysed by hematology analyzer. The water content in the lung tissue of mice was determined by calculating the ratio of dry weight to wet weight. Pathological damage to mouse lung tissue was evaluated using hematoxylin and eosin (H&E) staining, periodic acid-Schiff (PAS) staining and immunohistochemical staining (IHC), while structural damage was evaluated via micro-computed tomography (micro-CT). Synchronously, the Luminex 200 system was employed to analyze the levels of 23 chemokines and immune-inflammatory factors in serum, BALF and lung tissue of mice. Meanwhile, flow cytometry was utilized to determine the M1/M2 macrophage ratio, as well as the levels of B lymphocytes, dendritic cells (DCs), natural killer cells (NKs), T lymphocytes and their subtypes in the blood and lung tissues of mice with ALI. Additionally, the immunoregulatory and anti-inflammatory effects of NDK were validated using Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Set Enrichment Analysis (GSEA) in transcriptomic studies. The ultrastructural changes of blood vessels, alveoli and other lung tissue structures in ALI mice were measured by transmission electron microscopy (TEM). Finally, the gene and protein expression levels of Zonula Occludens-1 (ZO-1), Occludin (OCLN) and VE-cadherin in the lung tissues samples of ALI mice were detected by qRT-PCR, western blot (WB) and immunofluorescence.

RESULTS

Through the use of UPLC-Q-Exactive Orbitrap/MS, 95 chemical components in NDK were identified overall, which cover anti-inflammatory compounds such as salidroside, tyrosol and gallic acid. The results from WBP indicated that NDK intervention significantly ameliorated the aberrant changes in respiratory frequency (RF), minute volume (Mv), enhanced pause (Penh), and peak expiratory flow (PEF) observed in ALI mice, thereby improving their respiratory function. Furthermore, NDK was effective in reducing pulmonary water content and decreasing the levels of inflammatory cells in both the peripheral blood and BALF of ALI mice. Histological analysis via H&E staining revealed that NDK intervention markedly diminished inflammatory cell infiltration in lung tissue, reduced capillary congestion, and mitigated collagen fiber proliferation. PAS staining corroborated these findings, demonstrated that NDK effectively decreased mucus secretion in the lung tissue of ALI mice, thereby preserving normal pulmonary physiological functions. The IHC results also suggested that NDK had the effect of inhibiting the expression of α-SMA in lung alveolar interstitium and effectively inhibited the development of fibrosis in the later stage of ALI. Additionally, micro-CT imaging illustrated that the lung architecture of NDK-treated mice exhibited enhanced clarity and structural integrity. Marvelously, a high-dose NDK intervention (0.56 g/kg) effectively reduced the levels of chemokines and inflammatory mediators, including GM-CSF, TNF-α, IL-1α, IL-1β, IL-3, and KC, in the lung-tissue, serum and BALF samples of mice. Moreover, the administration of 0.56 g/kg NDK statistically plummeted the ratio of M1 macrophages in the peripheral blood and lung tissue of ALI mice, counteracted the upregulation of B lymphocytes, DCs, T lymphocytes, NKs and Th1/2/17 cells, and increased the levels of Treg cells. The findings from KEGG and GSEA enrichment analyses further corroborated that the anti-ALI effects of NDK are associated with the modulation of immune responses and the suppression of inflammatory responses. Furthermore, TEM results demonstrated that NDK intervention substantially reduced endothelial cell swelling and the number of neutrophils in lung tissue, while improving the stability of tight junction structures. Concurrently, results from qRT-PCR, Western blot and immunofluorescence assays further substantiated that 0.56 g/kg NDK effectively maintained the structural integrity of the pulmonary air-blood barrier via increasing the expression of ZO-1, OCLN and VE-cadherin, thereby supporting its therapeutic potential in ALI.

CONCLUSIONS

Our study innovatively revealed that NDK protected against ALI through a unique mechanism: beyond preserving pulmonary respiratory function and enhancing immune response, it specifically mitigated inflammatory factor-induced damage to the lung air-blood barrier. This distinct interplay of immune regulation and barrier protection, rarely reported previously, highlighted NDK as a novel therapeutic candidate and offered new insights into ALI pathogenesis.