Tubuloside A mitigates sepsis-induced splenic injury in mice by suppressing NOX4-associated oxidative stress,...

Cistanche deserticola Y.C.Ma is a well-known traditional medicinal herb widely used in traditional Chinese medicine for the treatment of kidney injury-related conditions, fatigue, infertility, and age-related disorders, as well as for improving immune function, and is traditionally prescribed for conditions associated with weakness and chronic inflammatory states.

To investigate the potential efficacy of Tubuloside A (TA), an active constituent of Cistanche deserticola Y.C.Ma, against sepsis-induced splenic injury, a sepsis-associated structural and functional impairment of the spleen characterized by disrupted splenic architecture, dysregulated immune-cell homeostasis, excessive inflammatory responses, and weakened host defense, and to clarify its underlying mechanism of action.

A murine cecal ligation and puncture (CLP) model and lipopolysaccharide (LPS)-stimulated J774A.1 macrophages were used to investigate the protective effects of TA against sepsis-induced splenic injury. Oxidative stress, antioxidant capacity, mitochondrial function, inflammatory responses, and apoptosis-related injury, splenic immune-cell composition, macrophage inflammatory phenotype, and F4/80/NOX4 colocalization were evaluated by biochemical assays, JC-1 staining, qPCR, Western blotting, flow cytometry, double immunofluorescence staining, and immunohistochemical analyses. Bone marrow-derived macrophages (BMDMs) were further used for supportive validation of macrophage-related inflammatory responses and NOX4 expression. Integrative network pharmacology and molecular docking were employed to identify candidate molecules potentially associated with TA-mediated protection, and NADPH oxidase 4 (NOX4) overexpression was used to further examine the involvement of NOX4-associated oxidative stress in vitro.

TA significantly alleviated splenic injury and improved survival in septic mice. TA reduced oxidative stress, as evidenced by decreased malondialdehyde and reactive oxygen species (ROS) levels and enhanced antioxidant defenses, including superoxide dismutase, catalase, glutathione, and total antioxidant capacity. TA restored mitochondrial membrane potential and improved mitochondrial homeostasis, accompanied by increased TOM20, GPX4, and PGC-1α expression and reduced Drp1 expression. In addition, TA suppressed pro-inflammatory mediators, including TNF-α, IL-1β, IL-6, and iNOS, increased anti-inflammatory IL-10 expression, and reduced Bax and cleaved caspase-3/9 levels, indicating inhibition of apoptosis-related injury. Flow cytometry and BMDM validation further showed that TA regulated splenic immune-cell alterations and macrophage inflammatory responses, while F4/80/NOX4 double immunofluorescence staining indicated that NOX4 expression was associated with F4/80-positive macrophages in splenic tissues. Mechanistically, network pharmacology and molecular docking suggested that NOX4-associated oxidative stress may be involved in TA-mediated protection, which was further supported by the marked induction of NOX4 during sepsis and by the finding that NOX4 overexpression significantly blunted the protective effects of TA in vitro.

This study demonstrates that TA exerts a protective effect against sepsis-induced splenic injury by suppressing NOX4-associated oxidative stress, preserving mitochondrial homeostasis, and limiting downstream inflammatory and apoptotic damage. These findings not only expand the pharmacological profile of TA, but also provide experimental support for the therapeutic potential of an active constituent from Cistanche deserticola Y.C.Ma in sepsis-related immune-organ injury, particularly through the regulation of oxidative stress, macrophage-associated inflammatory responses, and splenic immune-cell alterations.