Interleukin-35 Ameliorates Obesity-Associated Impairment of Endothelium-Dependent Vasodilation via the STAT3-MAPK/ERK-eNOS Signaling Axis.

BACKGROUND

Obesity impairs endothelial nitric oxide synthase (eNOS)-nitric oxide (NO) signaling, triggering chronic inflammation, endothelial dysfunction and defective vasodilation, which drives cardiometabolic diseases. Existing metabolic therapies provide only indirect vascular protection and fail to resolve residual inflammatory-mediated cardiovascular risk. As an anti-inflammatory interleukin 12 (IL-12) family cytokine, interleukin 35 (IL-35) is suppressed in obesity and exerts vascular protective effects under inflammatory conditions, yet its function and underlying mechanisms in obesity‑related endothelial vasodilator dysfunction remain undefined.

MATERIALS AND

METHODS

An obese mouse model was established by 8-week high-fat diet (HFD) feeding, followed by 4-week intraperitoneal administration of recombinant mouse IL-35. Aortic-ring assays evaluated vasodilatory function. Palmitic acid (PA)-treated human umbilical vein endothelial cells (HUVECs), recapitulate lipotoxic endothelial injury, were treated with recombinant human IL-35. Western blotting, immunofluorescence, 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA) detection and immunoprecipitation-mass spectrometry (IP-MS) were applied to detect IL-35-subunit (Epstein-Barr virus-induced gene 3 [EBI3] and interleukin-12 subunit alpha [IL-12A]) levels, eNOS Ser1177 phosphorylation, phosphorylation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling molecules (cellular rapidly accelerated fibrosarcoma [cRAF], mitogen-activated protein kinase kinase [MEK], and ERK) and signal transducer and activator of transcription 3 (STAT3) activation, NO production, and eNOS-binding partners. Rescue assays were performed using Raf or ERK specific inhibitors and STAT3 small interfering RNA (siRNA).

RESULTS

HFD mice displayed reduced aortic endothelial EBI3 expression, impaired endothelium-dependent vasorelaxation, decreased eNOS Ser1177 phosphorylation and lowered NO production; all these phenotypes were rescued by exogenous IL-35. IP-MS and immunoblotting demonstrated that IL-35 strengthened eNOS-ERK1 interaction and restored PA-repressed phosphorylation of cRAF, MEK, ERK and eNOS Ser1177, which were abrogated by Raf or ERK blockade. Furthermore, IL-35 recovered STAT3 phosphorylation suppressed by PA, while STAT3 knockdown blunted IL-35-driven MAPK/ERK activation, eNOS phosphorylation and subsequent NO production.

CONCLUSION

IL-35 alleviates obesity-induced impairment of endothelium-dependent vasodilation via the STAT3-MAPK/ERK-eNOS axis, which elevates eNOS Ser1177 phosphorylation and NO synthesis. This work supports IL-35 as a prospective therapeutic candidate for obesity-associated vascular complications.