Prunella vulgaris polyphenols alleviate liver injury-uveitis comorbidity by regulating acylcarnitine via the S100A9-PP2A-AMPK pathway

Abstract

Background: Liver injury and uveitis pose severe threats to human health. Owing to the close relationship of physiology and pathology between the liver and the eyes, cases in which both conditions occur simultaneously are not uncommon in clinical settings, significantly complicating treatment. However, no suitable comorbid animal model has been reported, and research on the pathological mechanisms of this comorbidity is lacking. Prunella vulgaris L., a well-known traditional Chinese medicine renowned for its liver-clearing and eye-brightening properties. Prunella vulgaris polyphenols (PVPs) hold promise for improving liver injury and uveitis. However, research exploring their dual therapeutic effects within a single organism remains lacking, leaving the key active components and mechanisms of action largely uninvestigated.

Purpose: This exploratory study aimed to establish a rat model of liver injury combined with uveitis and investigated its pathological mechanisms, evaluating the therapeutic efficacy of PVPs in alleviating liver injury combined with uveitis in rats. Additionally, it explored the mechanism of action and identified key active ingredients of PVPs, offering potential new directions for the development of clinical therapeutic drugs.

Methods: A rat model of liver injury with uveitis was established through intraperitoneal d-GalN/LPS injection. Metabolomics and proteomics were applied to investigate pathological mechanisms, followed by validation using acylcarnitine and S100A9 inhibitors. PVPs were administered to evaluate therapeutic effects and explore mechanisms involved in alleviating liver injury and uveitis. Network pharmacology combined with molecular docking identified critical active components in PVPs. Subsequent animal experiments verified the efficacy of the representative component in improving liver injury and uveitis.

Results: d-GalN/LPS (150 mg/kg : 1 mg/kg) induced significant liver injury and uveitis in rats. Metabolomics analysis pointed to acylcarnitine as a key metabolite, and its inhibition reduced inflammation. Proteomics analysis implicated S100A9 in inflammation and immunity. Then, we intervened with S100A9 inhibitors in the model rats. The results suggested that the pathological mechanism of liver injury and uveitis caused by d-GalN/LPS involved the upregulation of S100A9 expression, an increase in PP2A activity, the inhibition of AMPK phosphorylation, and the downregulation of CPT1A, leading to the accumulation of acylcarnitine and promoting the inflammatory response in the liver and retina. Further, experiments involving PVPs demonstrated dose-dependent improvements in liver injury and uveitis caused by d-GalN/LPS. The underlying mechanism of action involved suppression of S100A9 expression, reduction of PP2A activity, activation of AMPK, upregulation of CPT1A, and subsequent reduction in acylcarnitine accumulation in both the liver and retina. This mechanism effectively alleviated the inflammatory effects induced by d-GalN/LPS. Network pharmacology and molecular docking analyses pinpointed several key active components of PVPs-namely, rosmarinic acid, salviaflaside, esculetin, 2-hydroxycinnamic acid, 3,4-dihydroxybenzaldehyde, and 7,8-dihydroxycoumarin-that play significant roles in mitigating liver injury and uveitis. Follow-up experiments using the representative active component rosmarinic acid in rats confirmed its efficacy in improving symptoms of d-GalN/LPS-induced liver injury and uveitis, further validating the therapeutic potential of these key active components.

Conclusions: This study successfully established a rat model of liver injury combined with uveitis and confirmed the efficacy of PVPs in alleviating this condition. Furthermore, it determined that the underlying mechanism involves regulation of the S100A9-PP2A-AMPK pathway, with rosmarinic acid identified as a key active compound. These findings provide a basis for clinical studies on liver-eye comorbidities and offer critical evidence for further research and drug development of PVPs in liver-clearing and eye-brightening.

Keywords: Acylcarnitine; Liver injury, uveitis; Metabolomics; Proteomics; Prunella vulgaris polyphenols; S100A9-PP2A-AMPK pathway.