Jujing Formula Protects Retinal by Regulating Nrf2/Ho-1 Pathways and Sphingolipid Rheostat and its Key Protein Levels on Liver-Kidney Yin Deficient Retinal Damage Mice

Abstract

Ethnopharmacological relevanceDry age-related macular degeneration (AMD) has a high rate of blindness, which still lacks effective treatment. JuJing Formula (JJF) is an ancient prescription used to nourish the liver and kidneys and treat eye diseases. Prior studies indicated that JJF was effective in treating dry AMD. However, its potential mechanism of action against dry AMD remains undefined.Aim of the studyThe protective effect and mechanism of JJF on dry AMD were investigated in the liver-kidney Yin deficient retinal damage (LKYD-RD) model of mice. Materials and methodsFirstly, thyroxine, clipping the tail, and sodium iodate were combined to establish the LKYD-RD model. Secondly, improvement in the symptoms of Yin deficiency was examined by detecting biochemical indices and histopathologic changes. Meanwhile, the therapeutic efficacy on retinal damage was evaluated using optical coherence tomography (OCT), hematoxylin and eosin (H&E) staining, terminal deoxynucleotidyl (TUNEL) staining, and levels of oxidative stress markers (SOD, MDA, and GSH) and inflammatory factors (IL-1β, IL-6, and TNF-α). Furthermore, serum metabolomics studies were performed to predict the mechanisms of JJF on LKYD-RD mice. Eventually, ELISA, immunofluorescence analysis, RT-qPCR, and Western blotting assays were conducted to verify the mechanisms.ResultsJJF not only significantly ameliorated LKYD syndrome but also restored the retinal structure and function in LKYD-RD mice. Furthermore, JJF inhibited the inflammatory response and suppressed oxidative stress through the Nrf2/HO-1 signaling pathway. Metabolomics showed that sphingolipid signaling was the main regulatory pathway for JJF to improve LKYD-RD. Additionally, JJF significantly reduced the increased levels of ceramide and sphingosine and elevated sphingosine-1-phosphate in the eyes of mice. It also increased the expression of p-Sphk1/Sphk1, Asah1, and Bcl2 while decreasing the expression of Cers2, Cers6, Bax, cleaved-caspase 3, and Sgpp1. The findings suggested that regulation of the sphingolipid rheostat and its critical proteins is a potential mechanism of JJF resistance to dry AMD.ConclusionsIn conclusion, JJF demonstrates therapeutic effects in a mouse model of LKYD-RD, with its mechanism of action including the regulation of sphingolipid metabolic signaling pathways. This research provides a reference for dry AMD treatment in clinics.

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Funding declaration: This work received funding from the National Natural Science Foundation of China (U21A20408) and the Postgraduate Research and Practice Innovation Program of Jiangsu Province (No. KYCX23_2018).

Conflict of Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Ethical Approval: The protocols received formal approval from the Animal Ethical Committee at Nanjing University of Chinese Medicine (202302A073), ensuring all procedures adhered to ethical standards.

Keywords: JuJing Formula, Dry age-related macular degeneration, Liver-kidney Yin deficiency syndrome, Retinal damage, Sphingolipid rheostat, Nrf2/HO-1 pathways