Liver-eye axis in metabolic syndrome: integrated evidence from mendelian randomization, single-cell sequencing, and clinical sample validation.

Background: Non-alcoholic fatty liver disease (NAFLD) and diabetic retinopathy (DR) are common clinical complications of metabolic syndrome in different organ systems. However, the relationship between NAFLD and DR remains controversial, and more high-level evidence is needed to clarify the causal link between them. We aimed to investigate the causal relationship between NAFLD and DR across its progression stages, and to identify circulating proteins may mediate this association. Materials and methods: Based on genetic tools, a multi-stage network MR framework was constructed, integrating NAFLD/DR genome-wide association study data and three blood proteome-wide datasets. Causal effects of NAFLD on DR were assessed using inverse-variance weighted regression and multimodal models. Proteome-wide MR identified DR-associated proteins. Bidirectional two-sample MR and mediation models quantified regulatory weights in NAFLD-protein-DR pathways. In addition, the transcriptional expression levels of the identified mediator proteins in the liver were verified through single-cell transcriptome sequencing. The levels of the mediator protein in the serum samples of clinical patients were detected by ELISA. The levels of mediator proteins in the aqueous humor and vitreous humor samples of clinical patients were detected by Luminex liquid-phase protein chip. Results: NAFLD increased DR risk (OR = 1.04, 95% CI: 1.01-1.08) and specifically promoted non-proliferative DR (NPDR) progression (OR = 1.26, 95% CI: 1.05-1.52). Four proteins were identified as candidate mediators of NAFLD-DR association, among them, LTB (lymphotoxin-β, a TNF superfamily member involved in immune regulation and inflammatory responses) showed the strongest effect. In the liver of NAFLD model mice, the expression of the LTB gene was mainly elevated in T cells and B cells. The levels of LTB protein in the aqueous humor, vitreous humor and serum of NAFLD patients with DR were all higher than those of NAFLD patients without DR. Conclusions: NAFLD is causally associated with an increased risk of DR development, particularly during early NPDR stages. LTB may mediate the biological process by which NAFLD promotes DR. T cells and B cells in the liver of patients with NAFLD produce higher levels of LTB protein. In NAFLD patients with DR, LTB protein levels were elevated in serum, aqueous humor, and vitreous humor compared to NAFLD patients without DR, suggesting that LTB may play a role in the link between these two conditions.