Investigating the relationship between blood metabolites and diabetic retinopathy using two-sample mendelian randomization and in vivo validation

Abstract

We addressed fundamental questions about the influence of metabolites on the development of Diabetic retinopathy (DR), and explored the related pathological mechanism. Genome-wide association study (GWAS) database data for metabolites and DR were used to perform Mendelian randomization (MR) studies. The inverse variance weighting (IVW) was chosen as the primary analysis method. Sensitivity analysis was conducted using MR-PRESSO, leave-one-out and Cochran's Q test. Confounding factors were eliminated to ensure robustness. We also conducted metabolic pathway analysis. In vivo experimental validation was conducted using Sprague Dawley rats. The serum metabolites of the DR group rats and normal group rats were examined to evaluate the MR results. The screen identified eighteen metabolites associated with DR risk, twelve of which were known components. Seven metabolites were positively correlated with DR risk, while five could reduce it. Eight metabolites associated with proliferative DR (PDR) risk were identified, four of which are known components. Three of these were positively associated with PDR risk and one metabolite reduced PDR risk. Additionally, two possible metabolic pathways involved in the biological mechanism of DR were identified. The ELISA results showed that the serum levels of isoleucine and 4-HPA were significantly increased in DR rats, while the level of inosine was decreased. This study offers novel insights into the biological mechanisms underlying DR. Metabolites that are causally linked to DR may serve as promising biomarkers and therapeutic targets.

Keywords: Blood metabolites; Diabetic retinopathy; Hippurate; Inosine; Isoleucine; Mendelian randomization.

Fig. 1

Overview of the research workflow. Image created with BioRender.com, with permission.

Fig. 2

IVW, MR-Egger and WM methods for the causal relationship between blood metabolites and DR risk. DR diabetic retinopathy; IVW inverse-variance weighted; WM weighted median; OR odds ratio.

Fig. 3

Forest plot of the IVW analyses of the associations between metabolite and risk of DR. DR diabetic retinopathy, CI confidence interval, IVW inverse-variance weighted, OR odds ratio.

Fig. 4

Forest plot for the associations between metabolite and risk of DR after removal of confounders. DR diabetic retinopathy; CI confidence interval; IVW inverse-variance weighted; OR odds ratio.

Fig. 5

Forest plot for the associations between metabolite and risk of PDR after removal of confounders. PDR proliferative diabetic retinopathy; CI confidence interval; IVW inverse-variance weighted; OR odds ratio.

Fig. 6

Eight weeks after intraperitoneal injection, rats developed the typical pathological manifestations of DM and DR. (A) Comparison of appearance between the two groups of rats. The DR group rats exhibited a thin body shape, withered hair and diabetic cataracts. (B) Body weights; (C) random blood glucose levels. (D) Representative retinal morphology images of H&E staining. All data are presented as the mean ± SD; n = 6 per group. ****p < 0.0001, ***p < 0.001, **p < 0.005, *p < 0.05. Con control; DR diabetic retinopathy.

Fig. 7

Rat retinal cell apoptosis and blood metabolite level changes. A TUNEL immunoresponse staining showed that retinal cell apoptosis was significantly increased in the DR group. B Representation of the mean TUNEL-positive ratio in the retinas of rats. C, D,E Serum metabolites were detected by ELISA. The levels of isoleucine and 4-HPA were increased, and the levels of inosine were decreased in the DR group. All data are presented as the mean ± SD; n = 6 per group. ****p < 0.0001, ***p < 0.001, **p < 0.005, *p < 0.05. Con control; DR diabetic retinopathy.