A Study On The Correlation Of Diabetic Retinopathy With Choline And Its Metabolite,Trimethylamine-N-oxide

Part Ⅰ Association between dietary choline intake and diabetic retinopathyObjective:To explore the association between dietary choline intake and odds of diabetic retinopathy(DR).Methods:A cross-sectional study was conducted using the combined data of the National Health and Nutrition Examination Survey(NHANES)2005-2008.Diabetic patients aged 40 years and older who went through retinal exams were included in this study.Energy-adjusted choline intakes were calculated separately for men and women using the residual method.Participants were divided into low,moderate,and high choline-intake groups by the tertile cutoff values of energy-adjusted choline intake.Multivariable logistic regression analyses were performed to identify the association between dietary choline intake and DR.Results:1,272 diabetic patients were included in this study.The weighted population size was 9,339,124 for males and 10,109,553 for females.Female DR patients consumed more choline than non-DR patients(268.6 mg/d vs 250.9 mg/d;P=0.046).In multivariable logistic-regression models,the odds ratio(OR)of DR for female patients in the highest choline intake group was 2.14[95%confidence interval(CI),1.38-3.31;P=0.001]compared with those in the lowest intake group.This association was positive but not statistically significant in males.Conclusion:Higher intake of dietary choline is associated with increased odds of DR in females,but not in males.Further studies are warranted to investigate the direct role of choline in DR development and determine the recommended daily intake of choline for diabetic patients weighing the pros and cons of dietary choline consumption.Part Ⅱ Relationship between plasma trimethylamine-N-oxide levels and diabetic retinopathyObjective:To determine the relationship between plasma levels of trimethylamine-N-oxide(TMAO)and odds of DR.Methods:A cross-sectional study was conducted.Blood samples were obtained from type 2 diabetes mellitus(T2DM)patients with or without DR and analyzed using stable isotope dilution liquid chromatography tandem mass spectrometry.Multivariable logistic regression analyses were performed to identify the association between plasma TMAO and DR.The diagnostic value of plasma TMAO was assessed by the area under the receiver operating characteristic curve(AUROC)and integrated discrimination improvement(IDI).Results:We included 122 T2DM patients and 40 non-diabetes patients.In the T2DM patients,plasma levels of TMAO were significantly higher in patients with DR compared with those without DR(0.752 μmol/L vs 0.458μmol/L,P=0.001).As the increment of logarithmic(In)transformation of TMAO per each standard deviation(SD),the OR for DR was 2.31(95%Cl,1.30-4.12,P=0.005).In the diagnostic model,the addition of TMAO contributed to the improvement in AUROC from 0.646 to 0.734(P=0.043),and the IDI was 10.7%(P<0.001).Conclusion:Elevated levels of plasma TMAO were associated with higher odds of DR in T2DM patients,and further investigation are required for the causality of this association.Part Ⅲ The effects of trimethylamine-N-oxide on human retinal endothelial cells in vitroObjective:To address the effects of TMAO on the proliferation,migration,and tube formation abilities of human retinal endothelial cells(HRECs)and the underlying mechanisms.Methods:HRECs were cultured with or without TMAO under normal glucose(NG,5.5 mM)or high glucose(HG,25 mM)condition.Cell proliferation was detected using cell counting kit(CCK-8)assay.Migration of HRECs was examined by wound healing assay and Transwell assay.Tube formation of HRECs was examined using Matrigel assay.Real-Time PCR was used to detected the expression of ICAM-1,MCP-1,IL-1β,TNF-α,MMP-2,VEGF-A.Phosphorylation of AKT was detected by Western Blot.Results:Compared to NG group,HRECs cultured under NG+TMAO demonstrated significantly increasing ability of cell proliferation,migration,and tube formation.TMAO upregulated ICAM-1,MCP-1,IL-1β TNF-α,MMP-2,VEGF-A and enhanced the phosphorylation of AKT.The effects of TMAO mentioned above were more significant in cells cultured under HG.Conclusion:TMAO could enhance the inflammatory response and angiogenic activities of HRECs via activating AKT signaling pathway,and has a synergic effect with HG stimulation.