Diabetic retinopathy (DR) is one of the microvascular complications of diabetes mellitus causing severe visual impairment, and it is the main cause of blindness in adults. Metabolic abnormalities play an important role in the occurrence and development of DR, including the abnormal levels of glucose metabolism, lipid metabolism, amino acid metabolism and purine metabolism, which indicate that there are disorders of phosphopentose pathway, arginine metabolism pathway, polyol pathway and ascorbic acid pathway in the progression of DR. Metabolomics has great advantages in exploring the pathogenesis and diagnosis of DR, helping to identify the characteristic metabolic changes of DR And discover potential biomarkers. However, the existing metabolomics studies on DR have some limitations, such as the potential biomarkers found in some studies are difficult to verify in other studies due to differences in race, age, gender and sample size. There are few studies on biomarkers at different stages of DR. Therefore, in the future, multi-center and large-scale clinical studies are needed to screen out biomarkers with practical clinical diagnostic value.
ObjectiveTo observe the effects of RasGRP4 gene deletion on the structure and function of the retina in diabetic mice, and to explore the mechanism of RasGRP4 in diabetic retinopathy (DR) by transcriptome sequencing in conjunction with bioinformatics analysis. MethodsA total of 12 male C57BL/6J mice were divided into normal group, diabetic group (DM group), with 6 mice in each group. Six male RasGRP4 knockout mice were uesd as RasGRP4 knockout diabetic group (DM-KO group). Mice in the DM group and DM-KO group were fed with high-fat diet combined with intraperitoneal injection of streptozotocin to establish diabetic model and body weight and blood glucose were monitored regularly. Three months after modeling, optical coherence tomography (OCT) was used to detect the retinal thickness. Electroretinography (ERG) was used to detect the function of the retina in mice under dark-adapted conditions. Total RNA was extracted from the retinas of the DM group and DM-KO group for transcriptome sequencing and bioinformatics analysis. The relative expression of IL-8, TGF-β, IFN-γ, NLRP3, Caspase-1, and IL-1β mRNA in retina were detected by real-time quantitative polymerase chain reaction (qRT-PCR). One-way analysis of variance was used to compare groups. ResultsThere was no statistically significant difference in blood glucose and body weight between mice in the DM group and DM-KO group (t=0.12、2.02、0.22、0.10、0.59、0.41、1.35、0.31、1.12、1.58、1.47、1.20、1.24、0.39、0.66、0.14, P＞0.05). Compared with the normal group, the retinal thickness and ganglion cell layer thickness were significantly decreased in the DM group, while the retinal thickness and ganglion cell layer thickness were significantly increased in the DM-KO group compared with the DM group, with statistical significance (F=30.43、7.81, P＜0.0001、0.01). Compared with the normal group, the a-wave and b-wave amplitudes were significantly decreased in the DM group, while the a-wave and b-wave amplitudes were significantly increased in the DM-KO group compared with the DM group, with statistical significance (F=16.46、35.58, P＜0.001、0.0001). Compared with the DM group, 184 differential genes (DEG) were screened in the DM-KO group, among which 39 up-regulated and 145 down-regulated genes were detected, respectively. The results of the MCODE plug-in analysis showed that Col1a2, Fbln1, Fbn1, Col6a3, Fmod, Ogn, Tgfb, Mfap4, Vcan, Nid2, and Col18a1 were core genes in the DEG. Cytohubba plug-in analysis showed that Col1a2, Mrc1, Cd47, Fbn1, Cybb, Cd163, Fbln1, Fmod, Adgre1, and Col6a3 were the core genes in DEG. The results of the GO functional enrichment analysis showed that DEG was mainly involved in hemoglobin complexes, MHC class II protein complex, apical plasma membrane, inflammasome complex, immunological synapse, response to bacterium, inflammatory response, immune system processe, response to hypoxia, and cell adhesion were significantly enriched. qRT-PCR results showed that compared with the normal group, the relative expression levels of IL-8, TGF-β, IFN-γ, NLRP3, Caspase-1, and IL-1β mRNA in the retina of mice in DM group were significantly increased, while the relative expression levels of IL-8, TGF-β, IFN-γ, NLRP3, Caspase-1, and IL-1β mRNA in the retina of mice in DM-KO group were significantly decreased compared with the DM group, with statistical significance (F=12.43、15.41、70.09、29.04、11.79、41.28, P＜0.01). Conclusion RasGRP4 deficiency plays a therapeutic role in the development of DR through inhibition of inflammatory factor secretion and NLRP 3 inflammasome pathway activation.