Therapeutic effect of silencing <i>RasGRP4</i> gene on retinopathy in diabetic mice_Chinese Journal of Ocular Fundus Diseases

Authors：

Li Qingbo ¹ , Zhou Xu ¹ , Zhou Saijun ² , Shao Yan ¹ , Li Xiaorong ¹ ,  Liu Juping ¹

1. Tianjin Key Laboratory of Retinal Functions and Diseases, Tianjin Branch of National Clinical Research Center for Ocular Disease, Eye Institute and School of Optometry, Tianjin Medical University Eye Hospital, Tianjin 300384, China;
2. NHC Key Laboratory of Hormones and Development, Tianjin Key Laboratory of Metabolic Diseases, Chu Hsien-I Memorial Hospital & Tianjin Institute of Endocrinology, Tianjin Medical University, Tianjin 300134, China;

Corresponding author：

Liu Juping, Email: tydljp@126.com

Keywords：

Ras guanine nucleotide-releasing protein4; Diabetic retinopathy; Inflammation; NLRP3 inflammasome

DOI：

10.3760/cma.j.cn511434-20240428-00175

Video：

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Objective To 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. Methods A 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. Results There 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.

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11.	Gritsenko A, Green JP, Brough D, et al. Mechanisms of NLRP3 priming in inflammaging and age related diseases[J]. Cytokine Growth Factor Rev, 2020, 55: 15-25. DOI: 10.1016/j.cytogfr.2020.08.003.
12.	Gaidt MM, Hornung V. The NLRP3 inflammasome renders cell death pro-inflammatory[J]. J Mol Biol, 2018, 430(2): 133-141. DOI: 10.1016/j.jmb.2017.11.013.
13.	Campden RI, Zhang Y. The role of lysosomal cysteine cathepsins in NLRP3 inflammasome activation[J]. Arch Biochem Biophys, 2019, 670: 32-42. DOI: 10.1016/j.abb.2019.02.015.
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18.	Rübsam A, Parikh S, Fort PE. Role of inflammation in diabetic retinopathy[J]. Int J Mol Sci, 2018, 19(4): 942. DOI: 10.3390/ijms19040942.

1. Simó-Servat O, Hernández C, Simó R. Diabetic retinopathy in the context of patients with diabetes[J]. Ophthalmic Res, 2019, 62(4): 211-217. DOI: 10.1159/000499541.
2. Takino JI, Miyazaki S, Nagamine K, et al. The role of RASGRP2 in vascular endothelial cells-a mini review[J/OL]. Int J Mol Sci, 2021, 22(20): 11129[2021-10-15]. https://pubmed.ncbi.nlm.nih.gov/34681791/. DOI: 10.3390/ijms222011129.
3. Huang S, Wang J, Zhang L, et al. Ras guanine nucleotide-releasing protein-4 promotes renal inflammatory injury in type 2 diabetes mellitus[J/OL]. Metabolism, 2022, 131: 155177[2022-02-23]. https://pubmed.ncbi.nlm.nih.gov/35218794/. DOI: 10.1016/j.metabol.2022.155177.
4. Adachi R, Krilis SA, Nigrovic PA, et al. Ras guanine nucleotide-releasing protein-4 (RasGRP4) involvement in experimental arthritis and colitis[J]. J Biol Chem, 2012, 287(24): 20047-20055. DOI: 10.1074/jbc.M112.360388.
5. Suire S, Lécureuil C, Anderson KE, et al. GPCR activation of Ras and PI3Kc in neutrophils depends on PLCb2/b3 and the RasGEF RasGRP4[J]. EMBO J, 2012, 31(14): 3118-3129. DOI: 10.1038/emboj.2012.167.
6. Trapnell C, Roberts A, Goff L, et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks[J]. Nat Protoc, 2012, 7(3): 562-578. DOI: 10.1038/nprot.2012.016.
7. Zhao H, Wu L, Yan G, et al. Inflammation and tumor progression: signaling pathways and targeted intervention[J]. Signal Transduct Target Ther, 2021, 6(1): 263. DOI: 10.1038/s41392-021-00658-5.
8. Spencer BG, Estevez JJ, Liu E, et al. Pericytes, inflammation, and diabetic retinopathy[J]. Inflammopharmacology, 2020, 28(3): 697-709. DOI: 10.1007/s10787-019-00647-9.
9. Swanson KV, Deng M, Ting JP. The NLRP3 inflammasome: molecular activation and regulation to therapeutics[J]. Nat Rev Immunol, 2019, 19(8): 477-489. DOI: 10.1038/s41577-019-0165-0.
10. Sharma M, de Alba E. Structure, activation and regulation of NLRP3 and AIM2 inflammasomes[J]. Int J Mol Sci, 2021, 22(2): 872. DOI: 10.3390/ijms22020872.
11. Gritsenko A, Green JP, Brough D, et al. Mechanisms of NLRP3 priming in inflammaging and age related diseases[J]. Cytokine Growth Factor Rev, 2020, 55: 15-25. DOI: 10.1016/j.cytogfr.2020.08.003.
12. Gaidt MM, Hornung V. The NLRP3 inflammasome renders cell death pro-inflammatory[J]. J Mol Biol, 2018, 430(2): 133-141. DOI: 10.1016/j.jmb.2017.11.013.
13. Campden RI, Zhang Y. The role of lysosomal cysteine cathepsins in NLRP3 inflammasome activation[J]. Arch Biochem Biophys, 2019, 670: 32-42. DOI: 10.1016/j.abb.2019.02.015.
14. Paik S, Kim JK, Silwal P, et al. An update on the regulatory mechanisms of NLRP3 inflammasome activation[J]. Cell Mol Immunol, 2021, 18(5): 1141-1160. DOI: 10.1038/s41423-021-00670-3.
15. Yang Y, Jiang G, Huang R, et al. Targeting the NLRP3 inflammasome in diabetic retinopathy: from pathogenesis to therapeutic strategies[J/OL]. Biochem Pharmacol, 2023, 212: 115569[2023-04-25]. https://pubmed.ncbi.nlm.nih.gov/37100255/. DOI: 10.1016/j.bcp.2023.115569.
16. Chen H, Zhang X, Liao N, et al. Enhanced expression of NLRP3 inflammasome-related inflammation in diabetic retinopathy[J]. Invest Ophthalmol Vis Sci, 2018, 59(2): 978-985. DOI: 10.1167/iovs.17-22816.
17. Chai G, Liu S, Yang H, et al. NLRP3 blockade suppresses pro-inflammatory and pro-angiogenic cytokine secretion in diabetic retinopathy[J]. Diabetes Metab Syndr Obes, 2020, 13: 3047-3058. DOI: 10.2147/dmso.S264215.
18. Rübsam A, Parikh S, Fort PE. Role of inflammation in diabetic retinopathy[J]. Int J Mol Sci, 2018, 19(4): 942. DOI: 10.3390/ijms19040942.

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