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：

RasGRP4 gene; Diabetic retinopathy; Inflammation; NOd-like receptor thermal protein domain protein 3 inflammasome

DOI：

10.3760/cma.j.cn511434-20240428-00175

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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 was used to detect the retinal thickness and ganglion cell layer thickness. Electroretinography was used to detect the function of the retina in mice under dark-adapted conditions. Total RNA was extracted from the retinas of mice in DM group and DM-KO group, and transcriptomic sequencing was performed to screen differentially expressed genes (DEG). Core genes were screened using MCODE and Cytohubba plug-ins of Cytoscape v3.8.2 software. At the same time, the functional enrichment analysis of gene samples (GO) of the selected DEG was performed. The mRNA relative expression levels of interleukin-8, transforming growth factor-β (TGF-β), interferon-γ (IFN-γ), NOd-like receptor thermal protein domain protein 3 (NLRP3), Caspase-1 and IL-1β in each group were detected by real-time quantitative polymerase chain reaction. t test was used to compare the two groups. One-way analysis of variance was used to compare the three groups. Results Compared with the DM group, there was no significant difference in blood glucose and body weight in the DM-KO group with the extension of high-fat diet (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). The retinal thickness and ganglion cell layer thickness of mice in the three groups were significantly reduced in the DM group compared with the normal group, while DM-KO was significantly increased compared with the DM group, and the differences were statistically significant (F=30.43, 7.81; P＜0.000 1, 0.01). Comparison of a-wave and b-wave amplitudes among the three groups showed that the DM group was significantly lower than the normal group, while the DM-KO was significantly higher than the DM group, and the differences were statistically significant (F=16.46, 35.58; P＜0.001, 0.000 1). 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. Comparison of mRNA relative expression levels of IL-8, TGF-β, IFN-γ, NLRP3, Caspase-1 and IL-1β in the three groups showed that the DM group was significantly higher than the normal group, while the DM-KO was significantly lower than 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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