Interferon gene stimulating protein inhibitor improves leukocyte adhesion and glycolysis of retinal vascular endothelial cells_Chinese Journal of Ocular Fundus Diseases

Authors：

Wang Yong , Cao Jingjing , Li Hui , Hong Yaru , Liu Aihua ,  Dong Lijie

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 Wang Yong and Cao Jingjing are contributed equally to the article;

Corresponding author：

Dong Lijie, Email: aitaomubang@126.com

Keywords：

Diabetes mellitus, experimental; Stimulator of interferon genes; Leukocyte adhesion; Reactive oxygen species; Glycolysis

DOI：

10.3760/cma.j.cn511434-20220520-00312

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Objective To investigate the effects of interferon gene stimulating protein (STING) inhibitor (C176) on human retinal microvascular endothelial cells (hRMEC) under oxidative stress. Methods An animal experimental study. In vivo experiment: 48 healthy male C57BL/6J mice were randomly divided into wild type mice group (WT group) and diabetes (DM) group, with 24 mice in each group. DM mice were induced by streptozotocin to establish DM model. After successful modeling, DM group was divided into DM+dimethyl sulfoxide (DMSO) group and DM+C176 group, with 12 mice in each group. The mice in the DM+DMSO group were intraperitoneally injected with DMSO at the dose of 50 mg/kg. Mice in DM+C176 group were intraperitoneally injected with STING inhibitor C176 750 nmol at the dose of 50 mg/kg. Four weeks after modeling, immunohistochemical staining, Western blot and real-time fluorescence quantitative polymerase chain reaction were used to detect the expression of STING in the retina of WT and DM mice. The leukocyte adhesion test was used to detect the number of leukocytes adhering to hRMEC in mice with WT, DM+DMSO and DM+C176 groups. In vitro experiment: hRMEC was randomly divided into conventional culture cell group (N group), dimethyl sulfoxide (DMSO) group (with DMSO intervention) and C176 group (with C176 intervention). The cells were induced by 150 μg/ml glycation end products for each group. In vitro leukocyte adhesion test combined with 4', 6-diamino-2-phenylindole staining was used to detect the number of leukocytes adhering to hRMEC. The adherent leukocytes were quantitatively analyzed by flow cytometry; H₂DCFDA/reactive oxygen species (ROS) fluorescence probe was used to detect ROS expression in cells; Seahorse XFe96 cell energy metabolism analyzer was used to measure the level of intracellular glycolysis. t-test was used to compare the two groups; single factor analysis of variance was used to compare the three groups. Results In vivo experiment: compared with WT group, the expression level of STING (t=73.248) and the relative expression amount of mRNA (t=67.385) in the retina of DM group mice increased significantly (P＜0.05). Compared with WT group, the number of leukocytes adhering to the retinal vessels of mice in DM+DMSO group was significantly increased, while that in DM+C176 group was significantly decreased (F=84.352, P＜0.01). In vitro: compared with N group and DMSO group, the number of leukocyte adhesion on hRMEC in C176 group decreased significantly (F=35.251, P＜0.01). Compared with N group, the number of leukocytes adhering to hRMEC in DMSO group and C176 group decreased significantly (F=26.374, P＜0.01). The ROS level in hRMEC in C176 group was significantly lower than that in N group and C176 group (F=41.362, P＜0.01). Compared with N group and DMSO group, the glycolysis level of hRMEC in C176 group was significantly reduced, with a statistically significant difference (F=68.741, P＜0.01). Conclusion Inhibiting the expression of STING in retinal vascular endothelial cells can improve the progress of DM by inhibiting leukocyte adhesion, ROS production and glycolysis level.

Citation： Wang Yong, Cao Jingjing, Li Hui, Hong Yaru, Liu Aihua, Dong Lijie. Interferon gene stimulating protein inhibitor improves leukocyte adhesion and glycolysis of retinal vascular endothelial cells. Chinese Journal of Ocular Fundus Diseases, 2022, 38(12): 1013-1019. doi: 10.3760/cma.j.cn511434-20220520-00312 Copy

1.	Shao Y, Dong LJ, Takahashi Y, et al. MiRNA-451a regulates RPE function through promoting mitochondrial function in proliferative diabetic retinopathy[J/OL]. Am J Physiol Endocrinol Metab, 2019, 316(3): E443-452[2019-03-01]. https://pubmed.ncbi.nlm.nih.gov/30576241/. DOI: 10.1152/ajpendo.00360.2018.
2.	Hachana S, Fontaine O, Sapieha P, et al. The effects of anti-VEGF and kinin B(1) receptor blockade on retinal inflammation in laser-induced choroidal neovascularization[J]. Br J Pharmacol, 2020, 177(9): 1949-1966. DOI: 10.1111/bph.14962.
3.	Singh AD, Kulkarni YA. Vascular adhesion protein-1 and microvascular diabetic complications[J]. Pharmacol Rep, 2022, 74(1): 40-46. DOI: 10.1007/s43440-021-00343-y.
4.	Elmasry K, Ibrahim AS, Abdulmoneim S, et al. Bioactive lipids and pathological retinal angiogenesis[J]. Br J Pharmacol, 2019, 176(1): 93-109. DOI: 10.1111/bph.14507.
5.	Wan D, Jiang W, Hao J. Research advances in how the cGAS-STING pathway controls the cellular inflammatory response[J]. Front Immunol, 2020, 11: 615. DOI: 10.3389/fimmu.2020.00615.
6.	Hopfner KP, Hornung V. Molecular mechanisms and cellular functions of cGAS-STING signalling[J]. Nat Rev Mol Cell Biol, 2020, 21(9): 501-521. DOI: 10.1038/s41580-020-0244-x.
7.	Yu Y, Yang W, Bilotta AJ, et al. STING controls intestinal homeostasis through promoting antimicrobial peptide expression in epithelial cells[J]. FASEB J, 2020, 34(11): 15417-15430. DOI: 10.1096/fj.202001524R.
8.	Chen Q, Tang L, Zhang Y, et al. STING up-regulates VEGF expression in oxidative stress-induced senescence of retinal pigment epithelium via NF-κB/HIF-1α pathway[J/OL]. Life Sci, 2022, 293: 120089[2022-03-15]. https://pubmed.ncbi.nlm.nih.gov/35007563/. DOI: 10.1016/j.lfs.2021.120089.
9.	Han B, Wang X, Wu P, et al. Pulmonary inflammatory and fibrogenic response induced by graphitized multi-walled carbon nanotube involved in cGAS-STING signaling pathway[J/OL]. J Hazard Mater, 2021, 417: 125984[2021-09-05]. https://pubmed.ncbi.nlm.nih.gov/34020360/. DOI: 10.1016/j.jhazmat.2021.125984.
10.	Miyagi S, Watanabe T, Hara Y, et al. A STING inhibitor suppresses EBV-induced B cell transformation and lymphomagenesis[J]. Cancer Sci, 2021, 112(12): 5088-5099. DOI: 10.1111/cas.15152.
11.	Dong L, Zhang Z, Liu X, et al. RNA sequencing reveals BMP4 as a basis for the dual-target treatment of diabetic retinopathy[J]. J Mol Med (Berl), 2021, 99(2): 225-240. DOI: 10.1007/s00109-020-01995-8.
12.	Zhao J, Liu X, Lin J, et al. AKT2 identified as a potential target of mir-29a-3p via microRNA profiling of patients with high proliferation lacrimal gland adenoid cystic carcinoma[J/OL]. Exp Eye Res, 2022, 219:109067[2022-04-07]. https://linkinghub.elsevier.com/retrieve/pii/S0014-4835(22)00147-6. DOI: 10.1016/j.exer.2022.109067.
13.	Teng H, Hong Y, Cao J, et al. Senescence marker protein30 protects lens epithelial cells against oxidative damage by restoring mitochondrial function[J]. Bioengineered, 2022, 13(5): 12955-12971. DOI: 10.1080/21655979.2022.2079270.
14.	Peng Y, Zhuang J, Ying G, et al. Stimulator of IFN genes mediates neuroinflammatory injury by suppressing AMPK signal in experimental subarachnoid hemorrhage[J]. J Neuroinflammation, 2020, 17(1): 165. DOI: 10.1186/s12974-020-01830-4.
15.	Rom S, Heldt NA, Gajghate S, et al. Hyperglycemia and advanced glycation end products disrupt BBB and promote occludin and claudin-5 protein secretion on extracellular microvesicles[J/OL]. Sci Rep, 2020, 10(1): 7274[2020-04-29]. https://pubmed.ncbi.nlm.nih.gov/32350344/. DOI: 10.1038/s41598-020-64349-x.
16.	Crijns H, Vanheule V, Proost P. Targeting chemokine-glycosaminoglycan interactions to inhibit inflammation[J]. Front Immunol, 2020, 11: 483. DOI: 10.3389/fimmu.2020.00483.
17.	Dong L, Lin T, Li W, et al. Antioxidative effects of polypyrimidine tract-binding protein-associated splicing factor against pathological retinal angiogenesis through promotion of mitochondrial function[J]. J Mol Med (Berl), 2021, 99(7): 967-980. DOI: 10.1007/s00109-021-02069-z.
18.	Dunphy G, Flannery SM, Almine JF, et al. Non-canonical activation of the DNA sensing adaptor STING by ATM and IFI16 mediates NF-κB signaling after nuclear DNA damage[J]. Mol Cell, 2018, 71(5): 745-760. DOI: 10.1016/j.molcel.2018.07.034.
19.	Wang X, Rao H, Zhao J, et al. STING expression in monocyte-derived macrophages is associated with the progression of liver inflammation and fibrosis in patients with nonalcoholic fatty liver disease[J]. Lab Invest, 2020, 100(4): 542-552. DOI: 10.1038/s41374-019-0342-6.
20.	Zou M, Ke Q, Nie Q, et al. Inhibition of cGAS-STING by JQ1 alleviates oxidative stress-induced retina inflammation and degeneration[J]. Cell Death Differ, 2022, 29(9): 1816-1833. DOI: 10.1038/s41418-022-00967-4.
21.	Guo Y, Gu R, Gan D, et al. Mitochondrial DNA drives noncanonical inflammation activation via cGAS-STING signaling pathway in retinal microvascular endothelial cells[J]. Cell Commun Signal, 2020, 18(1): 172. DOI: 10.1186/s12964-020-00637-3.
22.	Dong L, Li W, Lin T, et al. PSF functions as a repressor of hypoxia-induced angiogenesis by promoting mitochondrial function[J]. Cell Commun Signal, 2021, 19(1): 14. DOI: 10.1186/s12964-020-00684-w.
23.	Saddala MS, Lennikov A, Huang H. Placental growth factor regulates the pentose phosphate pathway and antioxidant defense systems in human retinal endothelial cells[J/OL]. J Proteomics, 2020, 217: 103682[2020-04-15]. https://pubmed.ncbi.nlm.nih.gov/32058040/. DOI: 10.1016/j.jprot.2020.103682.

1. Shao Y, Dong LJ, Takahashi Y, et al. MiRNA-451a regulates RPE function through promoting mitochondrial function in proliferative diabetic retinopathy[J/OL]. Am J Physiol Endocrinol Metab, 2019, 316(3): E443-452[2019-03-01]. https://pubmed.ncbi.nlm.nih.gov/30576241/. DOI: 10.1152/ajpendo.00360.2018.
2. Hachana S, Fontaine O, Sapieha P, et al. The effects of anti-VEGF and kinin B(1) receptor blockade on retinal inflammation in laser-induced choroidal neovascularization[J]. Br J Pharmacol, 2020, 177(9): 1949-1966. DOI: 10.1111/bph.14962.
3. Singh AD, Kulkarni YA. Vascular adhesion protein-1 and microvascular diabetic complications[J]. Pharmacol Rep, 2022, 74(1): 40-46. DOI: 10.1007/s43440-021-00343-y.
4. Elmasry K, Ibrahim AS, Abdulmoneim S, et al. Bioactive lipids and pathological retinal angiogenesis[J]. Br J Pharmacol, 2019, 176(1): 93-109. DOI: 10.1111/bph.14507.
5. Wan D, Jiang W, Hao J. Research advances in how the cGAS-STING pathway controls the cellular inflammatory response[J]. Front Immunol, 2020, 11: 615. DOI: 10.3389/fimmu.2020.00615.
6. Hopfner KP, Hornung V. Molecular mechanisms and cellular functions of cGAS-STING signalling[J]. Nat Rev Mol Cell Biol, 2020, 21(9): 501-521. DOI: 10.1038/s41580-020-0244-x.
7. Yu Y, Yang W, Bilotta AJ, et al. STING controls intestinal homeostasis through promoting antimicrobial peptide expression in epithelial cells[J]. FASEB J, 2020, 34(11): 15417-15430. DOI: 10.1096/fj.202001524R.
8. Chen Q, Tang L, Zhang Y, et al. STING up-regulates VEGF expression in oxidative stress-induced senescence of retinal pigment epithelium via NF-κB/HIF-1α pathway[J/OL]. Life Sci, 2022, 293: 120089[2022-03-15]. https://pubmed.ncbi.nlm.nih.gov/35007563/. DOI: 10.1016/j.lfs.2021.120089.
9. Han B, Wang X, Wu P, et al. Pulmonary inflammatory and fibrogenic response induced by graphitized multi-walled carbon nanotube involved in cGAS-STING signaling pathway[J/OL]. J Hazard Mater, 2021, 417: 125984[2021-09-05]. https://pubmed.ncbi.nlm.nih.gov/34020360/. DOI: 10.1016/j.jhazmat.2021.125984.
10. Miyagi S, Watanabe T, Hara Y, et al. A STING inhibitor suppresses EBV-induced B cell transformation and lymphomagenesis[J]. Cancer Sci, 2021, 112(12): 5088-5099. DOI: 10.1111/cas.15152.
11. Dong L, Zhang Z, Liu X, et al. RNA sequencing reveals BMP4 as a basis for the dual-target treatment of diabetic retinopathy[J]. J Mol Med (Berl), 2021, 99(2): 225-240. DOI: 10.1007/s00109-020-01995-8.
12. Zhao J, Liu X, Lin J, et al. AKT2 identified as a potential target of mir-29a-3p via microRNA profiling of patients with high proliferation lacrimal gland adenoid cystic carcinoma[J/OL]. Exp Eye Res, 2022, 219:109067[2022-04-07]. https://linkinghub.elsevier.com/retrieve/pii/S0014-4835(22)00147-6. DOI: 10.1016/j.exer.2022.109067.
13. Teng H, Hong Y, Cao J, et al. Senescence marker protein30 protects lens epithelial cells against oxidative damage by restoring mitochondrial function[J]. Bioengineered, 2022, 13(5): 12955-12971. DOI: 10.1080/21655979.2022.2079270.
14. Peng Y, Zhuang J, Ying G, et al. Stimulator of IFN genes mediates neuroinflammatory injury by suppressing AMPK signal in experimental subarachnoid hemorrhage[J]. J Neuroinflammation, 2020, 17(1): 165. DOI: 10.1186/s12974-020-01830-4.
15. Rom S, Heldt NA, Gajghate S, et al. Hyperglycemia and advanced glycation end products disrupt BBB and promote occludin and claudin-5 protein secretion on extracellular microvesicles[J/OL]. Sci Rep, 2020, 10(1): 7274[2020-04-29]. https://pubmed.ncbi.nlm.nih.gov/32350344/. DOI: 10.1038/s41598-020-64349-x.
16. Crijns H, Vanheule V, Proost P. Targeting chemokine-glycosaminoglycan interactions to inhibit inflammation[J]. Front Immunol, 2020, 11: 483. DOI: 10.3389/fimmu.2020.00483.
17. Dong L, Lin T, Li W, et al. Antioxidative effects of polypyrimidine tract-binding protein-associated splicing factor against pathological retinal angiogenesis through promotion of mitochondrial function[J]. J Mol Med (Berl), 2021, 99(7): 967-980. DOI: 10.1007/s00109-021-02069-z.
18. Dunphy G, Flannery SM, Almine JF, et al. Non-canonical activation of the DNA sensing adaptor STING by ATM and IFI16 mediates NF-κB signaling after nuclear DNA damage[J]. Mol Cell, 2018, 71(5): 745-760. DOI: 10.1016/j.molcel.2018.07.034.
19. Wang X, Rao H, Zhao J, et al. STING expression in monocyte-derived macrophages is associated with the progression of liver inflammation and fibrosis in patients with nonalcoholic fatty liver disease[J]. Lab Invest, 2020, 100(4): 542-552. DOI: 10.1038/s41374-019-0342-6.
20. Zou M, Ke Q, Nie Q, et al. Inhibition of cGAS-STING by JQ1 alleviates oxidative stress-induced retina inflammation and degeneration[J]. Cell Death Differ, 2022, 29(9): 1816-1833. DOI: 10.1038/s41418-022-00967-4.
21. Guo Y, Gu R, Gan D, et al. Mitochondrial DNA drives noncanonical inflammation activation via cGAS-STING signaling pathway in retinal microvascular endothelial cells[J]. Cell Commun Signal, 2020, 18(1): 172. DOI: 10.1186/s12964-020-00637-3.
22. Dong L, Li W, Lin T, et al. PSF functions as a repressor of hypoxia-induced angiogenesis by promoting mitochondrial function[J]. Cell Commun Signal, 2021, 19(1): 14. DOI: 10.1186/s12964-020-00684-w.
23. Saddala MS, Lennikov A, Huang H. Placental growth factor regulates the pentose phosphate pathway and antioxidant defense systems in human retinal endothelial cells[J/OL]. J Proteomics, 2020, 217: 103682[2020-04-15]. https://pubmed.ncbi.nlm.nih.gov/32058040/. DOI: 10.1016/j.jprot.2020.103682.

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Chinese Journal of Ocular Fundus Diseases

Interferon gene stimulating protein inhibitor improves leukocyte adhesion and glycolysis of retinal vascular endothelial cells

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