Metformin inhibiting the activation of NLRP3 inflammasome and pyroptosis in diabetic retinal vascular endothelial cells_Chinese Journal of Ocular Fundus Diseases

Authors：

Jiao Mingfei , Cao Jingjing , Li Hui , Kou Zhenyu , Wu Guijia ,  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;

Corresponding author：

Dong Lijie, Email: aitaomubang@126.com

Keywords：

Metformin; Retinal vascular endothelial cells; NLRP3; Inflammasome; Pyroptosis; Animal experiment; Cell experiment

DOI：

10.3760/cma.j.cn511434-20220818-00463

Video：

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Objective To observe the effect of metformin (Met) on inflammatory bodies and focal death in human retinal microvascular endothelial cells (hRMEC) in diabetes mellitus (DM) microenvironment. Methods Experimental research was divided into in vivo animal experiment and in vitro cell experiment. In vivo animal experiments: 9 healthy C57BL/6J male mice were randomly divided into DM group, normal control group, and DM+Met group, with 3 mice in each group. DM group and DM+Met group mice were induced by streptozotocin to establish DM model, and DM+Met group was given Met 400 mg/ (kg · d) intervention. Eight weeks after modeling, the expression of NLRP3, cleaved-membrane perforating protein D (GSDMD) and cleaved-Caspase-1 in the retina of mice in the normal control group, DM group and DM+Met group were observed by immunohistochemical staining. In vitro cell experiments: hRMEC was divided into conventional culture cell group (N group), advanced glycation end products (AGE) group, and AGE+Met group. Joining the AGE, AGE+Met groups cells were induced by 150 μg/ml of glycation end products, and 2.0 mmol/L Met was added to the AGE+Met group. Pyroptosis was detected by flow cytometry; 2',7'-dichlorofluorescein diacetate (DCFH-DA) fluorescent probe was used to detect the expression of reactive oxygen species (ROS) in cells of each group. Real-time fluorescence quantitative polymerase chain reaction and Western blot were used to detect the relative mRNA and protein expression levels of NLRP3, cleaved-GSDMD, cleaved-Caspase-1 in each group of cells. Single factor analysis of variance was used for comparison among the three groups. Results In vivo animal experiments: compared with the DM group, the expression of NLRP3, cleaved-GSDMD, and cleaved-Caspase-1 in the retina of normal control group and DM+Met group mice was significantly reduced, with significant difference among the 3 groups (F=43.478, 36.643, 24.464; P＜0.01). In vitro cell experiment and flow cytometry showed that the pyroptosis rate of AGE group was significantly higher than that of N group and AGE+Met group (F=32.598, P＜0.01). The DCFH-DA detection results showed that the intracellular ROS levels in the N group and AGE+Met group were significantly lower than those in the AGE group, with the significant difference (F=47.267, P＜0.01). The mRNA (F=51.563, 32.192, 44.473; P＜0.01) and protein levels (F=63.372, 54.463, 48.412; P＜0.01) of NLRP3, cleaved-GSDMD, and cleaved-Caspase-1 in hRMEC of the AGE+Met group were significantly reduced compared to the N group. Conclusion Met can down regulate the expression of NLRP3 inflammatory body related factors in hRMEC and inhibit pyroptosis.

Citation： Jiao Mingfei, Cao Jingjing, Li Hui, Kou Zhenyu, Wu Guijia, Dong Lijie. Metformin inhibiting the activation of NLRP3 inflammasome and pyroptosis in diabetic retinal vascular endothelial cells. Chinese Journal of Ocular Fundus Diseases, 2023, 39(5): 408-414. doi: 10.3760/cma.j.cn511434-20220818-00463 Copy

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4.	Zhu M, Wang Y, Zhu L, et al. Crosstalk between rpe cells and choroidal endothelial cells via the ANXA1/FPR2/SHP2/NLRP3 inflammasome/pyroptosis axis promotes choroidal neovascularization[J]. Inflammation, 2022, 45(1): 414-427. DOI: 10.1007/s10753-021-01555-3.
5.	Ding H, Chen B, Lu Q, et al. Profilin-1 mediates microvascular endothelial dysfunction in diabetic retinopathy through HIF-1α-dependent pathway[J]. Int J Clin Exp Pathol, 2018, 11(3): 1247-1255.
6.	Ali N, El Hamdaoui S, et al. Improved glucometrics in people with type 1 diabetes 1 year into the COVID-19 pandemic[J/OL]. BMJ Open Diabetes Res Care, 2022, 10(3): e002789[2022-05-01]. https://europepmc.org/article/MED/35606020. DOI:10.1136/BMJDRC-2022-002789.
7.	刘勃实, 东莉洁, 黄亮瑜, 等. 氧诱导视网膜病变小鼠视网膜组织中miRNA的表达及分析[J]. 中华眼底病杂志, 2020, 36(7): 544-550. DOI: 10.3760/cma.j.cn511434-20201112-00545.Liu BS, Dong LJ, Huang LY, et al. Study on the mechanism of miRNA expression profile and enrichment analysis on p21 in oxygen-induced retinopathy mice[J]. Chin J Ocul Fundus Dis, 2020, 36(7): 544-550. DOI: 10.3760/cma.j.cn511434-20201112-00545.
8.	Shao Y, Dong LJ, Takahashi Y, et al. miRNA-451a regulates RPE function through promoting mitochondrial function in proliferative diabetic retinopathy[J]. Am J Physiol Endocrinol Metab, 2019, 316(3): 443-452. DOI: 10.1152/ajpendo.00360.2018.
9.	Wen D, Ren X, Li H, et al. Low expression of RBP4 in the vitreous humour of patients with proliferative diabetic retinopathy who underwent Conbercept intravitreal injection[J/OL]. Exp Eye Res, 2022, 225: 109197[2022-09-27]. https://linkinghub.elsevier.com/retrieve/pii/S0014-4835(22)00277-9. DOI:10.1016/j.exer.2022.109197.
10.	Jia Y, Cui R, Wang C, et al. Metformin protects against intestinal ischemia-reperfusion injury and cell pyroptosis via TXNIP-NLRP3-GSDMD pathway[J/OL]. Redox Biol, 2020, 32: 101534[2020-04-21]. https://linkinghub.elsevier.com/retrieve/pii/S2213-2317(20)30255-X. DOI:10.1016/j.redox.2020.101534.
11.	Yang Y, Liu PY, Bao W, et al. Hydrogen inhibits endometrial cancer growth via a ROS/NLRP3/caspase-1/GSDMD-mediated pyroptotic pathway[J]. BMC Cancer, 2020, 20(1): 28. DOI: 10.1186/s12885-019-6491-6.
12.	Silván JM, Reguero M, de Pascual-Teresa S. A protective effect of anthocyanins and xanthophylls on UVB-induced damage in retinal pigment epithelial cells[J]. Food Funct, 2016, 7(2): 1067-1076. DOI: 10.1039/c5fo01368b.
13.	Rui W, Li S, Xiao H, et al. Baicalein attenuates neuroinflammation by inhibiting NLRP3/caspase-1/GSDMD pathway in MPTP induced mice model of Parkinson's disease[J]. Int J Neuropsychopharmacol, 2020, 23(11): 762-773. DOI: 10.1093/ijnp/pyaa060.
14.	Yang F, Zhu W, Cai X, et al. Minocycline alleviates NLRP3 inflammasome-dependent pyroptosis in monosodium glutamate-induced depressive rats[J]. Biochem Biophys Res Commun, 2020, 526(3): 553-559. DOI: 10.1016/j.bbrc.2020.02.149.
15.	Zhang J, Huang L, Shi X, et al. Metformin protects against myocardial ischemia-reperfusion injury and cell pyroptosis via AMPK/NLRP3 inflammasome pathway[J]. Aging (Albany NY), 2020, 12(23): 24270-24287. DOI: 10.18632/aging.202143.
16.	Chowdhury AR, Zielonka J, Kalyanaraman B, et al. Mitochondria-targeted paraquat and metformin mediate ROS production to induce multiple pathways of retrograde signaling: a dose-dependent phenomenon[J/OL]. Redox Biol, 2020, 36: 101606[2020-06-21]. https://linkinghub.elsevier.com/retrieve/pii/S2213-2317(20)30811-9. DOI:10.1016/j.redox.2020.101606.
17.	Hsu SK, Cheng KC, Mgbeahuruike MO, et al. New insight into the effects of metformin on diabetic retinopathy, aging and cancer: nonapoptotic cell death, immunosuppression, and effects beyond the AMPK pathway[J]. Int J Mol Sci, 2021, 22(17): 9453. DOI: 10.3390/IJMS22179453.
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19.	Watanabe K, Asano D, Ushikubo H, et al. Metformin protects against NMDA-induced retinal injury through the MEK/ERK signaling pathway in rats[J]. Int J Mol Sci, 2021, 22(9): 4439. DOI: 10.3390/IJMS22094439.

1. 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.
2. Wang Y, Liu X, Shi H, et al. NLRP3 inflammasome, an immune-inflammatory target in pathogenesis and treatment of cardiovascular diseases[J]. Clin Transl Med, 2020, 10(1): 91-106. DOI: 10.1002/CTM2.13.
3. Zhang Y, Lv X, Hu Z, et al. Protection of Mcc950 against high-glucose-induced human retinal endothelial cell dysfunction[J]. Cell Death Dis, 2017, 8(7): 2941. DOI: 10.1038/CDDIS.2017.308.
4. Zhu M, Wang Y, Zhu L, et al. Crosstalk between rpe cells and choroidal endothelial cells via the ANXA1/FPR2/SHP2/NLRP3 inflammasome/pyroptosis axis promotes choroidal neovascularization[J]. Inflammation, 2022, 45(1): 414-427. DOI: 10.1007/s10753-021-01555-3.
5. Ding H, Chen B, Lu Q, et al. Profilin-1 mediates microvascular endothelial dysfunction in diabetic retinopathy through HIF-1α-dependent pathway[J]. Int J Clin Exp Pathol, 2018, 11(3): 1247-1255.
6. Ali N, El Hamdaoui S, et al. Improved glucometrics in people with type 1 diabetes 1 year into the COVID-19 pandemic[J/OL]. BMJ Open Diabetes Res Care, 2022, 10(3): e002789[2022-05-01]. https://europepmc.org/article/MED/35606020. DOI:10.1136/BMJDRC-2022-002789.
7. 刘勃实, 东莉洁, 黄亮瑜, 等. 氧诱导视网膜病变小鼠视网膜组织中miRNA的表达及分析[J]. 中华眼底病杂志, 2020, 36(7): 544-550. DOI: 10.3760/cma.j.cn511434-20201112-00545.Liu BS, Dong LJ, Huang LY, et al. Study on the mechanism of miRNA expression profile and enrichment analysis on p21 in oxygen-induced retinopathy mice[J]. Chin J Ocul Fundus Dis, 2020, 36(7): 544-550. DOI: 10.3760/cma.j.cn511434-20201112-00545.
8. Shao Y, Dong LJ, Takahashi Y, et al. miRNA-451a regulates RPE function through promoting mitochondrial function in proliferative diabetic retinopathy[J]. Am J Physiol Endocrinol Metab, 2019, 316(3): 443-452. DOI: 10.1152/ajpendo.00360.2018.
9. Wen D, Ren X, Li H, et al. Low expression of RBP4 in the vitreous humour of patients with proliferative diabetic retinopathy who underwent Conbercept intravitreal injection[J/OL]. Exp Eye Res, 2022, 225: 109197[2022-09-27]. https://linkinghub.elsevier.com/retrieve/pii/S0014-4835(22)00277-9. DOI:10.1016/j.exer.2022.109197.
10. Jia Y, Cui R, Wang C, et al. Metformin protects against intestinal ischemia-reperfusion injury and cell pyroptosis via TXNIP-NLRP3-GSDMD pathway[J/OL]. Redox Biol, 2020, 32: 101534[2020-04-21]. https://linkinghub.elsevier.com/retrieve/pii/S2213-2317(20)30255-X. DOI:10.1016/j.redox.2020.101534.
11. Yang Y, Liu PY, Bao W, et al. Hydrogen inhibits endometrial cancer growth via a ROS/NLRP3/caspase-1/GSDMD-mediated pyroptotic pathway[J]. BMC Cancer, 2020, 20(1): 28. DOI: 10.1186/s12885-019-6491-6.
12. Silván JM, Reguero M, de Pascual-Teresa S. A protective effect of anthocyanins and xanthophylls on UVB-induced damage in retinal pigment epithelial cells[J]. Food Funct, 2016, 7(2): 1067-1076. DOI: 10.1039/c5fo01368b.
13. Rui W, Li S, Xiao H, et al. Baicalein attenuates neuroinflammation by inhibiting NLRP3/caspase-1/GSDMD pathway in MPTP induced mice model of Parkinson's disease[J]. Int J Neuropsychopharmacol, 2020, 23(11): 762-773. DOI: 10.1093/ijnp/pyaa060.
14. Yang F, Zhu W, Cai X, et al. Minocycline alleviates NLRP3 inflammasome-dependent pyroptosis in monosodium glutamate-induced depressive rats[J]. Biochem Biophys Res Commun, 2020, 526(3): 553-559. DOI: 10.1016/j.bbrc.2020.02.149.
15. Zhang J, Huang L, Shi X, et al. Metformin protects against myocardial ischemia-reperfusion injury and cell pyroptosis via AMPK/NLRP3 inflammasome pathway[J]. Aging (Albany NY), 2020, 12(23): 24270-24287. DOI: 10.18632/aging.202143.
16. Chowdhury AR, Zielonka J, Kalyanaraman B, et al. Mitochondria-targeted paraquat and metformin mediate ROS production to induce multiple pathways of retrograde signaling: a dose-dependent phenomenon[J/OL]. Redox Biol, 2020, 36: 101606[2020-06-21]. https://linkinghub.elsevier.com/retrieve/pii/S2213-2317(20)30811-9. DOI:10.1016/j.redox.2020.101606.
17. Hsu SK, Cheng KC, Mgbeahuruike MO, et al. New insight into the effects of metformin on diabetic retinopathy, aging and cancer: nonapoptotic cell death, immunosuppression, and effects beyond the AMPK pathway[J]. Int J Mol Sci, 2021, 22(17): 9453. DOI: 10.3390/IJMS22179453.
18. Li Y, Gappy S, Liu X, et al. Metformin suppresses pro-inflammatory cytokines in vitreous of diabetes patients and human retinal vascular endothelium[J/OL]. PLoS One, 2022, 17(7): e0268451[2022-07-08]. https://europepmc.org/article/MED/35802672. DOI:10.1371/journal.pone.0268451.
19. Watanabe K, Asano D, Ushikubo H, et al. Metformin protects against NMDA-induced retinal injury through the MEK/ERK signaling pathway in rats[J]. Int J Mol Sci, 2021, 22(9): 4439. DOI: 10.3390/IJMS22094439.

Chinese Journal of Ocular Fundus Diseases

Metformin inhibiting the activation of NLRP3 inflammasome and pyroptosis in diabetic retinal vascular endothelial cells

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