LCN2 aggravates diabetic cataracts by promoting ferroptosis in lens epithelial cells

Jiayue Zhang; Liyao Sun; Xiaohan Yu; Chen Yang; Qi An; Chaoqun Wei; Hongyan Ge

doi:10.1515/fzm-2024-0018

LCN2 aggravates diabetic cataracts by promoting ferroptosis in lens epithelial cells

doi: 10.1515/fzm-2024-0018

Jiayue Zhang^{1, 2, 3},
Liyao Sun¹,
Xiaohan Yu¹,
Chen Yang¹,
Qi An¹,
Chaoqun Wei^{1, 2, 3},
Hongyan Ge^{1
, ,}

1.
Eye Hospital, First Affiliated Hospital, Harbin Medical University, Harbin 150001, China
2.
Key Laboratory of Ischemia-reperfusion, Harbin Medical University, Ministry of Education, Harbin 150001, China
3.
Experimental Animal Centre, the Second Affiliated Hospital, Harbin Medical University, Harbin 150001, China

Funds:

the Horizontal Scientific Research Project of Harbin Medical University 2022HX005

More Information

Corresponding author: Hongyan Ge, E-mail: gehongyan@hrbmu.edu.cn

摘要

Abstract: Background Cataracts are the leading cause of reversible blindness worldwide. Diabetic cataract (DC), a prevalent complication of diabetes mellitus, is characterized by its high occurrence, rapid progression, and severe impact. The prevalence of diabetes varies greatly between the northern and southern regions, with higher rates observed among northern residents. DC-induced lens opacity is mainly attributed to oxidative stress. However, it remains unclear whether ferroptosis, a form of regulated cell death, occurs in crystalline epithelial cells during the pathogenesis, which may represent a novel mechanism contributing to DC. Methods Transmission electron microscopy, quantitative assays for iron levels and reactive oxygen species (ROS), real-time quantitative polymerase chain reaction (RT-qPCR), western blotting, immunofluorescence, and immunohistochemistry were used to detect ferroptosis. Gene editing techniques were utilized to study the regulatory relationships among lipocalin 2 (LCN2), glutathione peroxidase 4 (GPX4), and ferritin heavy chain (FTH). Local knockdown of the LCN2 gene in B-3 cells and the eyes of Sprague Dawley (SD) rats was performed to verify and further explore the role and regulatory mechanisms of LCN2 in DC-associated ferroptosis. Results An in vitro model using high glucose levels and an in vivo model with streptozotocin-induced diabetes in SD rats were successfully established. Ferroptosis was observed in both in vitro and in vivo experiments. LCN2 protein was normally expressed in human and rat lens epithelial cells, but its expression significantly increased during ferroptosis. The ferroptosis inhibitor, ferrostatin-1 (Fer-1) effectively inhibited ferroptosis and reduced LCN2 protein expression. Notably, local knockdown of LCN2 via gene editing protected lens epithelial cells from ferroptosis in vitro and slowed the progression of DC in SD rats in vivo. Conclusion Our findings underscore the significant role of ferroptosis in the pathogenesis of DC, suggesting that selectively targeting LCN2 activation and enhancing ferroptosis resistance may offer a novel therapeutic approach for treating DC.
- lipocalin-2 /
- ferroptosis /
- diabetic cataracts
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Figure 1. High glucose induces ferroptosis in B-3 cells in a dose-dependent manner

(A) To analyze the effect of high glucose concentrations on B-3 cell viability, CCK-8 assays were performed after treating the cells with culture media containing glucose at concentrations of 5.5, 10, 20, 40, 60, 80, and 100 mmol/L. The results are presented from three independent experiments. (B-D) The results of RT-PCR and Western blot analysis of GPX4, FTH1 and SLC7A11 when B-3 cells were treated with glucose concentration of 5.5 mmol/L and glucose concentration of 5.5 mmol/L + mannitol concentration of 40 mmol/L. Results of RT-PCR and Western blot analysis of GPX4, FTH1, and SLC7A11 when B-3 cells were treated with glucose at concentrations of 10, 20, or 40 mmol/L. The results are presented from three independent experiments. (a: Compared with the control group, b: compared with the 40 mmol/L mannitol group, c: compared with the 10 mmol/L high glucose group, d: compared with the 20 mmol/L high glucose group, all P < 0. 05) (E-F) Iron levels (Fe²⁺/Fe³⁺) and ROS levels in B-3 cells treated with glucose at concentrations of 5.5, 10, 20, or 40 mmol/L and glucose at a concentration of 5.5 mmol/L plus mannitol at a concentration of 40 mmol/L. The results are presented from three independent experiments. (^**P < 0. 01; ^***P < 0. 005) (G) When B-3 cells were treated with glucose at concentrations of 5.5, 10, 20, or 40 mmol/L, the ultrastructure of the cells was photographed by TEM. (H-K) When B-3 cells were treated with 5.5 mmol/L glucose and 5.5 mmol/L glucose + 40 mmol/L mannitol, the relative fluorescence intensity of GPX4 and FTH1 was measured by IF staining. When B-3 cells were treated with glucose at concentrations of 10, 20, or 40 mmol/L, the relative fluorescence intensities of GPX4 and FTH1 were measured by the IF staining method. The results are presented from three independent experiments. (a: Compared with the control group, b: compared with the 10 mmol/L high glucose group c: compared with the 20 mmol/L high glucose group, all P < 0. 05).

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Figure 2. High glucose-induced differential expression of LCN2 in B-3 cells

(A-C) A blank control group and a high osmotic pressure control group were set up, and that result of RT-PCR and Western blot analysis of LCN2 are obtained when B-3 cells are treated with glucose concentration of 5.5 mmol/L, glucose concentration of 5.5 mmol/L + mannitol concentration of 40 mmol/L and glucose concentration of 40 mmol/L. The results are presented from three independent experiments. (D-E) A blank control group and a high osmotic pressure control group were set up. When B-3 cells were treated with glucose concentration of 5.5 mmol/L, glucose concentration of 5.5 mmol/L + mannitol concentration of 40 mmol/L and glucose concentration of 40 mmol/L, the relative fluorescence intensity of LCN2 was measured by if staining method. The results are presented from three independent experiments. (a: Compared with the control group, b: compared with the 40 mmol/L mannitol group, all P < 0. 05).

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Figure 3. Fer-1 rescues high glucose-induced ferroptosis in B-3 cells

(A-B) Iron levels (Fe²⁺/Fe³⁺) and ROS levels in B-3 cells treated with DMSO, high glucose at a concentration of 40 mmol/L or Fer-1. The results are presented from three independent experiments. (C-E) Results of RT-PCR and Western blot analysis of GPX4, FTH1, LCN2 and SLC7A11 in B-3 cells treated with DMSO, high glucose at a concentration of 40 mmol/L, Fer-1. The results are presented from three independent experiments. (a: Compared with the DMSO group, b: compared with the 40 mmol/L high glucose group, all P < 0. 05) (F-K) When B-3 cells were treated with DMSO, 40 mmol/L high glucose or Fer-1, the relative fluorescence intensity of GPX4, FTH1, and LCN2 was measured by IF staining method. The results are presented from three independent experiments. (a: Compared with the DMSO group, b: compared with the 40 mmol/L high glucose group, all P < 0. 05). (L) When B-3 cells were treated with glucose at concentrations of 40 mmol/L or Fer-1, the ultrastructure of the cells was photographed by TEM.

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Figure 4. Silencing of LCN2 alleviates ferroptosis in B-3 cells

(A-B) Results of Western blot analysis of GPX4, FTH1 and LCN2 of B-3 cells for four groups of si-Control, si-LCN2, 40 mmol/L high glucose, and si-LCN2 + 40 mmol/L high glucose. The results are presented from three independent experiments. (a: Compared with the si-Control group, b: Compared with the si-LCN2 group, c: Compared with the 40 mmol/L high glucose group, all P < 0. 05).

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Figure 5. Development of DC model in SD rats

(A-B) The results of fasting blood glucose (mmol/L) and body weight (g) were measured before modeling, 4 weeks after modeling, 8 weeks after modeling and 12 weeks after modeling in the three groups of SD rats, they were DMSO group, DC group and Fer-1 treatment group. (^**P < 0. 01; ^***P < 0. 005). (C) The eye images of SD rats in the DMSO group, DC group and Fer-1 treatment group were taken after pupil dilation at 12 weeks after modeling.

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Figure 6. Ferroptosis of lens epithelial cells isolated from SD rats with diabetes cataract

(A-B) They were three groups of SD rats (DMSO group, DC group and Fer-1 treatment group). The Western blot analysis results of GPX4, FTH1, LCN2, and SLC7A11 in the anterior capsule of their lenses. The results are presented from three independent experiments. (a: Compared with the DMSO group, b: : compared with the 40 mmol/L high glucose group, all P < 0. 05). (C) They were three groups of SD rats (DMSO group, DC group and Fer-1 treatment group). The results of iron levels (Fe²⁺/Fe³⁺) in the anterior capsule of their lenses. The results are presented from three independent experiments. (D) They were three groups of SD rats (DMSO group, DC group and Fer-1 treatment group). These are pictures of their complete lenses taken out. (E-F) They were three groups of SD rats (DMSO group, DC group and Fer-1 treatment group). The average optical density of GPX4, FTH1, and LCN2 in their anterior capsule was measured using the IHC method (compared to DMSO). The results are presented from three independent experiments. (^*P < 0. 05; ^**P < 0. 01; ^***P < 0. 005).

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Figure 7. LCN2 silencing inhibits ferroptosis in lens epithelial cells and delays the occurrence and development of diabetes cataract in anterior segment of SD rats

(A-B) The results of fasting blood glucose (mmol/L) and body weight (g) measured before modeling, 4 weeks after modeling, 8 weeks after modeling, and 12 weeks after modeling in SD rats of the Control and DC groups. (C-D) Western blot analysis of GPX4, FTH1, and LCN2 in the anterior capsule membrane of SD rats in four groups: si-Control, si-LCN2, DC, and si-LCN2+DC. The results are presented from three independent experiments. (a: Compared with the si-Control group, b: Compared with the si-LCN2 group, c: Compared with the DC group, all P < 0. 05). (E) Complete lens images of four groups of SD rats: si Control, si-LCN2, DC, and si-LCN2+DC. (FG) The average optical density of GPX4 and FTH1 was measured using IHC method for the anterior capsule of SD rats in four groups: si-Control, si-LCN2, DC, and siLCN2+DC (compared to the si-Control group). The results are presented from three independent experiments. (^*P < 0. 05; ^**P < 0. 01; ^***P < 0. 005).

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Table 1. All primer sequences

Primer	Nucleotide sequence (5′ - 3′)
GPX4	F-CGATACGCTGAGTGTGGTTT-
	R-CGGCGAACTCTTTGATCTCTT-
FTH1	F-TACCTGAATGAGCAGGTGAAAG-
	R-GATATTCCGCCAAGCCAGAT-
SLC7A11	F-GGTTGCCCTTTCCCTCTATTC-
	R-CCTGGGTTTCTTGTCCCATATAA-
LCN2	F-CCAGGACAACCAATTCCAGGG-
	R-GTGGCATACATCTTTTGCGGG-
GAPDH	F-CTGGGCTACACTGAGCACC-
	R-AAGTGGTCGTTGAGGGCAATG-
F, forward primer; R, reverse primer

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Table 2. Comparison of weight and fasting blood glucose values of SD rats

Index	Control (N = 20)	DC (N = 20)	DC+Fer-1 (N = 20)	F	P
Weight (g)
Before modeling	193.65±7.51	192.35±6. 04	190.7±4.51	1. 16	0. 321
4 weeks after modeling	330. 73 ±26.64^b	177.48±25. 25^{a, b}	180.75±24. 66^a	235. 18	< 0. 001
8 weeks after modeling	400. 82 ±25.26_{b, c}	159.94 ±23. 25^{a, b, c}	155.71 ±17. 39^{a, b, c}	797. 68	< 0. 001
12 weeks after modeling	465. 42 ±28.54^{b, c, d}	151.47 ± 21. 5^{a, b, c, d}	147.05 ± 17. 77^{a, b, c, d}	1255. 35	< 0. 001
Blood glucose (mmol/L)
Before modeling	4.50±0.36	4.51±0.42	4.48±0.35	0. 033	0. 968
4 weeks after modeling	4.71±0.36^b	24.11 ± 4. 13^ab	22. 46 ± 4.81^{a, b}	172. 15	< 0. 001
8 weeks after modeling	4.53±0.72	26.13 ± 4. 3^{a, b, c}	24. 72 ± 4.00^{a, b, c}	249. 85	< 0. 001
12 weeks after modeling	5.06±0.42^{b, c, d}	27.14 ± 3.63^{a, b, c}	26. 13 ± 3.42_{a, b, c}	372. 13	< 0. 001
^aCompared with the control group; ^bCompared with before modeling; ^cCompared with 4 weeks after modeling; ^dCompared to 8 weeks after modeling; SD, Sprague Dawley; DC, diabetic cataract

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Table 3. Comparison of weight and fasting blood glucose values of SD rats

Index	Control (N = 10)	DC (N = 10)	t	P
Weight (g)
Before modeling	196. 55 ± 7. 33	198.51 ± 6. 26	0. 642	0. 529
4 weeks after modeling	331. 68 ± 42. 47^a	178.15 ± 11. 03^a	11. 065	< 0. 001
8 weeks after modeling	405. 21 ± 29. 31^{a, b}	156.92 ± 16. 56^a	23. 323	< 0. 001
12 weeks after modeling	470. 93±32. 77^{a, b, c}	143.12 ± 14. 14^{a, b, c}	29. 048	< 0. 001
Blood glucose (mmol/L)
Before modeling	4.64±0.37	4.68±0.54	0. 194	0. 848
4 weeks after modeling	4.55±0.41	23. 85 ± 5. 02^a	12. 124	< 0. 001
8 weeks after modeling	4.61±0.46	25. 08 ± 4. 53^a	14. 224	< 0. 001
12 weeks after modeling	4.74±0.34	26. 31 ± 3. 73^a	18. 204	< 0. 001
^aCompared with before modeling; ^bCompared with 4 weeks after modeling; ^cCompared with 8 weeks after modeling

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