Cathepsin L inhibitor suppresses oxidative stress-induced apoptosis of retinal pigment epithelial cells by targeting mitochondria_Chinese Journal of Ocular Fundus Diseases

Authors：

He Zhen , Kou Zhenyu , Dong Lijie ,  Li Xiaorong

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：

Li Xiaorong, Email: lixiaorong@tmu.edu.cn

Keywords：

Cathepsin L; Retinal pigment epithelial cells; Cell apoptosis; Oxidative stress; Mitochondria; Cell experiment

DOI：

10.3760/cma.j.cn511434-20231207-00474

Video：

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Objective To explore the effect of cathepsin L (CTSL) inhibitor on apoptosis of retinal pigment epithelial (RPE) cells and mitochondrial oxidative stress. Methods RPE cells were cultured in vitro and divided into control group, hydrogen peroxide (H₂O₂) group, and H₂O₂+CTSL inhibitor group. The cells of H₂O₂ group and H₂O₂+CTSL inhibitor group were incubated in the medium containing 400 μmol/L H₂O₂ for 24 hours and 10 μmol/L CTSL inhibitor was added in H₂O₂+CTSL inhibitor group at the same time. The cells of normal group were routinely cultured cells. The follow-up experiment was carried out 24 hours after modeling. The rate of apoptosis was detected by flow cytometry. The expression of CTSL was detected by immunofluorescence staining, Western blot and real time-polymerase chain reaction. The level of mitochondrial super oxide was detected by MitoSOX fluorescent probe, and the mitochondrial structure was observed after MitoTracker staining, the average area, form factors, and branch of mitochondria were quantitatively analyzed. The two groups were compared using two-tailed Student t test, while numerous groups were compared using one-way ANOVA. Results Compared with control group, the rate of apoptosis in H₂O₂ group was significantly higher (t=3.307, P=0.029 7), the expression level of CTSL was significantly increased (t=19.950, 6.916, 14.220; P＜0.05). Compared with H₂O₂ group, the expression level of CTSL, the rate of apoptosis and the mitochondrial ROS level in H₂O₂+CTSL inhibitor group were significantly lower (t=11.940, 4.718, 16.680; P＜0.05). The mitochondria of H₂O₂+CTSL inhibitor group were elongated, oval-shaped or rod-shaped, while the mitochondria of H₂O₂ group lost their continuous contour shape and complete structure. The differences of the average area, form factors, and brach of mitochondria among 4 groups were statistically significant (F=251.700, 34.010, 60.500; P＜0.000 1). Conclusions H₂O₂ can significantly induce apoptosis in RPE cells and increase CTSL expression. CTSL inhibitor can inhibit the H₂O₂-induced apoptosis of RPE cells, lower the mitochondrial super oxide level, and successfully repair the mitochondrial structure.

Citation： He Zhen, Kou Zhenyu, Dong Lijie, Li Xiaorong. Cathepsin L inhibitor suppresses oxidative stress-induced apoptosis of retinal pigment epithelial cells by targeting mitochondria. Chinese Journal of Ocular Fundus Diseases, 2024, 40(5): 379-386. doi: 10.3760/cma.j.cn511434-20231207-00474 Copy

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5.	Qi N, Xing W, Li M, et al. Quercetin alleviates toxicity induced by high levels of copper in porcine follicular granulosa cells by scavenging reactive oxygen species and improving mitochondrial function[J]. Animals (Basel), 2023, 13(17): 2745. DOI: 10.3390/ani13172745.
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9.	Thirusangu P, Pathoulas CL, Ray U, et al. Quinacrine-induced autophagy in ovarian cancer triggers cathepsin-L mediated lysosomal/mitochondrial membrane permeabilization and cell death[J]. Cancers, 2021, 13(9): 2004. DOI: 10.3390/cancers 13092004.
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11.	Luo M, Su Z, Gao H, et al. Cirsiliol induces autophagy and mitochondrial apoptosis through the AKT/FOXO1 axis and influences methotrexate resistance in osteosarcoma[J]. J Transl Med, 2023, 21(1): 907. DOI: 10.1186/s12967-023-04682-7.
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15.	Hou X, Han L, An B, et al. Mitochondria and lysosomes participate in Vip3Aa-induced spodoptera frugiperda Sf9 cell apoptosis[J]. Toxins, 2020, 12(2): 116. DOI: 10.3390/toxins12020116.
16.	Chaudhry A, Shi R, Luciani DS. A pipeline for multidimensional confocal analysis of mitochondrial morphology, function, and dynamics in pancreatic β-cells[J/OL]. Am J Physiol Endocrinol Metab, 2020, 318(2): E87-101[2020-02-01]. https://pubmed.ncbi.nlm.nih.gov/31846372/. DOI: 10.1152/ajpendo.00457.2019.
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19.	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.
20.	Gomes CP, Fernandes DE, Casimiro F, et al. Cathepsin L in COVID-19: from pharmacological evidences to genetics[J/OL]. Front Cell Infect Microbiol, 2020, 10: 589505[2020-12-08]. https://pubmed.ncbi.nlm.nih.gov/33364201/. DOI: 10.3389/fcimb.2020.589505.
21.	Feng L, Liang L, Zhang S, et al. HMGB1 downregulation in retinal pigment epithelial cells protects against diabetic retinopathy through the autophagy-lysosome pathway[J]. Autophagy, 2022, 18(2): 320-339. DOI: 10.1080/15548627.2021.1926655.
22.	Khuanjing T, Maneechote C, Ongnok B, et al. Acetylcholinesterase inhibition protects against trastuzumab-induced cardiotoxicity through reducing multiple programmed cell death pathways[J]. Mol Med, 2023, 29(1): 123. DOI: 10.1186/s10020-023-00686-7.
23.	Mohamed AK, Ezhilarasan D, Shree HK. Coleus vettiveroides ethanolic root extract induces cytotoxicity by intrinsic apoptosis in HepG2 cells[J]. J Appl Toxicol, 2024, 44(2): 245-259. DOI: 10.1002/jat.4536.
24.	Gorospe CM, Carvalho G, Herrera Curbelo A, et al. Mitochondrial membrane potential acts as a retrograde signal to regulate cell cycle progression[J/OL]. Life Sci Alliance, 2023, 6(12): e202302091[2023-09-11]. https://pubmed.ncbi.nlm.nih.gov/37696576/. DOI: 10.26508/lsa.202302091.
25.	Siemers KM, Klein AK, Baack ML. Mitochondrial dysfunction in PCOS: insights into reproductive organ pathophysiology[J/OL]. Int J Mol Sci, 2023, 24(17): 13123[2023-08-23]. https://pubmed.ncbi.nlm.nih.gov/37685928/. DOI: 10.3390/ijms241713123.
26.	Fu G, Chen AF, Xu Q, et al. Cathepsin L deficiency results in reactive oxygen species (ROS) accumulation and vascular cells activation[J]. Free Radic Res, 2017, 51(11-12): 932-942. DOI: 10.1080/10715762.2017.1393665.
27.	Bock FJ, Tait S. Mitochondria as multifaceted regulators of cell death[J]. Nat Rev Mol Cell Biol, 2020, 21(2): 85-100. DOI: 10.1038/s41580-019-0173-8.
28.	Lankelma JM, Voorend DM, Barwari T, et al. Cathepsin L, target in cancer treatment?[J]. Life Sci, 2010, 86(7-8): 225-233. DOI: 10.1016/j.lfs.2009.11.016.
29.	Lopes De Faria JM, Duarte DA, Montemurro C, et al. Defective autophagy in diabetic retinopathy[J]. Invest Ophthalmol Vis Sci, 2016, 57(10): 4356-4366. DOI: 10.1167/iovs.16-19197.
30.	Choi SI, Woo JH, Kim EK. Lysosomal dysfunction of corneal fibroblasts underlies the pathogenesis of granular corneal dystrophy type 2 and can be rescued by TFEB[J]. J Cell Mol Med, 2020, 24(18): 10343-10355. DOI: 10.1111/jcmm.15646.
31.	Dana D, Pathak SK. A review of small molecule inhibitors and functional probes of human cathepsin L[J]. Molecules, 2020, 25(3): 698. DOI: 10.3390/molecules25030698.
32.	Niu R, Wang J, Geng C, et al. Tandem mass tag-based proteomic analysis reveals cathepsin-mediated anti-autophagic and pro-apoptotic effects under proliferative diabetic retinopathy[J]. Aging (Albany NY), 2020, 13(1): 973-990. DOI: 10.18632/aging.202217.

1. Shadrach KG, Rayborn ME, Hollyfield JG, et al. DJ-1-dependent regulation of oxidative stress in the retinal pigment epithelium (RPE)[J/OL]. PLoS One, 2013, 8(7): e67983[2013-07-02]. https://pubmed.ncbi.nlm.nih.gov/23844142/. DOI: 10.1371/journal.pone.0067983.
2. Bang E, Park C, Hwangbo H, et al. Spermidine attenuates high glucose-induced oxidative damage in retinal pigment epithelial cells by inhibiting production of ROS and NF-κB/NLRP3 inflammasome pathway[J/OL]. Int J Mol Sci, 2023, 24(13): 10550[2023-06-23]. https://pubmed.ncbi.nlm.nih.gov/37445726/. DOI: 10.3390/ijms241310550.
3. Tong Y, Wu Y, Ma J, et al. Comparative mechanistic study of RPE cell death induced by different oxidative stresses[J/OL]. Redox Biology, 2023, 65: 102840[2023-08-06]. https://pubmed.ncbi.nlm.nih.gov/37566944/. DOI: 10.1016/j.redox.2023.102840.
4. Tong S, Xia M, Xu Y, et al. Identification and validation of a novel prognostic signature based on mitochondria and oxidative stress related genes for glioblastoma[J]. J Transl Med, 2023, 21(1): 136. DOI: 10.1186/s12967-023-03970-6.
5. Qi N, Xing W, Li M, et al. Quercetin alleviates toxicity induced by high levels of copper in porcine follicular granulosa cells by scavenging reactive oxygen species and improving mitochondrial function[J]. Animals (Basel), 2023, 13(17): 2745. DOI: 10.3390/ani13172745.
6. Peyer SM, Kremer N, McFall Ngai MJ. Involvement of a host Cathepsin L in symbiont‐induced cell death[J/OL]. Microbiologyopen, 2018, 7(5): e00632[2018-04-24]. https://pubmed.ncbi.nlm.nih.gov/29692003/. DOI: 10.1002/mbo3.632.
7. Pan X, Yu Y, Chen Y, et al. Cathepsin L was involved in vascular aging by mediating phenotypic transformation of vascular cells[J/OL]. Arch Gerontol Geriatr, 2023, 104: 104828[2022-10-04]. https://pubmed.ncbi.nlm.nih.gov/36206719/. DOI: 10.1016/j.archger.2022.104828.
8. Shen X, Zhao Y, Xu S, et al. Cathepsin L induced PC-12 cell apoptosis via activation of B-Myb and regulation of cell cycle proteins[J]. Acta pharmacologica Sinica, 2019, 40(11): 1394-1403. DOI: 10.1038/s41401-019-0286-9.
9. Thirusangu P, Pathoulas CL, Ray U, et al. Quinacrine-induced autophagy in ovarian cancer triggers cathepsin-L mediated lysosomal/mitochondrial membrane permeabilization and cell death[J]. Cancers, 2021, 13(9): 2004. DOI: 10.3390/cancers 13092004.
10. Yamano K, Kinefuchi H, Kojima W. Mitochondrial quality control via organelle and protein degradation[J/OL]. J Biochem, 2023: E1(2023-12-07)[2023-12-15]. https://pubmed.ncbi.nlm.nih.gov/38102729/. DOI: 10.1093/jb/mvad106.
11. Luo M, Su Z, Gao H, et al. Cirsiliol induces autophagy and mitochondrial apoptosis through the AKT/FOXO1 axis and influences methotrexate resistance in osteosarcoma[J]. J Transl Med, 2023, 21(1): 907. DOI: 10.1186/s12967-023-04682-7.
12. Tian Z, Jiang S, Zhou J, et al. Copper homeostasis and cuproptosis in mitochondria[J/OL]. Life Sci, 2023, 334: 122223[2023-12-01]. https://pubmed.ncbi.nlm.nih.gov/38084674/. DOI: 10.1016/j.lfs.2023.122223.
13. Lee MH, Kim HL, Seo H, et al. A secreted form of chorismate mutase (Rv1885c) in mycobacterium bovis BCG contributes to pathogenesis by inhibiting mitochondria-mediated apoptotic cell death of macrophages[J]. J Biomed Sci, 2023, 30(1): 95. DOI: 10.1186/s12929-023-00988-2.
14. Renaud C, Trillet K, Jardine J, et al. The centrosomal protein 131 participates in the regulation of mitochondrial apoptosis[J]. Commun Biol, 2023, 6(1): 1271. DOI: 10.1038/s42003-023-05676-3.
15. Hou X, Han L, An B, et al. Mitochondria and lysosomes participate in Vip3Aa-induced spodoptera frugiperda Sf9 cell apoptosis[J]. Toxins, 2020, 12(2): 116. DOI: 10.3390/toxins12020116.
16. Chaudhry A, Shi R, Luciani DS. A pipeline for multidimensional confocal analysis of mitochondrial morphology, function, and dynamics in pancreatic β-cells[J/OL]. Am J Physiol Endocrinol Metab, 2020, 318(2): E87-101[2020-02-01]. https://pubmed.ncbi.nlm.nih.gov/31846372/. DOI: 10.1152/ajpendo.00457.2019.
17. Valente AJ, Maddalena LA, Robb EL, et al. A simple ImageJ macro tool for analyzing mitochondrial network morphology in mammalian cell culture[J]. Acta Histochem, 2017, 119(3): 315-326. DOI: 10.1016/j.acthis.2017.03.001.
18. Xu S, Zhang H, Yang X, et al. Inhibition of cathepsin L alleviates the microglia-mediated neuroinflammatory responses through caspase-8 and NF-κB pathways[J]. Neurobiol Aging, 2018, 62: 159-167. DOI: 10.1016/j.neurobiolaging.2017.09.030.
19. 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.
20. Gomes CP, Fernandes DE, Casimiro F, et al. Cathepsin L in COVID-19: from pharmacological evidences to genetics[J/OL]. Front Cell Infect Microbiol, 2020, 10: 589505[2020-12-08]. https://pubmed.ncbi.nlm.nih.gov/33364201/. DOI: 10.3389/fcimb.2020.589505.
21. Feng L, Liang L, Zhang S, et al. HMGB1 downregulation in retinal pigment epithelial cells protects against diabetic retinopathy through the autophagy-lysosome pathway[J]. Autophagy, 2022, 18(2): 320-339. DOI: 10.1080/15548627.2021.1926655.
22. Khuanjing T, Maneechote C, Ongnok B, et al. Acetylcholinesterase inhibition protects against trastuzumab-induced cardiotoxicity through reducing multiple programmed cell death pathways[J]. Mol Med, 2023, 29(1): 123. DOI: 10.1186/s10020-023-00686-7.
23. Mohamed AK, Ezhilarasan D, Shree HK. Coleus vettiveroides ethanolic root extract induces cytotoxicity by intrinsic apoptosis in HepG2 cells[J]. J Appl Toxicol, 2024, 44(2): 245-259. DOI: 10.1002/jat.4536.
24. Gorospe CM, Carvalho G, Herrera Curbelo A, et al. Mitochondrial membrane potential acts as a retrograde signal to regulate cell cycle progression[J/OL]. Life Sci Alliance, 2023, 6(12): e202302091[2023-09-11]. https://pubmed.ncbi.nlm.nih.gov/37696576/. DOI: 10.26508/lsa.202302091.
25. Siemers KM, Klein AK, Baack ML. Mitochondrial dysfunction in PCOS: insights into reproductive organ pathophysiology[J/OL]. Int J Mol Sci, 2023, 24(17): 13123[2023-08-23]. https://pubmed.ncbi.nlm.nih.gov/37685928/. DOI: 10.3390/ijms241713123.
26. Fu G, Chen AF, Xu Q, et al. Cathepsin L deficiency results in reactive oxygen species (ROS) accumulation and vascular cells activation[J]. Free Radic Res, 2017, 51(11-12): 932-942. DOI: 10.1080/10715762.2017.1393665.
27. Bock FJ, Tait S. Mitochondria as multifaceted regulators of cell death[J]. Nat Rev Mol Cell Biol, 2020, 21(2): 85-100. DOI: 10.1038/s41580-019-0173-8.
28. Lankelma JM, Voorend DM, Barwari T, et al. Cathepsin L, target in cancer treatment?[J]. Life Sci, 2010, 86(7-8): 225-233. DOI: 10.1016/j.lfs.2009.11.016.
29. Lopes De Faria JM, Duarte DA, Montemurro C, et al. Defective autophagy in diabetic retinopathy[J]. Invest Ophthalmol Vis Sci, 2016, 57(10): 4356-4366. DOI: 10.1167/iovs.16-19197.
30. Choi SI, Woo JH, Kim EK. Lysosomal dysfunction of corneal fibroblasts underlies the pathogenesis of granular corneal dystrophy type 2 and can be rescued by TFEB[J]. J Cell Mol Med, 2020, 24(18): 10343-10355. DOI: 10.1111/jcmm.15646.
31. Dana D, Pathak SK. A review of small molecule inhibitors and functional probes of human cathepsin L[J]. Molecules, 2020, 25(3): 698. DOI: 10.3390/molecules25030698.
32. Niu R, Wang J, Geng C, et al. Tandem mass tag-based proteomic analysis reveals cathepsin-mediated anti-autophagic and pro-apoptotic effects under proliferative diabetic retinopathy[J]. Aging (Albany NY), 2020, 13(1): 973-990. DOI: 10.18632/aging.202217.

Chinese Journal of Ocular Fundus Diseases

Cathepsin L inhibitor suppresses oxidative stress-induced apoptosis of retinal pigment epithelial cells by targeting mitochondria

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