Research of epigallocatechin gallate in delaying chondrocyte senescence_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

LUO Chao ^1,2 , ZHOU Weijun ¹ , ZHANG Huying ¹ , ZHU Jinwei ¹ , CHEN Lu ¹ ,  GU Yurong ¹

1. Department of Orthopedics, the Second Affiliated Hospital of Nanchang University, Nanchang Jiangxi, 330006, P. R. China;
2. Department of Orthopedics, Nanchang County People’s Hospital, Nanchang Jiangxi, 330200, P. R. China;

Corresponding author：

GU Yurong, Email: guyurong1646@163.com

Keywords：

Epigallocatechin gallate; chondrocytes; autophagy; senescence; PI3K/AKT/mTOR pathway

DOI：

10.7507/1002-1892.202210101

Video：

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Abstract Full text Figures/Tables Video References Cited by

Objective To investigate the effect of epigallocatechin gallate (EGCG) on chondrocyte senescence and its mechanism. Methods The chondrocytes were isolated from the articular cartilage of 4-week-old Sprague Dawley rats, and cultured with type Ⅱcollagenase and passaged. The cells were identified by toluidine blue staining, alcian blue staining, and immunocytochemical staining for type Ⅱ collagen. The second passage (P2) cells were divided into blank control group, 10 ng/mL IL-1β group, and 6.25, 12.5, 25.0, 50.0, 100.0, and 200.0 μmol/L EGCG+10 ng/mL IL-1β group. The chondrocyte activity was measured with cell counting kit 8 after 24 hours of corresponding culture, and the optimal drug concentration of EGCG was selected for the subsequent experiment. The P2 chondrocytes were further divided into blank control group (group A), 10 ng/mL IL-1β group (group B), EGCG+10 ng/mL IL-1β group (group C), and EGCG+10 ng/mL IL-1β+5 mmol/L 3-methyladenine (3-MA) group (group D). After cultured, the degree of cell senescence was detected by β-galactosidase staining, the autophagy by monodansylcadaverine method, and the expression levels of chondrocyte-related genes [type Ⅱ collagen, matrix metalloproteinase 3 (MMP-3), MMP-13] by real-time fluorescent quantitative PCR, the expression levels of chondrocyte-related proteins (Beclin-1, LC3, MMP-3, MMP-13, type Ⅱ collagen, P16, mTOR, AKT) by Western blot. Results The cultured cells were identified as chondrocytes. Compared with the blank control group, the cell activity of 10 ng/mL IL-1β group significantly decreased (P<0.05). Compared with the 10 ng/mL IL-1β group, the cell activity of EGCG+10 ng/mL IL-1β groups increased, and the 50.0, 100.0, and 200.0 μmol/L EGCG significantly promoted the activity of chondrocytes (P<0.05). The 100.0 μmol/L EGCG was selected for subsequent experiments. Compared with group A, the cells in group B showed senescence changes. Compared with group B, the senescence rate of chondrocytes in group C decreased, autophagy increased, the relative expression of type Ⅱ collagen mRNA increased, and relative expressions of MMP-3 and MMP-13 mRNAs decreased; the relative expressions of Beclin-1, LC3, and type Ⅱ collagen proteins increased, but the relative expressions of P16, MMP-3, MMP-13, mTOR, and AKT proteins decreased; the above differences were significant (P<0.05). Compared with group C, when 3-MA was added in group D, the senescence rate of chondrocytes increased, autophagy decreased, and the relative expressions of the target proteins and mRNAs showed an opposite trend (P<0.05). Conclusion EGCG regulates the autophagy of chondrocytes through the PI3K/AKT/mTOR signaling pathway and exerts anti-senescence effects.

Citation： LUO Chao, ZHOU Weijun, ZHANG Huying, ZHU Jinwei, CHEN Lu, GU Yurong. Research of epigallocatechin gallate in delaying chondrocyte senescence. Chinese Journal of Reparative and Reconstructive Surgery, 2023, 37(3): 308-315. doi: 10.7507/1002-1892.202210101 Copy

1.	Poliwoda S, Noor N, Mousa B, et al. A comprehensive review of intraarticular knee injection therapy, geniculate injections, and peripheral nerve stimulation for knee pain in clinical practice. Orthop Rev (Pavia), 2022, 14(4): 38676. doi: 10.52965/001c.38676.eCollection2022.
2.	Zheng T, Li Y, Zhang X, et al. Exosomes derived from miR-212-5p overexpressed human synovial mesenchymal stem cells suppress chondrocyte degeneration and inflammation by targeting ELF3. Front Bioeng Biotechnol, 2022, 10: 816209. doi: 10.3389/fbioe.2022.816209.
3.	Huang HT, Cheng TL, Yang CD, et al. Intra-articular injection of (-)-epigallocatechin 3-gallate (EGCG) ameliorates cartilage degeneration in guinea pigs with spontaneous osteoarthritis. Antioxidants (Basel), 2021, 10(2): 178. doi: 10.3390/antiox10020178.
4.	Snow AD, Cummings JA, Tanzi RE, et al. In vitro comparison of major memory-support dietary supplements for their effectiveness in reduction/inhibition of beta-amyloid protein fibrils and tau protein tangles: key primary targets for memory loss. Sci Rep, 2021, 11(1): 3001. doi: 10.1038/s41598-020-79275-1.
5.	Huang HT, Cheng TL, Ho CJ, et al. Intra-articular injection of (-)-Epigallocatechin 3-gallate to attenuate articular cartilage degeneration by enhancing autophagy in a post-traumatic osteoarthritis rat model. Antioxidants (Basel), 2020, 10(1): 8. doi: 10.3390/antiox10010008.
6.	Khan MI, Khan MZ, Shin JH, et al. Pharmacological approaches to attenuate inflammation and obesity with natural products formulations by regulating the associated promoting molecular signaling pathways. Biomed Res Int, 2021, 2021: 2521273. doi: 10.1155/2021/2521273.
7.	Albassam AA, Markowitz JS. An appraisal of drug-drug interactions with green tea (camellia sinensis). Planta Med, 2017, 83(6): 496-508.
8.	Wang M, Zhang WB, Zhu JH, et al. Breviscapine ameliorates hypertrophy of cardiomyocytes induced by high glucose in diabetic rats via the PKC signaling pathway. Acta Pharmacol Sin, 2009, 30(8): 1081-1091.
9.	Stefani M, Rigacci S. Beneficial properties of natural phenols: highlight on protection against pathological conditions associated with amyloid aggregation. Biofactors, 2014, 40(5): 482-493.
10.	Calgarotto AK, Maso V, Junior GCF, et al. Antitumor activities of quercetin and green tea in xenografts of human leukemia HL60 cells. Sci Rep, 2018, 8(1): 3459. doi: 10.1038/s41598-018-21516-5.
11.	Deretic V. Autophagy in inflammation, infection, and immunometabolism. Immunity, 2021, 54(3): 437-453.
12.	Wang M, Lv Q, Zhao L, et al. Metoprolol and bisoprolol ameliorate hypertrophy of neonatal rat cardiomyocytes induced by high glucose via the PKC/NF-κB/c-fos signaling pathway. Exp Ther Med, 2020, 19(2): 871-882.
13.	Thongrakard V, Titone R, Follo C, et al. Turmeric toxicity in A431 epidermoid cancer cells associates with autophagy degradation of anti-apoptotic and anti-autophagic p53 mutant. Phytother Res, 2014, 28(12): 1761-1769.
14.	Meng LS, Li B, Li DN, et al. Cyanidin-3-O-glucoside attenuates amyloid-beta (1-40)-induced oxidative stress and apoptosis in SH-SY5Y cells through a Nrf2 mechanism. Journal of Functional Foods, 2017, 38: 474-485.
15.	Meng XY, Li B, Liu S, et al. EGCG in green tea induces aggregation of HMGB1 protein through large conformational changes with polarized charge redistribution. Sci Rep, 2016, 6: 22128. doi: 10.1038/srep22128.
16.	Renaud J, Nabavi SF, Daglia M, et al. Epigallocatechin-3-gallate, a promising molecule for Parkinson’s disease? Rejuvenation Res, 2015, 18(3): 257-269.
17.	Chen C, Liu Q, Liu L, et al. Potential biological effects of (-)-epigallocatechin-3-gallate on the treatment of nonalcoholic fatty liver disease. Mol Nutr Food Res, 2018, 62(1): 1700483. doi: 10.1002/mnfr.201700483.
18.	Zhang YL, Zhang YQ, Lin HL, et al. Epigallocatechin-3-gallate increases autophagic activity attenuating TGF-β₁-induced transformation of human Tenon’s fibroblasts. Exp Eye Res, 2021, 204: 108447. doi: 10.1016/j.exer.2021.108447.
19.	Holczer M, Besze B, Zámbó V, et al. Epigallocatechin-3-gallate (EGCG) promotes autophagy-dependent survival via influencing the balance of mTOR-AMPK pathways upon endoplasmic reticulum stress. Oxid Med Cell Longev, 2018, 2018: 6721530. doi: 10.1155/2018/6721530.
20.	Blasiak J. Senescence in the pathogenesis of age-related macular degeneration. Cell Mol Life Sci, 2020, 77(5): 789-805.
21.	Wu X, Cai Y, Lu S, et al. Intra-articular injection of chloramphenicol reduces articular cartilage degeneration in a rabbit model of osteoarthritis. Clin Orthop Relat Res, 2019, 477(12): 2785-2797.
22.	Wu Y, Li X, Guo Y, et al. Pravastatin reduces matrix metalloproteinases expression and promotes cholesterol efflux in osteoarthritis chondrocytes. Evid Based Complement Alternat Med, 2022, 2022: 9666963. doi: 10.1155/2022/9666963.
23.	Deloney M, Smart K, Christiansen BA, et al. Thermoresponsive, hollow, degradable core-shell nanoparticles for intra-articular delivery of anti-inflammatory peptide. J Control Release, 2020, 323: 47-58.
24.	Debatin KM, Krammer PH. Death receptors in chemotherapy and cancer. Oncogene, 2004, 23(16): 2950-2966.
25.	Liu Z, Lang B, Gao M, et al. 3-(3-Methoxyphenyl)-6-(3-amino-4-methoxyphenyl)-7H-[1, 2, 4] triazolo [3, 4-b][1, 3, 4] thiadiazine, a novel tubulin inhibitor, evokes G2/M cell cycle arrest and apoptosis in SGC-7901 and HeLa cells. J Cell Biochem, 2020, 121(3): 2184-2196.
26.	Heimer S, Knoll G, Schulze-Osthoff K, et al. Raptinal bypasses BAX, BAK, and BOK for mitochondrial outer membrane permeabilization and intrinsic apoptosis. Cell Death Dis, 2019, 10(8): 556. doi: 10.1038/s41419-019-1790-z.
27.	Song D, Yang Q, Jiang X, et al. YY1 deficiency in β-cells leads to mitochondrial dysfunction and diabetes in mice. Metabolism, 2020, 112: 154353. doi: 10.1016/j.metabol.2020.154353.
28.	Tan S, Zheng Z, Liu T, et al. Schisandrin B induced ROS-mediated autophagy and Th1/Th2 imbalance via selenoproteins in Hepa1-6 cells. Front Immunol, 2022, 13: 857069. doi: 10.3389/fimmu.2022.857069.
29.	Rocha M, Apostolova N, Diaz-Rua R, et al. Mitochondria and T2D: Role of autophagy, ER stress, and inflammasome. Trends Endocrinol Metab, 2020, 31(10): 725-741.
30.	Wu L, Quan W, Yue G, et al. Identification of a novel six autophagy-related genes signature for the prognostic and a miRNA-related autophagy predictor for anti-PD-1 therapy responses in prostate cancer. BMC Cancer, 2021, 21(1): 15. doi: 10.1186/s12885-020-07725-0.
31.	Zhang Y, Gilmour A, Ahn YH, et al. The isothiocyanate sulforaphane inhibits mTOR in an NRF2-independent manner. Phytomedicine, 2021, 86: 153062. doi: 10.1016/j.phymed.2019.153062.
32.	Li Y, Xu Q, Shi M, et al. Low-level laser therapy induces human umbilical vascular endothelial cell proliferation, migration and tube formation through activating the PI3K/Akt signaling pathway. Microvasc Res, 2020, 129: 103959. doi: 10.1016/j.mvr.2019.103959.
33.	Chen X, Liu H, Chen K, et al. Inhibition of autophagy impairs free fatty acid-induced excessive lipid accumulation in hepatocellular carcinoma and hepatic cells. J Biosci, 2022, 47: 27. doi: 10.1007/s12038-022-00257-8.
34.	Lu H, Chen Y, Chen Y, et al. C/EBP α-mediated transcriptional activation of PIK3C2A regulates autophagy, matrix metalloproteinase expression, and phenotypic of vascular smooth muscle cells in aortic dissection. J Immunol Res, 2022, 2022: 7465353. doi: 10.1155/2022/7465353.
35.	Rao MV, Darji S, Stavrides PH, et al. Autophagy is a novel pathway for neurofilament protein degradation in vivo. Autophagy, 2022. doi: 10.1080/15548627.2022.2124500.
36.	Zhang Z, Huo Y, Zhou Z, et al. Hydroxysafflor yellow A (HSYA) protects endplate chondrocytes against IL-1β-induced injury through promoting autophagy. Evid Based Complement Alternat Med, 2022, 2022: 6326677. doi: 10.1155/2022/6326677.
37.	Liu Y, Hu L, Liu Q, et al. miR-3651 Participates in the growth cycle of hepatocellular carcinoma cells and promotes the malignant metastasis via the PI3K/AKT/mTOR signalling pathway. J Oncol, 2022, 2022: 5744999. doi: 10.1155/2022/5744999.

1. Poliwoda S, Noor N, Mousa B, et al. A comprehensive review of intraarticular knee injection therapy, geniculate injections, and peripheral nerve stimulation for knee pain in clinical practice. Orthop Rev (Pavia), 2022, 14(4): 38676. doi: 10.52965/001c.38676.eCollection2022.
2. Zheng T, Li Y, Zhang X, et al. Exosomes derived from miR-212-5p overexpressed human synovial mesenchymal stem cells suppress chondrocyte degeneration and inflammation by targeting ELF3. Front Bioeng Biotechnol, 2022, 10: 816209. doi: 10.3389/fbioe.2022.816209.
3. Huang HT, Cheng TL, Yang CD, et al. Intra-articular injection of (-)-epigallocatechin 3-gallate (EGCG) ameliorates cartilage degeneration in guinea pigs with spontaneous osteoarthritis. Antioxidants (Basel), 2021, 10(2): 178. doi: 10.3390/antiox10020178.
4. Snow AD, Cummings JA, Tanzi RE, et al. In vitro comparison of major memory-support dietary supplements for their effectiveness in reduction/inhibition of beta-amyloid protein fibrils and tau protein tangles: key primary targets for memory loss. Sci Rep, 2021, 11(1): 3001. doi: 10.1038/s41598-020-79275-1.
5. Huang HT, Cheng TL, Ho CJ, et al. Intra-articular injection of (-)-Epigallocatechin 3-gallate to attenuate articular cartilage degeneration by enhancing autophagy in a post-traumatic osteoarthritis rat model. Antioxidants (Basel), 2020, 10(1): 8. doi: 10.3390/antiox10010008.
6. Khan MI, Khan MZ, Shin JH, et al. Pharmacological approaches to attenuate inflammation and obesity with natural products formulations by regulating the associated promoting molecular signaling pathways. Biomed Res Int, 2021, 2021: 2521273. doi: 10.1155/2021/2521273.
7. Albassam AA, Markowitz JS. An appraisal of drug-drug interactions with green tea (camellia sinensis). Planta Med, 2017, 83(6): 496-508.
8. Wang M, Zhang WB, Zhu JH, et al. Breviscapine ameliorates hypertrophy of cardiomyocytes induced by high glucose in diabetic rats via the PKC signaling pathway. Acta Pharmacol Sin, 2009, 30(8): 1081-1091.
9. Stefani M, Rigacci S. Beneficial properties of natural phenols: highlight on protection against pathological conditions associated with amyloid aggregation. Biofactors, 2014, 40(5): 482-493.
10. Calgarotto AK, Maso V, Junior GCF, et al. Antitumor activities of quercetin and green tea in xenografts of human leukemia HL60 cells. Sci Rep, 2018, 8(1): 3459. doi: 10.1038/s41598-018-21516-5.
11. Deretic V. Autophagy in inflammation, infection, and immunometabolism. Immunity, 2021, 54(3): 437-453.
12. Wang M, Lv Q, Zhao L, et al. Metoprolol and bisoprolol ameliorate hypertrophy of neonatal rat cardiomyocytes induced by high glucose via the PKC/NF-κB/c-fos signaling pathway. Exp Ther Med, 2020, 19(2): 871-882.
13. Thongrakard V, Titone R, Follo C, et al. Turmeric toxicity in A431 epidermoid cancer cells associates with autophagy degradation of anti-apoptotic and anti-autophagic p53 mutant. Phytother Res, 2014, 28(12): 1761-1769.
14. Meng LS, Li B, Li DN, et al. Cyanidin-3-O-glucoside attenuates amyloid-beta (1-40)-induced oxidative stress and apoptosis in SH-SY5Y cells through a Nrf2 mechanism. Journal of Functional Foods, 2017, 38: 474-485.
15. Meng XY, Li B, Liu S, et al. EGCG in green tea induces aggregation of HMGB1 protein through large conformational changes with polarized charge redistribution. Sci Rep, 2016, 6: 22128. doi: 10.1038/srep22128.
16. Renaud J, Nabavi SF, Daglia M, et al. Epigallocatechin-3-gallate, a promising molecule for Parkinson’s disease? Rejuvenation Res, 2015, 18(3): 257-269.
17. Chen C, Liu Q, Liu L, et al. Potential biological effects of (-)-epigallocatechin-3-gallate on the treatment of nonalcoholic fatty liver disease. Mol Nutr Food Res, 2018, 62(1): 1700483. doi: 10.1002/mnfr.201700483.
18. Zhang YL, Zhang YQ, Lin HL, et al. Epigallocatechin-3-gallate increases autophagic activity attenuating TGF-β₁-induced transformation of human Tenon’s fibroblasts. Exp Eye Res, 2021, 204: 108447. doi: 10.1016/j.exer.2021.108447.
19. Holczer M, Besze B, Zámbó V, et al. Epigallocatechin-3-gallate (EGCG) promotes autophagy-dependent survival via influencing the balance of mTOR-AMPK pathways upon endoplasmic reticulum stress. Oxid Med Cell Longev, 2018, 2018: 6721530. doi: 10.1155/2018/6721530.
20. Blasiak J. Senescence in the pathogenesis of age-related macular degeneration. Cell Mol Life Sci, 2020, 77(5): 789-805.
21. Wu X, Cai Y, Lu S, et al. Intra-articular injection of chloramphenicol reduces articular cartilage degeneration in a rabbit model of osteoarthritis. Clin Orthop Relat Res, 2019, 477(12): 2785-2797.
22. Wu Y, Li X, Guo Y, et al. Pravastatin reduces matrix metalloproteinases expression and promotes cholesterol efflux in osteoarthritis chondrocytes. Evid Based Complement Alternat Med, 2022, 2022: 9666963. doi: 10.1155/2022/9666963.
23. Deloney M, Smart K, Christiansen BA, et al. Thermoresponsive, hollow, degradable core-shell nanoparticles for intra-articular delivery of anti-inflammatory peptide. J Control Release, 2020, 323: 47-58.
24. Debatin KM, Krammer PH. Death receptors in chemotherapy and cancer. Oncogene, 2004, 23(16): 2950-2966.
25. Liu Z, Lang B, Gao M, et al. 3-(3-Methoxyphenyl)-6-(3-amino-4-methoxyphenyl)-7H-[1, 2, 4] triazolo [3, 4-b][1, 3, 4] thiadiazine, a novel tubulin inhibitor, evokes G2/M cell cycle arrest and apoptosis in SGC-7901 and HeLa cells. J Cell Biochem, 2020, 121(3): 2184-2196.
26. Heimer S, Knoll G, Schulze-Osthoff K, et al. Raptinal bypasses BAX, BAK, and BOK for mitochondrial outer membrane permeabilization and intrinsic apoptosis. Cell Death Dis, 2019, 10(8): 556. doi: 10.1038/s41419-019-1790-z.
27. Song D, Yang Q, Jiang X, et al. YY1 deficiency in β-cells leads to mitochondrial dysfunction and diabetes in mice. Metabolism, 2020, 112: 154353. doi: 10.1016/j.metabol.2020.154353.
28. Tan S, Zheng Z, Liu T, et al. Schisandrin B induced ROS-mediated autophagy and Th1/Th2 imbalance via selenoproteins in Hepa1-6 cells. Front Immunol, 2022, 13: 857069. doi: 10.3389/fimmu.2022.857069.
29. Rocha M, Apostolova N, Diaz-Rua R, et al. Mitochondria and T2D: Role of autophagy, ER stress, and inflammasome. Trends Endocrinol Metab, 2020, 31(10): 725-741.
30. Wu L, Quan W, Yue G, et al. Identification of a novel six autophagy-related genes signature for the prognostic and a miRNA-related autophagy predictor for anti-PD-1 therapy responses in prostate cancer. BMC Cancer, 2021, 21(1): 15. doi: 10.1186/s12885-020-07725-0.
31. Zhang Y, Gilmour A, Ahn YH, et al. The isothiocyanate sulforaphane inhibits mTOR in an NRF2-independent manner. Phytomedicine, 2021, 86: 153062. doi: 10.1016/j.phymed.2019.153062.
32. Li Y, Xu Q, Shi M, et al. Low-level laser therapy induces human umbilical vascular endothelial cell proliferation, migration and tube formation through activating the PI3K/Akt signaling pathway. Microvasc Res, 2020, 129: 103959. doi: 10.1016/j.mvr.2019.103959.
33. Chen X, Liu H, Chen K, et al. Inhibition of autophagy impairs free fatty acid-induced excessive lipid accumulation in hepatocellular carcinoma and hepatic cells. J Biosci, 2022, 47: 27. doi: 10.1007/s12038-022-00257-8.
34. Lu H, Chen Y, Chen Y, et al. C/EBP α-mediated transcriptional activation of PIK3C2A regulates autophagy, matrix metalloproteinase expression, and phenotypic of vascular smooth muscle cells in aortic dissection. J Immunol Res, 2022, 2022: 7465353. doi: 10.1155/2022/7465353.
35. Rao MV, Darji S, Stavrides PH, et al. Autophagy is a novel pathway for neurofilament protein degradation in vivo. Autophagy, 2022. doi: 10.1080/15548627.2022.2124500.
36. Zhang Z, Huo Y, Zhou Z, et al. Hydroxysafflor yellow A (HSYA) protects endplate chondrocytes against IL-1β-induced injury through promoting autophagy. Evid Based Complement Alternat Med, 2022, 2022: 6326677. doi: 10.1155/2022/6326677.
37. Liu Y, Hu L, Liu Q, et al. miR-3651 Participates in the growth cycle of hepatocellular carcinoma cells and promotes the malignant metastasis via the PI3K/AKT/mTOR signalling pathway. J Oncol, 2022, 2022: 5744999. doi: 10.1155/2022/5744999.

Chinese Journal of Reparative and Reconstructive Surgery

Research of epigallocatechin gallate in delaying chondrocyte senescence

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