Expression of autophagy in rotator cuff tendon stem cells induced by oxidative stress

doi:10.12307/2022.954

Abstract

Abstract: BACKGROUND: The mechanism of rotator cuff tendon-bone healing has not yet been fully elucidated. Studies have shown that oxidative stress can lead to tissue cell damage, which is not conducive to wound healing. It is clinically found that the tendon-bone insertion point of rotator cuff injury often has a poor healing effect, and it is speculated that this may be related to the abnormal cell apoptosis in the wound under ischemia and hypoxia stress.
OBJECTIVE: To induce autophagy of rotator cuff tendon stem cells to promote cell survival as a breakthrough point by oxidative stress stimulation, it is envisaged that autophagy can alleviate cellular oxidative stress damage.
METHODS: The rotator cuff tendon stem cells were cultured in vitro and treated with 0, 50, 100, 200, 400 μmol/L H2O2 respectively for 24, 48 and 72 hours, followed by CCK-8 assay to detect the cell viability. The oxidative stress injury model of rotator cuff tendon stem cells in vitro was established with an appropriate concentration of H2O2. The cultured rotator cuff tendon stem cells at passage 3 were divided into three groups: blank group, H2O2 (50 μmol/L) intervention group, and 3-methyladenine pretreatment (5 mmol/L, 30 minutes)+H2O2 group. At 48 hours after intervention, autophagy was detected by MDC and Lyso-Tracker Red staining. Autophagy related genes were detected by gene expression microarray. Reactive oxygen species level was detected by DCFH-DA. Apoptosis was detected by Annexin V/PI. Protein expression levels of Beclin-1, p-mTOR, mTOR, LC3A/B and Caspase-3 were detected by western blot assay.
RESULTS AND CONCLUSION: (1) With the increase of H2O2 concentration, the proliferation activity of rotator cuff tendon stem cells significantly decreased (P < 0.05). When the concentration of H2O2 was 50 μmol/L, the proliferation activity of rotator cuff tendon stem cells was the strongest. (2) Compared with the blank group and 3-methyladenine+H2O2 group, the autophagy level of H2O2 group was significantly increased (P < 0.05). (3) Compared with the blank group and 3-methyladenine+H2O2 group, the level of reactive oxygen species was significantly increased (P < 0.05); the expression of Beclin-1 protein was up-regulated (P < 0.05); the expression of mTOR protein was down-regulated (P < 0.05); the expression of LC3A/B protein was up-regulated (P < 0.05); and the expression of Caspase-3 protein was up-regulated (P < 0.05) in the H2O2 group. (4) These results indicate that the low concentration of H2O2 can promote the proliferation of human tendon stem cells, and the degree of apoptosis and reactive oxygen species level also increase slightly in a certain range, which is a state of high cell transformation. Autophagy may play a cytoprotective role and promote this high transformation state, but the specific mechanism needs to be further studied.

Key words: rotator cuff, tendon stem cells, oxidative stress, autophagy, apoptosis, tendon bone healing

CLC Number:

Wei Hewei, Zheng Weipeng, Liu Zhijun, Zhao Guoyuan, Fang Weihua, Chen Sheng, Liao Zhihao, Wan Lei. Expression of autophagy in rotator cuff tendon stem cells induced by oxidative stress[J]. Chinese Journal of Tissue Engineering Research, 2022, 26(31): 4954-4961.

Figures/Tables 14

References

[1] 骆勇刚,陈俊,庄万强,等.肩关节镜下不同缝合固定修复技术治疗肩袖损伤的临床疗效[J].局解手术学杂志,2021,30(6):535-540.
[2] 段宝霖,鲍文强,黄佑庆,等.肩关节疼痛神经阻滞疗法中国专家共识(2020版)(第一部分)[J].中华疼痛学杂志,2020,16(5):349-360.
[3] 张阳洋,杨星光,赵金忠.伴有骨质疏松的肩袖损伤治疗进展[J].国际骨科学杂志,2016,37(4):214-218.
[4] 敖英芳.关节镜外科学[M].北京:北京大学医学出版社,2012.
[5] CHUNG SW, OH JH, GONG HS, et al. Factors affecting rotator cuff healing after arthroscopic repair: osteoporosis as one of the independent risk factors. Am J Sports Med. 2011;39(10):2099-2107.
[6] RUDZKI JR, ADLER RS, WARREN RF, et al. Contrast-enhanced ultrasound characterization of the vascularity of the rotator cuff tendon: age- and activity-related changes in the intact asymptomatic rotator cuff. J Shoulder Elbow Surg. 2008;17(1 Suppl):96S-100S.
[7] SUGAYA H, MAEDA K, MATSUKI K, et al. Repair integrity and functional outcome after arthroscopic double-row rotator cuff repair. A prospective outcome study. J Bone Joint Surg Am. 2007;89(5):953-960.
[8] WLASCHEK M, SCHARFFETTER-KOCHANEK K. Oxidative stress in chronic venous leg ulcers. Wound Repair Regen. 2005;13(5):452-461.
[9] 尚小可,郑君,余子杨,等.肩袖损伤的处理临床实践指南(2019年)解读[J].中华肩肘外科电子杂志,2021,9(2):103-111.
[10] LEE S, HWANG JT, LEE SS, et al. Greater Tuberosity Bone Mineral Density and Rotator Cuff Tear Size Are Independent Factors Associated With Cutting-Through in Arthroscopic Suture-Bridge Rotator Cuff Repair. Arthroscopy. 2021;37(7):2077-2086.
[11] YAMAGUCHI K, DITSIOS K, MIDDLETON WD, et al. The demographic and morphological features of rotator cuff disease. A comparison of asymptomatic and symptomatic shoulders. J Bone Joint Surg Am. 2006; 88(8):1699-1704.
[12] 邓炜聪,曾勤,洪钟源,等.富血小板血浆治疗肩袖损伤术后的疗效:随机对照试验Meta分析[J].创伤外科杂志,2021,23(4):276-284.
[13] 韦继南,李永刚,耿锐,等.LafosseⅠ型肩胛下肌损伤修复与否对前上方肩袖损伤修复疗效的影响[J].中华骨科杂志,2020,40(23): 1612-1622.
[14] SCHÄFER M, WERNER S. Oxidative stress in normal and impaired wound repair. Pharmacol Res. 2008;58(2):165-171.
[15] 冯加劲,向明,赵磊.全层肩袖损伤的分类、损伤机制及修复方法的研究进展[J].世界最新医学信息文摘,2019,19(20):132-133.
[16] 陈亚敏,文政芳,王国霞.褪黑素对缺氧缺血性脑损伤新生大鼠皮层氧化应激的影响[J].神经解剖学杂志,2021,37(3):305-309.
[17] 赵智龙,丁国萍,胡媛,等.银质针对大鼠肩袖冈上肌骨-肌腱结合部位损伤的修复作用[J].宁夏医学杂志,2021,43(6):535-538.
[18] 余静雅,戴燕,石玉兰.慢性伤口细菌生物膜的研究进展[J].华西医学,2021,36(5):686-690.
[19] 李凯群.高胆固酵通过激活ROS介导的NF-кB通路抑制肌腱干细胞的腱系分化[D].广州:南方医科大学,2019.
[20] 谭济阳. 肩关节镜下治疗合并骨质疏松的肩袖损伤的疗效分析[D].扬州:扬州大学,2019.
[21] LANG JY, MA K, GUO JX, et al. Oxidative stress induces B lymphocyte DNA damage and apoptosis by upregulating p66shc. Eur Rev Med Pharmacol Sci. 2018;22(4):1051-1060.
[22] TANG C, LIANG J, QIAN J, et al. Opposing role of JNK-p38 kinase and ERK1/2 in hydrogen peroxide-induced oxidative damage of human trophoblast-like JEG-3 cells. Int J Clin Exp Pathol. 2014;7(3):959-968.
[23] 宋慧芳,谈佳音,康毅,等.低氧预处理增强老年人骨髓间充质干细胞条件培养基对H9C2细胞氧化应激损伤的保护作用[J].中国组织工程研究,2022,26(1):1-6.
[24] 付国建,尹峰.干细胞治疗慢性巨大肩袖损伤的研究进展[J].同济大学学报(医学版),2019,40(6):877-883.
[25] 廖昊燃,余伟林,胡庆翔,等.生物学方法促进肩袖腱骨愈合研究进展[J].中华肩肘外科电子杂志,2021,9(2):183-186.
[26] 刘嘉鑫,安丽萍,张广瑞,等.促进肩袖止点腱骨愈合的研究进展[J].中国骨伤,2020,33(7):684-688.
[27] BI Y, EHIRCHIOU D, KILTS TM, et al. Identification of tendon stem/progenitor cells and the role of the extracellular matrix in their niche. Nat Med. 2007;13(10):1219-1227.
[28] LIANG C. Negative regulation of autophagy. Cell Death Differ. 2010; 17(12):1807-1815.
[29] XIE M, MORALES CR, LAVANDERO S, et al. Tuning flux: autophagy as a target of heart disease therapy. Curr Opin Cardiol. 2011;26(3):216-222.
[30] CHERRA SJ 3RD, KULICH SM, UECHI G, et al. Regulation of the autophagy protein LC3 by phosphorylation. J Cell Biol. 2010;190(4): 533-539.
[31] DJAVAHERI-MERGNY M, MAIURI MC, KROEMER G. Cross talk between apoptosis and autophagy by caspase-mediated cleavage of Beclin 1. Oncogene. 2010;29(12):1717-1719.
[32] 梁桂洪,黄和涛,潘建科,等.龙鳖胶囊含药血清对YAP抑制剂诱导人软骨细胞凋亡的保护作用及机制研究[J].中国药房,2021, 32(12):1442-1448.
[33] KANG R, ZEH HJ, LOTZE MT, et al. The Beclin 1 network regulates autophagy and apoptosis. Cell Death Differ. 2011;18(4):571-580.
[34] 杜洪,夏万松,韦四喜,等.长基因间非编码RNA963不同可变剪接体在胃癌细胞系中的表达和作用[J].贵州医科大学学报,2021, 46(7):759-765.
[35] LEVINE B, MIZUSHIMA N, VIRGIN HW. Autophagy in immunity and inflammation. Nature. 2011;469(7330):323-335.
[36] YOSHIMORI T. Autophagy: a regulated bulk degradation process inside cells. Biochem Biophys Res Commun. 2004;313(2):453-458.
[37] 于海,刘清华,肖培伦,等.Beclin-1,LC3A/B和mTOR在肝癌中的表达及临床意义[J].安徽医药,2018,22(2):246-249.
[38] PARGANLIJA D, KLINKENBERG M, DOMÍNGUEZ-BAUTISTA J, et al. Loss of PINK1 impairs stress-induced autophagy and cell survival. PLoS One. 2014;9(4):e95288.
[39] KAMINSKYY VO, ZHIVOTOVSKY B. Free radicals in cross talk between autophagy and apoptosis. Antioxid Redox Signal. 2014;21(1):86-102.
[40] YAN X, WANG L, YANG X, et al. Fluoride induces apoptosis in H9c2 cardiomyocytes via the mitochondrial pathway. Chemosphere. 2017; 182:159-165.