Mechanism of chronic circadian rhythm disorder on knee osteoarthritis-like cartilage injury in rats_West China Medical Journal

Authors：

YANG Chunsheng , CHEN Mi , WU Hengmin , ZHANG Tiecheng ,  WANG Baolan

Department of Rehabilitation Medicine, the First Affiliated Hospital of Xinjiang Medical University, Urumqi, Xinjiang 830000, P. R. China;

Corresponding author：

WANG Baolan, Email: wbl0308@163.com

Keywords：

Circadian rhythm; biological clock; Bmal1 gene; cell cycle; osteoarthritis; rat

DOI：

10.7507/1002-0179.202403266

Video：

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Objective To investigate the effect of circadian rhythm disorder on rat knee cartilage and the mechanism of basic helix-loop-helix ARNT like 1 (Bmal1) on the regulation of cell cycle-related genes. Methods Forty rats were randomly divided into normal group, circadian rhythm disorder group (disorder group), Bmal1 overexpression lentivirus infection circadian rhythm disorder group (Bmal1 up-regulated group) and Bmal1 overexpression lentivirus negative infection circadian rhythm disorder group (Bmal1 negative infection group), with 10 rats in each group. Saffron fast green staining, TdT-mediated dUTP nick-end labeling staining, immunohistochemical staining, reverse transcription polymerase chain reaction and Western blotting were used to compare the pathological changes of cartilage tissue, the apoptosis of chondrocytes, and the relative mRNA expression levels of Bmal1, WEE1 G₂ checkpoint kinase (Wee1), cyclin-dependent kinase 1 (Cdk1), cyclin B1 (Ccnb1), BCL2-associated X protein (Bax), apoptosis regulator 2 (Bcl2), interleukin 1 (Il1), interleukin 6 (Il6), tumor necrosis factor (Tnf) and matrix metallopeptidase 13 (Mmp13) among different groups. The relative expression levels of BMAL1, WEE1, CDK1, CCNB1, BAX and BCL2 proteins were detected, and correlation analysis was performed according to the relative expression of mRNA. Results Safranine fast green staining showed that the thickness of cartilage matrix in the normal group was normal and uniform red. The cartilage matrix in the disorder group and the Bmal1 negative infection group was destroyed, and the proteoglycan was lost obviously, showing uneven red. The thickness of cartilage matrix in the Bmal1 up-regulated group was basically normal, and the proteoglycan was not lost obviously, and the red was slightly less uniform. Compared with those of the normal group, the positive rates of apoptotic cells in articular cartilage of the disorder group and the Bmal1 negative infection group increased significantly, the mRNA and protein expression levels of Bmal1, Wee1, and Bcl2 were down-regulated, the mRNA and protein expression levels of Cdk1, Ccnb1, and Bax were up-regulated, the mRNA expression levels of Il1, Il6, Tnf and Mmp13 were up-regulated, the differences were statistically significant (P<0.05); there was no significant change in the positive rate of apoptotic cells in the articular cartilage of the Bmal1 up-regulated group, and there was no significant difference in the mRNA or protein expression of Bmal1, Wee1, Bcl2, Cdk1, Ccnb1 or Bax, nor the mRNA expression of Il1, Il6, Tnf or Mmp13 (P>0.05). Correlation analysis showed that Bmal1 was positively correlated with Wee1 and Bcl2 (r=0.84, 0.44; P<0.01), and negatively correlated with Cdk1, Ccnb1 and Bax (r=–0.55, –0.72, –0.41; P<0.01). Conclusion Chronic circadian rhythm disorder can cause the increase of chondrocyte apoptosis and osteoarthritis-like changes of articular cartilage through the expression changes of circadian clock gene Bmal1 and cell cycle-related genes and proteins.

Citation： YANG Chunsheng, CHEN Mi, WU Hengmin, ZHANG Tiecheng, WANG Baolan. Mechanism of chronic circadian rhythm disorder on knee osteoarthritis-like cartilage injury in rats. West China Medical Journal, 2024, 39(6): 891-898. doi: 10.7507/1002-0179.202403266 Copy

1.	Quevedo I, Zuniga AM. Low bone mineral density in rotating-shift workers. J Clin Densitom, 2010, 13(4): 467-469.
2.	Zhou M, Yang S, Guo Y, et al. Shift work and the risk of knee osteoarthritis among Chinese workers: a retrospective cohort study. Scand J Work Environ Health, 2020, 46(2): 152-160.
3.	胡海龙. 慢性昼夜节律紊乱对大鼠软骨及软骨细胞损伤作用机制的研究. 哈尔滨: 东北农业大学, 2021.
4.	Kc R, Li X, Voigt RM, et al. Environmental disruption of circadian rhythm predisposes mice to osteoarthritis-like changes in knee joint. J Cell Physiol, 2015, 230(9): 2174-2183.
5.	Ma Z, Jin X, Qian Z, et al. Deletion of clock gene Bmal1 impaired the chondrocyte function due to disruption of the HIF1α-VEGF signaling pathway. Cell Cycle, 2019, 18(13): 1473-1489.
6.	Huang L, Yuan H, Shi S, et al. CLOCK inhibits the proliferation of porcine ovarian granulosa cells by targeting ASB9. J Anim Sci Biotechnol, 2023, 14(1): 82.
7.	Jin M, Tavella F, Wang S, et al. In vitro cell cycle oscillations exhibit a robust and hysteretic response to changes in cytoplasmic density. Proc Natl Acad Sci U S A, 2022, 119(6): e2109547119.
8.	Naven MA, Zeef LAH, Li S, et al. Development of human cartilage circadian rhythm in a stem cell-chondrogenesis model. Theranostics, 2022, 12(8): 3963-3976.
9.	Yang N, Meng QJ. Circadian clocks in articular cartilage and bone: a compass in the sea of matrices. J Biol Rhythms, 2016, 31(5): 415-427.
10.	Hinoi E, Ueshima T, Hojo H, et al. Up-regulation of per mRNA expression by parathyroid hormone through a protein kinase A-CREB-dependent mechanism in chondrocytes. J Biol Chem, 2006, 281(33): 23632-26342.
11.	Luo B, Zhou X, Tang Q, et al. Circadian rhythms affect bone reconstruction by regulating bone energy metabolism. J Transl Med, 2021, 19(1): 410.
12.	Gossan N, Boot-Handford R, Meng QJ. Ageing and osteoarthritis: a circadian rhythm connection. Biogerontology, 2015, 16(2): 209-219.
13.	Chen G, Zhao H, Ma S, et al. Circadian rhythm protein Bmal1 modulates cartilage gene expression in temporomandibular joint osteoarthritis via the MAPK/ERK pathway. Front Pharmacol, 2020, 11: 527744.
14.	Dudek M, Gossan N, Yang N, et al. The chondrocyte clock gene Bmal1 controls cartilage homeostasis and integrity. J Clin Invest, 2016, 126(1): 365-376.
15.	郭镜, 高月. 华蟾素通过 wee1 蛋白调控卵巢癌细胞 SKOV3 的增殖与凋亡. 西安交通大学学报(医学版), 2021, 42(6): 857-861.
16.	Martin JC, Sims JR, Gupta A, et al. WEE1 inhibition augments CDC7 (DDK) inhibitor-induced cell death in Ewing sarcoma by forcing premature mitotic entry and mitotic catastrophe. Cancer Res Commun, 2022, 2(6): 471-482.
17.	Igarashi M, Nagata A, Jinno S, et al. Wee1(+)-like gene in human cells. Nature, 1991, 353(6339): 80-83.
18.	McGowan CH, Russell P. Human Wee1 kinase inhibits cell division by phosphorylating p34cdc2 exclusively on Tyr15. EMBO J, 1993, 12(1): 75-85.
19.	De Witt Hamer PC, Mir SE, Noske D, et al. WEE1 kinase targeting combined with DNA-damaging cancer therapy catalyzes mitotic catastrophe. Clin Cancer Res, 2011, 17(13): 4200-4207.
20.	Zhou L, Cai X, Han X, et al. CDK1 switches mitotic arrest to apoptosis by phosphorylating Bcl2/Bax family proteins during treatment with microtubule interfering agents. Cell Biol Int, 2014, 38(6): 737-746.
21.	Darweesh O, Al-Shehri E, Falquez H, et al. Identification of a novel Bax-Cdk1 signalling complex that links activation of the mitotic checkpoint to apoptosis. J Cell Sci, 2021, 134(8): jcs244152.
22.	Choi HJ, Zhu BT. Upregulated cyclin B1/CDK1 mediates apoptosis following 2-methoxyestradiol-induced mitotic catastrophe: role of Bcl-XL phosphorylation. Steroids, 2019, 150: 108381.
23.	Snelling SJ, Forster A, Mukherjee S, et al. The chondrocyte-intrinsic circadian clock is disrupted in human osteoarthritis. Chronobiol Int, 2016, 33(5): 574-579.
24.	Early JO, Menon D, Wyse CA, et al. Circadian clock protein BMAL1 regulates IL-1β in macrophages via NRF2. Proc Natl Acad Sci U S A, 2018, 115(36): E8460-E8468.
25.	Si T, Huang Z, Jiang Y, et al. Expression levels of three key genes CCNB1, CDC20, and CENPF in HCC are associated with antitumor immunity. Front Oncol, 2021, 11: 738841.
26.	Vince JE, De Nardo D, Gao W, et al. The mitochondrial apoptotic effectors BAX/BAK activate caspase-3 and -7 to trigger NLRP3 inflammasome and caspase-8 driven IL-1β activation. Cell Rep, 2018, 25(9): 2339-2353.e4.

1. Quevedo I, Zuniga AM. Low bone mineral density in rotating-shift workers. J Clin Densitom, 2010, 13(4): 467-469.
2. Zhou M, Yang S, Guo Y, et al. Shift work and the risk of knee osteoarthritis among Chinese workers: a retrospective cohort study. Scand J Work Environ Health, 2020, 46(2): 152-160.
3. 胡海龙. 慢性昼夜节律紊乱对大鼠软骨及软骨细胞损伤作用机制的研究. 哈尔滨: 东北农业大学, 2021.
4. Kc R, Li X, Voigt RM, et al. Environmental disruption of circadian rhythm predisposes mice to osteoarthritis-like changes in knee joint. J Cell Physiol, 2015, 230(9): 2174-2183.
5. Ma Z, Jin X, Qian Z, et al. Deletion of clock gene Bmal1 impaired the chondrocyte function due to disruption of the HIF1α-VEGF signaling pathway. Cell Cycle, 2019, 18(13): 1473-1489.
6. Huang L, Yuan H, Shi S, et al. CLOCK inhibits the proliferation of porcine ovarian granulosa cells by targeting ASB9. J Anim Sci Biotechnol, 2023, 14(1): 82.
7. Jin M, Tavella F, Wang S, et al. In vitro cell cycle oscillations exhibit a robust and hysteretic response to changes in cytoplasmic density. Proc Natl Acad Sci U S A, 2022, 119(6): e2109547119.
8. Naven MA, Zeef LAH, Li S, et al. Development of human cartilage circadian rhythm in a stem cell-chondrogenesis model. Theranostics, 2022, 12(8): 3963-3976.
9. Yang N, Meng QJ. Circadian clocks in articular cartilage and bone: a compass in the sea of matrices. J Biol Rhythms, 2016, 31(5): 415-427.
10. Hinoi E, Ueshima T, Hojo H, et al. Up-regulation of per mRNA expression by parathyroid hormone through a protein kinase A-CREB-dependent mechanism in chondrocytes. J Biol Chem, 2006, 281(33): 23632-26342.
11. Luo B, Zhou X, Tang Q, et al. Circadian rhythms affect bone reconstruction by regulating bone energy metabolism. J Transl Med, 2021, 19(1): 410.
12. Gossan N, Boot-Handford R, Meng QJ. Ageing and osteoarthritis: a circadian rhythm connection. Biogerontology, 2015, 16(2): 209-219.
13. Chen G, Zhao H, Ma S, et al. Circadian rhythm protein Bmal1 modulates cartilage gene expression in temporomandibular joint osteoarthritis via the MAPK/ERK pathway. Front Pharmacol, 2020, 11: 527744.
14. Dudek M, Gossan N, Yang N, et al. The chondrocyte clock gene Bmal1 controls cartilage homeostasis and integrity. J Clin Invest, 2016, 126(1): 365-376.
15. 郭镜, 高月. 华蟾素通过 wee1 蛋白调控卵巢癌细胞 SKOV3 的增殖与凋亡. 西安交通大学学报(医学版), 2021, 42(6): 857-861.
16. Martin JC, Sims JR, Gupta A, et al. WEE1 inhibition augments CDC7 (DDK) inhibitor-induced cell death in Ewing sarcoma by forcing premature mitotic entry and mitotic catastrophe. Cancer Res Commun, 2022, 2(6): 471-482.
17. Igarashi M, Nagata A, Jinno S, et al. Wee1(+)-like gene in human cells. Nature, 1991, 353(6339): 80-83.
18. McGowan CH, Russell P. Human Wee1 kinase inhibits cell division by phosphorylating p34cdc2 exclusively on Tyr15. EMBO J, 1993, 12(1): 75-85.
19. De Witt Hamer PC, Mir SE, Noske D, et al. WEE1 kinase targeting combined with DNA-damaging cancer therapy catalyzes mitotic catastrophe. Clin Cancer Res, 2011, 17(13): 4200-4207.
20. Zhou L, Cai X, Han X, et al. CDK1 switches mitotic arrest to apoptosis by phosphorylating Bcl2/Bax family proteins during treatment with microtubule interfering agents. Cell Biol Int, 2014, 38(6): 737-746.
21. Darweesh O, Al-Shehri E, Falquez H, et al. Identification of a novel Bax-Cdk1 signalling complex that links activation of the mitotic checkpoint to apoptosis. J Cell Sci, 2021, 134(8): jcs244152.
22. Choi HJ, Zhu BT. Upregulated cyclin B1/CDK1 mediates apoptosis following 2-methoxyestradiol-induced mitotic catastrophe: role of Bcl-XL phosphorylation. Steroids, 2019, 150: 108381.
23. Snelling SJ, Forster A, Mukherjee S, et al. The chondrocyte-intrinsic circadian clock is disrupted in human osteoarthritis. Chronobiol Int, 2016, 33(5): 574-579.
24. Early JO, Menon D, Wyse CA, et al. Circadian clock protein BMAL1 regulates IL-1β in macrophages via NRF2. Proc Natl Acad Sci U S A, 2018, 115(36): E8460-E8468.
25. Si T, Huang Z, Jiang Y, et al. Expression levels of three key genes CCNB1, CDC20, and CENPF in HCC are associated with antitumor immunity. Front Oncol, 2021, 11: 738841.
26. Vince JE, De Nardo D, Gao W, et al. The mitochondrial apoptotic effectors BAX/BAK activate caspase-3 and -7 to trigger NLRP3 inflammasome and caspase-8 driven IL-1β activation. Cell Rep, 2018, 25(9): 2339-2353.e4.

West China Medical Journal

Mechanism of chronic circadian rhythm disorder on knee osteoarthritis-like cartilage injury in rats

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