Low-intensity pulsed ultrasound regulates chondrocytes through primary cilia_West China Medical Journal

Authors：

WU Sha , ZHOU Haiqi , LING Huixian , LUO Ziyu , SUN Yuyan , Ngo Thai Nam Anh , ZHANG Changjie ,  KONG Ying

Department of Rehabilitation, the Second Xiangya Hospital, Central South University, Changsha, Hunan 410000, P. R. China;

Corresponding author：

KONG Ying, Email: kongying1502@csu.edu.cn

Keywords：

Low-intensity pulsed ultrasound; primary cilia; interleukin-1β; chondrocyte

DOI：

10.7507/1002-0179.202304182

Video：

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Objective To explore the effects of low-intensity pulsed ultrasound (LIPUS) on anabolism, apoptosis and intraflagellar transport 88 (IFT88) expression in mouse chondrocytes after interleukin (IL)-1β intervention, and the correlation of cartilage repairment by LIPUS with primary cilia. Methods IL-1β intervention, LIPUS intervention and lentiviral carrying IFT88-specifific short hairpin RNA (sh-IFT88) transfection were performed on mouse chondrocytes, respectively. The groups included: normal chondrocyte group (N group), chondrocyte after IL-1β intervention group (OA group), chondrocyte after IL-1β intervention+LIPUS group (OA+U group), sh-IFT88+IL-1β intervention chondrocyte group (KO+OA group), and sh-IFT88+LIPUS+IL-1β treated chondrocyte group (KO+OA+U group). Real-time polymerase chain reaction and immunofluorescence were used to determine the expression of collagen Ⅱ, aggrecan, and primary cilia, and apoptosis was measured by flow cytometry. All experimental data were statistically analyzed using the GraphPad Prism 9.5 software. Results The expression of collagen Ⅱ and aggrecan increased, the apoptosis decreased, and the incidence of primary cilia in chondrocytes of mice increased in the OA+U group compared with those in the OA group (P<0.05). The collagen Ⅱ and aggrecan expression decreased and the apoptosis increased in the KO+OA+U group compared with those in the OA+U group (P<0.05). Conclusion LIPUS can reduce the apoptosis of chondrocytes in C57 mice after IL-1β intervention, and increase the expression of collagen Ⅱ and aggrecan in chondrocyte matrix, and the effect is related to primary cilia.

Citation： WU Sha, ZHOU Haiqi, LING Huixian, LUO Ziyu, SUN Yuyan, Ngo Thai Nam Anh, ZHANG Changjie, KONG Ying. Low-intensity pulsed ultrasound regulates chondrocytes through primary cilia. West China Medical Journal, 2023, 38(6): 835-842. doi: 10.7507/1002-0179.202304182 Copy

1.	Hunter DJ, March L, Chew M. Osteoarthritis in 2020 and beyond: a Lancet commission. Lancet, 2020, 396(10264): 1711-1712.
2.	Courties A, Sellam J, Berenbaum F. Metabolic syndrome-associated osteoarthritis. Curr Opin Rheumatol, 2017, 29(2): 214-222.
3.	Scanzello CR. Role of low-grade inflammation in osteoarthritis. Curr Opin Rheumatol, 2017, 29(1): 79-85.
4.	Bierma-Zeinstra SM, van Middelkoop M. Osteoarthritis: in search of phenotypes. Nat Rev Rheumatol, 2017, 13(12): 705-706.
5.	Musumeci G, Aiello FC, Szychlinska MA, et al. Osteoarthritis in the XXIst century: risk factors and behaviours that influence disease onset and progression. Int J Mol Sci, 2015, 16(3): 6093-6112.
6.	Di Rosa M, Szychlinska MA, Tibullo D, et al. Expression of CHI3L1 and CHIT1 in osteoarthritic rat cartilage model. A morphological study. Eur J Histochem, 2014, 58(3): 2423.
7.	Li X, Li J, Cheng K, et al. Effect of low-intensity pulsed ultrasound on MMP-13 and MAPKs signaling pathway in rabbit knee osteoarthritis. Cell Biochem Biophys, 2011, 61(2): 427-434.
8.	Naito K, Watari T, Muta T, et al. Low-intensity pulsed ultrasound (LIPUS) increases the articular cartilage type Ⅱ collagen in a rat osteoarthritis model. J Orthop Res, 2010, 28(3): 361-369.
9.	Li X, Lin Q, Wang D, et al. The effects of low-intensity pulsed ultrasound and nanomagnet applications on the expressions of MMP-13 and MAPKs in rabbit knee osteoarthritis. J Nanosci Nanotechnol, 2013, 13(1): 722-727.
10.	Wann AK, Knight MM. Primary cilia elongation in response to interleukin-1 mediates the inflammatory response. Cell Mol Life Sci, 2012, 69(17): 2967-2977.
11.	Chang CF, Ramaswamy G, Serra R. Depletion of primary cilia in articular chondrocytes results in reduced Gli3 repressor to activator ratio, increased Hedgehog signaling, and symptoms of early osteoarthritis. Osteoarthritis Cartilage, 2012, 20(2): 152-161.
12.	Ishikawa H, Marshall WF. Ciliogenesis: building the cell’s antenna. Nat Rev Mol Cell Biol, 2011, 12(4): 222-234.
13.	Yang Q, Zhou Y, Cai P, et al. Up-regulated HIF-2α contributes to the osteoarthritis development through mediating the primary cilia loss. Int Immunopharmacol, 2019, 75: 105762.
14.	Muhammad H, Rais Y, Miosge N, et al. The primary cilium as a dual sensor of mechanochemical signals in chondrocytes. Cell Mol Life Sci, 2012, 69(13): 2101-2107.
15.	Thompson CL, Chapple JP, Knight MM. Primary cilia disassembly down-regulates mechanosensitive hedgehog signalling: a feedback mechanism controlling ADAMTS-5 expression in chondrocytes. Osteoarthritis Cartilage, 2014, 22(3): 490-498.
16.	Poole CA, Zhang ZJ, Ross JM. The differential distribution of acetylated and detyrosinated alpha-tubulin in the microtubular cytoskeleton and primary cilia of hyaline cartilage chondrocytes. J Anat, 2001, 199(Pt 4): 393-405.
17.	McGlashan SR, Knight MM, Chowdhury TT, et al. Mechanical loading modulates chondrocyte primary cilia incidence and length. Cell Biol Int, 2010, 34(5): 441-446.
18.	McGlashan SR, Cluett EC, Jensen CG, et al. Primary cilia in osteoarthritic chondrocytes: from chondrons to clusters. Dev Dyn, 2008, 237(8): 2013-2020.
19.	Rich DR, Clark AL. Chondrocyte primary cilia shorten in response to osmotic challenge and are sites for endocytosis. Osteoarthritis Cartilage, 2012, 20(8): 923-930.
20.	Goldring MB, Goldring SR. Osteoarthritis. J Cell Physiol, 2007, 213(3): 626-634.
21.	Kapoor M, Martel-Pelletier J, Lajeunesse D, et al. Role of proinflammatory cytokines in the pathophysiology of osteoarthritis. Nat Rev Rheumatol, 2011, 7(1): 33-42.
22.	Wang T, He C. Pro-inflammatory cytokines: the link between obesity and osteoarthritis. Cytokine Growth Factor Rev, 2018, 44: 38-50.
23.	Lin G, Reed-Maldonado AB, Lin M, et al. Effects and mechanisms of low-intensity pulsed ultrasound for chronic prostatitis and chronic pelvic pain syndrome. Int J Mol Sci, 2016, 17(7): 1057.
24.	Uddin SM, Richbourgh B, Ding Y, et al. Chondro-protective effects of low intensity pulsed ultrasound. Osteoarthritis Cartilage, 2016, 24(11): 1989-1998.
25.	Nishida T, Kubota S, Aoyama E, et al. Low-intensity pulsed ultrasound (LIPUS) treatment of cultured chondrocytes stimulates production of CCN family protein 2 (CCN2), a protein involved in the regeneration of articular cartilage: mechanism underlying this stimulation. Osteoarthritis Cartilage, 2017, 25(5): 759-769.
26.	Sekino J, Nagao M, Kato S, et al. Low-intensity pulsed ultrasound induces cartilage matrix synthesis and reduced MMP13 expression in chondrocytes. Biochem Biophys Res Commun, 2018, 506(1): 290-297.
27.	Pan YL, Ma Y, Guo Y, et al. Effects of Clematis chinensis Osbeck mediated by low-intensity pulsed ultrasound on transforming growth factor-β/Smad signaling in rabbit articular chondrocytes. J Med Ultrason (2001), 2019, 46(2): 177-186.
28.	Zhang S, Cheng J, Qin YX. Mechanobiological modulation of cytoskeleton and calcium influx in osteoblastic cells by short-term focused acoustic radiation force. PLoS One, 2012, 7(6): e38343.
29.	Kujawska T, Secomski W, Kruglenko E, et al. Determination of tissue thermal conductivity by measuring and modeling temperature rise induced in tissue by pulsed focused ultrasound. PLoS One, 2014, 9(4): e94929.
30.	Xiao H, Yan A, Li M, et al. LIPUS accelerates bone regeneration via HDAC6-mediated ciliogenesis. Biochem Biophys Res Commun, 2023, 641: 34-41.
31.	Tang L, Wu T, Zhou Y, et al. Study on synergistic effects of carboxymethyl cellulose and LIPUS for bone tissue engineering. Carbohydr Polym, 2022, 286: 119278.
32.	Sang F, Xu J, Chen Z, et al. Low-intensity pulsed ultrasound alleviates osteoarthritis condition through focal adhesion kinase-mediated chondrocyte proliferation and differentiation. Cartilage, 2021, 13(2_Suppl): 196S-203S.
33.	Nauli SM, Jin X, AbouAlaiwi WA, et al. Non-motile primary cilia as fluid shear stress mechanosensors. Methods Enzymol, 2013, 525: 1-20.
34.	Matsumoto K, Shimo T, Kurio N, et al. Low-intensity pulsed ultrasound stimulation promotes osteoblast differentiation through hedgehog signaling. J Cell Biochem, 2018, 119(6): 4352-4360.
35.	Sun JS, Yang DJ, Kinyua AW, et al. Ventromedial hypothalamic primary cilia control energy and skeletal homeostasis. J Clin Invest, 2021, 131(1): e138107.
36.	Huang X, Lin Z, Meng L, et al. Non-invasive low-intensity pulsed ultrasound modulates primary cilia of rat hippocampal neurons. Ultrasound Med Biol, 2019, 45(5): 1274-1283.
37.	Subramanian A, Budhiraja G, Sahu N. Chondrocyte primary cilium is mechanosensitive and responds to low-intensity-ultrasound by altering its length and orientation. Int J Biochem Cell Biol, 2017, 91(Pt A): 60-64.
38.	Oh S, Kim HM, Batsukh S, et al. High-intensity focused ultrasound induces adipogenesis via control of cilia in adipose-derived stem cells in subcutaneous adipose tissue. Int J Mol Sci, 2022, 23(16): 8866.
39.	Fu S, Thompson CL, Ali A, et al. Mechanical loading inhibits cartilage inflammatory signalling via an HDAC6 and IFT-dependent mechanism regulating primary cilia elongation. Osteoarthritis Cartilage, 2019, 27(7): 1064-1074.
40.	Moore ER, Mathews OA, Yao Y, et al. Prx1-expressing cells contributing to fracture repair require primary cilia for complete healing in mice. Bone, 2021, 143: 115738.
41.	Chen JC, Hoey DA, Chua M, et al. Mechanical signals promote osteogenic fate through a primary cilia-mediated mechanism. Faseb j, 2016, 30(4): 1504-1511.
42.	ter Haar G. Therapeutic applications of ultrasound. Prog Biophys Mol Biol, 2007, 93(1/2/3): 111-129.

1. Hunter DJ, March L, Chew M. Osteoarthritis in 2020 and beyond: a Lancet commission. Lancet, 2020, 396(10264): 1711-1712.
2. Courties A, Sellam J, Berenbaum F. Metabolic syndrome-associated osteoarthritis. Curr Opin Rheumatol, 2017, 29(2): 214-222.
3. Scanzello CR. Role of low-grade inflammation in osteoarthritis. Curr Opin Rheumatol, 2017, 29(1): 79-85.
4. Bierma-Zeinstra SM, van Middelkoop M. Osteoarthritis: in search of phenotypes. Nat Rev Rheumatol, 2017, 13(12): 705-706.
5. Musumeci G, Aiello FC, Szychlinska MA, et al. Osteoarthritis in the XXIst century: risk factors and behaviours that influence disease onset and progression. Int J Mol Sci, 2015, 16(3): 6093-6112.
6. Di Rosa M, Szychlinska MA, Tibullo D, et al. Expression of CHI3L1 and CHIT1 in osteoarthritic rat cartilage model. A morphological study. Eur J Histochem, 2014, 58(3): 2423.
7. Li X, Li J, Cheng K, et al. Effect of low-intensity pulsed ultrasound on MMP-13 and MAPKs signaling pathway in rabbit knee osteoarthritis. Cell Biochem Biophys, 2011, 61(2): 427-434.
8. Naito K, Watari T, Muta T, et al. Low-intensity pulsed ultrasound (LIPUS) increases the articular cartilage type Ⅱ collagen in a rat osteoarthritis model. J Orthop Res, 2010, 28(3): 361-369.
9. Li X, Lin Q, Wang D, et al. The effects of low-intensity pulsed ultrasound and nanomagnet applications on the expressions of MMP-13 and MAPKs in rabbit knee osteoarthritis. J Nanosci Nanotechnol, 2013, 13(1): 722-727.
10. Wann AK, Knight MM. Primary cilia elongation in response to interleukin-1 mediates the inflammatory response. Cell Mol Life Sci, 2012, 69(17): 2967-2977.
11. Chang CF, Ramaswamy G, Serra R. Depletion of primary cilia in articular chondrocytes results in reduced Gli3 repressor to activator ratio, increased Hedgehog signaling, and symptoms of early osteoarthritis. Osteoarthritis Cartilage, 2012, 20(2): 152-161.
12. Ishikawa H, Marshall WF. Ciliogenesis: building the cell’s antenna. Nat Rev Mol Cell Biol, 2011, 12(4): 222-234.
13. Yang Q, Zhou Y, Cai P, et al. Up-regulated HIF-2α contributes to the osteoarthritis development through mediating the primary cilia loss. Int Immunopharmacol, 2019, 75: 105762.
14. Muhammad H, Rais Y, Miosge N, et al. The primary cilium as a dual sensor of mechanochemical signals in chondrocytes. Cell Mol Life Sci, 2012, 69(13): 2101-2107.
15. Thompson CL, Chapple JP, Knight MM. Primary cilia disassembly down-regulates mechanosensitive hedgehog signalling: a feedback mechanism controlling ADAMTS-5 expression in chondrocytes. Osteoarthritis Cartilage, 2014, 22(3): 490-498.
16. Poole CA, Zhang ZJ, Ross JM. The differential distribution of acetylated and detyrosinated alpha-tubulin in the microtubular cytoskeleton and primary cilia of hyaline cartilage chondrocytes. J Anat, 2001, 199(Pt 4): 393-405.
17. McGlashan SR, Knight MM, Chowdhury TT, et al. Mechanical loading modulates chondrocyte primary cilia incidence and length. Cell Biol Int, 2010, 34(5): 441-446.
18. McGlashan SR, Cluett EC, Jensen CG, et al. Primary cilia in osteoarthritic chondrocytes: from chondrons to clusters. Dev Dyn, 2008, 237(8): 2013-2020.
19. Rich DR, Clark AL. Chondrocyte primary cilia shorten in response to osmotic challenge and are sites for endocytosis. Osteoarthritis Cartilage, 2012, 20(8): 923-930.
20. Goldring MB, Goldring SR. Osteoarthritis. J Cell Physiol, 2007, 213(3): 626-634.
21. Kapoor M, Martel-Pelletier J, Lajeunesse D, et al. Role of proinflammatory cytokines in the pathophysiology of osteoarthritis. Nat Rev Rheumatol, 2011, 7(1): 33-42.
22. Wang T, He C. Pro-inflammatory cytokines: the link between obesity and osteoarthritis. Cytokine Growth Factor Rev, 2018, 44: 38-50.
23. Lin G, Reed-Maldonado AB, Lin M, et al. Effects and mechanisms of low-intensity pulsed ultrasound for chronic prostatitis and chronic pelvic pain syndrome. Int J Mol Sci, 2016, 17(7): 1057.
24. Uddin SM, Richbourgh B, Ding Y, et al. Chondro-protective effects of low intensity pulsed ultrasound. Osteoarthritis Cartilage, 2016, 24(11): 1989-1998.
25. Nishida T, Kubota S, Aoyama E, et al. Low-intensity pulsed ultrasound (LIPUS) treatment of cultured chondrocytes stimulates production of CCN family protein 2 (CCN2), a protein involved in the regeneration of articular cartilage: mechanism underlying this stimulation. Osteoarthritis Cartilage, 2017, 25(5): 759-769.
26. Sekino J, Nagao M, Kato S, et al. Low-intensity pulsed ultrasound induces cartilage matrix synthesis and reduced MMP13 expression in chondrocytes. Biochem Biophys Res Commun, 2018, 506(1): 290-297.
27. Pan YL, Ma Y, Guo Y, et al. Effects of Clematis chinensis Osbeck mediated by low-intensity pulsed ultrasound on transforming growth factor-β/Smad signaling in rabbit articular chondrocytes. J Med Ultrason (2001), 2019, 46(2): 177-186.
28. Zhang S, Cheng J, Qin YX. Mechanobiological modulation of cytoskeleton and calcium influx in osteoblastic cells by short-term focused acoustic radiation force. PLoS One, 2012, 7(6): e38343.
29. Kujawska T, Secomski W, Kruglenko E, et al. Determination of tissue thermal conductivity by measuring and modeling temperature rise induced in tissue by pulsed focused ultrasound. PLoS One, 2014, 9(4): e94929.
30. Xiao H, Yan A, Li M, et al. LIPUS accelerates bone regeneration via HDAC6-mediated ciliogenesis. Biochem Biophys Res Commun, 2023, 641: 34-41.
31. Tang L, Wu T, Zhou Y, et al. Study on synergistic effects of carboxymethyl cellulose and LIPUS for bone tissue engineering. Carbohydr Polym, 2022, 286: 119278.
32. Sang F, Xu J, Chen Z, et al. Low-intensity pulsed ultrasound alleviates osteoarthritis condition through focal adhesion kinase-mediated chondrocyte proliferation and differentiation. Cartilage, 2021, 13(2_Suppl): 196S-203S.
33. Nauli SM, Jin X, AbouAlaiwi WA, et al. Non-motile primary cilia as fluid shear stress mechanosensors. Methods Enzymol, 2013, 525: 1-20.
34. Matsumoto K, Shimo T, Kurio N, et al. Low-intensity pulsed ultrasound stimulation promotes osteoblast differentiation through hedgehog signaling. J Cell Biochem, 2018, 119(6): 4352-4360.
35. Sun JS, Yang DJ, Kinyua AW, et al. Ventromedial hypothalamic primary cilia control energy and skeletal homeostasis. J Clin Invest, 2021, 131(1): e138107.
36. Huang X, Lin Z, Meng L, et al. Non-invasive low-intensity pulsed ultrasound modulates primary cilia of rat hippocampal neurons. Ultrasound Med Biol, 2019, 45(5): 1274-1283.
37. Subramanian A, Budhiraja G, Sahu N. Chondrocyte primary cilium is mechanosensitive and responds to low-intensity-ultrasound by altering its length and orientation. Int J Biochem Cell Biol, 2017, 91(Pt A): 60-64.
38. Oh S, Kim HM, Batsukh S, et al. High-intensity focused ultrasound induces adipogenesis via control of cilia in adipose-derived stem cells in subcutaneous adipose tissue. Int J Mol Sci, 2022, 23(16): 8866.
39. Fu S, Thompson CL, Ali A, et al. Mechanical loading inhibits cartilage inflammatory signalling via an HDAC6 and IFT-dependent mechanism regulating primary cilia elongation. Osteoarthritis Cartilage, 2019, 27(7): 1064-1074.
40. Moore ER, Mathews OA, Yao Y, et al. Prx1-expressing cells contributing to fracture repair require primary cilia for complete healing in mice. Bone, 2021, 143: 115738.
41. Chen JC, Hoey DA, Chua M, et al. Mechanical signals promote osteogenic fate through a primary cilia-mediated mechanism. Faseb j, 2016, 30(4): 1504-1511.
42. ter Haar G. Therapeutic applications of ultrasound. Prog Biophys Mol Biol, 2007, 93(1/2/3): 111-129.

West China Medical Journal

Low-intensity pulsed ultrasound regulates chondrocytes through primary cilia

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