Effects of icariin on autophagy and exosome production of bone microvascular endothelial cells_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

ZHANG Qingyu ^1,2 , GAO Fuqiang ² , CHENG Liming ² , LIU Lihua ^1,2 ,  SUN Wei ^1,2,3 , LI Zirong ²

1. Graduate School of Peking Union Medical College, Beijing, 100730, P.R.China;
2. Department of Orthopedics, China-Japan Friendship Hospital, Beijing, 100029, P.R.China;
3. Center for Osteonecrosis and Joint Preserving & Reconstruction, China-Japan Friendship Hospital, Beijing, 100029, P.R.China;

Corresponding author：

SUN Wei, Email: sun887@163.com

Keywords：

Femoral head; microvascular endothelial cells; glucocorticoid; icariin; exosome; autophagy

DOI：

10.7507/1002-1892.201811009

Video：

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Objective To evaluate the effects of icariin on autophagy induced by low-concentration of glucocorticoid and exosome production in bone microvascular endothelial cells (BMECs).Methods BMECs were isolated from femoral heads resected in total hip arthroplasty and then intervened with hydrocortisone of low concentration (0, 0.03, 0.06, 0.10 mg/mL), which were set as groups A, B, C, and D, respectively. On the basis of hydrocortisone intervention, 5×10⁻⁵ mol/L of icariin was added to each group (set as groups A1, B1, C1 and D1, respectively). Western blot was used to detect the expressions of microtubule-associated protein 1 light chain 3B (LC3B) and dead bone slice 1 (p62) after 24 hours. Exosomes were extracted from BMECs treated with icariin (intervention group) and without icariin (non-intervention group), and the diameter and concentration of exosomes were evaluated by nanoparticle tracking analysis technique. The total protein content of exosomes was detected by BCA method, and the expressions of proteins carried by exosomes including CD9, CD81, transforming growth factor β₁ (TGF-β₁), and vascular endothelial growth factor A (VEGFA) were assessed by Western blot. The BMECs were further divided into three groups: BMECs in the experimental group and the control group were co-cultured with exosomes secreted by BMECs treated with or without icariin, respectively; the blank control group was BMECs without exosome intervention. The three groups were treated with hydrocortisone and Western blot was used to detect the expressions of LC3B and p62. The scratching assay was used to detect cell migration ability; angiogenic ability of BMECs was also assessed.Results With the increase of hydrocortisone concentration, the protein expression of LC3B-Ⅱ increased gradually, and the protein expression of p62 decreased gradually (P<0.01). Compared with group with same concentration of hydrocortisone, the protein expression of LC3B-Ⅱ decreased and the protein expression of p62 increased after the administration of icariin (P<0.01). The concentration of exosomes in the intervention group was significantly higher than that in the non-intervention group (t=−10.191, P=0.001); and there was no significant difference in exosome diameter and total protein content between the two groups (P>0.05). CD9 and CD81 proteins were highly expressed in the non-intervention group and the intervention group, and the relative expression ratios of VEGFA/CD9 and TGF-β₁/CD9 proteins in the intervention group were significantly higher than those in the non-intervention group (P<0.01). After co-culture of exosomes, the protein expression of p62 increased in blank control group, control group, and experimental group, while the protein expression of LC3B-Ⅱ decreased. There were significant differences among groups (P<0.05). When treated with hydrocortisone for 12 and 24 hours, the scratch closure rate of the control group and experimental group was significantly higher than that of the blank control group (P<0.05), and the scratch closure rate of the experimental group was significantly higher than that of the control group (P<0.05). When treated with hydrocortisone for 4 and 8 hours, the number of lumens, number of sprouting vessels, and length of tubule branches in the experimental group and the control group were significantly greater than those in the blank control group (P<0.05); the length of tubule branches and the number of lumens in the experimental group were significantly greater than those in the control group (P<0.05).Conclusion Icariin and BMECs-derived exosomes can improve the autophagy of BMECs induced by low concentration of glucocorticoid.

Citation： ZHANG Qingyu, GAO Fuqiang, CHENG Liming, LIU Lihua, SUN Wei, LI Zirong. Effects of icariin on autophagy and exosome production of bone microvascular endothelial cells. Chinese Journal of Reparative and Reconstructive Surgery, 2019, 33(5): 568-577. doi: 10.7507/1002-1892.201811009 Copy

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19.	Xia X, Kar R, Gluhak-Heinrich J, et al. Glucocorticoid-induced autophagy in osteocytes. J Bone Miner Res, 2010, 25(11): 2479-2488.
20.	杨雨润, 娄晋宁, 李子荣, 等. 糖皮质激素对骨髓微血管内皮细胞活性氧代谢影响的实验研究. 中国修复重建外科杂志, 2011, 25(5): 533-537.
21.	Chen R, Jiang M, Li B, et al. The role of autophagy in pulmonary hypertension: a double-edge sword. Apoptosis, 2018, 23(9-10): 459-469.
22.	Wu YY, Chen C, Yu X, et al. Effects of tiaozhi granule on regulation of autophagy levels in HUVECs. Evid Based Complement Alternat Med, 2018, 2018: 1765731.
23.	Li C, Li Q, Mei Q, et al. Pharmacological effects and pharmacokinetic properties of icariin, the major bioactive component in Herba Epimedii. Life Sci, 2015, 126: 57-68.
24.	Huang Z, Cheng C, Cao B, et al. Icariin protects against glucocorticoid-induced osteonecrosis of the femoral head in rats. Cell Physiol Biochem, 2018, 47(2): 694-706.
25.	Zhao J, Ohba S, Shinkai M, et al. Icariin induces osteogenic differentiation in vitro in a BMP- and Runx2-dependent manner. Biochem Biophys Res Commun, 2008, 369(2): 444-448.
26.	Chen KM, Ge BF, Liu XY, et al. Icariin inhibits the osteoclast formation induced by RANKL and macrophage-colony stimulating factor in mouse bone marrow culture. Pharmazie, 2007, 62(5): 388-391.
27.	Hu Y, Li H, Liu K, et al. Protective effects of icariin on human vascular endothelial cells induced by oxidized low-density lipoprotein via modulating caspase-3 and Bcl-2. Mol Med Rep, 2018, 17(5): 6835-6839.
28.	王佰亮, 李子荣, 娄晋宁, 等. 淫羊藿苷对糖皮质激素诱导的骨微血管内皮细胞损伤的保护作用. 中国微循环, 2009, 13(6): 461-464, 封2.
29.	Mi B, Wang J, Liu Y, et al. Icariin activates autophagy via down-regulation of the NF-κB signaling-mediated apoptosis in chondrocytes. Front Pharmacol, 2018, 9: 605.
30.	Jiang S, Chang H, Deng S, et al. Icariin inhibits autophagy and promotes apoptosis in SKVCR cells through mTOR signal pathway. Cell Mol Biol (Noisy-le-grand), 2018, 64(6): 4-10.
31.	Hua W, Zhang Y, Wu X, et al. Icariin attenuates interleukin-1β-induced inflammatory response in human nucleus pulposus cells. Curr Pharm Des, 2018, 23(39): 6071-6078.
32.	Zeng L, Wang W, Rong XF, et al. Chondroprotective effects and multi-target mechanisms of Icariin in IL-1 beta-induced human SW 1353 chondrosarcoma cells and a rat osteoarthritis model. Int Immunopharmacol, 2014, 18(1): 175-181.
33.	Ristorcelli E, Beraud E, Mathieu S, et al. Essential role of Notch signaling in apoptosis of human pancreatic tumoral cells mediated by exosomal nanoparticles. Int J Cancer, 2009, 125(5): 1016-1026.
34.	张静, 易阳艳, 阳水发, 等. 脂肪干细胞来源的外泌体对人脐静脉血管内皮细胞增殖、迁移及管样分化的影响. 中国修复重建外科杂志, 2018, 32(10): 1351-1357.
35.	Zhao L, Luo H, Li X, et al. Exosomes derived from human pulmonary artery endothelial cells shift the balance between proliferation and apoptosis of smooth muscle cells. Cardiology, 2017, 137(1): 43-53.
36.	Melo SA, Sugimoto H, O’Connell JT, et al. Cancer exosomes perform cell-independent microRNA biogenesis and promote tumorigenesis. Cancer Cell, 2014, 26(5): 707-721.
37.	Castañeda-Cabral JL, Beas-Zárate C, Rocha-Arrieta LL, et al. Increased protein expression of VEGF-A, VEGF-B, VEGF-C and their receptors in the temporal neocortex of pharmacoresistant temporal lobe epilepsy patients. J Neuroimmunol, 2019, 328: 68-72.
38.	Wang L, Wang J, Wang F, et al. VEGF-mediated cognitive and synaptic improvement in chronic cerebral hypoperfusion rats involves autophagy process. Neuromolecular Med, 2017, 19(2-3): 423-435.
39.	Domigan CK, Warren CM, Antanesian V, et al. Autocrine VEGF maintains endothelial survival through regulation of metabolism and autophagy. J Cell Sci, 2015, 128(12): 2236-2248.
40.	Mao YQ, Fan XM. Autophagy: A new therapeutic target for liver fibrosis. World J Hepatol, 2015, 7(16): 1982-1986.
41.	Tong H, Yin H, Hossain MA, et al. Starvation-induced autophagy promotes the invasion and migration of human bladder cancer cells via TGF-β1/Smad3-mediated epithelial-mesenchymal transition activation. J Cell Biochem, 2019, 120(4): 5118-5127.

1. Kusumbe AP, Ramasamy SK, Adams RH. Coupling of angiogenesis and osteogenesis by a specific vessel subtype in bone. Nature, 2014, 507(7492): 323-328.
2. Chandler FA. Coronary disease of the hip. 1949. Clin Orthop Relat Res, 2001, (386): 7-10.
3. Weinstein RS. Glucocorticoid-induced osteoporosis and osteonecrosis. Endocrinol Metab Clin North Am, 2012, 41(3): 595-611.
4. 魏秋实, 杨帆, 陈哓俊, 等. 激素性与酒精性股骨头坏死患者骨标本坏死区域病理与显微结构特点分析. 中国修复重建外科杂志, 2018, 32(7): 866-872.
5. Kim KA, Shin D, Kim JH, et al. Role of autophagy in endothelial damage and blood-brain barrier disruption in ischemic stroke. Stroke, 2018, 49(6): 1571-1579.
6. Grootaert MOJ, Roth L, Schrijvers DM, et al. Defective autophagy in atherosclerosis: to die or to senesce? Oxid Med Cell Longev, 2018, 2018: 7687083.
7. Zhu XR, Du JH. Autophagy: a potential target for the treatment of intraocular neovascularization. Int J Ophthalmol, 2018, 11(4): 695-698.
8. Tanida I, Ueno T, Kominami E. LC3 conjugation system in mammalian autophagy. Int J Biochem Cell Biol, 2004, 36(12): 2503-2518.
9. de Jong OG, Verhaar MC, Chen Y, et al. Cellular stress conditions are reflected in the protein and RNA content of endothelial cell-derived exosomes. J Extracell Vesicles, 2012, 1. doi: 10.3402/jev.v1i0.18396.eCollection 2012.
10. Hromada C, Mühleder S, Grillari J, et al. Endothelial extracellular vesicles-promises and challenges. Front Physiol, 2017, 8: 275.
11. Jiang Y, Xie H, Tu W, et al. Exosomes secreted by HUVECs attenuate hypoxia/reoxygenation-induced apoptosis in neural cells by suppressing miR-21-3p. Am J Transl Res, 2018, 10(11): 3529-3541.
12. Liao Y, Zhang P, Yuan B, et al. Pravastatin protects against avascular necrosis of femoral head via autophagy. Front Physiol, 2018, 9: 307.
13. Mata-Greenwood E, Jackson PN, Pearce WJ, et al. Endothelial glucocorticoid receptor promoter methylation according to dexamethasone sensitivity. J Mol Endocrinol, 2015, 55(2): 133-146.
14. Kadmiel M, Cidlowski JA. Glucocorticoid receptor signaling in health and disease. Trends Pharmacol Sci, 2013, 34(9): 518-530.
15. Huang W, Glass CK. Nuclear receptors and inflammation control: molecular mechanisms and pathophysiological relevance. Arterioscler Thromb Vasc Biol, 2010, 30(8): 1542-1549.
16. Jia J, Yao W, Guan M, et al. Glucocorticoid dose determines osteocyte cell fate. FASEB J, 2011, 25(10): 3366-3376.
17. Luo P, Gao F, Han J, et al. The role of autophagy in steroid necrosis of the femoral head: a comprehensive research review. Int Orthop, 2018, 42(7): 1747-1753.
18. Ezzat S, Louka ML, Zakaria ZM, et al. Autophagy in osteoporosis: Relation to oxidative stress. J Cell Biochem, 2018. [Epub ahead of print].
19. Xia X, Kar R, Gluhak-Heinrich J, et al. Glucocorticoid-induced autophagy in osteocytes. J Bone Miner Res, 2010, 25(11): 2479-2488.
20. 杨雨润, 娄晋宁, 李子荣, 等. 糖皮质激素对骨髓微血管内皮细胞活性氧代谢影响的实验研究. 中国修复重建外科杂志, 2011, 25(5): 533-537.
21. Chen R, Jiang M, Li B, et al. The role of autophagy in pulmonary hypertension: a double-edge sword. Apoptosis, 2018, 23(9-10): 459-469.
22. Wu YY, Chen C, Yu X, et al. Effects of tiaozhi granule on regulation of autophagy levels in HUVECs. Evid Based Complement Alternat Med, 2018, 2018: 1765731.
23. Li C, Li Q, Mei Q, et al. Pharmacological effects and pharmacokinetic properties of icariin, the major bioactive component in Herba Epimedii. Life Sci, 2015, 126: 57-68.
24. Huang Z, Cheng C, Cao B, et al. Icariin protects against glucocorticoid-induced osteonecrosis of the femoral head in rats. Cell Physiol Biochem, 2018, 47(2): 694-706.
25. Zhao J, Ohba S, Shinkai M, et al. Icariin induces osteogenic differentiation in vitro in a BMP- and Runx2-dependent manner. Biochem Biophys Res Commun, 2008, 369(2): 444-448.
26. Chen KM, Ge BF, Liu XY, et al. Icariin inhibits the osteoclast formation induced by RANKL and macrophage-colony stimulating factor in mouse bone marrow culture. Pharmazie, 2007, 62(5): 388-391.
27. Hu Y, Li H, Liu K, et al. Protective effects of icariin on human vascular endothelial cells induced by oxidized low-density lipoprotein via modulating caspase-3 and Bcl-2. Mol Med Rep, 2018, 17(5): 6835-6839.
28. 王佰亮, 李子荣, 娄晋宁, 等. 淫羊藿苷对糖皮质激素诱导的骨微血管内皮细胞损伤的保护作用. 中国微循环, 2009, 13(6): 461-464, 封2.
29. Mi B, Wang J, Liu Y, et al. Icariin activates autophagy via down-regulation of the NF-κB signaling-mediated apoptosis in chondrocytes. Front Pharmacol, 2018, 9: 605.
30. Jiang S, Chang H, Deng S, et al. Icariin inhibits autophagy and promotes apoptosis in SKVCR cells through mTOR signal pathway. Cell Mol Biol (Noisy-le-grand), 2018, 64(6): 4-10.
31. Hua W, Zhang Y, Wu X, et al. Icariin attenuates interleukin-1β-induced inflammatory response in human nucleus pulposus cells. Curr Pharm Des, 2018, 23(39): 6071-6078.
32. Zeng L, Wang W, Rong XF, et al. Chondroprotective effects and multi-target mechanisms of Icariin in IL-1 beta-induced human SW 1353 chondrosarcoma cells and a rat osteoarthritis model. Int Immunopharmacol, 2014, 18(1): 175-181.
33. Ristorcelli E, Beraud E, Mathieu S, et al. Essential role of Notch signaling in apoptosis of human pancreatic tumoral cells mediated by exosomal nanoparticles. Int J Cancer, 2009, 125(5): 1016-1026.
34. 张静, 易阳艳, 阳水发, 等. 脂肪干细胞来源的外泌体对人脐静脉血管内皮细胞增殖、迁移及管样分化的影响. 中国修复重建外科杂志, 2018, 32(10): 1351-1357.
35. Zhao L, Luo H, Li X, et al. Exosomes derived from human pulmonary artery endothelial cells shift the balance between proliferation and apoptosis of smooth muscle cells. Cardiology, 2017, 137(1): 43-53.
36. Melo SA, Sugimoto H, O’Connell JT, et al. Cancer exosomes perform cell-independent microRNA biogenesis and promote tumorigenesis. Cancer Cell, 2014, 26(5): 707-721.
37. Castañeda-Cabral JL, Beas-Zárate C, Rocha-Arrieta LL, et al. Increased protein expression of VEGF-A, VEGF-B, VEGF-C and their receptors in the temporal neocortex of pharmacoresistant temporal lobe epilepsy patients. J Neuroimmunol, 2019, 328: 68-72.
38. Wang L, Wang J, Wang F, et al. VEGF-mediated cognitive and synaptic improvement in chronic cerebral hypoperfusion rats involves autophagy process. Neuromolecular Med, 2017, 19(2-3): 423-435.
39. Domigan CK, Warren CM, Antanesian V, et al. Autocrine VEGF maintains endothelial survival through regulation of metabolism and autophagy. J Cell Sci, 2015, 128(12): 2236-2248.
40. Mao YQ, Fan XM. Autophagy: A new therapeutic target for liver fibrosis. World J Hepatol, 2015, 7(16): 1982-1986.
41. Tong H, Yin H, Hossain MA, et al. Starvation-induced autophagy promotes the invasion and migration of human bladder cancer cells via TGF-β1/Smad3-mediated epithelial-mesenchymal transition activation. J Cell Biochem, 2019, 120(4): 5118-5127.

Chinese Journal of Reparative and Reconstructive Surgery

Effects of icariin on autophagy and exosome production of bone microvascular endothelial cells

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