Protective effect of Kaempferol on endothelial cell injury in glucocorticoid induced osteonecrosis of the femoral head_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

XU Xin ¹ , FAN Xiaoyu ² , WU Xinjie ² , SHI Lijun ¹ , WANG Peixu ¹ , GAO Fuqiang ³ ,  SUN Wei ^1,3 , LI Zirong ³

1. Department of Orthopedics, China-Japan Friendship Hospital, Graduate School of Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, 100029, P. R. China;
2. Department of Orthopedics, Peking University China-Japan Friendship School of Clinical Medicine, 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: sunwei@zryhyy.com.cn

Keywords：

Kaempferol; dexamethasone; osteonecrosis of the femoral head; bone microvascular endothelial cells

DOI：

10.7507/1002-1892.202204028

Video：

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Objective To explore the effect of Kaempferol on bone microvascular endothelial cells (BMECs) in glucocorticoid induced osteonecrosis of the femoral head (GIONFH) in vitro. Methods BMECs were isolated from cancellous bone of femoral head or femoral neck donated voluntarily by patients with femoral neck fracture. BMECs were identified by von Willebrand factor and CD31 immunofluorescence staining and tube formation assay. The cell counting kit 8 (CCK-8) assay was used to screen the optimal concentration and the time point of dexamethasone (Dex) to inhibit the cell activity and the optimal concentration of Kaempferol to improve the inhibition of Dex. Then the BMECs were divided into 4 groups, namely, the cell group (group A), the cells treated with optimal concentration of Dex group (group B), the cells treated with optimal concentration of Dex+1 μmol/L Kaempferol group (group C), and the cells treated with optimal concentration of Dex+5 μmol/L Kaempferol group (group D). EdU assay, in vitro tube formation assay, TUNEL staining assay, Annexin Ⅴ/propidium iodide (PI) staining assay, Transwell migration assay, scratch healing assay, and Western blot assay were used to detect the effect of Kaempferol on the proliferation, tube formation, apoptosis, migration, and protein expression of BMECs treated with Dex. Results The cultured cells were identified as BMECs. CCK-8 assay showed that the optimal concentration and the time point of Dex to inhibit cell activity was 300 μmol/L for 24 hours, and the optimal concentration of Kaempferol to improve the inhibitory activity of Dex was 1 μmol/L. EdU and tube formation assays showed that the cell proliferation rate, tube length, and number of branch points were significantly lower in groups B-D than in group A, and in groups B and D than in group C (P<0.05). TUNEL and Annexin V/PI staining assays showed that the rates of TUNEL positive cells and apoptotic cells were significantly higher in groups B-D than in group A, and in groups B and D than in group C (P<0.05). Scratch healing assay and Transwell migration assay showed that the scratch healing rate and the number of migration cells were significantly lower in groups B-D than in group A, and in groups B and D than in group C (P<0.05). Western blot assay demonstrated that the relative expressions of Cleaved Caspase-3 and Bax proteins were significantly higher in groups B-D than in group A, and in groups B and D than in group C (P<0.05); the relative expressions of matrix metalloproteinase 2, Cyclin D1, Cyclin E1, VEGFA, and Bcl2 proteins were significantly lower in groups B-D than in group A, and in groups B and D than in group C (P<0.05). Conclusion Kaempferol can alleviate the damage and dysfunction of BMECs in GIONFH.

Citation： XU Xin, FAN Xiaoyu, WU Xinjie, SHI Lijun, WANG Peixu, GAO Fuqiang, SUN Wei, LI Zirong. Protective effect of Kaempferol on endothelial cell injury in glucocorticoid induced osteonecrosis of the femoral head. Chinese Journal of Reparative and Reconstructive Surgery, 2022, 36(10): 1277-1287. doi: 10.7507/1002-1892.202204028 Copy

1.	Xu H, Wang C, Liu C, et al. Cotransplantation of mesenchymal stem cells and endothelial progenitor cells for treating steroid-induced osteonecrosis of the femoral head. Stem Cells Transl Med, 2021, 10(5): 781-796.
2.	Shang G, Wang Y, Xu Y, et al. Long non-coding RNA TCONS_00041960 enhances osteogenesis and inhibits adipogenesis of rat bone marrow mesenchymal stem cell by targeting miR-204-5p and miR-125a-3p. J Cell Physiol, 2018, 233(8): 6041-6051.
3.	Fu F, Huang Z, Ye H, et al. Mechanisms and molecular targets of the Tao-Hong-Si-Wu-Tang formula for treatment of osteonecrosis of femoral head: A network pharmacology study. Evid Based Complement Alternat Med, 2020, 2020: 7130105. doi: 10.1155/2020/7130105.
4.	Kuang MJ, Huang Y, Zhao XG, et al. Exosomes derived from Wharton’s jelly of human umbilical cord mesenchymal stem cells reduce osteocyte apoptosis in glucocorticoid-induced osteonecrosis of the femoral head in rats via the miR-21-PTEN-AKT signalling pathway. Int J Biol Sci, 2019, 15(9): 1861-1871.
5.	Johnston JC, Haile A, Wang D, et al. Dexamethasone treatment alters function of adipocytes from a mesenchymal stromal cell line. Biochem Biophys Res Commun, 2014, 451(4): 473-479.
6.	Sheng H, Sheng CJ, Cheng XY, et al. Pathomorphological changes of bone marrow adipocytes in process of steroid-associated osteonecrosis. Int J Clin Exp Pathol, 2013, 6(6): 1046-1050.
7.	Weinstein RS. Clinical practice. Glucocorticoid-induced bone disease. N Engl J Med, 2011, 365(1): 62-70.
8.	Mont MA, Salem HS, Piuzzi NS, et al. Nontraumatic osteonecrosis of the femoral head: Where do we stand today?: A 5-year update. J Bone Joint Surg (Am), 2020, 102(12): 1084-1099.
9.	Weinstein RS, Wan C, Liu Q, et al. Endogenous glucocorticoids decrease skeletal angiogenesis, vascularity, hydration, and strength in aged mice. Aging Cell, 2010, 9(2): 147-161.
10.	Li Z, Yang A, Yin X, et al. Mesenchymal stem cells promote endothelial progenitor cell migration, vascularization, and bone repair in tissue-engineered constructs via activating CXCR2-Src-PKL/Vav2-Rac1. FASEB J, 2018, 32(4): 2197-2211.
11.	张庆宇, 高福强, 程立明, 等. 淫羊藿苷对骨微血管内皮细胞自噬及外泌体产生的影响. 中国修复重建外科杂志, 2019, 33(5): 568-577.
12.	Yu H, Liu P, Zuo W, et al. Decreased angiogenic and increased apoptotic activities of bone microvascular endothelial cells in patients with glucocorticoid-induced osteonecrosis of the femoral head. BMC Musculoskelet Disord, 2020, 21(1): 277. doi: 10.1186/s12891-020-03225-1.
13.	Nishimura T, Matsumoto T, Nishino M, et al. Histopathologic study of veins in steroid treated rabbits. Clin Orthop Relat Res, 1997, (334): 37-42.
14.	Kerachian MA, Séguin C, Harvey EJ. Glucocorticoids in osteonecrosis of the femoral head: a new understanding of the mechanisms of action. J Steroid Biochem Mol Biol, 2009, 114(3-5): 121-128.
15.	Huang C, Wen Z, Niu J, et al. Steroid-induced osteonecrosis of the femoral head: Novel insight into the roles of bone endothelial cells in pathogenesis and treatment. Front Cell Dev Biol, 2021, 9: 777697. doi: 10.3389/fcell.2021.777697.
16.	Yu H, Yue J, Wang W, et al. Icariin promotes angiogenesis in glucocorticoid-induced osteonecrosis of femoral heads: In vitro and in vivo studies. J Cell Mol Med, 2019, 23(11): 7320-7330.
17.	Cheng CF, Chien-Fu Lin J, Tsai FJ, et al. Protective effects and network analysis of natural compounds obtained from Radix dipsaci, Eucommiae cortex, and Rhizoma drynariae against RANKL-induced osteoclastogenesis in vitro. J Ethnopharmacol, 2019, 244: 112074. doi: 10.1016/j.jep.2019.112074.
18.	Yang L, Li H, Yang M, et al. Exploration in the mechanism of Kaempferol for the treatment of gastric cancer based on network pharmacology. Biomed Res Int, 2020, 2020: 5891016. doi: 10.1155/2020/5891016.
19.	Wong SK, Chin KY, Ima-Nirwana S. The osteoprotective effects of Kaempferol: The evidence from in vivo and in vitro studies. Drug Des Devel Ther, 2019, 13: 3497-3514.
20.	Xie B, Zeng Z, Liao S, et al. Kaempferol ameliorates the inhibitory activity of dexamethasone in the osteogenesis of MC3T3-E1 cells by JNK and p38-MAPK pathways. Front Pharmacol, 2021, 12: 739326. doi: 10.3389/fphar.2021.739326.
21.	Wu X, Wang Y, Fan X, et al. Extracorporeal shockwave relieves endothelial injury and dysfunction in steroid-induced osteonecrosis of the femoral head via miR-135b targeting FOXO1: in vitro and in vivo studies. Aging (Albany NY), 2022, 14(1): 410-429.
22.	An F, Zhang L, Gao H, et al. Variants in RETN gene are associated with steroid-induced osteonecrosis of the femoral head risk among Han Chinese people. J Orthop Surg Res, 2020, 15(1): 96. doi: 10.1186/s13018-020-1557-3.
23.	Kim TW, Lee SY, Kim M, et al. Kaempferol induces autophagic cell death via IRE1-JNK-CHOP pathway and inhibition of G9a in gastric cancer cells. Cell Death Dis, 2018, 9(9): 875. doi: 10.1038/s41419-018-0930-1.
24.	Wu W, Yang B, Qiao Y, et al. Kaempferol protects mitochondria and alleviates damages against endotheliotoxicity induced by doxorubicin. Biomed Pharmacother, 2020, 126: 110040. doi: 10.1016/j.biopha.2020.110040.
25.	Yao H, Sun J, Wei J, et al. Kaempferol protects blood vessels from damage induced by oxidative stress and inflammation in association with the Nrf₂/HO-1 signaling pathway. Front Pharmacol, 2020, 11: 1118. doi: 10.3389/fphar.2020.01118.
26.	Adhikary S, Choudhary D, Ahmad N, et al. Dietary flavonoid Kaempferol inhibits glucocorticoid-induced bone loss by promoting osteoblast survival. Nutrition, 2018, 53: 64-76.
27.	Tao SC, Yuan T, Rui BY, et al. Exosomes derived from human platelet-rich plasma prevent apoptosis induced by glucocorticoid-associated endoplasmic reticulum stress in rat osteonecrosis of the femoral head via the Akt/Bad/Bcl-2 signal pathway. Theranostics, 2017, 7(3): 733-750.
28.	Wang C, Xu H, Liu C, et al. CaO₂/gelatin oxygen slow-releasing microspheres facilitate tissue engineering efficiency for the osteonecrosis of femoral head by enhancing the angiogenesis and survival of grafted bone marrow mesenchymal stem cells. Biomater Sci, 2021, 9(8): 3005-3018.
29.	Zhang B, Yi J, Zhang CL, et al. MiR-146a inhibits proliferation and induces apoptosis in murine osteoblastic MC3T3-E1 by regulating Bcl₂. Eur Rev Med Pharmacol Sci, 2017, 21(17): 3754-3762.
30.	Sun F, Zhou JL, Wei SX, et al. Glucocorticoids induce osteonecrosis of the femoral head in rats via PI3K/AKT/FOXO1 signaling pathway. PeerJ, 2022, 10: e13319. doi: 10.7717/peerj.13319.
31.	Peng P, Nie Z, Sun F, et al. Glucocorticoids induce femoral head necrosis in rats through the ROS/JNK/c-Jun pathway. FEBS Open Bio, 2021, 11(1): 312-321.
32.	Ma X, Su P, Yin C, et al. The roles of FoxO transcription factors in regulation of bone cells function. Int J Mol Sci, 2020, 21(3): 692. doi: 10.3390/ijms21030692.
33.	Gan L, Leng Y, Min J, et al. Kaempferol promotes the osteogenesis in rBMSCs via mediation of SOX2/miR-124-3p/PI3K/Akt/mTOR axis. Eur J Pharmacol, 2022, 927: 174954. doi: 10.1016/j.ejphar.2022.174954.
34.	Chen XJ, Shen YS, He MC, et al. Polydatin promotes the osteogenic differentiation of human bone mesenchymal stem cells by activating the BMP2-Wnt/β-catenin signaling pathway. Biomed Pharmacother, 2019, 112: 108746. doi: 10.1016/j.biopha.2019.108746.
35.	Park JH, Seo JH, Jeon HY, et al. Lentivirus-mediated VEGF knockdown suppresses gastric cancer cell proliferation and tumor growth in vitro and in vivo. Onco Targets Ther, 2020, 13: 1331-1341.
36.	Mitsiogianni M, Koutsidis G, Mavroudis N, et al. The role of isothiocyanates as cancer chemo-preventive, chemo-therapeutic and anti-melanoma agents. Antioxidants (Basel), 2019, 8(4): 106. doi: 10.3390/antiox8040106.
37.	Hu WH, Wang HY, Xia YT, et al. Kaempferol, a major flavonoid in ginkgo folium, potentiates angiogenic functions in cultured endothelial cells by binding to vascular endothelial growth factor. Front Pharmacol, 2020, 11: 526. doi: 10.3389/fphar.2020.00526.

1. Xu H, Wang C, Liu C, et al. Cotransplantation of mesenchymal stem cells and endothelial progenitor cells for treating steroid-induced osteonecrosis of the femoral head. Stem Cells Transl Med, 2021, 10(5): 781-796.
2. Shang G, Wang Y, Xu Y, et al. Long non-coding RNA TCONS_00041960 enhances osteogenesis and inhibits adipogenesis of rat bone marrow mesenchymal stem cell by targeting miR-204-5p and miR-125a-3p. J Cell Physiol, 2018, 233(8): 6041-6051.
3. Fu F, Huang Z, Ye H, et al. Mechanisms and molecular targets of the Tao-Hong-Si-Wu-Tang formula for treatment of osteonecrosis of femoral head: A network pharmacology study. Evid Based Complement Alternat Med, 2020, 2020: 7130105. doi: 10.1155/2020/7130105.
4. Kuang MJ, Huang Y, Zhao XG, et al. Exosomes derived from Wharton’s jelly of human umbilical cord mesenchymal stem cells reduce osteocyte apoptosis in glucocorticoid-induced osteonecrosis of the femoral head in rats via the miR-21-PTEN-AKT signalling pathway. Int J Biol Sci, 2019, 15(9): 1861-1871.
5. Johnston JC, Haile A, Wang D, et al. Dexamethasone treatment alters function of adipocytes from a mesenchymal stromal cell line. Biochem Biophys Res Commun, 2014, 451(4): 473-479.
6. Sheng H, Sheng CJ, Cheng XY, et al. Pathomorphological changes of bone marrow adipocytes in process of steroid-associated osteonecrosis. Int J Clin Exp Pathol, 2013, 6(6): 1046-1050.
7. Weinstein RS. Clinical practice. Glucocorticoid-induced bone disease. N Engl J Med, 2011, 365(1): 62-70.
8. Mont MA, Salem HS, Piuzzi NS, et al. Nontraumatic osteonecrosis of the femoral head: Where do we stand today?: A 5-year update. J Bone Joint Surg (Am), 2020, 102(12): 1084-1099.
9. Weinstein RS, Wan C, Liu Q, et al. Endogenous glucocorticoids decrease skeletal angiogenesis, vascularity, hydration, and strength in aged mice. Aging Cell, 2010, 9(2): 147-161.
10. Li Z, Yang A, Yin X, et al. Mesenchymal stem cells promote endothelial progenitor cell migration, vascularization, and bone repair in tissue-engineered constructs via activating CXCR2-Src-PKL/Vav2-Rac1. FASEB J, 2018, 32(4): 2197-2211.
11. 张庆宇, 高福强, 程立明, 等. 淫羊藿苷对骨微血管内皮细胞自噬及外泌体产生的影响. 中国修复重建外科杂志, 2019, 33(5): 568-577.
12. Yu H, Liu P, Zuo W, et al. Decreased angiogenic and increased apoptotic activities of bone microvascular endothelial cells in patients with glucocorticoid-induced osteonecrosis of the femoral head. BMC Musculoskelet Disord, 2020, 21(1): 277. doi: 10.1186/s12891-020-03225-1.
13. Nishimura T, Matsumoto T, Nishino M, et al. Histopathologic study of veins in steroid treated rabbits. Clin Orthop Relat Res, 1997, (334): 37-42.
14. Kerachian MA, Séguin C, Harvey EJ. Glucocorticoids in osteonecrosis of the femoral head: a new understanding of the mechanisms of action. J Steroid Biochem Mol Biol, 2009, 114(3-5): 121-128.
15. Huang C, Wen Z, Niu J, et al. Steroid-induced osteonecrosis of the femoral head: Novel insight into the roles of bone endothelial cells in pathogenesis and treatment. Front Cell Dev Biol, 2021, 9: 777697. doi: 10.3389/fcell.2021.777697.
16. Yu H, Yue J, Wang W, et al. Icariin promotes angiogenesis in glucocorticoid-induced osteonecrosis of femoral heads: In vitro and in vivo studies. J Cell Mol Med, 2019, 23(11): 7320-7330.
17. Cheng CF, Chien-Fu Lin J, Tsai FJ, et al. Protective effects and network analysis of natural compounds obtained from Radix dipsaci, Eucommiae cortex, and Rhizoma drynariae against RANKL-induced osteoclastogenesis in vitro. J Ethnopharmacol, 2019, 244: 112074. doi: 10.1016/j.jep.2019.112074.
18. Yang L, Li H, Yang M, et al. Exploration in the mechanism of Kaempferol for the treatment of gastric cancer based on network pharmacology. Biomed Res Int, 2020, 2020: 5891016. doi: 10.1155/2020/5891016.
19. Wong SK, Chin KY, Ima-Nirwana S. The osteoprotective effects of Kaempferol: The evidence from in vivo and in vitro studies. Drug Des Devel Ther, 2019, 13: 3497-3514.
20. Xie B, Zeng Z, Liao S, et al. Kaempferol ameliorates the inhibitory activity of dexamethasone in the osteogenesis of MC3T3-E1 cells by JNK and p38-MAPK pathways. Front Pharmacol, 2021, 12: 739326. doi: 10.3389/fphar.2021.739326.
21. Wu X, Wang Y, Fan X, et al. Extracorporeal shockwave relieves endothelial injury and dysfunction in steroid-induced osteonecrosis of the femoral head via miR-135b targeting FOXO1: in vitro and in vivo studies. Aging (Albany NY), 2022, 14(1): 410-429.
22. An F, Zhang L, Gao H, et al. Variants in RETN gene are associated with steroid-induced osteonecrosis of the femoral head risk among Han Chinese people. J Orthop Surg Res, 2020, 15(1): 96. doi: 10.1186/s13018-020-1557-3.
23. Kim TW, Lee SY, Kim M, et al. Kaempferol induces autophagic cell death via IRE1-JNK-CHOP pathway and inhibition of G9a in gastric cancer cells. Cell Death Dis, 2018, 9(9): 875. doi: 10.1038/s41419-018-0930-1.
24. Wu W, Yang B, Qiao Y, et al. Kaempferol protects mitochondria and alleviates damages against endotheliotoxicity induced by doxorubicin. Biomed Pharmacother, 2020, 126: 110040. doi: 10.1016/j.biopha.2020.110040.
25. Yao H, Sun J, Wei J, et al. Kaempferol protects blood vessels from damage induced by oxidative stress and inflammation in association with the Nrf₂/HO-1 signaling pathway. Front Pharmacol, 2020, 11: 1118. doi: 10.3389/fphar.2020.01118.
26. Adhikary S, Choudhary D, Ahmad N, et al. Dietary flavonoid Kaempferol inhibits glucocorticoid-induced bone loss by promoting osteoblast survival. Nutrition, 2018, 53: 64-76.
27. Tao SC, Yuan T, Rui BY, et al. Exosomes derived from human platelet-rich plasma prevent apoptosis induced by glucocorticoid-associated endoplasmic reticulum stress in rat osteonecrosis of the femoral head via the Akt/Bad/Bcl-2 signal pathway. Theranostics, 2017, 7(3): 733-750.
28. Wang C, Xu H, Liu C, et al. CaO₂/gelatin oxygen slow-releasing microspheres facilitate tissue engineering efficiency for the osteonecrosis of femoral head by enhancing the angiogenesis and survival of grafted bone marrow mesenchymal stem cells. Biomater Sci, 2021, 9(8): 3005-3018.
29. Zhang B, Yi J, Zhang CL, et al. MiR-146a inhibits proliferation and induces apoptosis in murine osteoblastic MC3T3-E1 by regulating Bcl₂. Eur Rev Med Pharmacol Sci, 2017, 21(17): 3754-3762.
30. Sun F, Zhou JL, Wei SX, et al. Glucocorticoids induce osteonecrosis of the femoral head in rats via PI3K/AKT/FOXO1 signaling pathway. PeerJ, 2022, 10: e13319. doi: 10.7717/peerj.13319.
31. Peng P, Nie Z, Sun F, et al. Glucocorticoids induce femoral head necrosis in rats through the ROS/JNK/c-Jun pathway. FEBS Open Bio, 2021, 11(1): 312-321.
32. Ma X, Su P, Yin C, et al. The roles of FoxO transcription factors in regulation of bone cells function. Int J Mol Sci, 2020, 21(3): 692. doi: 10.3390/ijms21030692.
33. Gan L, Leng Y, Min J, et al. Kaempferol promotes the osteogenesis in rBMSCs via mediation of SOX2/miR-124-3p/PI3K/Akt/mTOR axis. Eur J Pharmacol, 2022, 927: 174954. doi: 10.1016/j.ejphar.2022.174954.
34. Chen XJ, Shen YS, He MC, et al. Polydatin promotes the osteogenic differentiation of human bone mesenchymal stem cells by activating the BMP2-Wnt/β-catenin signaling pathway. Biomed Pharmacother, 2019, 112: 108746. doi: 10.1016/j.biopha.2019.108746.
35. Park JH, Seo JH, Jeon HY, et al. Lentivirus-mediated VEGF knockdown suppresses gastric cancer cell proliferation and tumor growth in vitro and in vivo. Onco Targets Ther, 2020, 13: 1331-1341.
36. Mitsiogianni M, Koutsidis G, Mavroudis N, et al. The role of isothiocyanates as cancer chemo-preventive, chemo-therapeutic and anti-melanoma agents. Antioxidants (Basel), 2019, 8(4): 106. doi: 10.3390/antiox8040106.
37. Hu WH, Wang HY, Xia YT, et al. Kaempferol, a major flavonoid in ginkgo folium, potentiates angiogenic functions in cultured endothelial cells by binding to vascular endothelial growth factor. Front Pharmacol, 2020, 11: 526. doi: 10.3389/fphar.2020.00526.

Chinese Journal of Reparative and Reconstructive Surgery

Protective effect of Kaempferol on endothelial cell injury in glucocorticoid induced osteonecrosis of the femoral head

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