Expression and significance of hypoxia-inducible factor 1α in endplate chondrocytes of rats_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

HUANG Zhigang ¹ , WANG Yao ² ,  MA Ke ¹

1. Department of Orthopedics, the Third People’s Hospital of Shenzhen, Shenzhen Guangdong, 518112, P.R.China;
2. Department of Ultrasound, the Third People’s Hospital of Shenzhen, Shenzhen Guangdong, 518112, P.R.China;

Corresponding author：

MA Ke, Email: 13602517196@qq.com

Keywords：

Hypoxia-inducible factor 1α; endplate chondrocytes; apoptosis; rat

DOI：

10.7507/1002-1892.201611129

Video：

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Objective To explore the expression and significance of hypoxia-inducible factor 1α (HIF-1α) in endplate chondrocytes, and to study the relations between HIF-1α expression and endplate chondrocytes apoptosis. Methods Eight Sprague Dawley rats were selected to obtain the L_1-5 intervertebral disc endplate; the endplate chondrocytes were isolated by enzyme digestion method, and the endplate chondrocytes at passage 3 were cultured under 20% O₂ condition (group A), and under 0.5% O₂ condition (group B). Cell morphology was observed by inverted phase contrast microscope and cell apoptosis was detected using flow cytometry after cultured for 24 hours; the mRNA expression of HIF-1α was detected by real-time fluorescent quantitative PCR, the protein expressions of HIF-1α, Bax, and Bcl-2 by Western blot. Gene clone technology to design and synthesize two siRNAs based on the sequence of HIF-1α mRNA. HIF-1α specific RNAi sequence compound was constructed and transfected into cells. The transfected endplate chondrocytes at passage 3 were cultured under 0.5% O₂ condition in group C and group D (HIF-1α gene was silenced). After cultured for 24 hours, cells were observed via immunofluorescence staining of HIF-1α, and cell apoptosis was detected using flow cytometry. Meanwhile, the mRNA expressions of HIF-1α, collagen type II (COL II), Aggrecan, and SOX9 were detected by real-time fluorescent quantitative PCR, and the protein expressions of HIF-1α, Bax, and Bcl-2 by Western blot. Results At 24 hours after culture, small amount of vacuoles necrotic cells could be observed in group A and group B; there was no significant difference in apoptosis rate between groups A and B (t=1.026,P=0.471), and HIF-1α mRNA and protein expressions in group B were significantly higher than those in group A (t=22.672,P=0.015;t=18.396,P=0.013), but, there was no significant difference in protein expressions of Bax and Bcl-2 between groups A and B (t=0.594,P=0.781;t=1.251,P=0.342). The number of vacuolar necrosis cells in group D was significantly higher than that in group C, and HIF-1α positive cells were observed in group D. The apoptosis rate of group D was significantly higher than that of group C (t=27.143,P=0.002). The mRNA expressions of HIF-1α, COL II, Aggrecan, and SOX9 in group D were significantly lower than those in group C (t=21.097,P=0.015;t=34.829,P=0.002;t=18.673,P=0.022;t=31.949,P=0.007). The protein expressions of HIF-1α and Bcl-2 in group D were significantly lower than those in group C (t=37.648,P=0.006;t=16.729,P=0.036), but the protein expression of Bax in group D was significantly higher than that in group C (t=25.583,P=0.011). Conclusion HIF-1α mRNA expression is up-regulated under hypoxia condition, which will increase the hypoxia tolerance of endplate chondrocytes. Cell apoptosis is suppressed by the activation of HIF-1α in endplate chondrocytes under hypoxia condition.

Citation： HUANG Zhigang, WANG Yao, MA Ke. Expression and significance of hypoxia-inducible factor 1α in endplate chondrocytes of rats. Chinese Journal of Reparative and Reconstructive Surgery, 2017, 31(3): 351-356. doi: 10.7507/1002-1892.201611129 Copy

1.	DeLucca JF, Cortes DH, Jacobs NT,et al. Human cartilage endplate permeability varies with degeneration and intervertebral disc site. J Biomech, 2016, 49(4): 550-557.
2.	Lauing KL, Cortes M, Domowicz MS,et al. Aggrecan is required for growth plate cytoarchitecture and differentiation. Dev Biol, 2014, 396(2): 224-236.
3.	Yang L, Shen L, Li G,et al. Silencing of hypoxia inducible factor-1α gene attenuated angiotensin II-induced abdominal aortic aneurysm in apolipoprotein E-deficient mice. Atherosclerosis, 2016, 252: 40-49.
4.	Pfander D, Kobayashi T, Knight MC,et al. Deletion of Vhlh in chondrocytes reduces cell proliferation and increases matrix deposition during growth plate development. Development, 2004, 131(10): 2497-2508.
5.	Ding Y, Jiang J, Zhou J,et al. The effects of osteoporosis and disc degeneration on vertebral cartilage endplate lesions in rats. Eur Spine J, 2014, 23(9): 1848-1855.
6.	倪嘉延, 吴裕丹, 许林峰, 等. HIF-1α 基因表达沉默对肝癌细胞 PI3K/AKT 信号转导通路影响的研究. 中华肿瘤防治杂志, 2014, 21(5): 356-359.
7.	Tu C, Wang S, Hu X,et al. Lipopolysaccharide induces TREM-1-dependent HIF-1α expression in human keratinocyte cell line. Cell Biol Int, 2016, 40(12): 1357-1365.
8.	Ghodsi SM, Rouhani R, Abdollahzade S,et al. Frequency of vertebral endplate modic changes in patients with unstable lumbar spine and its effect on surgical outcome. Asian Spine J, 2015, 9(5): 737-740.
9.	He Z, Pu L, Yuan C,et al. Nutrition deficiency promotes apoptosis of cartilage endplate stem cells in a caspase-independent manner partially through upregulating BNIP3. Acta Biochim Biophys Sin (Shanghai), 2017, 49(1): 25-32.
10.	Wu Y, Cisewski SE, Wegner N,et al. Region and strain-dependent diffusivities of glucose and lactate in healthy human cartilage endplate. J Biomech, 2016, 49(13): 2756-2762.
11.	Schipani E, Ryan HE, Didrickson S,et al. Hypoxia in cartilage: HIF-1alpha is essential for chondrocyte growth arrest and survival. Genes Dev, 2001, 15(21): 2865-2876.
12.	Vasconcelos MG, Vasconcelos RG, Pereira de Oliveira DH,et al. Distribution of hypoxia-inducible factor-1α and glucose transporter-1 in human tongue cancers. J Oral Maxillofac Surg, 2015, 73(9): 1753-1760.
13.	Risbud MV, Schoepflin ZR, Mwale F,et al. Defining the phenotype of young healthy nucleus pulposus cells: recommendations of the Spine Research Interest Group at the 2014 annual ORS meeting. J Orthop Res, 2015, 33(3): 283-293.
14.	Xiao L, Xu HG, Wang H,et al. Intermittent cyclic mechanical tension promotes degeneration of endplate cartilage via the nuclear factor-κB signaling pathway: an in vivo study. Orthop Surg, 2016, 8(3): 393-399.
15.	Acosta Rodriguez EV, Zuniga EI, Montes CL,et al. Trypanosoma cruzi infection beats the B-cell compartment favouring parasite establishment: can we strike first? Scand J Immunol, 2007, 66(2-3): 137-142.
16.	Gong X, Duan R, Ao JE,et al. Metformin suppresses intrahepatic coagulation activation in mice with lipopolysaccharide/D-galactosamine-induced fulminant hepatitis. Mol Med Rep, 2015, 12(4): 6384-6390.
17.	Li Q, Hakimi P, Liu X,et al. Cited2, a transcriptional modulator protein, regulates metabolism in murine embryonic stem cells. J Biol Chem, 2014, 289(1): 251-263.
18.	Xu B, Doughman Y, Turakhia M,et al. Partial rescue of defects in Cited2-deficient embryos by HIF-1alpha heterozygosity. Dev Biol, 2007, 301(1): 130-140.
19.	Li Q, Pan H, Guan L,et al. CITED2 mutation links congenital heart defects to dysregulation of the cardiac gene VEGF and PITX2C expression. Biochem Biophys Res Commun, 2012, 423(4): 895-899.

1. DeLucca JF, Cortes DH, Jacobs NT,et al. Human cartilage endplate permeability varies with degeneration and intervertebral disc site. J Biomech, 2016, 49(4): 550-557.
2. Lauing KL, Cortes M, Domowicz MS,et al. Aggrecan is required for growth plate cytoarchitecture and differentiation. Dev Biol, 2014, 396(2): 224-236.
3. Yang L, Shen L, Li G,et al. Silencing of hypoxia inducible factor-1α gene attenuated angiotensin II-induced abdominal aortic aneurysm in apolipoprotein E-deficient mice. Atherosclerosis, 2016, 252: 40-49.
4. Pfander D, Kobayashi T, Knight MC,et al. Deletion of Vhlh in chondrocytes reduces cell proliferation and increases matrix deposition during growth plate development. Development, 2004, 131(10): 2497-2508.
5. Ding Y, Jiang J, Zhou J,et al. The effects of osteoporosis and disc degeneration on vertebral cartilage endplate lesions in rats. Eur Spine J, 2014, 23(9): 1848-1855.
6. 倪嘉延, 吴裕丹, 许林峰, 等. HIF-1α 基因表达沉默对肝癌细胞 PI3K/AKT 信号转导通路影响的研究. 中华肿瘤防治杂志, 2014, 21(5): 356-359.
7. Tu C, Wang S, Hu X,et al. Lipopolysaccharide induces TREM-1-dependent HIF-1α expression in human keratinocyte cell line. Cell Biol Int, 2016, 40(12): 1357-1365.
8. Ghodsi SM, Rouhani R, Abdollahzade S,et al. Frequency of vertebral endplate modic changes in patients with unstable lumbar spine and its effect on surgical outcome. Asian Spine J, 2015, 9(5): 737-740.
9. He Z, Pu L, Yuan C,et al. Nutrition deficiency promotes apoptosis of cartilage endplate stem cells in a caspase-independent manner partially through upregulating BNIP3. Acta Biochim Biophys Sin (Shanghai), 2017, 49(1): 25-32.
10. Wu Y, Cisewski SE, Wegner N,et al. Region and strain-dependent diffusivities of glucose and lactate in healthy human cartilage endplate. J Biomech, 2016, 49(13): 2756-2762.
11. Schipani E, Ryan HE, Didrickson S,et al. Hypoxia in cartilage: HIF-1alpha is essential for chondrocyte growth arrest and survival. Genes Dev, 2001, 15(21): 2865-2876.
12. Vasconcelos MG, Vasconcelos RG, Pereira de Oliveira DH,et al. Distribution of hypoxia-inducible factor-1α and glucose transporter-1 in human tongue cancers. J Oral Maxillofac Surg, 2015, 73(9): 1753-1760.
13. Risbud MV, Schoepflin ZR, Mwale F,et al. Defining the phenotype of young healthy nucleus pulposus cells: recommendations of the Spine Research Interest Group at the 2014 annual ORS meeting. J Orthop Res, 2015, 33(3): 283-293.
14. Xiao L, Xu HG, Wang H,et al. Intermittent cyclic mechanical tension promotes degeneration of endplate cartilage via the nuclear factor-κB signaling pathway: an in vivo study. Orthop Surg, 2016, 8(3): 393-399.
15. Acosta Rodriguez EV, Zuniga EI, Montes CL,et al. Trypanosoma cruzi infection beats the B-cell compartment favouring parasite establishment: can we strike first? Scand J Immunol, 2007, 66(2-3): 137-142.
16. Gong X, Duan R, Ao JE,et al. Metformin suppresses intrahepatic coagulation activation in mice with lipopolysaccharide/D-galactosamine-induced fulminant hepatitis. Mol Med Rep, 2015, 12(4): 6384-6390.
17. Li Q, Hakimi P, Liu X,et al. Cited2, a transcriptional modulator protein, regulates metabolism in murine embryonic stem cells. J Biol Chem, 2014, 289(1): 251-263.
18. Xu B, Doughman Y, Turakhia M,et al. Partial rescue of defects in Cited2-deficient embryos by HIF-1alpha heterozygosity. Dev Biol, 2007, 301(1): 130-140.
19. Li Q, Pan H, Guan L,et al. CITED2 mutation links congenital heart defects to dysregulation of the cardiac gene VEGF and PITX2C expression. Biochem Biophys Res Commun, 2012, 423(4): 895-899.

Chinese Journal of Reparative and Reconstructive Surgery

Expression and significance of hypoxia-inducible factor 1α in endplate chondrocytes of rats

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