Expression and correlation analysis of hypoxia inducible factor 1α and autophagy related molecules in rat nucleus pulposus cells under hypoxia_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

CHENG Ao , HE Pengjie , ZHANG Junyu , ZHENG Weiwei ,  YANG Min

Department of Orthopedics and Traumatology, Yijishan Hospital Affiliated to Wannan Medical College, Wuhu Anhui, 241001, P.R.China;

Corresponding author：

YANG Min, Email: pkuyang@hotmail.com

Keywords：

Nucleus pulposus cells; hypoxia inducible factor 1α; autophagy; hypoxia; rat

DOI：

10.7507/1002-1892.201908088

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Objective To investigate the expression and correlation of hypoxia inducible factor 1α (HIF-1α) and autophagy related molecules (Beclin1 and LC3B) in rat nucleus pulposus cells under hypoxia in vitro.Methods The nucleus pulposus cells were extracted from the nucleus pulposus of healthy adult Sprague Dawley rats and passaged. The 3rd generation cells were identified by HE staining and collagenase type Ⅱ immunofluorescence staining and randomly divided into 4 groups. The cells in group A were cultured for 8 hours under normal oxygen condition (37℃, 5%CO₂, 20%O₂); the cells in group B were cultured for 8 hours under hypoxia condition (37℃, 5%CO₂, 1%O₂); the cells in group C were transfected with HIF-1α-small interfering RNA and cultured for 8 hours under hypoxia condition; and the cells in group D were cultured with autophagy inhibitor 3-MA for 8 hours under hypoxia condition. Western blot and real-time fluorescence quantitative PCR (qRT-PCR) were used to detect the expressions of HIF-1α and autophagy related molecules (Beclin1 and LC3B) in all groups.Results HE staining of the 3rd generation nucleus pulposus cells showed that the cytoplasm was light pink and the nucleus was blue black, and the collagenase type Ⅱ immunofluorescence staining was positive. Western blot and qRT-PCR results showed that the relative expressions of HIF-1α, Beclin1, and LC3B proteins and genes in group B were significantly higher than those in group A (P<0.05); the relative expressions of HIF-1α, Beclin1, and LC3B proteins and genes in group C were significantly lower than those in group B (P<0.05). There was no significant difference in the relative expression of HIF-1α protein and gene between groups B and D (P>0.05); while the relative expressions of Beclin1 and LC3B proteins and genes in group D were significant lower than those in group B (P<0.05).Conclusion Hypoxia can induce the expressions of HIF-1α and autophagy related molecules (Beclin1 and LC3B) in rat nucleus pulposus cells, and HIF-1α in rat nucleus pulposus cells under hypoxia is related to the expression of autophagy related molecules, that is, down-regulation of HIF-1α can significantly reduce the expression of autophagy related molecules, while the down-regulation of autophagy levels under hypoxia has no or little effect on the expression of HIF-1α.

Citation： CHENG Ao, HE Pengjie, ZHANG Junyu, ZHENG Weiwei, YANG Min. Expression and correlation analysis of hypoxia inducible factor 1α and autophagy related molecules in rat nucleus pulposus cells under hypoxia. Chinese Journal of Reparative and Reconstructive Surgery, 2020, 34(3): 318-322. doi: 10.7507/1002-1892.201908088 Copy

1.	Middendorp M, Vogl TJ, Kollias K, et al. Association between intervertebral disc degeneration and the Oswestry Disability Index. J Back Musculoskelet Rehabil, 2017, 30(4): 819-823.
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6.	Agrawal A, Guttapalli A, Narayan S, et al. Normoxic stabilization of HIF-1alpha drives glycolytic metabolism and regulates aggrecan gene expression in nucleus pulposus cells of the rat intervertebral disk. Am J Physiol Cell Physiol, 2007, 293(2): C621-C631.
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8.	王良梅, 司进, 周萍, 等. HIF-1α siRNA 对非小细胞肺癌 A549 细胞 TIMP1、MMP1 表达的影响. 中华全科医学, 2018, 16(3): 339-342, 359.
9.	Dong Y, Wu Y, Zhao GL, et al. Inhibition of autophagy by 3-MA promotes hypoxia-induced apoptosis in human colorectal cancer cells. Eur Rev Med Pharmacol Sci, 2019, 23(3): 1047-1054.
10.	朱厚毅, 王运涛, 王锋, 等. 大鼠不同节段椎间盘髓核细胞分离鉴定及生物特性的对比研究. 中国修复重建外科杂志, 2015, 29(10): 1295-1300.
11.	Junaid M, Rashid MU, Afsheen A, et al. Analysis of 1058 lumbar prolapsed intervertebral disc cases in two tertiary care hospitals of pakistan. J Ayub Med Coll Abbottabad, 2016, 28(2): 281-284.
12.	Martinez-Outschoorn UE, Trimmer C, Lin Z, et al. Autophagy in cancer associated fibroblasts promotes tumor cell survival: Role of hypoxia, HIF1 induction and NFκB activation in the tumor stromal microenvironment. Cell Cycle, 2010, 9(17): 3515-3533.
13.	Ke Q, Costa M. Hypoxia-inducible factor-1 (HIF-1). Mol Pharmacol, 2006, 70(5): 1469-1480.
14.	Ohsumi Y. Historical landmarks of autophagy research. Cell Res, 2014, 24(1): 9-23.
15.	Schaaf MB, Keulers TG, Vooijs MA, et al. LC3/GABARAP family proteins: autophagy-(un)related functions. FASEB J, 2016, 30(12): 3961-3978.
16.	Chen Y, Sun L, Guo D, et al. Co-delivery of hypoxia inducible factor-1α small interfering RNA and 5-fluorouracil to overcome drug resistance in gastric cancer SGC-7901 cells. J Gene Med, 2017, 19(12).
17.	McCarty MF, DiNicolantonio JJ, O’Keefe JH. Ketosis may promote brain macroautophagy by activating Sirt1 and hypoxia-inducible factor-1. Med Hypotheses, 2015, 85(5): 631-639.
18.	陈琪, 马龙冰, 杨曦, 等. 缺氧诱导因子对椎间盘调控作用的研究现状. 中国修复重建外科杂志, 2016, 30(10): 1295-1300.
19.	葛礼豪, 于德水, 苏瑞超, 等. 缺氧诱导因子 1α 对人羊膜间充质干细胞耐受缺氧能力影响的实验研究. 中国修复重建外科杂志, 2018, 32(3): 264-269.

1. Middendorp M, Vogl TJ, Kollias K, et al. Association between intervertebral disc degeneration and the Oswestry Disability Index. J Back Musculoskelet Rehabil, 2017, 30(4): 819-823.
2. Muftuler LT, Jarman JP, Yu HJ, et al. Association between intervertebral disc degeneration and endplate perfusion studied by DCE-MRI. Eur Spine J, 2015, 24(4): 679-685.
3. Suyama K, Silagi ES, Choi H, et al. Circadian factors BMAL1 and RORα control HIF-1α transcriptional activity in nucleus pulposus cells: implications in maintenance of intervertebral disc health. Oncotarget, 2016, 7(17): 23056-23071.
4. Semenza GL. Hypoxia-inducible factor 1 and cardiovascular disease. Annu Rev Physiol, 2014, 76: 39-56.
5. Lu N, Li X, Tan R, et al. HIF-1α/Beclin1-mediated autophagy is involved in neuroprotection induced by hypoxic preconditioning. J Mol Neurosci, 2018, 66(2): 238-250.
6. Agrawal A, Guttapalli A, Narayan S, et al. Normoxic stabilization of HIF-1alpha drives glycolytic metabolism and regulates aggrecan gene expression in nucleus pulposus cells of the rat intervertebral disk. Am J Physiol Cell Physiol, 2007, 293(2): C621-C631.
7. 陈琪, 石芳芳, 杨曦, 等. 不同代次髓核细胞生物学特性的比较研究. 中国修复重建外科杂志, 2018, 32(6): 660-667.
8. 王良梅, 司进, 周萍, 等. HIF-1α siRNA 对非小细胞肺癌 A549 细胞 TIMP1、MMP1 表达的影响. 中华全科医学, 2018, 16(3): 339-342, 359.
9. Dong Y, Wu Y, Zhao GL, et al. Inhibition of autophagy by 3-MA promotes hypoxia-induced apoptosis in human colorectal cancer cells. Eur Rev Med Pharmacol Sci, 2019, 23(3): 1047-1054.
10. 朱厚毅, 王运涛, 王锋, 等. 大鼠不同节段椎间盘髓核细胞分离鉴定及生物特性的对比研究. 中国修复重建外科杂志, 2015, 29(10): 1295-1300.
11. Junaid M, Rashid MU, Afsheen A, et al. Analysis of 1058 lumbar prolapsed intervertebral disc cases in two tertiary care hospitals of pakistan. J Ayub Med Coll Abbottabad, 2016, 28(2): 281-284.
12. Martinez-Outschoorn UE, Trimmer C, Lin Z, et al. Autophagy in cancer associated fibroblasts promotes tumor cell survival: Role of hypoxia, HIF1 induction and NFκB activation in the tumor stromal microenvironment. Cell Cycle, 2010, 9(17): 3515-3533.
13. Ke Q, Costa M. Hypoxia-inducible factor-1 (HIF-1). Mol Pharmacol, 2006, 70(5): 1469-1480.
14. Ohsumi Y. Historical landmarks of autophagy research. Cell Res, 2014, 24(1): 9-23.
15. Schaaf MB, Keulers TG, Vooijs MA, et al. LC3/GABARAP family proteins: autophagy-(un)related functions. FASEB J, 2016, 30(12): 3961-3978.
16. Chen Y, Sun L, Guo D, et al. Co-delivery of hypoxia inducible factor-1α small interfering RNA and 5-fluorouracil to overcome drug resistance in gastric cancer SGC-7901 cells. J Gene Med, 2017, 19(12).
17. McCarty MF, DiNicolantonio JJ, O’Keefe JH. Ketosis may promote brain macroautophagy by activating Sirt1 and hypoxia-inducible factor-1. Med Hypotheses, 2015, 85(5): 631-639.
18. 陈琪, 马龙冰, 杨曦, 等. 缺氧诱导因子对椎间盘调控作用的研究现状. 中国修复重建外科杂志, 2016, 30(10): 1295-1300.
19. 葛礼豪, 于德水, 苏瑞超, 等. 缺氧诱导因子 1α 对人羊膜间充质干细胞耐受缺氧能力影响的实验研究. 中国修复重建外科杂志, 2018, 32(3): 264-269.

Chinese Journal of Reparative and Reconstructive Surgery

Expression and correlation analysis of hypoxia inducible factor 1α and autophagy related molecules in rat nucleus pulposus cells under hypoxia

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