Tetramethylpyrazine protects CoCl2-induced apoptosis in human umbilical vein endothelial cells by regulating the PHD2/HIF/1α-VEGF pathway

Yang,Cheng; Xu,Yue; Zhou,Huanjiao; Yang,Lu; Yu,Shanshan; Gao,Yi; Huang,Yongsheng; Lu,Lin; Liang,Xiaoling

doi:10.3892/mmr.2015.4679

Tetramethylpyrazine protects CoCl2-induced apoptosis in human umbilical vein endothelial cells by regulating the PHD2/HIF/1α-VEGF pathway

Authors:
- Cheng Yang
- Yue Xu
- Huanjiao Zhou
- Lu Yang
- Shanshan Yu
- Yi Gao
- Yongsheng Huang
- Lin Lu
- Xiaoling Liang
View Affiliations

Affiliations: State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat‑sen University, Guangzhou, Guangdong 510000, P.R. China
Published online on: December 11, 2015 https://doi.org/10.3892/mmr.2015.4679
Pages: 1287-1296

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Tetramethylpyrazine (TMP), one of the active ingredients isolated from a Chinese herbal prescription, possesses protective effects against apoptosis in endothelial cells. However, the underlying mechanism of its protective effects in endothelial cells remains to be elucidated. Using human umbilical vein endothelial cells (HUVECs), the present study assessed the protective effects of TMP on CoCl2‑induced apoptosis. Following pre‑incubation with CoCl2 (150 µM/ml) for 4 h, the HUVECs were treated with TMP at different concentrations (50, 100 and 200 µM/ml) for 8 h. TMP upregulated the expression of prolyl hydroxylase (PHD)2, reduced the protein and mRNA expression levels of vascular endothelial growth factor (VEGF), and reduced the expression of HIF‑1α only at the protein level, not at the mRNA level in HUVECs, in a concentration‑dependent manner. Furthermore, silencing of the PHD2 gene with small interfering (si)RNAs abolished the reduction in the expression of hypoxia‑inducible factor (HIF)‑1α and VEGF by TMP. In addition, TMP protected CoCl2‑induced HUVEC injury via an apoptosis pathway, as characterized by the increased ratio of cell viability and the reduced percentage of apoptotic and terminal deoxynucleotidyl transferase dUTP nick end labeling‑positive HUVECs, activation of caspase‑3, ‑8 and ‑9, B‑cell lymphoma (Bcl)‑2/Bcl‑2‑activated X protein expression, as well as the release of cytochrome c. The protective properties of TMP were partially attributed to the mRNA and protein expression levels of PHD, since silencing of the PHD2 gene with siRNAs abolished these effects. The present study demonstrated that the antiapoptotic effect of TMP in CoCl2‑induced HUVECs was, at least in part, via the regulation of the PHD2/HIF-1α signaling pathway.

View Figures

View References

1	Nyengaard JR, Ido Y, Kilo C and Williamson JR: Interactions between hyperglycemia and hypoxia: Implications for diabetic retinopathy. Diabetes. 53:2931–2938. 2004. View Article : Google Scholar : PubMed/NCBI
2	Rosenfeld PJ, Brown DM, Heier JS, Boyer DS, Kaiser PK, Chung CY and Kim RY; MARINA Study Group: Ranibizumab for neovascular age-related macular degeneration. N Engl J Med. 355:1419–1431. 2006. View Article : Google Scholar : PubMed/NCBI
3	Arjamaa O and Nikinmaa M: Oxygen-dependent diseases in the retina: Role of hypoxia-inducible factors. Exp Eye Res. 83:473–483. 2006. View Article : Google Scholar : PubMed/NCBI
4	Forooghian F, Razavi R and Timms L: Hypoxia-inducible factor expression in human RPE cells. Br J Ophthalmol. 91:1406–1410. 2007. View Article : Google Scholar : PubMed/NCBI
5	Forsythe JA, Jiang BH, Iyer NV, Agani F, Leung SW, Koos RD and Semenza GL: Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Mol Cell Biol. 16:4604–4613. 1996. View Article : Google Scholar : PubMed/NCBI
6	Liu LZ, Jing Y, Jiang LL, Jiang XE, Jiang Y, Rojanasakul Y and Jiang BH: Acacetin inhibits VEGF expression, tumor angiogenesis and growth through AKT/HIF-1α pathway. Biochem Biophys Res Commun. 413:299–305. 2011. View Article : Google Scholar : PubMed/NCBI
7	Lin M, Chen Y, Jin J, Hu Y, Zhou KK, Zhu M, Le YZ, Ge J, Johnson RS and Ma JX: Ischaemia-induced retinal neovascularisation and diabetic retinopathy in mice with conditional knockout of hypoxia-inducible factor-1 in retinal Müller cells. Diabetologia. 54:1554–1566. 2011. View Article : Google Scholar : PubMed/NCBI
8	Forooghian F and Das B: Anti-angiogenic effects of ribonucleic acid interference targeting vascular endothelial growth factor and hypoxia-inducible factor-1alpha. Am J Ophthalmol. 144:761–768. 2007. View Article : Google Scholar : PubMed/NCBI
9	Yanyan C, Guoxian Q, Yang G and Leting W: Mechanism of hypoxia-induced factor 1alpha expression in endothelial cells of the human umbilical vein and its induction of apoptosis. Mol Biol Rep. 35:285–290. 2008. View Article : Google Scholar
10	Zhu X, Zhou W, Cui Y, Zhu L, Li J, Feng X, Shao B, Qi H, Zheng J, Wang H and Chen H: Pilocarpine protects cobalt chloride-induced apoptosis of RGC-5 cells: Involvement of muscarinic receptors and HIF-1 alpha pathway. Cell Mol Neurobiol. 30:427–435. 2010. View Article : Google Scholar
11	Jin Y, An X, Ye Z, Cully B, Wu J and Li J: RGS5, a hypoxia-inducible apoptotic stimulator in endothelial cells. J Biol Chem. 284:23436–23443. 2009. View Article : Google Scholar : PubMed/NCBI
12	Toffoli S and Michiels C: Intermittent hypoxia is a key regulator of cancer cell and endothelial cell interplay in tumours. FEBS J. 275:2991–3002. 2008. View Article : Google Scholar : PubMed/NCBI
13	Mazure NM, Brahimi-Horn MC, Berta MA, Benizri E, Bilton RL, Dayan F, Ginouvès A, Berra E and Pouysségur J: HIF-1: Master and commander of the hypoxic world. A pharmacological approach to its regulation by siRNAs. Biochem Pharmacol. 68:971–980. 2004. View Article : Google Scholar : PubMed/NCBI
14	Li SY, Jia YH, Sun WG, Tang Y, An GS, Ni JH and Jia HT: Stabilization of mitochondrial function by tetramethylpyrazine protects against kainate-induced oxidative lesions in the rat hippocampus. Free Radic Biol Med. 48:597–608. 2010. View Article : Google Scholar
15	Li WM, Liu HT, Li XY, Wu JY, Xu G, Teng YZ, Ding ST and Yu C: The effect of tetramethylpyrazine on hydrogen peroxide-induced oxidative damage in human umbilical vein endothelial cells. Basic Clin Pharmacol Toxicol. 106:45–52. 2010.
16	Liao SL, Kao TK, Chen WY, Lin YS, Chen SY, Raung SL, Wu CW, Lu HC and Chen CJ: Tetramethylpyrazine reduces ischemic brain injury in rats. Neurosci Lett. 372:40–45. 2004. View Article : Google Scholar : PubMed/NCBI
17	Zou Y, Jiang W and Chiou GC: Effect of tetramethylpyrazine on rat experimental choroidal neovascularization in vivo and endothelial cell cultures in vitro. Curr Eye Res. 32:71–75. 2007. View Article : Google Scholar : PubMed/NCBI
18	Liang X, Zhou H, Ding Y, Li J, Yang C, Luo Y, Li S, Sun G, Liao X and Min W: TMP prevents retinal neovascularization and imparts neuroprotection in an oxygen-induced retinopathy model. Invest Ophthalmol Vis Sci. 53:2157–2169. 2012. View Article : Google Scholar : PubMed/NCBI
19	Li M, Zhao MQ, Kumar Durairajan SS, Xie LX, Zhang HX, Kum WF, Goto S and Liao FL: Protective effect of tetramethylpyrazine and salvianolic acid B on apoptosis of rat cerebral microvascular endothelial cell under high shear stress. Clin Hemorheol Microcirc. 38:177–187. 2008.PubMed/NCBI
20	Yang J, Yang S and Yuan YJ: Integrated investigation of lipidome and related signaling pathways uncovers molecular mechanisms of tetramethylpyrazine and butylidenephthalide protecting endothelial cells under oxidative stress. Mol Biosyst. 8:1789–1797. 2012. View Article : Google Scholar : PubMed/NCBI
21	Ou Y, Dong X, Liu XY, Cheng XC, Cheng YN, Yu LG and Guo XL: Mechanism of tetramethylpyrazine analogue CXC195 inhibition of hydrogen peroxide-induced apoptosis in human endothelial cells. Biol Pharm Bull. 33:432–438. 2010. View Article : Google Scholar : PubMed/NCBI
22	Kang Y, Hu M, Zhu Y, Gao X and Wang MW: Antioxidative effect of the herbal remedy Qin Huo Yi Hao and its active component tetramethylpyrazine on high glucose-treated endothelial cells. Life Sci. 84:428–436. 2009. View Article : Google Scholar : PubMed/NCBI
23	Chen L, Lu Y, Wu JM, Xu B, Zhang LJ, Gao M, Zheng SZ, Wang AY, Zhang CB, Zhang WW and Lei N: Ligustrazine inhibits B16F10 melanoma metastasis and suppresses angiogenesis induced by vascular endothelial growth factor. Biochem Biophys Res Commun. 386:374–379. 2009. View Article : Google Scholar : PubMed/NCBI
24	de Martin R, Vanhove B, Cheng Q, Hofer E, Csizmadia V, Winkler H and Bach FH: Cytokine-inducible expression in endothelial cells of an I kappaB alpha-like gene is regulated by NF kappa B. EMBO J. 12:2773–2779. 1993.PubMed/NCBI
25	Dai H, Yu Z, Fan X, Liu N, Yan M, Chen Z, Lo EH, Hajjar KA and Wang X: Dysfunction of annexin A2 contributes to hyperglycaemia-induced loss of human endothelial cell surface fibrinolytic activity. Thromb Haemost. 109:1070–1078. 2013. View Article : Google Scholar : PubMed/NCBI
26	Hedenbjörk-Lager A, Bjørndal L, Gustafsson A, Sorsa T, Tjäderhane L, Åkerman S and Ericson D: Caries correlates strongly to salivary levels of matrix metalloproteinase-8. Caries Res. 49:1–8. 2015. View Article : Google Scholar
27	Gottlieb E, Armour SM, Harris MH and Thompson CB: Mitochondrial membrane potential regulates matrix configuration and cytochrome c release during apoptosis. Cell Death Differ. 10:709–717. 2003. View Article : Google Scholar : PubMed/NCBI
28	Zhang Z, Wei T, Hou J, Li G, Yu S and Xin W: Iron-induced oxidative damage and apoptosis in cerebellar granule cells: Attenuation by tetramethylpyrazine and ferulic acid. Eur J Pharmacol. 467:41–47. 2003. View Article : Google Scholar : PubMed/NCBI
29	Dang SC, Zhang JX, Qu JG, Wang XQ and Fan X: Ligustrazine alleviates gastric mucosal injury in a rat model of acute necrotizing pancreatitis. Hepatobiliary Pancreat Dis Int. 6:213–218. 2007.PubMed/NCBI
30	Guo SK, Chen KJ, Qian ZH, Weng WL and Qian MY: Tetramethylpyrazine in the treatment of cardiovascular and cerebrovascular diseases. Planta Med. 47:891983. View Article : Google Scholar : PubMed/NCBI
31	Brahimi-Horn MC and Pouysségur J: HIF at a glance. J Cell Sci. 122:1055–1057. 2009. View Article : Google Scholar : PubMed/NCBI
32	Semenza GL: HIF-1 and mechanisms of hypoxia sensing. Curr Opin Cell Biol. 13:167–171. 2001. View Article : Google Scholar : PubMed/NCBI
33	Semenza GL: Hypoxia-inducible factor 1: Oxygen homeostasis and disease pathophysiology. Trends Mol Med. 7:345–350. 2001. View Article : Google Scholar : PubMed/NCBI
34	Lee C, Cheng W, Chang M, Su Y, Chen C and Hsieh F: Hypoxia-induced apoptosis in endothelial cells and embryonic stem cells. Apoptosis. 10:887–894. 2005. View Article : Google Scholar
35	Matsushita H, Morishita R, Nata T, Aoki M, Nakagami H, Taniyama Y, Yamamoto K, Higaki J, Yasufumi K and Ogihara T: Hypoxia-induced endothelial apoptosis through nuclear factor-kappaB (NF-kappaB)-mediated bcl-2 suppression: In vivo evidence of the importance of NF-kappaB in endothelial cell regulation. Circ Res. 86:974–981. 2000. View Article : Google Scholar : PubMed/NCBI
36	Wang D, Weng Q, Zhang L, He Q and Yang B: VEGF and Bcl-2 interact via MAPKs signaling pathway in the response to hypoxia in neuroblastoma. Cell Mol Neurobiol. 29:391–401. 2009. View Article : Google Scholar
37	Chen N, Chen X, Huang R, Zeng H, Gong J, Meng W, Lu Y, Zhao F, Wang L and Zhou Q: BCL-xL is a target gene regulated by hypoxia-inducible factor-1{alpha}. J Biol Chem. 284:10004–10012. 2009. View Article : Google Scholar : PubMed/NCBI
38	Carmeliet P, Dor Y, Herbert JM, Fukumura D, Brusselmans K, Dewerchin M, Neeman M, Bono F, Abramovitch R, Maxwell P, et al: Role of HIF-1alpha in hypoxia-mediated apoptosis, cell proliferation and tumour angiogenesis. Nature. 394:485–490. 1998. View Article : Google Scholar : PubMed/NCBI