Naringin protects H9C2 cardiomyocytes from chemical hypoxia‑induced injury by promoting the autophagic flux via the activation of the HIF‑1α/BNIP3 signaling pathway

Li,Shanghai; Jiang,Jiamei; Fang,Junyu; Li,Xingyue; Huang,Chunyan; Liang,Weijun; Wu,Keng

doi:10.3892/ijmm.2021.4935

Naringin protects H9C2 cardiomyocytes from chemical hypoxia‑induced injury by promoting the autophagic flux via the activation of the HIF‑1α/BNIP3 signaling pathway

Authors:
- Shanghai Li
- Jiamei Jiang
- Junyu Fang
- Xingyue Li
- Chunyan Huang
- Weijun Liang
- Keng Wu
View Affiliations

Affiliations: Department of Cardiology, Affiliated Hospital of Guangdong Medical University, Zhanjiang, Guangdong 524001, P.R. China, Department of Neurology, The First Affiliated Hospital of University of South China, Hengyang, Hunan 421001, P.R. China
Published online on: April 15, 2021 https://doi.org/10.3892/ijmm.2021.4935
Article Number: 102

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

Naringin, a natural bioflavonoid, has been shown to exert protective effects in multiple cardiovascular diseases; however, the protective effects of naringin against hypoxic/ischemia‑induced myocardial are not yet fully understood. Autophagy is a vital factor involved in the pathogenesis of myocardial injury. The aim of the present study was to investigate the protective effects of naringin on H9c2 cells against chemical hypoxia [cobalt chloride (CoCl₂)]‑induced injury. The role of autophagy and the hypoxia‑inducible factor‑1α (HIF‑1α)/Bcl‑2/BCL2 interacting protein 3 (BNIP3) signaling pathway in the protective effects of naringin were also assessed. The results revealed that naringin pre‑treatment significantly attenuated the CoCl₂‑induced cytotoxicity and apoptosis, and also decreased caspase‑3 activity, which had been increased by CoCl₂. In addition, CoCl₂ increased Beclin‑1 expression, enhanced the IL3B‑II/IL3B‑I ratio and increased p62 expression in the H9C2 cells. Treatment with 3‑methyladenine (3‑MA), a selective inhibitor of autophagy, also blocked CoCl₂‑induced cytotoxicity and apoptosis. Notably, treatment with bafilomycin A1 (Baf A1), an inhibitor of the vacuolar H+ ATPase of lysosomes, resulted in an increase in the upregulation of the LC3B‑II/LC3B‑I ratio, but did not further increase the LC3B‑II/LC3B‑I ratio compared with CoCl₂ treatment. These results suggested that CoCl₂ inhibited the autophagic flux, which resulted in myocardial cell damage. Furthermore, naringin pre‑treatment exacerbated Beclin 1 expression and the increased IL3B‑II/IL3B‑I ratio, and reduced p62 expression in CoCl₂‑treated H9C2 cells. 3‑MA and Baf A1 both reversed the protective effects of naringin against CoCl₂‑induced injury, indicating that naringin attenuated CoCl₂‑induced myocardial cell injury by the increasing autophagic flux. Moreover, naringin treatment resulted in upregulated expression levels of HIF‑1α and BNIP3 in the CoCl₂‑treated H9C2 cells. The inhibition of the HIF‑1α/BNIP3 signaling pathway using 3‑(5'‑hydroxymethyl‑2'‑furyl)‑1‑benzylindazole (an inhibitor of HIF‑1α) prevented the effects of naringin on the autophagic flux and reversed its protective effects against CoCl₂‑induced injury. Taken together, these results suggest that naringin protects the H9C2 cells against CoCl₂‑induced injury by enhancing the autophagic flux via the activation of the HIF‑1α/BNIP3 signaling pathway.

View Figures

View References

1	GBD 2017 Causes of Death Collaborators: Global, regional, and national age-sex-specific mortality for 282 causes of death in 195 countries and territories, 1980-2017: A systematic analysis for the global burden of disease study 2017. Lancet. 392:1736–1788. 2018. View Article : Google Scholar
2	Aouiss A, Anka Idrissi D, Kabine M and Zaid Y: Update of inflammatory proliferative retinopathy: Ischemia, hypoxia and angiogenesis. Curr Res Transl Med. 67:62–71. 2019. View Article : Google Scholar : PubMed/NCBI
3	Cordova Martinez A, Pascual Fernandez J, Fernandez Lazaro D and Alvarez Mon M: Muscular and heart adaptations of execise in hypoxia. Is training in slow hypoxy healthy? Med Clin (Barc). 148:469–474. 2017.In English, Spanish.
4	Rodgers JL, Iyer D, Rodgers LE, Vanthenapalli S and Panguluri SK: Impact of hyperoxia on cardiac pathophysiology. J Cell Physiol. 234:12595–12603. 2019. View Article : Google Scholar : PubMed/NCBI
5	Den Hartogh DJ and Tsiani E: Antidiabetic properties of naringenin: A citrus fruit polyphenol. Biomolecules. 9:992019. View Article : Google Scholar :
6	Kwatra M, Kumar V, Jangra A, Mishra M, Ahmed S, Ghosh P, Vohora D and Khanam R: Ameliorative effect of naringin against doxorubicin-induced acute cardiac toxicity in rats. Pharm Biol. 54:637–647. 2016. View Article : Google Scholar
7	Ming H, Chuang Q, Jiashi W, Bin L, Guangbin W and Xianglu J: Naringin targets Zeb1 to suppress osteosarcoma cell proliferation and metastasis. Aging (Albany NY). 10:4141–4151. 2018. View Article : Google Scholar
8	Salehi B, Fokou PVT, Sharifi-Rad M, Zucca P, Pezzani R, Martins N and Sharifi-Rad J: The therapeutic potential of naringenin: A review of clinical trials. Pharmaceuticals (Basel). 12:112019. View Article : Google Scholar
9	Adebiyi OA, Adebiyi OO and Owira PM: Naringin reduces hyperglycemia-induced cardiac fibrosis by relieving oxidative stress. PLoS One. 11:e01498902016. View Article : Google Scholar : PubMed/NCBI
10	Park JH, Ku HJ, Kim JK, Park JW and Lee JH: Amelioration of high fructose-induced cardiac hypertrophy by naringin. Sci Rep. 8:94642018. View Article : Google Scholar : PubMed/NCBI
11	Jian CY, Ouyang HB, Xiang XH, Chen JL, Li YX, Zhou X, Wang JY, Yang Y, Zhong EY, Huang WH and Zhang HW: Naringin protects myocardial cells from doxorubicin-induced apoptosis partially by inhibiting the p38MAPK pathway. Mol Med Rep. 16:9457–9463. 2017. View Article : Google Scholar : PubMed/NCBI
12	Chen RC, Sun GB, Wang J, Zhang HJ and Sun XB: Naringin protects against anoxia/reoxygenation-induced apoptosis in H9c2 cells via the Nrf2 signaling pathway. Food Funct. 6:1331–1344. 2015. View Article : Google Scholar : PubMed/NCBI
13	Cai CC, Zhu JH, Ye LX, Dai YY, Fang MC, Hu YY, Pan SL, Chen S, Li PJ, Fu XQ and Lin ZL: Glycine protects against hypoxic-ischemic brain injury by regulating mitochondria-mediated autophagy via the AMPK pathway. Oxid Med Cell Longev. 2019:42485292019. View Article : Google Scholar : PubMed/NCBI
14	Zhang Z, Wang C, Lin J, Jin H, Wang K, Yan Y, Wang J, Wu C, Nisar M, Tian N, et al: Therapeutic potential of naringin for intervertebral disc degeneration: Involvement of autophagy against oxidative stress-induced apoptosis in nucleus pulposus cells. Am J Chin Med. Oct 4–2018.Epub ahead of print. View Article : Google Scholar
15	Sousa Fialho MD, Abd Jamil AH, Stannard GA and Heather LC: Hypoxia-inducible factor 1 signalling, metabolism and its therapeutic potential in cardiovascular disease. Biochim Biophys Acta Mol Basis Dis. 1865:831–843. 2019. View Article : Google Scholar
16	Bravo-San Pedro JM, Kroemer G and Galluzzi L: Autophagy and mitophagy in cardiovascular disease. Circ Res. 120:1812–1824. 2017. View Article : Google Scholar : PubMed/NCBI
17	Sciarretta S, Maejima Y, Zablocki D and Sadoshima J: The role of autophagy in the heart. Annu Rev Physiol. 80:1–26. 2018. View Article : Google Scholar
18	Qiao J, Huang J, Zhou M, Cao G and Shen H: Inhibition of HIF-1α restrains fracture healing via regulation of autophagy in a rat model. Exp Ther Med. 17:1884–1890. 2019.PubMed/NCBI
19	Zimmerman MA, Biggers CD and Li PA: Rapamycin treatment increases hippocampal cell viability in an mTOR-independent manner during exposure to hypoxia mimetic, cobalt chloride. BMC Neurosci. 19:822018. View Article : Google Scholar : PubMed/NCBI
20	Gallo S, Gatti S, Sala V, Albano R, Costelli P, Casanova E, Comoglio PM and Crepaldi T: Agonist antibodies activating the Met receptor protect cardiomyoblasts from cobalt chloride-induced apoptosis and autophagy. Cell Death Dis. 5:e11852014. View Article : Google Scholar : PubMed/NCBI
21	Lee JW, Bae SH, Jeong JW, Kim SH and Kim KW: Hypoxia-inducible factor (HIF-1)alpha: Its protein stability and biological functions. Exp Mol Med. 36:1–12. 2004. View Article : Google Scholar : PubMed/NCBI
22	Wang H, Zhang D, Jia S, Huang S, Xiao L, Ma L, Liu G, Gong K and Xu L: Effect of sustained hypoxia on autophagy of genioglossus muscle-derived stem cells. Med Sci Monit. 24:2218–2224. 2018. View Article : Google Scholar : PubMed/NCBI
23	Zhang Y, Liu D, Hu H, Zhang P, Xie R and Cui W: HIF-1α/BNIP3 signaling pathway-induced-autophagy plays protective role during myocardial ischemia-reperfusion injury. Biomed Pharmacother. 120:1094642019. View Article : Google Scholar
24	Liu XW, Lu MK, Zhong HT, Wang LH and Fu YP: Panax notoginseng saponins attenuate myocardial ischemia-reperfusion injury through the HIF-1α/BNIP3 pathway of autophagy. J Cardiovasc Pharmacol. 73:92–99. 2019. View Article : Google Scholar
25	Wu H, Huang S, Chen Z, Liu W, Zhou X and Zhang D: Hypoxia-induced autophagy contributes to the invasion of salivary adenoid cystic carcinoma through the HIF-1α/BNIP3 signaling pathway. Mol Med Rep. 12:6467–6474. 2015. View Article : Google Scholar : PubMed/NCBI
26	Wang K, Peng S, Xiong S, Niu A, Xia M, Xiong X, Zeng G and Huang Q: Naringin inhibits autophagy mediated by PI3K-Akt-mTOR pathway to ameliorate endothelial cell dysfunction induced by high glucose/high fat stress. Eur J Pharmacol. 874:1730032020. View Article : Google Scholar : PubMed/NCBI
27	Manu TM, Anand T, Pandareesh MD, Kumar PB and Khanum F: Terminalia arjuna extract and arjunic acid mitigate cobalt chloride-induced hypoxia stress-mediated apoptosis in H9c2 cells. Naunyn Schmiedebergs Arch Pharmacol. 392:1107–1119. 2019. View Article : Google Scholar : PubMed/NCBI
28	Warbrick I and Rabkin SW: Hypoxia-inducible factor 1-alpha (HIF-1α) as a factor mediating the relationship between obesity and heart failure with preserved ejection fraction. Obes Rev. 20:701–712. 2019. View Article : Google Scholar : PubMed/NCBI
29	Hescheler J, Meyer R, Plant S, Krautwurst D, Rosenthal W and Schultz G: Morphological, biochemical, and electrophysiological characterization of a clonal cell (H9c2) line from rat heart. Circ Res. 69:1476–1486. 1991. View Article : Google Scholar : PubMed/NCBI
30	Haider N, Narula N and Narula J: Apoptosis in heart failure represents programmed cell survival, not death, of cardiomyocytes and likelihood of reverse remodeling. J Card Fail. 8(6 Suppl): S512–S517. 2002. View Article : Google Scholar
31	Semenza GL: Hypoxia-inducible factor 1: Regulator of mitochondrial metabolism and mediator of ischemic preconditioning. Biochim Biophys Acta. 1813:1263–1268. 2011. View Article : Google Scholar
32	Yadav AK, Yadav PK, Chaudhary GR, Tiwari M, Gupta A, Sharma A, Pandey AN, Pandey AK and Chaube SK: Autophagy in hypoxic ovary. Cell Mol Life Sci. 76:3311–3322. 2019. View Article : Google Scholar : PubMed/NCBI
33	Lamark T, Svenning S and Johansen T: Regulation of selective autophagy: The p62/SQSTM1 paradigm. Essays Biochem. 61:609–624. 2017. View Article : Google Scholar : PubMed/NCBI
34	Mallet RT, Manukhina EB, Ruelas SS, Caffrey JL and Downey HF: Cardioprotection by intermittent hypoxia conditioning: Evidence, mechanisms, and therapeutic potential. Am J Physiol Heart Circ Physiol. 315:H216–H232. 2018. View Article : Google Scholar : PubMed/NCBI
35	Cheng CI, Lee YH, Chen PH, Lin YC, Chou MH and Kao YH: Cobalt chloride induces RhoA/ROCK activation and remodeling effect in H9c2 cardiomyoblasts: Involvement of PI3K/Akt and MAPK pathways. Cell Signal. 36:25–33. 2017. View Article : Google Scholar : PubMed/NCBI
36	Munoz-Sanchez J and Chanez-Cardenas ME: The use of cobalt chloride as a chemical hypoxia model. J Appl Toxicol. 39:556–570. 2019. View Article : Google Scholar
37	Shi YN, Zhang XQ, Hu ZY, Zhang CJ, Liao DF, Huang HL and Qin L: Genistein protects H9c2 cardiomyocytes against chemical hypoxia-induced injury via inhibition of apoptosis. Pharmacology. 103:282–290. 2019. View Article : Google Scholar : PubMed/NCBI
38	Caglayan C: The effects of naringin on different cyclophosphamide-induced organ toxicities in rats: Investigation of changes in some metabolic enzyme activities. Environ Sci Pollut Res Int. 26:26664–26673. 2019. View Article : Google Scholar : PubMed/NCBI
39	You Q, Wu Z, Wu B, Liu C, Huang R, Yang L, Guo R, Wu K and Chen J: Naringin protects cardiomyocytes against hyperglycemia-induced injuries in vitro and in vivo. J Endocrinol. 230:197–214. 2016. View Article : Google Scholar : PubMed/NCBI
40	Moosavi MA and Djavaheri-Mergny M: Autophagy: New insights into mechanisms of action and resistance of treatment in acute promyelocytic leukemia. Int J Mol Sci. 20:35592019. View Article : Google Scholar :
41	Ravanan P, Srikumar IF and Talwar P: Autophagy: The spotlight for cellular stress responses. Life Sci. 188:53–67. 2017. View Article : Google Scholar : PubMed/NCBI
42	Gao C, Wang R, Li B, Guo Y, Yin T, Xia Y, Zhang F, Lian K, Liu Y, Wang H, et al: TXNIP/Redd1 signaling and excessive autophagy: A novel mechanism of myocardial ischemia/reperfusion injury in mice. Cardiovasc Res. 116:645–657. 2019. View Article : Google Scholar
43	McCormick J, Knight RA, Barry SP, Scarabelli TM, Abounit K, Latchman DS and Stephanou A: Autophagy in the stress-induced myocardium. Front Biosci (Elite Ed). 4:2131–2141. 2012. View Article : Google Scholar
44	Ryter SW, Lee SJ, Smith A and Choi AM: Autophagy in vascular disease. Proc Am Thorac Soc. 7:40–47. 2010. View Article : Google Scholar : PubMed/NCBI
45	Chen Y, Yan Q, Xu Y, Ye F, Sun X, Zhu H and Wang H: BNIP3-mediated autophagy induced inflammatory response and inhibited VEGF expression in cultured retinal pigment epithelium cells under hypoxia. Curr Mol Med. 19:395–404. 2019. View Article : Google Scholar : PubMed/NCBI
46	Glick D, Barth S and Macleod KF: Autophagy: Cellular and molecular mechanisms. J Pathol. 221:3–12. 2010. View Article : Google Scholar : PubMed/NCBI
47	Maiuri MC, Zalckvar E, Kimchi A and Kroemer G: Self-eating and self-killing: Crosstalk between autophagy and apoptosis. Nat Rev Mol Cell Biol. 8:741–752. 2007. View Article : Google Scholar : PubMed/NCBI
48	Kaizuka T, Morishita H, Hama Y, Tsukamoto S, Matsui T, Toyota Y, Kodama A, Ishihara T, Mizushima T and Mizushima N: An autophagic flux probe that releases an internal control. Mol Cell. 64:835–849. 2016. View Article : Google Scholar : PubMed/NCBI
49	Wu J and Lipinski MM: Autophagy in neurotrauma: Good, bad, or dysregulated. Cells. 8:6932019. View Article : Google Scholar :
50	Feng J, Chen X, Lu S, Li W, Yang D, Su W, Wang X and Shen J: Naringin attenuates cerebral ischemia-reperfusion injury through inhibiting peroxynitrite-mediated mitophagy activation. Mol Neurobiol. 55:9029–9042. 2018. View Article : Google Scholar : PubMed/NCBI
51	Raha S, Yumnam S, Hong GE, Lee HJ, Saralamma VV, Park HS, Heo JD, Lee SJ, Kim EH, Kim JA and Kim GS: Naringin induces autophagy-mediated growth inhibition by downregulating the PI3K/Akt/mTOR cascade via activation of MAPK pathways in AGS cancer cells. Int J Oncol. 47:1061–1069. 2015. View Article : Google Scholar : PubMed/NCBI
52	Adams JM, Difazio LT, Rolandelli RH, Luján JJ, Haskó G, Csóka B, Selmeczy Z and Németh ZH: HIF-1: A key mediator in hypoxia. Acta Physiol Hung. 96:19–28. 2009. View Article : Google Scholar : PubMed/NCBI
53	Chinnadurai G, Vijayalingam S and Gibson SB: BNIP3 subfamily BH3-only proteins: Mitochondrial stress sensors in normal and pathological functions. Oncogene. 27(Suppl 1): S114–S127. 2008. View Article : Google Scholar
54	Lu N, Li X, Tan R, An J, Cai Z, Hu X, Wang F, Wang H, Lu C and Lu H: HIF-1α/beclin1-mediated autophagy is involved in neuroprotection induced by hypoxic preconditioning. J Mol Neurosci. 66:238–250. 2018. View Article : Google Scholar : PubMed/NCBI
55	Chen R, Jiang T, She Y, Xu J, Li C, Zhou S, Shen H, Shi H and Liu S: Effects of cobalt chloride, a hypoxia-mimetic agent, on autophagy and atrophy in skeletal C2C12 myotubes. Biomed Res Int. 2017:70975802017.PubMed/NCBI
56	Qian X, Zhu M, Qian W and Song J: Vitamin D attenuates myocardial ischemia-reperfusion injury by inhibiting inflammation via suppressing the RhoA/ROCK/NF-κB pathway. Biotechnol Appl Biochem. 66:850–857. 2019. View Article : Google Scholar : PubMed/NCBI
57	Vinten-Johansen J, Jiang R, Reeves JG, Mykytenko J, Deneve J and Jobe LJ: Inflammation, proinflammatory mediators and myocardial ischemia-reperfusion Injury. Hematol Oncol Clin North Am. 21:123–145. 2007. View Article : Google Scholar : PubMed/NCBI
58	Zhao H, Liu M, Liu H, Suo R and Lu C: Naringin protects endothelial cells from apoptosis and inflammation by regulating the hippo-YAP pathway. Biosci Rep. 40:BSR201934312020. View Article : Google Scholar : PubMed/NCBI
59	Sun LJ, Qiao W, Xiao YJ, Cui L, Wang X and Ren WD: Naringin mitigates myocardial strain and the inflammatory response in sepsis-induced myocardial dysfunction through regulation of PI3K/AKT/NF-κB pathway. Int Immunopharmacol. 75:1057822019. View Article : Google Scholar