Shikonin attenuates H<sub>2</sub>O<sub>2</sub>‑induced oxidative injury in HT29 cells via antioxidant activities and the inhibition of mitochondrial pathway‑mediated apoptosis

Zhong,Yu; Qi,Ao; Liu,Lulu; Huang,Qionglin; Zhang,Junjie; Cai,Kangrong; Cai,Chun

doi:10.3892/etm.2021.10552

Shikonin attenuates H₂O₂‑induced oxidative injury in HT29 cells via antioxidant activities and the inhibition of mitochondrial pathway‑mediated apoptosis

Authors:
- Yu Zhong
- Ao Qi
- Lulu Liu
- Qionglin Huang
- Junjie Zhang
- Kangrong Cai
- Chun Cai
View Affiliations

Affiliations: Southern Marine Science and Engineering Guangdong Laboratory, Zhanjiang, Guangdong 524023, P.R. China
Published online on: August 4, 2021 https://doi.org/10.3892/etm.2021.10552
Article Number: 1118
Copyright: © Zhong et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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

Shikonin, a natural naphthoquinone extracted from the roots of Lithospermumery throrhizon, possesses multiple pharmacological properties, including antioxidant, anti‑inflammatory and antitumor effects. It has been hypothesized that the properties of shikonin are associated with its oxygen free radical scavenging abilities. However, the mechanism underlying the antioxidant activity of shikonin is not completely understood. The aim of the present study was to investigate the effect of shikonin against H₂O₂‑induced oxidative injury in HT29 cells and to explore the underlying molecular mechanism. The concentration and duration of H₂O₂ treatment to cause maximal damage, and the effects of shikonin (2.5, 5 or 10 µg/ml) on the activity of H₂O₂‑induced HT29 cells were determined by MTT assay. The apoptotic rate in HT29 cells was determined by annexin V/propidium iodide staining. HT29 cell cycle alteration was also analyzed by propidium iodide staining. Reactive oxygen species (ROS) production was assessed by monitoring 2',7'‑dichlorofluorescin in diacetate fluorescence. Mitochondrial membrane potentials were determined by JC‑1 staining. The activities of malondialdehyde, superoxide dismutase, caspase‑9 and caspase‑3 were measured using spectrophotometric assays. The expression levels of Bcl‑2, Bax and cytochrome c were determined by western blotting. The results suggested that shikonin increased cell viability, reduced cell apoptosis and increased the proliferation index in H₂O₂‑treated HT29 cells. Shikonin also significantly inhibited increases in intracellular reactive oxygen species (ROS), restored the mitochondrial membrane potential, prevented the release of lactic dehydrogenase and decreased the levels of superoxide dismutase and malondialdehyde in H₂O₂‑induced HT29 cells. Furthermore, shikonin significantly decreased caspase‑9 and caspase‑3 activities, increased Bcl‑2 expression and decreased Bax and cytochrome c expression levels in H₂O₂‑induced HT29 cells. The results indicated that shikonin protected against H₂O₂‑induced oxidative injury by removing ROS, ameliorating mitochondrial dysfunction, attenuating DNA oxidative damage and inhibiting mitochondrial pathway‑mediated apoptosis.

View Figures

View References

1	Sinha N and Dabla PK: Oxidative stress and antioxidants in hypertension-a current review. Curr Hypertens Rev. 11:132–142. 2015.PubMed/NCBI View Article : Google Scholar
2	Guo H, Sun J, Li D, Hu Y, Yu X, Hua H, Jing X, Chen F, Jia Z and Xu J: Shikonin attenuates acetaminophen-induced acute liver injury via inhibition of oxidative stress and inflammation. Biomed Pharmacother. 112(108704)2019.PubMed/NCBI View Article : Google Scholar
3	Pistollato F, Iglesias RC, Ruiz R, Aparicio S, Crespo J, Lopez LD, Manna PP, Giampieri F and Battino M: Nutritional patterns associated with the maintenance of neurocognitive functions and the risk of dementia and Alzheimer's disease: A focus on human studies. Pharmacol Res. 131:32–43. 2018.PubMed/NCBI View Article : Google Scholar
4	Yan H, Wang H, Zhang X, Li X and Yu J: Ascorbic acid ameliorates oxidative stress and inflammation in dextran sulfate sodium-induced ulcerative colitis in mice. Int J Clin Exp Med. 8:20245–20253. 2015.PubMed/NCBI
5	Mrowicka M, Mrowicki J, Mik M, Wojtczak R, Dziki L, Dziki A and Majsterek I: Association between SOD1, CAT, GSHPX1 polymorphisms and the risk of inflammatory bowel disease in the Polish population. Oncotarget. 8:109332–109339. 2017.PubMed/NCBI View Article : Google Scholar
6	Shalkami AS, HAssan M and Bakr AG: Anti-inflammatory, antioxidant and anti-apoptotic activity of diosmin in acetic acid-induced ulcerative colitis. Hum Exp Toxicol. 37:78–86. 2018.PubMed/NCBI View Article : Google Scholar
7	Kruidenier L and Verspaget HW: Review article: Oxidative stress as a pathogenic factor in inflammatory bowel disease-radicals or ridiculous? Aliment Pharmacol Ther. 16:1997–2015. 2002.PubMed/NCBI View Article : Google Scholar
8	Zhu H and Li YR: Oxidative stress and redox signaling mechanisms of inflammatory bowel disease: Updated experimental and clinical evidence. Exp Biol Med (Maywood). 237:474–480. 2012.PubMed/NCBI View Article : Google Scholar
9	Sakthivel KM and Guruvayoorappan C: Amentoflavone inhibits iNOS, COX-2 expression and modulates cytokine profile, NF-κB signal transduction pathways in rats with ulcerative colitis. Int Immunopharmacol. 17:907–916. 2013.PubMed/NCBI View Article : Google Scholar
10	Zhong Y, Yu W, Feng J, Fan Z and Li J: Curcumin suppresses tumor necrosis factor-alpha-induced matrix metalloproteinase-2 expression and activity in rat vascular smooth muscle cells via the NF-κB pathway. Exp Ther Med. 7:1653–1658. 2014.PubMed/NCBI View Article : Google Scholar
11	An S, Park YD, Paik YK, Jeong TS and Lee WS: Human ACAT inhibitory effects of shikonin derivatives from Lithospermum erythrorhizon. Bioorg Med Chem Lett. 17:1112–1116. 2007.PubMed/NCBI View Article : Google Scholar
12	Zhang Z, Yang L, Wang B, Zhang L, Zhang Q, Li D, Zhang S, Gao H and Wang X: Protective role of liriodendrin in mice with dextran sulphate sodium-induced ulcerative colitis. Int Immunopharmacol. 52:203–210. 2017.PubMed/NCBI View Article : Google Scholar
13	Liang W, Cai A, Chen G, Xi H, Wu X, Cui J, Zhang K, Zhao X, Yu J, Wei B and Chen L: Shikonin induces mitochondria-mediated apoptosis and enhances chemotherapeutic sensitivity of gastric cancer through reactive oxygen species. Sci Rep. 6(38267)2016.PubMed/NCBI View Article : Google Scholar
14	Gao D, Kakuma M, Oka S, Sugino K and Sakurai H: Reaction of beta-alkannin (shikonin) with reactive oxygen species: Detection of beta-alkannin free radicals. Bioorg Med Chem. 8:2561–2569. 2000.PubMed/NCBI View Article : Google Scholar
15	Tong Y, Bai L, Gong R, Chuan J, Duan X and Zhu Y: Shikonin protects PC12 cells against β-amyloid peptide-induced cell injury through antioxidant and antiapoptotic activities. Sci Rep. 8(26)2018.PubMed/NCBI View Article : Google Scholar
16	Esmaeilzadeh E, gardaneh M, Gharib E and Sabouni F: Shikonin protects dopaminergic cell line PC12 against 6-hydroxydopamine-mediated neurotoxicity via both glutathione-dependent and independent pathways and by inhibiting apoptosis. Neurochem Res. 38:1590–1604. 2013.PubMed/NCBI View Article : Google Scholar
17	Tong Y, Chuan J, Bai L, Shi J, Zhong L, Duan X and Zhu Y: The protective effect of shikonin on renal tubular epithelial cell injury induced by high glucose. Biomed Pharmacother. 98:701–708. 2018.PubMed/NCBI View Article : Google Scholar
18	Liu T, Zhang Q, Mo W, Yu Q, Xu S, Li J, Li S, Feng J, Wu L, Lu X, et al: The protective effects of shikonin on hepatic ischemia/reperfusion injury are mediated by the activation of the PI3K/Akt pathway. Sci Rep. 7(44785)2017.PubMed/NCBI View Article : Google Scholar
19	Wang Z, Liu T, Gan L, Wang T, Yuan X, Zhang B, Chen H and Zheng Q: Shikonin protects mouse brain against cerebral ischemia/reperfusion injury through its antioxidant activity. Eur J Pharmacol. 643:211–217. 2010.PubMed/NCBI View Article : Google Scholar
20	Moore LD, Le T and Fan G: DNA methylation and its basic function. Neuropsychopharmacology. 38:23–38. 2013.PubMed/NCBI View Article : Google Scholar
21	Sena P, Mancini S, Benincasa M, Mariani F, Palumbo C and Roncucci L: Metformin induces apoptosis and alters cellular responses to oxidative stress in Ht29 colon cancer cells: Preliminary findings. Int J Mol Sci. 19(1478)2018.PubMed/NCBI View Article : Google Scholar
22	Bai J, Yu J, Wang J, Xue B, He N, Tian Y, Yang L, Wang Y, Wang Y and Tang Q: DNA methylation of miR-122 aggravates oxidative stress in colitis targeting SELENBP1 partially by p65NF-κ B signaling. Oxid Med Cell Longev. 2019(5294105)2019.
23	Zhao X, Fang J, Li S, Gaur U, Xing X, Wang H and Zheng W: Artemisinin attenuated hydrogen peroxide (H2O2)-induced oxidative injury in SH-SY5Y and hippocampal neurons via the activation of AMPK pathway. Int J Mol Sci. 20(2680)2019.PubMed/NCBI View Article : Google Scholar
24	Akbay E, Arbag H, Uyar Y and Ozturk K: Oxidative stress and antioxidant factors in pathophysiology of allergic rhinitis. Kulak Burun Bogaz Ihtis Derg. 17:189–1896. 2007.PubMed/NCBI(In Turkish).
25	Bresciani G, DA CI and González-Gallego J: Manganese superoxide dismutase and oxidative stress modulation. Adv Clin Chem. 68:87–130. 2015.PubMed/NCBI View Article : Google Scholar
26	Jablonski RP, Kim SJ, Cheresh P, Williams DB, Morales-Nebreda L, Cheng Y, Yeldandi A, Bhorade S, Pardo A, Selman M, et al: SIRT3 deficiency promotes lung fibrosis by augmenting alveolar epithelial cell mitochondrial DNA damage and apoptosis. FASEB J. 31:2520–2532. 2017.PubMed/NCBI View Article : Google Scholar
27	Chen XH, Zhou X, Yang XY, Zhou ZB, Lu DH, Tang Y, Ling ZM, Zhou LH and Feng X: Propofol protects against H2O2-induced oxidative injury in differentiated PC12 cells via inhibition of Ca(2+)-dependent NADPH oxidase. Cell Mol Neurobiol. 36:541–551. 2016.PubMed/NCBI View Article : Google Scholar
28	Wagener FA, Dekker D, Berden JH, Scharstuhl A and van der Vlag J: The role of reactive oxygen species in apoptosis of the diabetic kidney. Apoptosis. 14:1451–1458. 2009.PubMed/NCBI View Article : Google Scholar
29	Yang H, Villani RM, Wang H, Simpson MJ, Roberts MS, Tang M and Liang X: The role of cellular reactive oxygen species in cancer chemotherapy. J Exp Clin Cancer Res. 37(266)2018.PubMed/NCBI View Article : Google Scholar
30	Cavallucci V, D'amelio M and Cecconi F: Abeta toxicity in Alzheimer's disease. Mol Neurobiol. 45:366–378. 2012.PubMed/NCBI View Article : Google Scholar
31	Li C, Jiang W, Liu ZG, Liang PQ and Hu R: Role of reactive oxygen species in GDC-0152-induced apoptosis and autophagy of NB4 cells. Zhongguo Shi Yan Xue Ye Xue Za Zhi. 27:1786–1793. 2019.PubMed/NCBI View Article : Google Scholar : (In Chinese).
32	Prasad S, Gupta SC and Tyagi AK: Reactive oxygen species (ROS) and cancer: Role of antioxidative nutraceuticals. Cancer Lett. 387:95–105. 2017.PubMed/NCBI View Article : Google Scholar
33	Kehrer JP and Klotz LO: Free radicals and related reactive species as mediators of tissue injury and disease: Implications for Health. Crit Rev Toxicol. 45:765–798. 2015.PubMed/NCBI View Article : Google Scholar
34	He L, He T, Farrar S, Ji L, Liu T and Ma X: Antioxidants maintain cellular redox homeostasis by elimination of reactive oxygen species. Cell Physiol Biochem. 44:532–553. 2017.PubMed/NCBI View Article : Google Scholar
35	Tsikas D: Assessment of lipid peroxidation by measuring malondialdehyde (MDA) and relatives in biological samples: Analytical and biological challenges. Anal Biochem. 524:13–30. 2017.PubMed/NCBI View Article : Google Scholar
36	Del RD, Stewart AJ and Pellegrini N: A review of recent studies on malondialdehyde as toxic molecule and biological marker of oxidative stress. Nutr Metab Cardiovasc Dis. 15:316–328. 2005.PubMed/NCBI View Article : Google Scholar
37	Gallo M, Sapio L, Spina A, Naviglio D, Calogero A and Naviglio S: Lactic dehydrogenase and cancer: An overview. Front Biosci (Landmark Ed). 20:1234–1249. 2015.PubMed/NCBI View Article : Google Scholar
38	Chong CM and Zheng W: Artemisinin protects human retinal pigment epithelial cells from hydrogen peroxide-induced oxidative damage through activation of ERK/CREB signaling. Redox Biol. 9:50–56. 2016.PubMed/NCBI View Article : Google Scholar
39	Li S, Chaudhary SC, Zhao X, Gaur U, Fang J, Yan F and Zheng W: Artemisinin protects human retinal pigmented epithelial cells against hydrogen peroxide-induced oxidative damage by enhancing the activation of AMP-active protein kinase. Int J Biol Sci. 15:2016–2028. 2019.PubMed/NCBI View Article : Google Scholar
40	Green DR and Llambi F: Cell death signaling. Cold Spring Harb Perspect Biol. 7(a006080)2015.PubMed/NCBI View Article : Google Scholar
41	Maddika S, Ande SR, Panigrahi S, Paranjothy T, Weglarczyk K, Zuse A, Eshraghi M, Manda KD, Wiechec E and Los M: Cell survival, cell death and cell cycle pathways are interconnected: Implications for cancer therapy. Drug Resist Updat. 10:13–29. 2007.PubMed/NCBI View Article : Google Scholar
42	Cory S and Adams JM: The Bcl2 family: Regulators of the cellular life-or-death switch. Nat Rev Cancer. 2:647–656. 2002.PubMed/NCBI View Article : Google Scholar
43	Lin HH, Chen JH, Huang CC and Wang CJ: Apoptotic effect of 3,4-dihydroxybenzoic acid on human gastric carcinoma cells involving JNK/p38 MAPK signaling activation. Int J Cancer. 120:2306–2316. 2007.PubMed/NCBI View Article : Google Scholar
44	Pistritto G, Trisciuoglio D, Ceci C, Garufi A and D'orazi G: Apoptosis as anticancer mechanism: Function and dysfunction of its modulators and targeted therapeutic strategies. Aging (Albany NY). 8:603–169. 2016.PubMed/NCBI View Article : Google Scholar
45	Maulik N, Goswami S, Galang N and Das DK: Differential regulation of Bcl-2, AP-1 and NF-kappaB on cardiomyocyte apoptosis during myocardial ischemic stress adaptation. FEBS Lett. 443:331–336. 1999.PubMed/NCBI View Article : Google Scholar
46	Cohen GM: Caspases: The executioners of apoptosis. Biochem J. 326:1–16. 1997.PubMed/NCBI View Article : Google Scholar
47	Khalilzadeh B, Shadjou N, Kanberoglu GS, Afsharan H, de la Guardia M, Charoudeh HN, Ostadrahimi A and Rashidi MR: Advances in nanomaterial based optical biosensing and bioimaging of apoptosis via caspase-3 activity: A review. Mikrochim Acta. 185(434)2018.PubMed/NCBI View Article : Google Scholar