Vinblastine an antimitotic agent is used to treat hematological malignancies and in combination cancer chemotherapy. Vinblastine produces second malignancies in the survivors which can be reduced/prevented in cancer patients by intervention with natural products. Naringin a grapefruit bioflavonoid was tested for its chemoprotective activity in cultured V79 cells. The V79 cells were treated with 1, 2, 5, 10, 25, 50, 100, and 200 µg/mL of naringin 1 h before exposure to 10 µg/mL of vinblastine. The DNA damage was evaluated by micronucleus assay at 22 h after vinblastine exposure in mononucleate, binucleate, and trinucleate cells. Vinblastine treatment increased the frequency of micronuclei significantly in both the mononucleate and binucleate V79 cells whereas different concentrations of naringin significantly reduced the formation of vinblastine-induced micronuclei. A maximum reduction in micronuclei formation was recorded at 200 µg/mL naringin in both mononucleate and binucleate cells. The determination of cell proliferation indicates that naringin treatment impacted the cell proliferation, especially at 2 to 25 µg/mL which increased at 200 µg/mL in both the naringin and  Naringin+Vinblastine groupsgroup as indicated by a rise in binucleate and trinucleate cell numbers. The present study indicates that naringin protects the V79 cells against vinblastine-induced DNA damage indicated by significant attrition in micronuclei formation and 200 µg/mL naringin was highly effective.

Keywords: V79 cells, vinblastine, naringin, micronuclei, DNA damage

Vinblastine is a bisindole alkaloid isolated from Madagascar periwinkle plant Vinca rosea (Catharanthus roseus) in the 1950s by the Canadian scientists Robert Noble and Charles Beer.¹ It has been used to treat various cancers including Hodgkin's disease, Kaposi's sarcoma, certain types of lymphomas, testicular cancer, lung cancer, renal cell carcinoma, breast cancer, and choriocarcinoma, anaplastic large cell lymphoma either alone or in combination with other chemotherapeutic agents.^2–7 Vinblastine therapy in cancer patients leads to encephalopathy, seizures, visual loss, bone marrow, gastrointestinal, renal, pulmonary, liver, and cardiac toxicity.⁷ This indicates the need to reduce the adverse effects of vinblastine treatment. The name naringin (Figure 1) finds its origin in the Sanskrit word Narangi which stands for oranges. The grapefruit contains approximately 10% of naringin in respect to its dry weight and 800 mg/L of naringin is present in one liter of grapefruit juice. The red grapefruit contains only 332 mg/L of naringin.^8–9 The naringin is transformed into naringenin in the human gut by intestinal flora and it is an aglycone form of naringin.¹⁰ Several reports suggest that naringin acts as an antiviral, antiatherogenic, anti-inflammatory, antimutagenic, cardioprotective, cholesterol-lowering, neuroprotective, and radioprotective agent.^11–20 The antimutagenic and genotoxic effects of naringin have been reported against doxorubicin, and bleomycin in vitro and in vivo.^19–22 It also declines tumor formation in the forestomach of mice treated with benzo-a-pyrene.²³ Naringin alleviated iron-induced biochemical stress in vitro and in vivo.^24,25  It has been reported to reduce doxorubicin-induced cardiotoxicity without compromising the antineoplastic action of doxorubicin in tumor-bearing mice.¹⁵ Naringin protects against bleomycin-induced bone loss induced by sciatic neurectomy, dyslipidemia, lung fibrosis, and lipodystrophy in rats.^26,27 A single dose of 16 g/kg and 1.250 mg/kg body weight naringin once daily for 13 weeks orally has been reported to be nontoxic in rats.²⁸ Naringin is an effective treatment for obese patients because it reduces lipid profile.²⁹ Therefore, the present study was undertaken to evaluate the protective effect of naringin (7-(2-O-(6-deoxy-alpha-L-mannopyranosyl)-β-D-gluco-pyranosyloxy)-2,3-dihydro-4',5,7-trihydroxyflavone) on vinblastine-induced DNA damage in cultured V79 cells.

Chemicals

The grapefruit bioflavonoid naringin (Figure 1) was procured from Across Organics, Geel, Belgium, and vinblastine sulfate was a kind gift by Eli Lilly and Company, Indiana, Indianapolis, USA. Fetal calf serum, minimum essential medium (MEM), L-glutamine, gentamicin sulfate, and cytochalasin-B were supplied by Sigma Chemical, St Louis, USA. Dimethylsulfoxide and other routine chemicals were requisitioned from Merck India, Mumbai.

Figure 1 Chemical structure of naringin, 7-(2-O-(6-deoxy-alpha-L-mannopyranosyl)-β-D-glucopyranosyloxy)-2,3-dihydro-4',5,7-trihydroxyflavone.

Cell line and culture

Mycoplasma-free V79, Chinese hamster lung fibroblast cells were supplied by the National Center for Cell Sciences, Pune, India. The doubling time of V79 cells is about 16 to 20 h. V79 cells were grown in 25 cm² Falcon (Beckton Dickinson, USA) culture flasks filled with 5 mL MEM containing 10% fetal calf serum, 1% L-glutamine, and 50 μg mL⁻¹ gentamycin sulfate at 37°C in a CO₂ incubator (NuAire, Plymouth, MN, USA) in a humidified atmosphere of 5% CO₂ in 95% air.

Experimental

 Usually, 5X10⁵ V79 cells were inoculated into several 5 mL culture flasks and allowed to grow overnight. The flasks containing V79 cells were divided into the following groups.

Naringin: The cells of this group were treated with 0, 1, 2, 5, 10, 25, 50, 100, and 200 µg/mL naringin for 1 h.

Naringin+Vinblastine: This group of cells were exposed to 0, 1, 2, 5, 10, 25, 50, 100, and 200 µg/mL naringin for 1 h. The medium was removed and the cells were washed twice with phosphate buffered saline (PBS) before treatment with 10 µg/mL vinblastine.

Micronucleus assay

Six- hours after treatment with vinblastine the cells were washed with PBS and treated with 3 µg/mL cytochalasin-B to block the cytokinesis. Sixteen hours after cytochalasin-B treatment the cells were washed with PBS and dislodged from the culture flasks by trypsin-EDTA treatment for micronucleus assay.²¹ The micronucleus assay was carried out as described earlier with minor modifications.³⁰ The cells from individual cultures were collected in prelabelled separate tubes, centrifuged, kept in hypotonic (0.75% ammonium oxalate) solution at 37°C and centrifuged once again. The cell pellet was disturbed and a small amount of Carnoy’s (3:1 methanol and acetic acid) fixative was added to fix the cells. The cells were centrifuged and suspended in a small volume of fixative and placed on the precleaned coded slides for blinded observation. The cells were stained in acridine orange in Sorensen's buffer (pH 6.4), mounted in the buffer and observed under a Carl Zeiss Photomicroscope III (Gottingen, Germany) fitted with epifluorescence system. The mononucleate cells, binucleate cells and trinucleate cells were counted in such a way so as to get 1000 binucleate cells per culture for the presence of micronuclei in three individual cultures. The data on cell proliferation was also collected. The results were confirmed by repeating the experiment.

Statistical analysis

The data were analyzed using nonparametric Kruskal-Wallis one-way ANOVA followed by Dunn’s post hoc test to test the significance between the treatments and within the group using Origin Pro 8.5 (Origin Lab Corporation, Northampton, MA, USA) statistical software.

The results are expressed as percent micronucleated cells ± standard error of the mean (SEM) and cell proliferation (%) in Tables 1­­­ & 2 and Figures 2–5.

Figure 2 Alteration in the frequency of micronuclei in mononucleate V79 cells treated with different concentrations of naringin before exposure to 10 µg/mL vinblastine. NIN: Naringin alone.

Figure 3 Alteration in the frequency of micronuclei in binucleate V79 cells treated with different concentrations of naringin before exposure to 10 µg/mL vinblastine. NIN: Naringin alone.

Figure 4 Alteration in the frequency of micronuclei in trinucleate V79 cells treated with different concentrations of naringin before exposure to 10 µg/mL vinblastine. NIN: Naringin alone.

Figure 5 Cell proliferation kinetics of V79 cells treated with different concentrations of naringin before treatment with 10 µg/mL of vinblastine. a. Naringin alone and Naringin+vinblastine.

The frequency of mononucleate micronucleated cells declined concentration-dependently in the naringin treated group when compared to untreated cells (0 µg/mL). However, the difference among all concentrations of naringin was statistically non-significant (Table 1 and Figure 2–5). The vinblastine treatment alone significantly elevated the frequency of micronucleated mononucleate cells including the cells bearing two and more than two micronuclei (Table 1 and Figure 2). Exposure of V79 cells with 1, 2, 5, 10, 25, 50, 100, and 200 µg/mL naringin before vinblastine treatment significantly reduced the cells bearing one and two micronuclei depending on the naringin concentration, and maximum attrition in micronucleated cells was recorded at 200 µg/mL where this decline was 8.6 folds when compared to vinblastine treatment alone (Table 1).

The scoring of micronuclei in binucleate cells revealed that naringin treatment alone non-significantly reduced the spontaneous DNA damage in V79 cells at all concentrations (Table 1). Vinblastine treatment alone significantly elevated the frequency of binucleate cells with one, two, and greater than two micronuclei (Table 1). Treatment of V79 cells with naringin before vinblastine exposure led to a significant but dose-dependent decline in binucleated cells bearing one, two, and more than two micronuclei, and the greatest alleviation was detected at 200 µg/mL naringin where the micronuclei formation was 12.5 folds lower than vinblastine treatment alone. More than two micronuclei were completely absent in the 200 µg/mL naringin pretreated group (Table 1 and Figure 3).

The scoring of micronuclei in second division cells (trinucleate cells), indicated a significant rise in the micronucleated trinucleate cells after vinblastine treatment and treatment of naringin before vinblastine inoculation significantly reduced the trinucleate cells bearing one, two, and more than two micronuclei with a maximum depletion at 200 µg/mL naringin and this decline was 9.8 folds when compared to vinblastine treatment alone (Table 1 and Figure 4). The cells with two and more than two micronuclei were absent at 100 and 200 µg/mL naringin (Table 1 and Figure 4).

Cell proliferation

The naringin treatment arrested cell proliferation, especially at 2 to 100 µg/mL, and an increase was detected at 200 µg/mL however, the differences were non-significant (Table 2 & Figure 5). An almost similar pattern in cell proliferation was recorded for the Naringin+Vinblastine group (Table 2 & Figure 5)

The adverse effects of vinblastine treatment are of major concern in the survivors and natural products like naringin may help to neutralize these effects. Therefore, a study was planned to evaluate the effect of naringin on the DNA damage induced by vinblastine in V79 cells. Vinblastine produced micronuclei formation in binucleate and second-division trinucleate V79 cells in Naringin + Vinblastine groups, whereas naringin pretreatment significantly reduced the vinblastine-induced micronuclei formation.  Vinblastine is an antimitotic agent that inhibits tubulin formation and inhibits spindle formation required for mitosis.³¹ Vinblastine treatment was cytotoxic to mouse sperms.³² Vindesine, a semisynthetic derivative of vinblastine has been reported to increase the frequency of micronuclei in V79 cells dose-dependently.³³ Vinblastine has been reported to induce clastogenic effect by producing micronuclei in the bone marrow cells of mice.³⁴ Likewise, other antimitotic agent taxol also increased the micronuclei in V79 cells.³⁰

The micronuclei formation was consistently detected in mononucleate cells in both the Naringin and Naringin + Vinblastine groups. The presence of micronuclei in mononucleate cells seems to be due to the escape of these cells from cytochalasin-B labeling or the failure of mononucleate cells to divide due to high genomic loss. Very few investigators have scored micronuclei in mononucleate cells after cytokinesis blockage of cells.^35,36 The vinblastine also induced the formation of mononucleate, binucleate, and trinucleate cells with two and > two micronuclei indicating that it induced complex multiply sites of DNA damage and cells were unable to repair the complex DNA damage.³⁷ The presence of micronuclei in trinucleate cells is due to nuclear division of binucleate cells and the presence of cytochalasin B did not allow the cell separation. Since vinblastine is a tubulin inhibitor the formation of micronuclei in V79 cells is due to spindle dysfunction, lagging chromosome during cell division, chromatid condensation, fragmentation of chromosomes like acentric fragments, DNA double strand breaks generated due to pre-mitotic DNA replication stress, and mis-repair of DNA breaks.^38–40

Naringin significantly reduced the formation of vinblastine-induced micronuclei in mononucleate, binucleate, and trinucleate cells. Naringin also alleviated the spontaneous micronuclei formation in binucleate cells indicating that it protected the DNA from daily wear and tear. There are no reports on the reduction of vinblastine-induced DNA damage by naringin. However, naringin protected V79 cells against bleomycin-induced micronuclei formation and molecular DNA damage.²¹ The exact mechanism of action of naringin in reducing the vinblastine-induced DNA damage is not known. However, free radical formation and increased oxidative stress play a pivotal role in inducing DNA damage and reduction of micronuclei by naringin is due to its ability to neutralize free radical formation and reduce oxidative stress.¹² Vinblastine induces 8-hydroxy-2-deoxyguanosine (8-OHdG) DNA adducts in human lymphocytes and naringin reduces the formation of these adducts due to its ability to elevate catalase, glutathione peroxidase, glutathione-s-transferase, and superoxide dismutase and reduced glutathione (GSH) in vivo.^15,41–44 This increase in antioxidants by nariginin is due to its ability to upregulate Nrf2.^45–47 The reduction in micronuclei in the present study is due to the activation of DNA repair pathways by naringin that may seal the DNA SSBs and DNA DSBs by suppressing the overactivation of poly (ADP-ribose) polymerases (PARPs) as their overactivation triggers genomic instability.^15,48,49 The overactivation of PARP triggers nuclear translocation of NF-κB and its transcriptional activation leads to DNA damage and inhibition of NF-κB and COX-2 by naringin seems to protect against it.^50–53 The cell culture techniques do not provide information on long-term genomic instability due to the use of cytochalasin-B which provides information on DNA damage after one cell division thereafter the DNA damage gets diluted due to cell multiplication or death of highly damaged cells.

Vinblastine treatment increased the frequency of mononucleate, binucleate, and trinucleate V79 cells bearing one two, and more than two micronuclei whereas naringin pretreatment significantly reduced the mononucleate, binucleate, and trinucleate cells with micronuclei. This reduction in DNA damage in the form of micronuclei may be due to free radical scavenging, an increase in antioxidant status, and Nrf2 followed by a decline in PARP, NF-κB, and COX-II activation. The naringin treatment also efficiently reduced spontaneous DNA damage in V79 cells.  The present study demonstrates that the grapefruit bioflavonoid naringin can reduce genotoxicity in V79 cells.

This work was supported by the Indian Council of Medical Research, New Delhi, India vide grant No. 58/8/2000~BMS.

There is no conflicts of interest.

1. Duffin J. Poisoning the spindle: serendipity and discovery of the anti–tumor properties of the Vinca alkaloids. Can Bull Med Hist. 2000;17(1–2):155–192.
2. Bleehen NM, Jelliffe AM. Vinblastine sulphate in the treatment of malignant disease. Br J Anaesth. 1965;19:268–272.
3. Le Deley MC, Rosolen A, Williams DM, et al. Vinblastine in children and adolescents with high–risk anaplastic large–cell lymphoma: results of the randomized ALCL99–vinblastine trial. J Clin Oncol. 2010;28:3987–3993.
4. Ceccarelli M, Facciolà A, Taibi R, et al. The treatment of Kaposi’s sarcoma: present and future options, a review of the literature. Eur Rev Med Pharmacol Sci. 2019;23(17):7488–7497.
5. Lagerï I, Fohlin H, Enblad G, et al. Limited, but not eliminated, excess long–term morbidity in stage I–IIA Hodgkin lymphoma treated with doxorubicin, bleomycin, vinblastine, and dacarbazine and limited–field radiotherapy. J Clin Oncol. 2022;40(13):1487–1496.
6. Bröckelmann PJ,  Bühnen I, Meissner J, et al. Nivolumab and doxorubicin, vinblastine, and dacarbazine in early–stage unfavorable Hodgkin lymphoma: final analysis of the randomized German Hodgkin study group phase II NIVAHL trial. J Clin Oncol .2023;41(6):1193–1199.
7. Arora RD, Menenezes RG. Vinca alkaloid toxicity. 2023.
8. Zhang J. Flavonoids in grapefruit and commercial grapefruit juices : Concentration, distribution, and potential health benefits. Proc Fla State Hort Soc. 2007;120:288–294.
9. Diaconu C, Vlase L, Cuciureanu M, Filip L, et al. Assessment of flavonoids content in citrus juices using a LC/MS method. Farmacia 2017;65:92–96
10. Fuhr U, Kummert AL. The fate of naringin in humans: A key to grapefruit juice–drug interactions? Clin Pharmacol Ther. 1995;58(4):365–373.
11. Lee CH, Jeong TS, Choi YK, et al. Anti–atherogenic effect of citrus flavonoids, naringin and naringenin, associated with hepatic ACAT and aortic VCAM–1 and MCP–1 in high cholesterol–fed rabbits. Biochem Biophys Res Commun. 2001;284(3):681–688.
12. Jagetia GC, Venkatesha VA, Reddy TK. Naringin, a citrus flavonone, protects against radiation–induced chromosome damage in mouse bone marrow. Mutagenesis. 2003;18(4):337–343.
13. Jagetia GC, Reddy TK. The grapefruit flavanone naringin protects against the radiation–induced genomic instability in the mice bone marrow: A micronucleus study. Mutat Res – Genet Toxicol Environ Mutagen. 2002;519:37–48.
14. Jagetia GC, Reddy TK. Modulation of radiation–induced alteration in the antioxidant status of mice by naringin. Life Sci. 2005;77(7):780–794.
15. Jageta GC, Reddy TK.  The grape fruit flavonone naringin protects mice against doxorubicin–induced cardiotoxicity. J Mol Biochem. 2014;3:34–49.
16. Nie YC, Wu H, Li PB, et al. Anti–inflammatory effects of naringin in chronic pulmonary neutrophilic inflammation in cigarette smoke–exposed rats. J Med Food. 2012;15(10):894–900.
17. Kim HD, Jeong KH, Jung UJ, Kim SR. Naringin treatment induces neuroprotective effects in a mouse model of Parkinson’s disease in vivo, but not enough to restore the lesioned dopaminergic system. J Nutr Biochem. 2016;28:140–146.
18. Kwatra M, Kumar V, Jangra A, et al. Ameliorative effect of naringin against doxorubicin–induced acute cardiac toxicity in rats. Pharm Biol. 2016;54(4):637–647.
19. Jagetia GC. The citrus bioflavonoid naringin mitigates doxorubicin induced tissue toxicity: A review. J Allergy Res. 2019;1(2):25–33.
20. 20. Clementi N, Scagnolari C, D’Amore A, et al. Naringenin is a powerful inhibitor of SARS–CoV–2 infection in vitro. Pharmacol Res 2021;163:105255.
21. Jagetia A, Jagetia GC, Jha S. Naringin, a grapefruit flavanone, protects V79 cells against the bleomycin–induced genotoxicity and decline in survival. J Appl Toxicol. 2007;27(2):122–132.
22. Jagetia GC, Reddy TK. The grape fruit bioflavonoid naringin protects against the doxorubicin–induced micronuclei formation in mouse bone marrow. Int J Mol Biol. 2016;1(1):48–55.
23. Jagetia GC, Reddy TK. Chemopreventive effect of naringin on the benzo (a) pyrene–induced forestomach carcinoma in mice. Int J Cancer Prev. 2004;6:429–444
24. Jagetia GC, Reddy TK, Venkatesha VA, Kedlaya R. Influence of naringin on ferric iron induced oxidative damage in vitro. Clin Chim Acta. 2004;347(1–2):189–197.
25. Jagetia GC, Reddy TK. Alleviation of iron induced oxidative stress by the grape fruit flavanone naringin in vitro. Chem Biol Interact. 2011;190(2–3):121–128.
26. Turgut NH, Kara H, Elagoz S, et al. The protective effect of naringin against bleomycin–induced pulmonary fibrosis in Wistar rats. Pulm Med. 2016; 2016:7601393.
27. Ma X, Lv J, Sun X, et al.  Naringin ameliorates bone loss induced by sciatic neurectomy and increases Semaphorin 3A expression in denervated bone. Sci Rep. 2016;6:24562.
28. Li P, Wang S, Guan X, et al. Acute and 13 weeks subchronic toxicological evaluation of naringin in Sprague–Dawley rats. Food Chem Toxicol. 2013;60:1–9.
29. Barajas–Vega JL, Raffoul–Orozco AK, Hernandez–Molina D, et al. Naringin reduces body weight, plasma lipids and increases adiponectin levels in patients with dyslipidemia. Int J Vitam Nutr Res. 2020;9:1–7.
30. Jagetia GC, Adiga SK. Influence of various concentrations of taxol on cell survival, micronuclei induction, and LDH activity in cultured V79 cells. Cancer Lett. 1995;96(2):195–200.
31. Chagas CM, Alisaraie L. Metabolites of vinca alkaloid vinblastine: Tubulin binding and activation of nausea–associated receptors. ACS omega. 2019;4(6):9784–9799.
32. Jagetia GC, Krishnamurthy H, Jyothi P. Evaluation of cytotoxic effects of different doses of vinblastine on mouse spermatogenesis by flow cytometry. Toxicology. 1996;112(3):227–36.
33. Jagetia GC, Adiga SK. Influence of vindesine exposure on the micronucleus formation and cell survival in V79 cells. Mutat Res – Fundam Mol Mech Mutagen. 1998;421(1):55–63.
34. Jagetia GC, Jacob PS. Vinblastine treatment induces dose–dependent increases in the frequency of micronuclei in mouse bone marrow. Mutat Res Toxicol. 1992;280(2):87–92.
35. Rosefort C, Fauth E, Zankl H. Micronuclei induced by aneugens and clastogens in mononucleate and binucleate cells using the cytokinesis block assay. Mutagenesis. 2004;19(4):277–284.
36. Lourenço ED, Do Amaral VS, Lehmann M. Micronuclei induced by reverse transcriptase inhibitors in mononucleated and binucleated cells as assessed by the cytokinesis–block micronucleus assay. Genet Mol Biol. 2010;33(4):756–760.
37. Nickoloff JA, Sharma N, Taylor L. Clustered DNA double–strand breaks: Biological effects and relevance to cancer radiotherapy. Genes (Basel). 2020;11(1):99.
38. Ye CJ, Sharpe Z, Alemara S. Micronuclei and genome chaos: Changing the system inheritance. Genes (Basel). 2019;10(5):366.
39. Gómez–Herreros F. DNA Double strand breaks and chromosomal translocations induced by DNA topoisomerase II. Front Mol Biosci. 2019;6:141.
40. McKie SJ, Maxwell A, Neuman KC. Mapping DNA topoisomerase binding and cleavage genome wide using next‐generation sequencing techniques. Genes (Basel). 2020;11(1):92.
41. Mhaidat NM, Alzoubi KH, Khabour OF. Assessment of genotoxicity of vincristine, vinblastine and vinorelbine in human cultured lymphocytes: a comparative study. Balkan J Med Genet. 2016;19(1):13–20.
42. Jagetia GC, Lalnuntluangi V. The citrus flavanone naringin enhances antioxidant status in the albino rat liver treated with doxorubicin. Biochem Mol Biol J. 2016;2(2):9.
43. Jagetia GC, Lalrinengi C. Treatment of mice with naringin alleviates the doxorubicin–induced oxidative stress in the liver of Swiss albino mice. MOJ Anat Physiol. 2017;4(2):267‒278.
44. Jagetia GC, Lalrinengi C. Naringin, a grape fruit bioflavonoid protects mice bone marrow cells against the doxorubicin–induced oxidative stress. SOJ Biochem. 2017;3(1):1–19.
45. Manna K, Khan A, Biswas S. Naringin ameliorates radiation–induced hepatic damage through modulation of Nrf2 and NF–κB pathways. RSC Adv. 2016;6(27):23058–23073.
46. Lv Z, Wu W, Ge S. Naringin protects against perfluorooctane sulfonate–induced liver injury by modulating NRF2 and NF–κB in mice. Int Immunopharmacol. 2018;65:140–147.
47. Zhai X, Dai T, Chi Z. Naringin alleviates acetaminophen–induced acute liver injury by activating Nrf2 via CHAC2 upregulation. Environ Toxicol. 2022;37(6):1332–1342.
48. Pacher P, Szabó C. Role of poly(ADP–ribose) polymerase 1 (PARP–1) in cardiovascular diseases: The therapeutic potential of PARP inhibitors. Cardiovasc. Drug Rev. 2007;25(3):235–260.
49. Palazzo L, Mikolčević P, Mikoč A, Ahel I. ADP–ribosylation signalling and human disease. Open Biol. 2019;9(4):190041.
50. Vuong B, Hogan–Cann ADJ, Alano CC. NF–κB transcriptional activation by TNF–α requires phospholipase C, extracellular signal–regulated kinase 2 and poly(ADP–ribose) polymerase–1. J Neuroinflammation. 2015;12:229.
51. Zeng L, Zhen Y, Chen Y. Naringin inhibits growth and induces apoptosis by a mechanism dependent on reduced activation of NF–κB/COX–2–caspase–1 pathway in HeLa cervical cancer cells. Int J Oncol. 2014;45(5):1929–1936.
52. Vabeiryureilai M, Lalrinzuali K, Jagetia GC. NF–κB and COX–2 repression with topical application of hesperidin and naringin hydrogels augments repair and regeneration of deep dermal wounds. Burns. 2022;48(1):132–145.
53. Gao G, Chang F, Zhang T. Naringin protects against interleukin 1β (IL–1β)–induced human nucleus pulposus cells degeneration via downregulation nuclear factor kappa B (NF–κB) pathway and p53 expression. Med Sci Monit. 2019;25:9963–9972.