Isopulegol mitigates high glucose-induced oxidative stress in HK-2 cells via activation of the Nrf2/ARE pathway

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Research Paper 04/11/2025
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Isopulegol mitigates high glucose-induced oxidative stress in HK-2 cells via activation of the Nrf2/ARE pathway

Mathew Maria Caroline Rebellow, Ravishankar Sarumathi, Chandrasekaran Sankaranarayanan
Int. J. Biosci. 27(5), 6-15, November 2025.
Copyright Statement: Copyright 2025; The Author(s).
License: CC BY-NC 4.0

Abstract

Diabetic nephropathy (DN) represents a major contributor to chronic kidney disease, primarily resulting from oxidative stress and injury to renal tubules triggered by persistent hyperglycemia. This study evaluated the protective effects of isopulegol (IPG), a monoterpene on high glucose (HG)-induced cytotoxicity in HK-2 cell lines. Cytotoxicity assays (MTT and CCK-8) revealed the effect of IPG on cell viability in HK-2 cells under HG conditions, with an IC₅₀ of 45 µM. Treatment with IPG significantly improved the enzymatic antioxidants [superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx)] and glutathione (GSH) level, while reducing lipid peroxidation (LPO). Additionally, IPG promoted expression of Nrf2, activating the antioxidant response element (ARE) pathway. These findings suggest that IPG confers cytoprotective effects by enhancing antioxidant defense, reducing oxidative stress, highlighting its therapeutic potential in preventing oxidative renal damage in diabetic nephropathy.

Al Kury LT, Abdoh A, Ikbariah K, Sadek B, Mahgoub M. 2022. In vitro and in vivo antidiabetic potential of monoterpenoids: An update. Molecules 27(1), 182. https://doi.org/10.3390/molecules27010182

Alicic RZ, Rooney MT, Tuttle KR. 2017. Diabetic kidney disease: challenges, progress, and possibilities. Clinical Journal of the American Society of Nephrology 12(12), 2032–2045.

Bellezza I, Giambanco I, Minelli A, Donato R. 2018. Nrf2–Keap1 signaling in oxidative and reductive stress. Biochimica et Biophysica Acta (Molecular Cell Research) 1865(5), 721–733.

Chen W, Zhao M, Zhao S, Lu Q, Ni L, Zou C, Lu L, Xu X, Guan H, Zheng Z, Qiu Q. 2017. Activation of the TXNIP/NLRP3 inflammasome pathway contributes to inflammation in diabetic retinopathy: a novel inhibitory effect of minocycline. Inflammation Research 66(2), 157–166.

Darenskaya M, Kolesnikov S, Semenova N, Kolesnikova L. 2023. Diabetic nephropathy: Significance of determining oxidative stress biomarkers in the pathogenesis and treatment. International Journal of Molecular Sciences 24(15), 12391.

Ellman GL. 1959. Tissue sulfhydryl groups. Archives of Biochemistry and Biophysics 82(1), 70–77.

Fan J, Schiemer T, Vaska A, Jahed V, Klavins K. 2024. Cell via cell viability assay changes cellular metabolic characteristics by intervening with glycolysis and pentose phosphate pathway. Chemical Research in Toxicology 37(2), 208–211.

Finkel T. 2011. Signal transduction by reactive oxygen species. Journal of Cell Biology 194(1), 7–15.

Fonsêca DV, Salgado PRR, Silva-Alves KS, Siqueira RMP, Almeida RN, Sousa DP. 2020. Pharmacological potential of the monoterpene isopulegol in inflammatory and oxidative stress conditions. Phytomedicine 67, 153164. DOI:10.1016/j.phymed.2019.153164

Forbes JM, Thorburn DR. 2018. Mitochondrial dysfunction in diabetic kidney disease. Nature Reviews Nephrology 14(5), 291–312.

Habig WH, Pabst MJ, Jakoby WB. 1974. Glutathione S-transferases: The first enzymatic step in mercapturic acid formation. Journal of Biological Chemistry 249(22), 7130–7139.

Hashemi M, Zandieh MA, Ziaolhagh S. 2023. Nrf2 signaling in diabetic nephropathy, cardiomyopathy and neuropathy: Therapeutic targeting, challenges and future perspectives. European Journal of Pharmacology 947(1), 175667.

Hernandez LF, Eguchi N, Whaley D, Alexander M, Tantisattamo E, Ichii H. 2022. Anti-oxidative therapy in diabetic nephropathy. Frontiers in Pharmacology 13(1), 925406.

Jiang M, Zhang H, Zhai L, Ye B, Cheng Y, Zhai C. 2017. ALA/LA ameliorates glucose toxicity on HK-2 cells by attenuating oxidative stress and apoptosis through the ROS/p38/TGF-β1 pathway. Lipids in Health and Disease 16(1), 216.

Jin Q, Liu T, Qiao Y, Liu D, Yang L, Mao H, Ma F, Wang Y, Peng L, Zhan Y. 2023. Oxidative stress and inflammation in diabetic nephropathy: role of polyphenols. Frontiers in Immunology 14(1), 1185317.

Kakkar P, Das B, Viswanathan PN. 1984. A modified spectrophotometric assay of superoxide dismutase. Indian Journal of Biochemistry & Biophysics 21(2), 130–132.

Kalaivani K, Sankaranarayanan C. 2019. Modulatory effect of isopulegol on hepatic key enzymes of glucose metabolism in high-fat diet/streptozotocin-induced diabetic rats. Archives of Physiology and Biochemistry 127(4), 318–326.

Kehrer JP, Klotz LO. 2015. Free radicals and related reactive species as mediators of tissue injury and disease: implications for health. Critical Reviews in Toxicology 45(9), 765–798.

Kim HJ, Vaziri ND. 2010. Contribution of impaired Nrf2-Keap1 pathway to oxidative stress and inflammation in chronic renal failure. American Journal of Physiology–Renal Physiology 298(3), F662–F671.

Kruger NJ. 2009. The Bradford method for protein quantitation. In: Walker, J.M. (Ed.), The Protein Protocols Handbook. Humana Press, Totowa, NJ, pp. 17–24.

Li KX, Ji MJ, Sun HJ. 2021. An updated pharmacological insight of resveratrol in the treatment of diabetic nephropathy. Gene 780(1), 145532.

Lin Y, Zhong L, Li H, Xu Y, Li X, Zheng D. 2020. Psoralen alleviates high glucose-induced HK-2 cell injury by inhibition of Smad2 signaling via upregulation of microRNA-874. BMC Pharmacology and Toxicology 21(1), 40.

Lu Y, Zhong Y, Liu H, Song W, Xu M, Liu Y. 2019. Activation of Nrf2 ameliorates oxidative stress and mitochondrial dysfunction in diabetic nephropathy. Journal of Cellular Physiology 234(10), 16154–16165.

Ma Q. 2013. Role of Nrf2 in oxidative stress and toxicity. Annual Review of Pharmacology and Toxicology 53(1), 401–426.

Ma Z, Zhu L, Wang S, Guo X, Sun B, Wang Q, Chen L. 2022. Berberine protects diabetic nephropathy by suppressing epithelial-to-mesenchymal transition involving the inactivation of the NLRP3 inflammasome. Renal Failure 44(1), 923–932.

Mosmann T. 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of Immunological Methods 65(1–2), 55–63.

Niehaus WG, Samuelsson B. 1968. Formation of malonaldehyde from phospholipid arachidonate during microsomal lipid peroxidation. European Journal of Biochemistry 6(1), 126–130.

Ou M-C, Liu Y-H, Sun Y-W, Chan C-F. 2015. The composition, antioxidant and antibacterial activities of cold-pressed and distilled essential oils of Citrus paradisi and Citrus grandis (L.) Osbeck. Journal of Chemistry, 804091. https://doi.org/10.1155/2015/804091

Rotruck JT, Pope AL, Ganther HE, Swanson AB, Hafeman DG, Hoekstra WG. 1973. Selenium: Biochemical role as a component of glutathione peroxidase. Science 179(4073), 588–590.

Roy S, Ghosh A, Majie A, Karmakar V, Das S, Dinda SC, Bose A, Gorain B. 2024. Terpenoids as potential phytoconstituent in the treatment of diabetes: From preclinical to clinical advancement. Phytomedicine: International Journal of Phytotherapy and Phytopharmacology 129(1), 155638.

Salem MZM, Elansary HO, Ali HM, El-Settawy AA, Elshikh MS, Abdel-Salam EM, Skalicka-Woźniakp K. 2018. Bioactivity of essential oils extracted from Cupressus macrocarpa branchlets and Corymbia citriodora leaves grown in Egypt. BMC Complementary and Alternative Medicine 18(1), 2085. https://doi.org/10.1186/s12906-018-2085-0

Sinha AK. 1972. Colorimetric assay of catalase. Analytical Biochemistry 47(2), 389–394.

Sousa LR, Barbosa CO, Viana NR, Coelho AG, Barros DSL, Martins MCC, Arcanjo DDR. 2023. Use of monoterpenes as potential therapeutics in diabetes mellitus: A prospective review. Advances in Pharmacological and Pharmaceutical Sciences 2023(1), 1512974.

Suliman Y, Omar A, Ali D, Alarifi S, Harrath AH, Mansour L, Alwasel SH. 2015. Evaluation of cytotoxic, oxidative stress, proinflammatory and genotoxic effect of silver nanoparticles in human lung epithelial cells. Environmental Toxicology 30(2), 149–160.

Tan SM, De Haan JB. 2014. Combating oxidative stress in diabetic complications with Nrf2 activators: How much is too much? Redox Report 19(3), 107–117.

Tesauro M, Nisticò S, Noce A. 2015. The possible role of glutathione-S-transferase activity in diabetic nephropathy. International Journal of Immunopathology and Pharmacology 28(1), 129–133.

Tonelli C, Chio IIC, Tuveson DA. 2018. Transcriptional regulation by Nrf2. Antioxidants and Redox Signaling 29(17), 1727–1745.

Tong Y, Shan Y. 2019. Ethyl vanillin protects against kidney injury in diabetic nephropathy by inhibiting oxidative stress and apoptosis. Oxidative Medicine and Cellular Longevity 2019(1), 2129350.

Uddandrao VVS, Brahmanaidu P, Ravindarnaik R, Suresh P, Vadivukkarasi S, Saravanan G. 2019. Restorative potentiality of S-allylcysteine against diabetic nephropathy through attenuation of oxidative stress and inflammation. European Journal of Nutrition 58(6), 2425–2437.

Wang J, Guo HM. 2019. Astragaloside IV ameliorates high glucose induced HK-2 cell apoptosis and oxidative stress by regulating the Nrf2/ARE signaling pathway. Experimental and Therapeutic Medicine 17(6), 4409–4416.

Xue R, Xiao H, Kumar V. 2024. The molecular mechanism of renal tubulointerstitial inflammation promoting diabetic nephropathy. International Journal of Nephrology and Renovascular Disease 17(1), 45–59.

Yang Y, Ma X, Dai W, Su Z, Yang Y. 2017. Mechanisms of diabetic kidney disease: Oxidative stress and epigenetic perspective. Cell Signalling 44(1), 31–37.

Yao L, Liang X, Qiao Y, Chen B, Wang P, Liu Z. 2022. Mitochondrial dysfunction in diabetic tubulopathy. Metabolism 131(1), 155195.

Zhan M, Usman IM, Sun L, Kanwar YS. 2021. Disruption of renal tubular mitochondrial quality control by Myo-inositol oxygenase in diabetic kidney disease. Journal of the American Society of Nephrology 32(3), 685–700.

Zhang J, Zhao X, Zhu H, Wang J, Ma J, Gu M. 2018. Crocin protects the renal tubular epithelial cells against high glucose-induced injury and oxidative stress via regulation of the SIRT1/Nrf2 pathway. Iranian Journal of Basic Medical Sciences 21(2), 193–197.

Zhang L, Chen Z, Gong W, Zou Y, Xu F, Chen L. 2018. Paeonol ameliorates diabetic renal fibrosis through promoting the activation of the Nrf2/ARE pathway via up-regulating Sirt1. Frontiers in Pharmacology 9(1), 512.

Zhao Y, Wang Y. 2023. Nrf2 signaling in diabetic nephropathy, cardiomyopathy, and other diabetic complications. European Journal of Pharmacology 948(1), 175687.

Zuo MH, Tang J, Xiang MM. 2018. Clinical observation of the reduced glutathione in the treatment of diabetic chronic kidney disease. Journal of Cellular Biochemistry 1(1), 1–9.

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