Rosmarinic acid exerts anti-inflammatory effect and relieves oxidative stress via Nrf2 activation in carbon tetrachloride-induced liver damage

Yue-hong Lu; Yue Hong; Tian-yang Zhang; You-xia Chen; Zhao-jun Wei; Chun-yan Gao

doi:10.29219/fnr.v66.8359

Yue-hong Lu College of Bioscience and Bioengineering, North Minzu University, Yinchuan, China
Yue Hong School of Public Health, Dali University, Dali, China
Tian-yang Zhang School of Public Health, Dali University, Dali, China
You-xia Chen School of Public Health, Dali University, Dali, China
Zhao-jun Wei College of Bioscience and Bioengineering, North Minzu University, Yinchuan, China; and School of Food Science and Engineering, Hefei University of Technology, Hefei, China
Chun-yan Gao College of Bioscience and Bioengineering, North Minzu University, Yinchuan, China

DOI: https://doi.org/10.29219/fnr.v66.8359

Keywords: Rosmarinic acid; Hepatoprotective effect; Nrf2 pathway; Oxidative stress; Inflammation

Abstract

Background: Rosmarinic acid (RA) has biological and pharmaceutical properties and shows hepatoprotective potential. However, the hepatoprotective mechanism of RA needs to be further elucidated in vivo and in vitro.

Objective: This study was aimed to evaluate the protective effect of RA on carbon tetrachloride (CCl₄)-induced liver injury and elucidate the hepatoprotective mechanism of RA in vivo and in vitro.

Design: In vivo, the mice were orally administrated with RA (10, 20, and 40 mg/kg bw) daily for 28 consecutive days, and 1% CCl₄ (5 mL/kg bw, dissolved in peanut oil) was used to induce liver injury. In vitro, the big rat liver (BRL) hepatocytes were pretreated with RA (0.2, 0.4, and 0.8 mg/mL) for 3 h, and then the hepatocytes were treated with CC1₄ (final concentration, 14 mM) for 3 h to induce cell injury. The related indexes, including hepatic function, oxidative stress, protein expression of nuclear-factor erythroid 2-related factor 2 (Nrf2) pathway, inflammation, histopathological change, hepatocyte apoptosis, and mitochondrial membrane potential, were evaluated.

Results: Oral administration of RA to mice considerably decreased the CCl₄-induced elevation of serum alanine aminotransferase (ALT), alkaline phosphatase (ALP), triacylglycerols (TG), total cholesterol (TC), total bilirubin (TBIL), hepatic reactive oxygen species (ROS), malondialdehyde (MDA), nitric oxide (NO), 8-hydroxydeoxyguanosine (8-OHdG), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-8 (IL-8). RA also increased the levels of hepatic glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT) and the protein expressions of Nrf2, quinine oxidoreductase (NQO1), and heme oxygenease-1 (HO-1). Histopathological examinations indicated that RA (20 and 40 mg/kg bw) alleviated the liver tissue injury induced by CCl₄. Moreover, RA inhibited the hepatocyte apoptosis caused by CCl₄ based on TUNEL assay. In vitro, RA pretreatment remarkably recovered the cell viability and reduced the CCl₄-induced elevation of AST, ALT, lactate dehydrogenase (LDH), ROS, and 8-OHdG. Immunohistochemistry staining demonstrated that pretreatment with RA markedly inhibited the expression of IL-6, inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and Caspase-3 in CCl₄-treated hepatocytes. Additionally, RA pretreatment significantly decreased the elevation of mitochondrial membrane potential in CCl₄-treated hepatocytes.

Conclusions: RA exerted a protective effect against CCl₄-induced liver injury in mice through activating Nrf2 signaling pathway, reducing antioxidant damage, suppressing inflammatory response, and inhibiting hepatocyte apoptosis. RA could attenuate BRL hepatocyte ROS production, DNA oxidative damage, inflammatory response, and apoptosis induced by CCl₄ exposure.

Downloads

Download data is not yet available.

References

1.
Xiao J, Wang F, Wong NK, He J, Zhang R, Sun R, et al. Global liver disease burdens and research trends: analysis from a China perspective. J Hepatol 2019; 71(1): 212–21. doi: 10.1016/j.jhep.2019.03.004

2.
Bhatia V, Singhal A, Panda SK, Acharya SK, A 20-year single-center experience with acute liver failure during pregnancy: is the prognosis really worse? Hepatology 2008; 48(5): 1577–85. doi: 10.1002/hep.22493

3.
Byass P. The global burden of liver disease: a challenge for methods and for public health. BMC Med 2014; 12: 159. doi: 10.1186/s12916-014-0159-5

4.
Sun F, Hamagawa E, Tsutsui C, Ono Y, Kojo S. Evaluation of oxidative stress during apoptosis and necrosis caused by carbon tetrachloride in rat liver. BBA-Mol Basis Dis 2001; 1535(2): 186–91. doi: 10.1016/S0925-4439(00)00098-3

5.
Bayram HM, Majoo FM, Ozturkcan A. Polyphenols in the prevention and treatment of non-alcoholic fatty liver disease: an update of preclinical and clinical studies. Clin Nutr ESPEN 2021; 44: 1–14. doi: 10.1016/j.clnesp.2021.06.026

6.
Maalej A, Mahmoudi A, Bouallagui Z, Fki I, Marrekchi R, Sayadi S. Olive phenolic compounds attenuate deltamethrin-induced liver and kidney toxicity through regulating oxidative stress, inflammation and apoptosis. Food Chem Toxicol 2017; 106: 455–65. doi: 10.1016/j.fct.2017.06.010

7.
Lu YH, Tian CR, Gao CY, Wang WJ, Yang WY, Kong X, et al. Protective effect of free phenolics from Lycopus lucidus Turcz. root on carbon tetrachloride-induced liver injury in vivo and in vitro. Food Nutr Res 2018; 62: 1398. doi: 10.29219/fnr.v62.1398

8.
Quitete FT, Almeida Santos GM, de Oliveira Ribeiro L, Aguiar da Costa C, Freitas SP, Martins da Matta V, et al. Phenolic-rich smoothie consumption ameliorates non-alcoholic fatty liver disease in obesity mice by increasing antioxidant response. Chem-Biol Interact 2021; 336: 109369. doi: 10.1016/j.cbi.2021.109369

9.
Marchev AS, Vasileva LV, Amirova KM, Savova MS, Koycheva IK, Balcheva-Sivenova ZP, et al. Rosmarinic acid – from bench to valuable applications in food industry. Trends Food Sci Tech 2021; 177: 182–93. doi: 10.1016/j.tifs.2021.03.015

10.
Fachel FNS, Prá MD, Azambuja JH, Endres M, Braganhol E. Glioprotective effect of chitosan-coated rosmarinic acid nanoemulsions against lipopolysaccharide-induced inflammation and oxidative stress in rat astrocyte primary cultures. Cell Mol Neurobiol 2020; 40(1): 123–39. doi: 10.1007/s10571-019-00727-y

11.
Fachel FNS, Schuh RS, Veras KS, Bassani VL, Koester LS, Henriques AT, et al. An overview of the neuroprotective potential of rosmarinic acid and its association with nanotechnology-based delivery systems: a novel approach to treating neurodegenerative disorders. Neurochem Int 2019; 122: 47–58. doi: 10.1016/j.neuint.2018.11.003

12.
Amoah SK, Sandjo LP, Kratz JM, Biavatti MW. Rosmarinic acid-pharmaceutical and clinical aspects. Planta Med 2016; 82(5): 388–406. doi: 10.1055/s-0035-1568274

13.
Luo C, Zou L, Sun H, Peng J, Gao C, Bao L, et al. A review of the anti-inflammatory effects of Rosmarinic acid on inflammatory diseases. Front Pharmacol 2020; 11: 153. doi: 10.3389/fphar.2020.00153

14.
Chhabra P, Chauhan G, Kumar A. Augmented healing of full thickness chronic excision wound by rosmarinic acid loaded chitosan encapsulated graphene nanopockets. Drug Dev Ind Pharm 2020; 46(6): 878–88. doi: 10.1080/03639045.2020.1762200

15.
Wang SJ, Chen Q, Liu MY, Yu HY, Wang T. Regulation effects of rosemary (Rosmarinus officinalis Linn.) on hepatic lipid metabolism in OA induced NAFLD rats. Food Funct 2019; 10(11): 7356–65. doi: 10.1039/C9FO01677E

16.
Li Z, Feng H, Wang Y, Shen B, Tian Y, Wu L, et al. Rosmarinic acid protects mice from lipopolysaccharide/d-galactosamine-induced acute liver injury by inhibiting MAPKs/NF-κB and activating Nrf2/HO-1 signaling pathways. Int Immunopharmacol 2019; 67: 465–72. doi: 10.1016/j.intimp.2018.12.052

17.
Lin SY, Wang YY, Chen WY, Liao SL, Chou ST, Yang CP, et al. Hepatoprotective activities of rosmarinic acid against extrahepatic cholestasis in rats. Food Chem Toxicol 2017; 108: 214–23. doi: 10.1016/j.fct.2017.08.005

18.
Lou K, Yang M, Erdan D, Zhao J, Cong Y, Zhang R, et al. Rosmarinic acid stimulates liver regeneration through the mTOR pathway. Phytomedicine 2016; 23(13): 1574–82. doi: 10.1016/j.phymed.2016.09.010

19.
Weber LW, Boll M, Stampfl A. Hepatotoxicity and mechanism of action of haloalkanes: carbon tetrachloride as a toxicological model. Crit Rev Toxicol 2003; 33: 105–36. doi: 10.1080/713611034

20.
Cho BO, Ryu HW, So Y, Jin CH, Baek JY, Park KH, et al. Hepatoprotective effect of 2,3-dehydrosilybin on carbon tetrachloride-induced liver injury in rats. Food Chem 2013; 138: 107–15. doi: 10.1016/j.foodchem.2012.10.026

21.
Huang QF, Zhang SJ, Zheng L, He M, Huang RB, Lin X. Hepatoprotective effects of total saponins isolated from Taraphochlamys affinis against carbon tetrachloride induced liver injury in rats. Food Chem Toxicol 2012; 50: 713–8. doi: 10.1016/j.fct.2011.12.009

22.
Subash P, Gurumurthy P, Sarasabharathi A, Cherian KM. Urinary 8-ohdg: a marker of oxidative stress to DNA and total antioxidant status in essential hypertension with south Indian population. Indian J Clin Bioche 2010; 25(2): 127–32. doi: 10.1007/s12291-010-0024-z

23.
Szymonik-Lesiuk S, Czechowska G, Stryjecka-Zimmer M, Slomka M, Madro A, Celinski K, et al. Catalase, superoxide dismutase, and glutathione peroxidase activities in various rat tissues after carbon tetrachloride intoxication. J Hepatobiliary Pancreat Surg 2003; 10: 309–15. doi: 10.1007/s00534-002-0824-5

24.
Gao CY, Tian CR, Zhou R, Zhang RG, Lu YH. Phenolic composition, DNA damage protective activity and hepatoprotective effect of free phenolic extract from Sphallerocarpus gracilis seeds. Int Immunopharmacol 2014; 20: 238–47. doi: 10.1016/j.intimp.2014.03.002

25.
Lee KJ, Choi HJ, Jeong HG. Hepatoprotective and antioxidant effects of the coffee diterpenes kahweol and cafestol on carbon tetrachloride-induced liver damage in mice. Food Chem Toxicol 2007; 45: 2118–25. doi: 10.1016/j.fct.2007.05.010

26.
Klaassen CD, Reisman SA. Nrf2 the rescue: effects of the antioxidative/electrophilic response on the liver. Toxicol Appl Pharmacol 2010; 244: 57–65. doi: 10.1016/j.taap.2010.01.013

27.
Vasiliou V, Ross D, Nebert DW. Update of the NAD(P)H:quinone oxidoreductase (NQO1) gene family. Hum Genomics 2006; 2: 329–35. doi: 10.1186/1479-7364-2-5-329

28.
Su C, Xia X, Shi Q, Song X, Fu J, Xiao C, et al. Neohesperidin dihydrochalcone against CCl₄-induced hepatic injury through different mechanisms: the implication of free radical scavenging and Nrf2 activation. J Agr Food Chem 2015; 63(22): 5468–75. doi: 10.1021/acs.jafc.5b01750

29.
Niu XF, Liu F, Li WF, Zhi WB, Yao Q, Zhao JM, et al. Hepatoprotective effect of fraxin against carbon tetrachloride-induced hepatotoxicity in vitro and in vivo through regulating hepatic antioxidant, inflammation response and the MAPK-NF-κB signaling pathway. Biomed Pharmacother 2017; 95: 1091–102. doi: 10.1016/j.biopha.2017.09.029

30.
Morio LA, Chiu H, Sprowles KA, Zhou P, Heck DE, Gordon MK, et al. Distinct roles of tumor necrosis factor-alpha and nitric oxide in acute liver injury induced by carbon tetrachloride in mice. Toxicol Appl Pharm 2001; 172: 44–51. doi: 10.1006/taap.2000.9133

31.
Zhang S, Lu BN, Han X, Xu LN, Qi Y, Yin LH, et al. Protection of the flavonoid fraction from Rosa laevigata Michx fruit against carbon tetrachloride-induced acute liver injury in mice. Food Chem Toxicol 2013; 55: 60–9. doi: 10.1016/j.fct.2012.12.041

32.
Kim H, Park J, Lee K, Lee D, Kwak J, Kim YS, et al. Ferulic acid protects against carbon tetrachloride-induced liver injury in mice. Toxicol 2011; 282: 104–11. doi: 10.1016/j.tox.2011.01.017

33.
Li Y, Jiang X, Xu HJ, Lv JY, Zhang GN, Dou XJ, et al. Acremonium terricola culture plays anti-inflammatory and antioxidant roles by modulating MAPK signaling pathways in rats with lipopolysaccharide-induced mastitis. Food Nutr Res 2020; 64: 3649. doi: 10.29219/fnr.v64.3649

34.
Ross D. Quinone reductases multitasking in the metabolic world. Drug Metab Rev 2004; 36(3–4): 639–54. doi: 10.1081/DMR-200033465

35.
Hu L, Li LR, Xu DM, Xia XM, Pi RX, Xu D, et al. Protective effects of neohesperidin dihydrochalcone against carbon tetrachloride-induced oxidative damage in vivo and in vitro. Chem-Biol Interact 2014; 213: 51–9. doi: 10.1016/j.cbi.2014.02.003

36.
Knockaert L, Berson A, Ribault C, Prost PE, Fautrel A, Pajaud J, et al. Carbon tetrachloride-mediated lipid peroxidation induces early mitochondrial alterations in mouse liver. Lab Invest 2012; 92: 396–410. doi: 10.1038/labinvest.2011.193

37.
Tian ZX, Jia HY, Jin YZ, Wang MH, Kou JJ, Wang CL, et al. Chrysanthemum extract attenuates hepatotoxicity via inhibiting oxidative stress in vivo and in vitro. Food Nutr Res 2019; 63: 1667. doi: 10.29219/fnr.v63.1667

38.
Lu YH, Tian CR, Gao CY, Zhang RG. Nutritional profiles, phenolics and DNA damage protective effect of Lycopus lucidus Turcz. root at different harvest times. Int J Food Prop 2017; 20: S3062–77. doi: 10.1080/10942912.2017.1402030