Quercetin Ameliorates Lipid and Apolipoprotein Profile in High-Dose Glucocorticoid Treated Rats

Abstract

Background

Glucocorticoids (GCs) are widely prescribed for the treatment of numerous clinical disorders due to their anti-inflammatory and immune-modulatory properties and one of the most common untoward effects of these drugs is dyslipidemia.

Objective

To evaluate the effect of quercetin, a plant-derived flavonoid, on the lipid profile of high-dose glucocorticoid treated rats.

Methods

A total of 32 Sprague-Dawley rats, were randomly distributed among four groups (8 rats per group) and treated for 6 weeks with one of the following: (i) normal saline; (ii) 40 mg/kg methylprednisolone sodium succinate (MP); (iii) MP + 50 mg/kg quercetin; (iv) MP + 150 mg/kg quercetin. MP was injected subcutaneously, and quercetin was administered by oral gavage 3 days a week. At the end of the study, the animals’ lipid profile was measured by enzymatic kits. Data were analyzed and statistical significance was set at p<0.05.

Results

The mean serum total cholesterol (TC), triglyceride (TG) and LDL levels were drastically increased in GC-treated animals compared with the control group. Both doses of quercetin (50 and 150 mg/kg) ameliorated TC (43% and 45%), LDL (56% and 56%) and TG (46% and 55% respectively). Apo B/A1 ratio decreased more than 20% following quercetin intake and the decline in TC/HDL, TG/HL, LDL/HDL ratios were significant.

Conclusions

These data suggest that quercetin intake with both doses of 50 and 150 mg/kg could be considered as a protective agent for glucocorticoid-induced dyslipidemia. (Arq Bras Cardiol. 2020; 115(1):102-108.)

Rats, Sprague-Dawley; Anti-Inflammatory Agents; Quercetin; Glucocorticoids; Dyslipidemias; Triglycerides; Cholesterol

Resumo

Fundamento

Os glicocorticóides (GCs) são amplamente prescritos para o tratamento de numerosos distúrbios clínicos devido às suas propriedades anti-inflamatórias e imunomoduladoras, e um dos efeitos indesejáveis mais comuns desses medicamentos é a dislipidemia.

Objetivo

Avaliar o efeito da quercetina, um flavonoide derivado de plantas, no perfil lipídico de ratos tratados com glicocorticóides em altas doses.

Métodos

Um total de 32 ratos Sprague-Dawley foram distribuídos aleatoriamente entre quatro grupos (8 ratos por grupo) e tratados por 6 semanas com uma das seguintes opções : (i) solução salina normal; (ii) 40 mg/kg de succinato sódico de metilprednisolona (MP); (iii) MP + 50 mg/kg de quercetina; (iv) MP + 150 mg/kg de quercetina. O MP foi injetado por via subcutânea e a quercetina foi administrada por gavagem oral 3 dias por semana. No final do estudo, o perfil lipídico dos animais foi medido através de kits enzimáticos. Os dados foram analisados e a significância estatística foi estabelecida em p <0,05.

Resultados

Os níveis séricos médios de colesterol total (CT), triglicerídeos (TG) e LDL aumentaram drasticamente em animais tratados com GC em comparação com o grupo controle. Ambas as doses de quercetina (50 e 150 mg/kg) melhoraram o CT (43% e 45%), LDL (56% e 56%) e TG (46% e 55%, respectivamente). A razão Apo B/A1 diminuiu mais de 20% após a ingestão de Anti-Inflamatory Agents.

Conclusões

Esses dados sugerem que a ingestão de quercetina Quercetin; induzida por glicocorticóides. (Arq Bras Cardiol. 2020; 115(1):102-108)

Ratos Sprague-Dawley; Anti-inflamatórios; Queraracina; Glicocorticoides; Dislipidemias; Triglicérides; Colesterol

Introduction

Glucocorticoids such as prednisone, methylprednisolone, and dexamethasone are widely prescribed for the treatment of numerous clinical disorders, including pulmonary, gastrointestinal, hematological, skin, and renal diseases, as well as organ transplants, particularly due to their anti-inflammatory and immune-modulatory properties.¹ Although these drugs have such benefits, their adverse effects such as hyperglycemia, hypertension, hyperlipidemia, osteoporosis, muscle atrophy and obesity must be taken seriously.² Impaired lipid metabolism, as one of the most common undesirable reactions, in high-dose or long-term GC users, resembles Cushing’s syndrome. In other words, hypercholesterolemia and hypertriglyceridemia are highly prevalent in patients undergoing GC therapy for prolonged periods and may ultimately lead to risks for atherosclerosis.³ - ⁴ However, when the administration of these immunosuppressive drugs is inevitable, one should look for some drugs or natural products to minimize their untoward effects.

Quercetin, 3,3’,4’,5,7-Pentahydroxyflavone, 2-(3,4-Dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one, C15H10O7, is a plant-derived flavonoid, isolated from onions, apples, grapes, leafy vegetables and tea.⁵ , ⁶ This naturally occurring polyphenol compound is generally known for its antioxidant and anti-inflammatory properties and is reported to enhance the antioxidant defense system, and decrease the incidence of cardiovascular, neoplastic and inflammatory diseases.⁷ - ⁹ Since the oxidant-antioxidant balance and inflammation status play an important role the etiology of many diseases, flavonoid compounds have been in the spotlight as natural preventive or therapeutic agents.¹⁰ , ¹¹ In addition, some previous studies reported the beneficial impact of quercetin on metabolic syndrome and lipid metabolism.¹² , ¹³ The aim of this study is to evaluate the effect of quercetin on lipid profile of rats treated with high-dose glucocorticoid.

Materials and methods

Animals

A total of 32 Sprague-Dawley rats, aged 6-7 months, weighing 210±30 grams were obtained from the Razi Institute (Karaj, Iran). The animals were acclimatized to the standard laboratory conditions (temperature 20-25˚C, and a 12-h light/dark cycle) for 10 days before the beginning of the main experiment. Clean water and pelleted standard chow diet (Danbehparvar, Thran, Iran) were provided ad libitum . The experimental protocol was in accordance with the Principles of Laboratory Animal Care.¹⁴ The sample size was calculated with 80% power, using a two-sided test at the 5% significance level and based on the effect size of 0.5.

Chemicals

Methylprednisolone sodium succinate (MP) was used as the glucocorticoid (SOLU-MEDROL, Pfizer Pharmaceuticals, NY, U.S.A) for generating GC-induced dyslipidemia.¹⁵ Quercetin, with a purity of 95%, was obtained from Sigma-Aldrich Chemicals (St. Louis, MO, U.S.A) and the quercetin suspension was prepared by adding quercetin to 0.05% aqueous carboxymethyl cellulose (CMC) solution immediately before being administered by oral gavage.

Experimental procedure

Thirty-two animals were randomly distributed into four groups, using the block randomization scheme. Each experimental group contained eight rats, which were treated for six weeks. All groups were injected subcutaneously (s.c.) with MP (40 mg/kg body weight), except the control group, which received normal saline solution three days a week. Each of the three glucocorticoid-injected groups received one of the following treatments: CMC as placebo, 50 mg/kg quercetin or 150 mg/kg quercetin. All treatments were given three days a week per os . At the end of the study all animals were anesthetized with an intra-peritoneal (i.p.) injection of ketamine together with xylazine (50 mg/kg and 30 mg/kg respectively).¹⁵ , ¹⁶ Blood samples were collected by cardiac puncture and were immediately centrifuged at 3000 rpm for 10 min for serum isolation and stored at -80˚C until analysis of the lipid profile. The rats were fasted for 12-14 hours and all blood samples were collected between 8 and 10 am. Commercially available enzymatic kits were used to measure the serum concentrations of total cholesterol (TC), high density lipoprotein (HDL), and triglycerides (TG) in duplicate tests (Pars Azmoon Co., Tehran, Iran) and Apo A and Apo B were measured by immunoturbidimetric methods (biorexfars LTD, Iran). Low-density lipoprotein (LDL) level was calculated using the Friedewald equation.¹⁷ Animals were weighed at the beginning and end of the study.

Statistical analysis

All data were presented as mean ± standard deviation (SD) and analyzed by the Statistical Package for Social Sciences (version 23.0; SPSS Inc., Chicago, USA). The Kolmogorov-Smirnov test was used to assess the normality of the data. Statistical differences between groups were evaluated using analysis of variance (one-way ANOVA) followed by Bonferroni post hoc test. Statistical significance was set at p < 0.05.

Results

Although the average body weight of rats was the same in all groups at the beginning of the experiment, after six weeks of intervention, all glucocorticoid-treated animals showed a significant weight reduction compared with their own initial weights and with their age-matched controls ( Table 1 ).

Table 1
– Initial and final body weight (gram) of experimental groups

Body weight	Control	MP	MP+Q50	MP+Q150
Initial	212±29	212±27	210±28	212±28
Final	214±30†	182±22*,‡	185±20*,‡	180±16*,‡

• ^†‡

Following six weeks of methylprednisolone injection, the mean plasma cholesterol and triglyceride levels were drastically increased in glucocorticoid-treated animals compared with the control group. Both doses of quercetin (50 and150 mg/kg) improved the hypercholesterolemia and hypertriglyceridemia in comparison with the MP group, and the same trend was observed for LDL levels. In addition, the MP injection caused a moderate increase in HDL levels, which was not significantly changed following quercetin supplementation. However, the reduction in TC/HDL, TG/HDL and LDL/HDL ratios were statistically and clinically significant. Moreover, Apo B/A1 ratio decreased more than 20% following quercetin intake ( Table 2 ; Figures 1-3 ). It seems that a higher dose of quercetin does not have a conspicuous superiority for cholesterol and apolipoprotein level improvement. However, a negative correlation was found between the quercetin dose and TG, as well as TC/HDL (-0.87 and -0.75 respectively).

Table 2
– Lipid profile of experimental groups after six weeks of intervention

	Control	MP	MP+Q50	MP+Q150	p value
TC (mg/dl)	89.12±3.35	193.50±12.77*.‡	108.75±15.47*.†	105.87±11.25*.†	<0.001
HDL (mg/dl)	34.25±3.69	41.37±5.75*	38.25±4.77	39.00±4.07	=0.03
LDL (mg/dl)	41.87±3.79	119.22±12.70*.‡	52.72±15.15†	52.22±10.87†	<0.001
TG (mg/dl)	65.00±4.34	164.50±9.36*.‡	88.87±12.93*.†	73.25±11.33†.‡	<0.001
TC/HDL	2.62±0.27	4.76±0.86*.‡	2.86±0.42†	2.74±0.47†.‡	<0.001
TG/HDL	1.92±0.29	4.05±0.71*.‡	2.33±0.34†	1.92±0.51†	<0.001
LDL/HDL	1.24±0.23	2.95±0.73*.‡	1.39±0.43†	1.36±0.38†	<0.001
Apo B/ Al	0.93±0.16	1.63±0.19*.‡	1.25±0.30†	1.06±0.28†	<0.001

• ^*†‡

Figure 1
– Mean of the total cholesterol to HDL ratio in the different groups. Data presented as Mean±SD. N=8 for all groups. MP: methylprednisolone; Q50: quercetin 50 mg/kg; Q150: quercetin 150 mg/kg; TC: total cholesterol; HDL: high-density lipoprotein;^*p<0.05 compared with control group,^†p<0.05 compared with MP group,^‡p<0.05 compared with MP+Q50.

Discussion

Our findings revealed that the administration of high-dose glucocorticoid for 6 weeks drastically increased serum concentrations of total cholesterol, LDL and triglycerides. However, oral supplementation with two different doses of quercetin, as a naturally occurring flavone that was previously reported to be beneficial in metabolic syndrome, conspicuously reversed the undesirable effects of methylprednisolone. Different doses of quercetin were chosen, since the lower one can be provided by a quercetin-rich diet and the higher one might be taken as commercially available supplements.¹⁸ Needless to say, the different metabolic rates of rats and humans were taken into account for dose determination.¹⁹ The final results indicated that 150 mg/kg quercetin were not much more effective than 50 mg/kg to improve lipid profile, except for TG concentrations, which decreased to the control level as a result of high dose quercetin administration. Methylprednisolone also caused a moderate increase in HDL levels, which was not significantly changed following quercetin supplementation.

Although the hyperlipidemic impact of GCs has been noticed for the last decades, the molecular mechanisms are not well recognized yet. Some in vitro and in vivo studies demonstrated that these anti-inflammatory drugs can directly increase hepatic HDL production, up-regulate lipoprotein lipase activity and impair LDL catabolism by reducing hepatic LDL receptors expression and activity.¹⁵ , ²⁰ Consequently, they contribute to fatty liver development by increasing fatty acid synthesis and decreasing β oxidation.²¹

On the other hand, flavonoids have been described as lipid metabolism modulators. They mostly act through the inhibition of phosphodiesterase, alteration of hepatic cholesterol absorption and triglyceride production and secretion.²² - ²⁵ In addition, quercetin as a potent antioxidant distributed in both the lipid bilayer and aqueous phase of the cell, can suppress lipid peroxidation by radical scavenging activity.²⁶ Large studies have shown that ApoB/Al ratio is superior to the total cholesterol and TG for cardiovascular risk prediction in both genders and at all age ranges.²⁷ Given that the ApoB/Al ratio is a measurement of the number of ApoB atherogenic particles over the number of ApoAl anti-atherogenic particles, there is also a possibility that it is a more important factor than the amount of lipids carried per particle. In the present study, quercetin intake significantly decreased ApoB/AI ratio, which might be an important indicator of lower cardiovascular risk in the future.²⁷ , ²⁸

At the end of intervention, all glucocorticoid-treated animals showed a significant weight reduction compared to their controls, which might be due to glucocorticoid-induced anorexia in rats, which has been previously reported,²⁹ or to severe proteolysis and muscle loss.³⁰ One of the limitations of this study was the lack of precise data about the animals’ food intake, which could be very useful for the interpretation of GC-induced weight loss in rats. Overall, our findings are in accordance with previous studies reporting the beneficial effects of flavonoids on lipid metabolism.³¹ This is the first research evaluating the impact of quercetin on GC-induced hyperlipidemia. However, the hypolipidemic effect of some other flavonoids has been reported in GC-treated rats.³² Other favorable properties of quercetin in improving bone density and modifying blood glucose, make this flavonoid an excellent choice to control glucocorticoid side effects.³³

Conclusion

Quercetin administration, at both doses of 50 and 150 mg/kg, was able to reverse the untoward effects of high-dose glucocorticoids on the lipid profile of rats, and might be considered for combination therapy with GCs to minimize the resulting dyslipidemia.

Figure 2
– Mean of triglycerides to HDL ratio in the different groups. Data presented as Mean±SE. n=8 for all groups. MP: methylprednisolone; Q50: quercetin 50 mg/kg; Q150: quercetin 150 mg/kg; TG; triglyceride; HDL: high-density lipoprotein;^*p<0.05 compared with control group,^†p<0.05 compared with MP group,^‡p<0.05 compared with MP+Q50.

Figure 3
– Mean of apolipoprotein B to apolipoprotein Al ratio in the different groups. Data presented as Mean±SE. n=8 for all groups. MP: methylprednisolone; Q50: quercetin 50 mg/kg; Q150: quercetin 150 mg/kg; ApoB/Al: apolipoprotein B to apolipoprotein Al ratio;^*p<0.05 compared with control group,^†p<0.05 compared with MP group,^‡p<0.05 compared with MP+Q50.

Referências

• ¹
  Arafah BM. Pharmacology of glucocorticoids. In: Levine A, editor. Adrenal disorders. Contemporary endocrinology. Cham: Humana Press; 2018. p. 67-81.
• ²
  Moghadam-Kia S, Werth VP. Prevention and treatment of systemic glucocorticoid side effects. Int J Dermatol. 2010;49(3):239-48.
• ³
  Staels B, van Tol A, Chan L, Verhoeven G, Auwerx J. Variable effects of different corticosteroids on plasma lipids, apolipoproteins, and hepatic apolipoprotein mRNA levels in rats. Arterioscler Thromb. 1991;11(3):760-9.
• ⁴
  Yadav A, Jahan A, Yadav TP, Sachdev N, Chitkara A, Asare R. Effect of glucocorticoids on serum lipid profile and endothelial function and arterial wall mechanics. Indian J Pediatr. 2013;80(12):1007-14.
• ⁵
  Bentz AB. A Review of quercetin: chemistry, antioxident properties, and bioavailability. J Young Investig. 2009 apr.
• ⁶
  Shah PM, Priya VV, Gayathri R. Quercetin-a flavonoid: a systematic review. J Pharm Sci Res. 2016;8(8):878-80.
• ⁷
  D’Andrea G. Quercetin: a flavonol with multifaceted therapeutic applications? Fitoterapia. 2015 Oct;106:256-71.
• ⁸
  Li Y, Yao J, Han C, Yang J, Chaudhry MT, Wang S, et al. Quercetin, inflammation and immunity. Nutrients. 2016;8(3):167.
• ⁹
  David AVA, Arulmoli R, Parasuraman S. Overviews of biological importance of quercetin: a bioactive flavonoid. Pharmacogn Rev. 2016;10(20):84-9.
• ¹⁰
  Ginwala R, Bhavsar R, Chigbu DI, Jain P, Khan ZK. Potential role of flavonoids in treating chronic inflammatory diseases with a special focus on the anti-inflammatory activity of apigenin. Antioxidants. 2019;8(2):pii:E35.
• ¹¹
  Javanbakht MH, Djalali M, Daneshpazhooh M, Zarei M, Eshraghian MR, Derakhshanian H, et al. Evaluation of antioxidant enzyme activity and antioxidant capacity in patients with newly diagnosed pemphigus vulgaris. Clin Exp Dermatol. 2015;40(3):313-7.
• ¹²
  Santhakumar AB, Battino M, Alvarez-Suarez JM. Dietary polyphenols: structures, bioavailability and protective effects against atherosclerosis. Food Chem Toxicol. 2018 Mar;113:49-65.
• ¹³
  Sahebkar A. Effects of quercetin supplementation on lipid profile: a systematic review and meta-analysis of randomized controlled trials. Crit Rev Food Sci Nutr. 2017;57(4):666-76.
• ¹⁴
  National Research Council. Guide for the care and use of laboratory animals. 8th ed. Washington (DC): National Academies Press; 2011.
• ¹⁵
  Hazra A, Pyszczynski NA, DuBois DC, Almon RR, Jusko WJ. Modeling of corticosteroid effects on hepatic low-density lipoprotein receptors and plasma lipid dynamics in rats. Pharm Res. 2008;25(4):769-80.
• ¹⁶
  Green C, Knight J, Precious S, Simpkin S. Ketamine alone and combined with diazepam or xylazine in laboratory animals: a 10 year experience. Lab Anim. 1981;15(2):163-70.
• ¹⁷
  Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin Chem. 1972;18(6):499-502.
• ¹⁸
  de Vrie JH, Janssen PL, Hollman PC, van Staveren WA, Katan MB. Consumption of quercetin and kaempferol in free-living subjects eating a variety of diets. Cancer Lett. 1997;114(1-2):141-4.
• ¹⁹
  Nair AB, Jacob S. A simple practice guide for dose conversion between animals and human. J Basic Clin Pharm. 2016;7(2):27-31.
• ²⁰
  Sholter DE, Armstrong PW. Adverse effects of corticosteroids on the cardiovascular system. Can J Cardiol. 2000;16(4):505-11.
• ²¹
  Van De Wier B, Koek GH, Bast A, Haenen GR. The potential of flavonoids in the treatment of non-alcoholic fatty liver disease. Crit Rev Food Sci Nutr. 2017;57(4):834-55.
• ²²
  Seiva FR, Chuffa LG, Braga CP, Amorim JP, Fernandes AA. Quercetin ameliorates glucose and lipid metabolism and improves antioxidant status in postnatally monosodium glutamate-induced metabolic alterations. Food Chem Toxicol. 2012;50(10):3556-61.
• ²³
  Peluso MR. Flavonoids attenuate cardiovascular disease, inhibit phosphodiesterase, and modulate lipid homeostasis in adipose tissue and liver. Exp Biol Med. 2006;231(8):1287-99.
• ²⁴
  Rivera L, Morón R, Sánchez M, Zarzuelo A, Galisteo M. Quercetin ameliorates metabolic syndrome and improves the inflammatory status in obese Zucker rats. Obesity. 2008;16(9):2081-7.
• ²⁵
  Mbikay M, Mayne J, Sirois F, Fedoryak O, Raymond A, Noad J, et al. Mice fed a high-cholesterol diet supplemented with quercetin-3-glucoside show attenuated hyperlipidemia and hyperinsulinemia associated with differential regulation of PCSK9 and LDLR in their liver and pancreas. Mol Nutr Food Res. 2018;62(9):1700729.
• ²⁶
  Ishisaka A, Ichikawa S, Sakakibara H, Piskula MK, Nakamura T, Kato Y, et al. Accumulation of orally administered quercetin in brain tissue and its antioxidative effects in rats. Free Radic Biol Med. 2011;51(7):1329-36.
• ²⁷
  Sathe CA, Chogle SA, Bharadwaj D. Apo B/Apo A1 ratio: a risk marker in patients with cardiovascular disease. Int J Res Med. 2017;6(2):20-3.
• ²⁸
  Panayiotou A, Griffin M, Georgiou N, Bond D, Tyllis T, Tziakouri-Shiakalli C, et al. ApoB/ApoA1 ratio and subclinical atherosclerosis. Int Angiol. 2008;27(1):74-80.
• ²⁹
  Simpson CW, Dicara LV, Wolf G. Glucocorticoid anorexia in rats. Pharmacol Biochem Behav. 1974;2(1):19-25.
• ³⁰
  Löfberg E, Gutierrez A, Wernerman J, Anderstam B, Mitch WE, Price SR, et al. Effects of high doses of glucocorticoids on free amino acids, ribosomes and protein turnover in human muscle. Eur J Clin Invest. 2002;32(5):345-53.
• ³¹
  Sangeetha KSS, Reddy CUM, Kalkura SN. Flavonoids: therapeutic potential of natural pharmacological agents. Int J Pharm Sci Res. 2016;7(10):3924-30.
• ³²
  Dugani AM, Alkhetally WI, Elghedafi EO, Alkayed FW. Effects of the aqueous extract from Abelmoschus esculentus L peel on hyperglycemia and hyperlipidemia induced by dexamethasone in rats. Libyan Int. Med. Univ. J. 2018;3(1):3-7.
• ³³
  Derakhshanian H, Djalali M, Djazayery A, Nourijelyani K, Ghadbeigi S, Pishva H, et al. Quercetin prevents experimental glucocorticoid-induced osteoporosis: a comparative study with alendronate. Can J Physiol Pharmacol. 2013;91(5):380-5.

• Study Association
  This article is part of the thesis of Master submitted by Hoda Derakhshanian, from Tehran University of Medical Sciences.
• Ethics approval and consent to participate
  This study was approved by the Ethics Committee of the Tehran University of Medical Sciences under the protocol number 11157. All the procedures in this study were in accordance with the 1975 Helsinki Declaration, updated in 2013.

• Sources of Funding
  This study was funded by Tehran University of Medical Sciences.

• ERRATUM

  July 2020 Issue, vol. 115 (1), pages 102-108
  In the Original Article “Quercetin Ameliorates Lipid and Apolipoprotein Profile in High-Dose Glucocorticoid Treated Rats”, with DOI number: https://doi.org/10.36660/abc.20180397, published in the periodical Arquivos Brasileiros de Cardiologia, 115(1):102-108, on page 102, add one more affiliation for the author Ahmad Reza Dehpour. Include the institution: Experimental Medicine Research Center, Tehran University of Medical Sciences, Tehran, Iran.

Publication Dates

• Publication in this collection
  07 Aug 2020
• Date of issue
  July 2020

History

• Received
  05 Dec 2018
• Reviewed
  30 June 2019
• Accepted
  14 Aug 2019