Activation of Cardiac TNF-α in Altered Thyroid State-Induced Cardiometabolic Disorder

Ajayi, Akhigbe , and Ajayi: Activation of Cardiac TNF-α in Altered Thyroid State-Induced Cardiometabolic Disorder

Authors

INTRODUCTION

Thyroid hormones are catabolic hormones which are known to play essential roles in various metabolic processes. Altered thyroid states have been reported to be associated with metabolic disorders as well as cardiovascular diseases.1,2 Hypothyroidism has been reported to impair cardiac contractility, decreased cardiac output, increased vascular resistance, reduced chronotropy, cardiac atrophy, and cardiac failure.3-5 The effect of hypothyroidism on cardiac function is linked with attendant dyslipidaemia and atherosclerosis.6-11 On the other hand, hyperthyroidism-induced cardiovascular dysfunction is due to the hyper metabolic state and accelerated free radical production in the mitochondria with resultant changes in the antioxidant defense system caused by high levels of thyroid hormones.12,13

Even within the normal range of thyroid-stimulating hormone (TSH), total cholesterol, triglycerides and LDL increase while HDL reduces with increasing level of TSH.14 Thyroid hormones stimulate the first step of cholesterol synthesis by inducing 3-hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) reductase. Triiodothyronine (T3) controls LDL receptor gene activation by the direct binding of T3 to specific thyroid hormone responsive elements (TREs).15 T3 also controls sterol regulatory element-binding protein-2 (SREBP-2), thus regulating LDL receptor’s gene expression.16 Faure et al. also reported that T3 protects LDL from oxidation.17 Thyroid hormones has been documented to affect HDL metabolism by enhancing cholesteryl ester transfer protein (CETP) activity which is involved in the conversion of cholesteryl esters from HDL2 to the very low density lipoproteins (VLDL) and TGs to the opposite direction.18 Thyroid hormones have also been shown to induce lipoprotein lipase which catabolizes the TG-rich lipoproteins, and the hepatic lipase (HL).19,20 They also up-regulate apolipoprotein AV (ApoAV), which plays a major role in TG regulation.21

Besides the effect of thyroid hormones on plasma lipids, their effects on other metabolic parameters which are associated with cardiovascular diseases include their influence on adipocyte metabolism and the production of adipokines.22-24 Lipid peroxidation is also affected by thyroid function with studies however showing controversial outcomes.17-25

This study thus determines the effect of dysthyroidism on lipid profile, cardiac TNF-α and lipid peroxidation, and whether the effect is estradiol-dependent.

MATERIALS AND METHODS

Experimental animals

Male white New Zealand (Oryctolagus cuniculus huxley) rabbits of comparable weights were used for the study. Experimental animals were obtained from the Animal Holdings of the Department of Physiology, Ladoke Akintola University of Technology, Nigeria. They were fed with standard rabbit chow and water ad’libitum. Animals were handled under standard laboratory conditions of a 12:12 hour light/dark cycle in a temperature and humidity controlled room (temperature: 28 °C–31 °C, humidity of 45%–50%).26,27

Treatment

Rabbits were randomly allocated to one of the three experimental groups of 10 animals each. Hypothyroidism was induced by administration of 5 mg/250 g body weight of carbimazole, while hyperthyroidism was induced by administration of 5 μg/100 g body weight of levothyroxine. Treatment lasted for 35 days. Doses of carbimazole and levothyroxine were based on previous studies.28,29 Animal maintenance and handling was performed according to the NIH Guide for the Care and Use of Laboratory Animals (NIH publication No 86-23, revised 1985 and 1991) and the UK requirements for ethics of animal experimentation (Animals Scientific Procedures, Act 1986).

Biochemical assays

At the end of the 35-day experiment, blood samples were collected via cardiac puncture, serum was obtained, and biochemical parameters were assayed. Fasting plasma levels of total cholesterol (TC), triglyceride (TG), and HDL-cholesterol (HDL-C) were assayed by standard enzymatic method coupled with spectrophotometry using assay kit (Randox Lab Ltd., UK). LDL-cholesterol (LDL-C) was calculated using Friedewald’s formula.27-30 Plasma glucose and 17β-estradiol were also determined using standard laboratory kit (Randox Lab Ltd., UK).31,32

Lipid peroxidation index, malondialdehyde (MDA), reduced glutathione (GSH) and catalase were evaluated as previously documented.33,34

TNF-α immunoassay

This assay employs the quantitative sandwich enzyme immunoassay technique. An antibody specific for TNF-α was pre-coated into a microplate. All reagents and working standards were prepared and 100μL of dilution buffer was added to the blank wells. 100μL of standard and samples were added per well and covered with the plate sealer. This was incubated for 2 hours on a microplate shaker at room temperature. Each well is aspirated and washed four times. Washing is done by filling each well with 1x wash buffer (300μL) using a squirt bottle. After the last washing, any remaining wash buffer is removed by aspirating or decanting. The plate is then inverted and blotted against a clean paper towel. 100μL of detection antibody working solution was then added to each well and covered with the plate sealer. This was incubated for another 2 hours on a microplate shaker at room temperature. The aspiration/wash procedure was repeated. 100μL of Avidin-HRP conjugate working solution was now added to each well and incubated for 1 hour on a microplate shaker at room temperature. It was protected from light. The aspiration/wash procedure was again repeated. 100μL of Substrate solution was added to each well, incubated for 4 minutes at room temperature and protected from light. 100μL of stop solution was added to each well, which caused the colour in the wells to change from blue to yellow. The optical density of each well was determined using the micro-plate reader set to 450nm wavelength.

A standard line is constructed by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and a best line of fit is drawn through the points on the graph. Using the best line of fit, the TNF-alpha concentration of the heart tissue is deduced.

Statistical analysis

Statistical analysis was performed using SPSS. Results were reported as mean ± standard error of mean (S.E.M.). One way analysis of variance (ANOVA) was used to analyze for the significance of differences between means followed by post hoc Duncan’s multiple range test. Statistical significance was assigned at a p-value of less than 0.05.

RESULTS

Effect of dysthyroidism on plasma glucose and lipids

Plasma glucose level was altered by dysthyroidism; it was reduced in hyperthyroid rats but raised in hypothyroid rabbits, although the rise observed in the hypothyroid rabbits was marginal. Cholesterol and triglyceride were significantly higher in hypothyroidism and lower in hyperthyroidism respectively. HDL-cholesterol was significantly lowered by dysthyroidism. The reduction observed was more in hyperthyroid rabbits. LDL-cholesterol was significantly elevated by dysthyroidism (Figure 1).

Effect of dysthyroidism on hemorheological parameters and serum electrolytes

Although, dysthyroidism led to elevation of hemorheological parameters, this was marginal (Figure 2). Serum electrolytes, indices of renal function, were also not significantly altered (Figure 3).

Effect of dysthyroidism on cardiac TNF-α

Cardiac TNF-α was significantly raised in dysthyroid rabbits. Hyperthyroidism led to 46.4% rise, while hypothyroidism caused 78.4% rise (Figure 4).

Effect of dysthyroidism on indices of lipid peroxidation

Dysthyroidism led to significant rise in cardiac and renal MDA. The rise was observed to be more in hypothyroid rabbits. Cardiac catalase and renal catalase activities were significantly reduced in dysthyroidism. Serum MDA was significantly elevated in dysthyroidism when compared to the control. Serum catalase activity was significantly reduced in dysthyroidism. This was more in hyperthyroid rabbits. Cardiac GSH, renal GSH and serum GSH activities were comparable in all groups (Figure 5).

Effect of dysthyroidism on 17β-estradiol

Figure 6 illustrate the circulating level of plasma 17β-estradiol. It was observed that 17β-estradiol level was reduced by about 11% in hypothyroid rabbits and 9% in hyperthyroid rabbits (Figure 6).

DISCUSSION

The key novel finding of the present study is the activation of cardiac TNF-α in dysthyroidism and its association with oxidative stress and estradiol. In addition, we confirmed the observation that altered thyroid state led to dyslipidaemia. According to our knowledge, this is the first study to report the association in dysthyroidism between oxidative stress, inflammatory molecule and estradiol. Recognizing the public health challenges associated with altered thyroid state, the findings of our study may provide essential information about the complex associations between hormonal milieu and inflammatory mechanisms.

Figure 1

Effect of dysthyroidism on plasma levels of glucose and lipids Bars carrying different letters on the same parameter are statistically different at P<0.05

https://s3-us-west-2.amazonaws.com/jourdata/jcdr/JCardiovascRes.8.151-g001.jpg
Figure 2

Effect of dysthyroidism on plasma proteins Bars carrying different letters on the same parameter are statistically different at P<0.05

https://s3-us-west-2.amazonaws.com/jourdata/jcdr/JCardiovascRes.8.151-g002.jpg
Figure 3

Effect of dysthyroidism on renal function Bars carrying different letters on the same parameter are statistically different at P<0.05

https://s3-us-west-2.amazonaws.com/jourdata/jcdr/JCardiovascRes.8.151-g003.jpg
Figure 4

Effect of dysthyroidism on cardiac TNF-α Bars carrying different letters are statistically different at P<0.05

https://s3-us-west-2.amazonaws.com/jourdata/jcdr/JCardiovascRes.8.151-g004.jpg
Figure 5

Effect of dysthyroidism on lipid peroxidation indices Bars carrying different letters are statistically different at P<0.05

https://s3-us-west-2.amazonaws.com/jourdata/jcdr/JCardiovascRes.8.151-g005.jpg
Figure 6

Effect of dysthyroidism on plasma estradiol Bars carrying different letters are statistically different at P<0.05

https://s3-us-west-2.amazonaws.com/jourdata/jcdr/JCardiovascRes.8.151-g006.jpg

Glucose and lipid metabolisms are very sensitive to altered thyroid states; hence optimal levels of thyroid hormones are important for glucose and lipid metabolism. Although, plasma glucose level was similar in the control and hypothyroid animals, it was significantly reduced in hyperthyroid state. Hypothyroidism also led to increase in cholesterol and triglyceride levels while these lipids were observed to be reduced in hyperthyroid state. Alteration of thyroid state led to significant rise in LDL-cholesterol and decline in HDL-cholesterol. These findings are in tandem with previous studies that also reported dyslipidaemia in a similar trend in association with altered thyroid state.35,36,37 These changes are due to the regulatory effect of thyroid hormones on lipoprotein metabolism. Thyroid hormone stimulates hepatic de novo cholesterol synthesis by inducing the 3-hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) reductase that catalyzes the conversion of HMG-CoA to mevalonate.38 This causes a rise in intracellular cholesterol concentration in hyperthyroidism and a decline in hypothyroidism. Thyroid hormone also stimulates CETP and LPL.19,20 Dyslipidaemia seen in altered thyroid state has been reported to predispose to atherosclerotic coronary artery disease (CAD).39,40 High LDL-cholesterol and low HDL-cholesterol is a factor in the aetiopathogenesis of atherosclerosis. It reduces endothelial synthesis or bioavailability of nitric oxide which is a potent vasodilator.27 However, high HDL-cholesterol enhanced endothelial function by augmenting nitric oxide biosynthesis and bioavailability, and suppressing formation of free radicals, thereby preventing the deleterious effect of LDL-cholesterol in vasculature.27-41

There are accumulating data to support the role of plasma proteins, especially albumin, as hem rheological factors that play essential role in the development of cardio metabolic disorders.27 They have been shown to modulate blood and plasma viscosity.42 Our present results revealed that dysthyroidism is associated with rise in plasma proteins; however, the changes observed in these hemorheological factors were marginal. Similarly, no significant change was observed in the renal function of dysthyroid rabbits.

Tumour necrosis factor-alpha (TNF-α) is a 157 amino acid cytokine and is produced in response to inflammatory or infectious stimuli by macrophages, lymphocytes, neutrophils and structural cells like fibroblasts and smooth muscle cells.43 Due to the large population of macrophages, the liver and spleen remain major sources of TNF-α,44 although the myocardium also synthesizes TNF-α (45) in response to endotoxin, pressure or volume overload, ischaemia/reperfusion and oxidative stimulus through nuclear factor kappa B (NFkB) mediated mechanism.46,47 The present study revealed that dysthyroidism led to a rise in cardiac TNF-α. Though this seems to be the first study that documents the effect of dysthyroidism on cardiac TNF-α, this observation is in complete agreement with previous studies that documented a rise in serum TNF-α in hyperthyroidism and hypothyroidism. The higher cardiac TNF-α observed in dysthyroidism may account for cardiometabolic disturbances seen in patients with altered thyroid states. TNF-α has been reported to cause apoptosis of myocardial cells via mechanisms of cell death.48,49,50 The VEST trial documented that TNF-α is directly related to the New York Heart Association (NYHA) functional stage of patients with HF and that the highest levels of TNF-α were associated with a worse prognosis.51 This was reported to be due to negative inotropic action and a disturbance of beta-adrenergic receptor sensitivity caused by TNF-α via the inducible nitric oxide synthase (iNOs) system.51 Increased TNF-α level is associated with a disturbance of left atrial function and an advanced degree of left ventricular diastolic and systolic dysfunction.52 Although a low level of TNF-α may confer a short-term cardio protection,53,54 a prolonged exposure and high level of TNF-α have deleterious effects on the heart.55

Several mechanisms might explain the connection between dysthyroidism and cardiometabolic disorder. In the present study, although altered thyroid states did not affect GSH, they led to a higher level of lipid peroxidation index, MDA, and a significant decline in catalase activities in the serum, heart and kidneys. This also seems to be the first study to report the association between dysthyroidism-induced cardiometabolic disorders with cardiac redox status. Our observations were in consonance with the results of Torun et al.56 and Akinci et al.57 that documented a similar trend in the serum levels of MDA and catalase. Oxidative stress, which is characterized by an imbalance between free radicals and antioxidants in favour of radicals, participates in the pathogenesis of many diseases and their complications.58,59 The levels of ROS are controlled by antioxidant enzymes, such as superoxide dismutase (SOD), glutathione peroxidase (GSH-Px).27-59,60 Malondialdehyde (MDA) is a useful index for lipid peroxidation and thus used as an indicator for oxidative damage in cells and tissues.59 The altered redox state seen in dysthyroidism possibly accounts for the activation of cardiac TNF-α with resultant cardio metabolic disorder.

Also, it is worthy of note that the present study also observed a decline in the circulating level of 17β-estradiol. This is another key finding of the present study. It is a known fact that estradiol is cardio protective. A decline in estradiol thus has a deleterious effect on the cardiac function. 17β-estradiol has been reported to have lipid-lowering effect by inhibiting cholesterol accumulation, influencing hepatic lipid metabolism, elevating the circulatory levels of HDL and preventing LDL-oxidation.61 The cardioprotective effect of 17β-estradiol has also been related to its anti-atherogenic effect on monocyte chemo-attractant protein 1 (MCP-1), a chemokine expressed in atherosclerotic lesions.62,63,64 17β-estradiol inhibits monocyte chemo-attractant protein 1 (MCP-1) by lowering the production of TNF-α.65 The decline in 17β-estradiol circulatory level observed in dysthyroidism in the study also accounts for the associated dyslipidaemia and elevated cardiac TNF-α.

Conclusively, this study expands on the documentation of previous studies that reported dyslipidaemia and elevated levels of serum TNF-α in altered thyroid states by demonstrating increased lipid peroxidation, and reduced antioxidants and estradiol levels in relation to activation of cardiac TNF-α in dysthyroidism. These new data provide an approach to understanding the pathogenesis of cardio metabolic disorders associated with altered thyroid states, with possible prognostic implications.

Notes

[1] Conflicts of interest CONFLICT OF INTEREST All authors declared no conflict of interest.

REFERENCES

1. 

Ichiki T , author. Thyroid hormone and atherosclerosis. Vascul Pharmacol. 2010;52:151–6

2. 

Steinberg AD , author. Myxedema and coronary artery disease–a comparative autopsy study. Ann Intern Med. 1968;68(2):338–44

3. 

Biondi B, Palmieri EA, Lombardi G, Fazio S , authors. Effects of subclinical thyroid dysfunction on the heart. Ann Intern Med. 2002;137(11):904–14

4. 

Klein I , author. Thyroid hormone and cardiac contractility. Am J Cardiol. 2003;91:1331–2

5. 

Liu Z, Gerdes AM , authors. Influence of hypothyroidism and the reversal of hypothyroidism on hemodynamics and cell size in the adult rat heart. J Mol Cell Cardiol. 1990;22:1339–48

6. 

Auer J, Berent R, Weber T, Lassnig E, Eber B , authors. Thyroid function is associated with presence and severity of coronary atherosclerosis. Clin Cardiol. 2003;26(12):569–73

7. 

Tang YD, Kuzman JA, Said S, Anderson BE, Wang X, Gerdes AM , authors. Low thyroid function leads to cardiac atrophy with chamber dilatation, impaired myocardial blood flow, loss of arterioles, and severe systolic dysfunction. Circulation. 2005;112(20):3122–30

8. 

Bengel FM, Nekolla SG, Ibrahim T, Weniger C, Ziegler SI, Schwaiger M , authors. Effect of thyroid hormones on cardiac function, geometry, and oxidative metabolism assessed noninvasively by positron emission tomography and magnetic resonance imaging. J Clin Endocrinol Metab. 2000;85(5):1822–7

9. 

Chen WJ, Lin KH, Lee YS , authors. Molecular characterization of myocardial fibrosis during hypothyroidism: evidence for negative regulation of the pro-alpha1(I) collagen gene expression by thyroid hormone receptor. Molecular and cellular endocrinology. 2000;162(1):45–55

10. 

Keating FR Jr.; Parkin TW, Selby JB, Dickinson LS , authors. Treatment of heart disease associated with myxedema. Prog Cardiovasc Dis. 1961;3(4):364–81

11. 

Ojamaa K, Klemperer JD, MacGilvray SS, Klein I, Samarel A , authors. Thyroid hormone and hemodynamic regulation of beta-myosin heavy chain promoter in the heart. Endocrinology. 1996;137(3):802–8

12. 

Asayama K, Dobashi K, Hayashibe H, Megata Y, Kato K , authors. Lipid peroxidation and free radical scavengers in thyroid dysfunction in the rat: a possible mechanism of injury to heart and skeletal muscle in hyperthyroidism. Endocrinology. 1987;121(6):2112–8

13. 

Pereira B, Rosa LF, Safi DA, Bechara EJ, Curi R , authors. Control of superoxide dismutase, catalase and glutathione peroxidase activities in rat lymphoid organs by thyroid hormones. J Endocrinol. 1994;140(1):73–7

14. 

Asvold BO, Vatten LJ, Nilsen TI, Bjoro T , authors. The association between TSH within the reference range and serum lipid concentrations in a population-based study. The HUNT Study. Eur J Endocrinol. 2007;156(2):181–6

15. 

Bakker O, Hudig F, Meijssen S, Wiersinga WM , authors. Effects of triiodothyronine and amiodarone on the promoter of the human LDL receptor gene. Biochem Biophys Res Commun. 1998;249(2):517–21

16. 

Shin DJ, Osborne TF , authors. Thyroid hormone regulation and cholesterol metabolism are connected through Sterol Regulatory Element- Binding Protein-2 (SREBP-2). J Biol Chem. 2003;278(36):34114–8

17. 

Faure P, Oziol L, Artur Y, Chomard P , authors. Thyroid hormone (T3) and its acetic derivative (TA3) protect low-density lipoproteins from oxidation by different mechanisms. Biochimie. 2004;86(6):411–8

18. 

Lagrost L , author. Regulation of cholesteryl ester transfer protein (CETP) activity: review of in vitro and in vivo studies. Biochim BiophysActa. 1994;1215(3):209–36

19. 

Kuusi T, Saarinen P, Nikkila EA , authors. Evidence for the role of hepatic endothelial lipase in the metabolism of plasma high density lipoprotein2 in man. Atherosclerosis. 1980;36(4):589–93

20. 

Santamarina-Fojo S, Gonzalez-Navarro H, Freeman L, Wagner E, Nong Z , authors. Hepatic lipase, lipoprotein metabolism, and atherogenesis. Arterioscler Thromb Vasc Biol. 2004;24(10):1750–4

21. 

Prieur X, Huby T, Coste H, Schaap FG, Chapman MJ, Rodriguez JC , authors. Thyroid hormone regulates the hypotriglyceridemic gene APOA5. J Biol Chem. 2005;280(30):27533–43

22. 

Iglesias P, Diez JJ , authors. Influence of thyroid dysfunction on serum concentrations of adipocytokines. Cytokine. 2007;40(2):61–70

23. 

Viguerie N, Millet L, Avizou S, Vidal H, Larrouy D, Langin D , authors. Regulation of human adipocyte gene expression by thyroid hormone. J Clin Endocrinol Metab. 2002;87:630–4

24. 

Hsieh CJ, Wang PW , authors. Serum concentrations of adiponectin in patients with hyperthyroidism before and after control of thyroid function. Endocr J. 2008;55:489–94

25. 

Venditti P, Di Meo S , authors. Thyroid hormone-induced oxidative stress. Cell Mol Life Sci. 2006;63(4):414–34

26. 

Akhigbe R.E, Olatunji L.A, Soladoye A.O, Oyeyipo I.P , authors. Effect of angiotensin 1-converting enzyme inhibitor, captopril, on body weight and food and water consumption in oral contraceptive-treated rats. American Journal of Biochemistry and Molecular Biology. 2011;1(2011):95–100

27. 

Ige SF, Akhigbe RE , authors. Common onion (Allium cepa) extract reverses cadmiuminduced organ toxicity and dyslipidaemia via redox alteration in rats. Pathophysiology. 2013;20(4):269–74

28. 

Ajayi AF, Akhigbe RE , authors. Implication of altered thyroid state on liver function. Thyroid Res Pract. 2012;9(3):84–7

29. 

Ajayi A.F, Akhigbe R.E, Ajayi L.O , authors. Hypothalamic-pituitary-ovarian axis in thyroid dysfunction. West Indian Medical Journal. 2013;62(9):835–8

30 

Friedewald WT, Levy RI, Fredrickson DS , authors. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clinical Chemistry. 1972;18(6):499–502

31. 

Ajayi A. F.; Akhigbe R. E, Adewumi O. M.; Okeleji L. O.; Mujaidu K. B.; Olaleye S. B. , authors. 2012. Effect of ethanolic extract of Cryptolepis sanguinolenta stem on in vivo and in vitro glucose absorption and transport: Mechanism of its antidiabetic activity. Indian J Endocr Metab. 2012;16:S91–6

32. 

Ajayi AF, Akhigbe RE , authors. Trona Causes Fetal Loss in Pregnant Rat via an Oestrogen-dependent Mechanism. West Indian Med J. 2016;65(1):83DOI: 10.7727/wimj.2014.254

33. 

Ige S.F, Akhigbe RE , authors. The role of Allium cepa on aluminium-induced reproductive dysfunction in experimental male rat models. J Hum Reprod Sci. 2012;5(2):200–5

34. 

Ige SF, Olaleye SB, Akhigbe RE, Akanbi TA, Oyekunle OA, Udoh US , authors. Testicular toxicity and sperm quality following cadmium exposure in rats: Ameliorative potentials of Allium cepa. J Hum Reprod Sci. 2012;5(1):37–42

35. 

Hapon M.B, Varas S.M, Jahn G.A, Giménez M.S , authors. Effects of hypothyroidism on mammary and liver lipid metabolism in virgin and late-pregnant rats. Journal of Lipid Research. 2005;46(6):1320–30

36. 

Prakash A, Lal AK , authors. Serum lipids in hypothyroidism: our experience. Indian Journal of Clinical Biochemistry. 2006;21(2):153–5

37. 

Liberopoulos E.N, Elisaf M.S , authors. Dyslipidemia in patients with thyroid disorders. Hormone. 2002;1(4):218–3

38. 

Ness GC, Dugan RE, Lakshmanan MR, Nepokroeff CM, Porter JW , authors. Stimulation of hepatic â-hydroxy-methylglutaryl Coenzyme A reductase in hypophysectomized rats by L-triiodothyronine. Proc Natl Acad Sci USA. 1973;70(12):3839–42

39. 

Tunbridge WMG, Evered DC, Hall R , authors. Lipid profiles and cardiovascular disease in the Wickham areas with particular reference to thyroid failure. Clin Endocrinol. 1977;7(6):495–508

40. 

Pucci E, Chiovato L, Pinchera A , authors. Thyroid and lipid metabolism. Int J Obesity. 2000;24:109–112

41. 

O’Connel B.J, Genest J , authors. High-density lipoproteins and endothelial function. Circulation. 2001;104(16):1978–83

42. 

Akhigbe R.E, Azeez M.O, Ige S.F, Oyeyipo I.P, Ajao F.O, Soladoye A.O , authors. Hemorheological effect of long-term administration of oral contraceptive in rats. Int J Pharmacol. 2008;4(5):403–06

43. 

Henriksen P A, Newby DE , authors. Therapeutic inhibition of tumor necrosis factor a in patients with heart failure: cooling an inflamed heart. Heart. 2003;89(1):14–8

44. 

Meldrum D. R , author. Tumor necrosis factor in the heart. Amer J Physiol. 1998;274(3):R577–95

45. 

Kapadia SR, Oral H, Lee J, Nakano M, Taffet GE, Mann DL , authors. Hemodynamic regulation of tumor necrosis factor-a gene and protein expression in adult feline myocardium. Circ Res. 1997;81:187–95

46. 

Cain BS, Harken AH, Meldrum DR , authors. Therapeutic strategies to reduce TNF-a mediated cardiac contractile depression following ischemia and reperfusion. J Mol Cell Cardiol. 1999;31(5):931–47

47. 

Cairns CB, Panacek EA, Harken AH, Banerjee A , authors. Bench to bedside: tumor necrosis factor-alpha: from inflammation to resuscitation. Acad Emerg Med. 2000;7(8):930–41

48. 

Fernandez V, Videla LA, Tapia G, Israel Y , authors. Increases in tumor necrosis factor-alpha in response to thyroid hormone-induced liver oxidative stress in the rat. Free Radic Res. 2002 Jul;36(7):719–25

49. 

Díez JJ, Hernanz A, Medina S, Bayón C, Iglesias P , authors. Serum concentrations of tumour necrosis factor-alpha (TNF-alpha) and soluble TNF-alpha receptor p55 in patients with hypothyroidism and hyperthyroidism before and after normalization of thyroid function. Clin Endocrinol (Oxf). 2002;57(4):515–21

50. 

Haudek SB, Taffet GE, Schneider MD, Mann DL , authors. TNF provokes cardiomyocyte apoptosis and cardiac remodeling through activation of multiple cell death pathways. J Clin Invest. 2007;117(9):2692–01

51. 

Deswal A, Petersen NJ, Feldman AM, Young JB, White BG, Mann DL , authors. Cytokines and cytokine receptors in advanced heart failure: an analysis of the cytokine database from the Vesnarinone trial (VEST). Circulation. 2001;103(16):2055–9

52. 

Chrysohoou C, Pitsavos C, Barbetseas J , authors. Chronic systemic inflammation accompanies impaired ventricular diastolic function, detected by Doppler imaging, in patients with newly diagnosed systolic heart failure (Hellenic Heart Failure Study). Heart Vessels. 2009;24(1):22–6

53. 

Nakano M, Knowlton A. A, Dibbs Z, Mann D. L , authors. Tumor necrosis factor-a confers resistance to hypoxic injury in the adult mammalian cardiac myocyte. Circulation. 1998;97(14):1392–400

54. 

Kurrelmeyer K. M, Michael L. H, Baumgarten G, Taffet G. E, Peschon J. J, Sivasubramanian N, Entman M. L, Mann D. L , authors. Endogenous tumor necrosis factor protects the adult cardiac myocyte against ischemic-induced apoptosis in a murine model of acute myocardial infarction. Proc. Natl. Acad. Sci. 2000;97:5456–61

55. 

Sarzi-Puttini P, Atzeni F, Doria A, Iaccarino L, Turiel M , authors. Tumor necrosis factor-alpha, biologic agents and cardiovascular risk. Lupus. 2005;14(9):780–4

56. 

Torun AN, Kulaksizoglu S, Kulaksizoglu M, Pamuk BO, Isbilen E, Tutuncu NB , authors. Serum total antioxidant status and lipid peroxidation marker malondialdehyde levels in overt and subclinical hypothyroidism. Clin Endocrinol (Oxf). 2009;70(3):469–74

57. 

Akinci M, Kosova F, Cetin B, Sepici A, Altan N, Aslan S, Cetin A , authors. Oxidant/antioxidant balance in patients with thyroid cancer. Acta Cir Bras. 2008;23(6):551–4

58. 

Bankson DD, Kestin M, Rifai N , authors. Role of free radicals in cancer and atherosclerosis. Clin Lab Med. 1993;13(2):463–80

59. 

Akhigbe R.E , author. Discordant Results in Plant Toxicity Studies in Africa: Attempt of Standardization. Toxicological Survey of African Medicinal Plants. Elsevier; Victor Kuete , editor. 2014. p. 53–61

60. 

Akinci M, Kosova F, Ҫetin B, Sepici A, Altan N, Aslan S, Ҫetin A , authors. Oxidant/antioxidant balance in patients with thyroid cancer. Acta Cir Bras. 2008;23(6):551–4

61. 

Barton M , author. Cholesterol and atherosclerosis: modulation by oestrogen. Curr Opin Lipidol. 2013;24(3):214–0

62. 

Yla-Herttuala S, Lipton BA, Rosenfeld ME, Sarkioja T, Yoshimura T, Leonard EJ, Witztum JL, Steinberg D , authors. Expression of monocyte chemoattractant protein-1 in macrophage rich areas of human and rabbit atherosclerotic lesions. Proc Natl Acad Sci U S A. 1991;88(12):5252–6

63. 

Yu X, Dluz S, Graves DT, Zhang L, Antoniades HN, Hollander W, Prusty S, Valente AJ, Schwartz CJ, Sonenshein GE , authors. Elevated expression of monocyte chemoattractant protein-1 by vascular smooth muscles in hypercholesterolemic primates. Proc Natl Acad Sci U S A. 1992;89(15):6953–57

64. 

Nelken NA, Coughlin SR, Gordon D, Wilcox JN , authors. Monocyte chemoattractant protein-1 in human atheromatous plaques. J Clin Invest. 1991;88(4):1121–7

65. 

Ralston SH, Russell RG, Gowen M , authors. Estrogen inhibits release of tumor necrosis factor from peripheral blood mononuclear cells in postmenopausal women. J Bone Miner Res. 1990;5:983–88