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Reduction in nitric oxide bioavailability shifts serum lipid content towards atherogenic lipoprotein in rats
Biomedicine & Pharmacotherapy 101 (2018) 792–797
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Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha
Reduction in nitric oxide bioavailability shifts serum lipid content towards atherogenic lipoprotein in rats
T
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Esther Oluwasola Alukoa, , Temidayo Olutayo Omobowaleb, Ademola Adetokunbo Oyagbemic, Olumuyiwa Abiola Adejumobib, Temitayo Olabisi Ajibadec, Adesoji Adedipe Fasanmaded a
Department of Physiology, Faculty of Basic Medical Sciences, University of Uyo, Uyo, Akwa-Ibom State, Nigeria Department of Veterinary Medicine, Faculty of Veterinary Medicine, University of Ibadan, Nigeria c Department of Veterinary Physiology, Biochemistry and Pharmacology, Faculty of Veterinary Medicine, University of Ibadan, Nigeria d Department of Physiology, Faculty of Basic Medical Sciences, University of Ibadan, Nigeria b
A R T I C LE I N FO
A B S T R A C T
Keywords: Nitric oxide synthase inhibitor Blood pressure Lipid contents ACE inhibitor
Nitric oxide (NO) is major endothelial relaxing factor and reduction in its bioavailabilty has been linked to hypertension. Furthermore, high lipid content is a strong risk factor predisposing to cardiovascular diseases. The principal focus of this study was to investigate the effect of blockade of nitric oxide synthase (NOS) on serum lipid content in rats. Male Wistar rats (150–170 g, n = 15) were randomly divided into two groups designated control (n = 5), and L-Name group (n = 10) and were gavage with distilled water and 60 mg/kg of L-NAME respectively daily for three weeks. After 3 weeks, the L-NAME group was sub-divided into two sub-groups (n = 5 each): L-NAME (60 mg/kg of L-NAME), and L-NAME plus ramipril (LR) (60 mg/kg of L-NAME plus 20 mg/kg of ramipril) and were treated daily for another three weeks. The blood pressure (BP) of the conscious rats was measured by tail-cuff method at the onset, at the third and at the sixth weeks of the experiment; while serum lipid contents and NO were measured at the third and sixth weeks. At the end of the experiment blood sample was drawn by ocular puncture for evaluation of lipid profile and NO, and the animals were later euthanized by overdose of anaesthesia. Data were analyzed using ANOVA at p < 0.05. There was a significant increase in BP, triglyceride, total cholesterol, low density lipoprotein-cholesterol, and atherogenic indices in L-NAME group compared to the control and LR group (p < 0.05); NO and high density lipoprotein-cholesterol was significant lower in the L-NAME group compared to control and LR (p < 0.05). In conclusion, reduction in NO bioavailability alters lipid metabolism, which was rectified by ramipril.
1. Introduction Hypertension and hyperlipidemia are major risk factors for cardiovascular diseases. In addition, the combined occurrence of these two risk factors such as seen in metabolic syndrome augments greatly the risk for cardiovascular complications [1]. Nitric oxide (NO) is a main local mediator released by the endothelium and serves as a key signaling molecule in various physiological processes. It is a major endothelial relaxing factor essential for dilating the blood vessels to facilitate blood flow. Reduction in nitric oxide (NO) bioavailability plays an important role in blood pressure elevation [2] and several diseases associated with vascular dysfunction such as atherosclerosis, diabetes mellitus, hypertension, or preeclampsia are associated with altered NO signaling [3]. Nitric oxide has been documented to be a principal factor involved in the anti-atherosclerotic properties of the endothelium [4] and inhibition of NO synthase causes accelerated atherosclerosis in experimental models [5]. ⁎
Studies have suggested the role of nitric oxide in the regulation of lipid metabolism and endothelial and inducible nitric oxide synthase (NOS) have been shown to be present in adipose tissue of the rat [6], suggesting that adipose tissue may be a potential source of NO production. NO exerts beneficial effects on lipid metabolism through activation of hepatic sterol regulatory element-binding protein (SREBP)-2 [7]. On the contrary, the renin angiotensin aldosterone system (RAAS) plays major role in blood pressure regulation, and excessive activation of this system, is implicated in the pathogenesis of hypertension and end-organ damage associated with hypertension [8]. Furthermore, angiotensin II has been reported to inhibit the endothelium-dependent relaxation by decreasing nitric oxide bioavailability. Angiotensin converting enzyme has also been shown to inhibit kallikrein–kinin-bradykinin system, which is an important system involved in nitric oxide production [9]. The adipose tissue has been shown to express renin-angiotensin
Corresponding author at: Physiology Department, Faculty of Basic Medical Sciences, University of Ibadan, Ibadan, Nigeria. E-mail address: [email protected] (E.O. Aluko).
https://doi.org/10.1016/j.biopha.2018.03.001 Received 29 January 2018; Received in revised form 28 February 2018; Accepted 2 March 2018 0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.
Biomedicine & Pharmacotherapy 101 (2018) 792–797
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system components and angiotensin II receptors; moreover, adipocyte lipid metabolism has been reported to be influenced by angiotensin II. Angiotensin II has also been documented to increase inflammatory gene expression and decrease adiponectin expression [10]. Adiponectin is a protein produced by the adipose tissue that has beneficial effects on insulin resistance, atherosclerosis, and inflammation [11]. The use of combination therapies that tackle hyperlipidemia and hypertension has been recommended and predicted to be an effective approach to significantly reduce the occurrence of cardiovascular complications in metabolic syndrome [12]. Therefore, this study is aimed at assessing the effect of ramipril an ACE inhibitor on reduction in nitric oxide bio-availability in rats.
Nigeria. The rats were housed under standard laboratory conditions, fed with normal rat chow and were given access to clean water ad libitum. They were allowed to acclimatize for a period of two (2) weeks before the commencement of the experiment. The experimental protocols were approved by the Animal Ethics Committee of the University of Ibadan, Oyo State, Nigeria.
Diagram illustrating the inter-connections between nitric oxide, renin-angiotensin II-aldosterone system and kallikrein–kinin-bradykinin system. iNOS- inducible nitric oxide synthase and cNOS- constitutive nitric oxide synthase.
in nitric oxide bioavailability was induced by daily oral administration of 60 mg/kg of L-NAME for three (3) weeks. After three weeks, the LNAME group was sub-divided into two sub-groups (n = 5):
2.4. Experimental design Fifteen (15) male Wistar rats (150–170 g) were used for the experiment. These were divided into two groups: the control group (n = 5) and L-NAME group (n = 10). In the L-NAME group, reduction
• L-NAME group: received 60 mg/kg/daily of L-NAME. • L-NAME plus ramipril group: received 60 mg/kg/daily of L-NAME plus 20 mg/kg/daily ramipril. • Administration was done orally for another three (3) weeks.
2. Materials and methods 2.1. List of assay kits used Total cholesterol (TC), Triglycerides (TAG), High-density lipoprotein cholesterol (HDL-C)
2.5. Measurement of blood pressure The blood pressure of the conscious rats was measured non-invasively by using a volume pressure recording sensor through the occlusion tail-cuff method (CODA, Kent Scientific, USA). The rats were placed in a holding device mount on a thermostatically control warming plate with mini-cuffs fix around the tails to detect the artery pulsations. The rats were allowed to acclimate to the cuffs for 10–15 min before the recording session. The data were recorded thrice per session and average values were taken [13]. The blood pressure was measured at the onset, at the third and at the sixth weeks of the experiment.
2.2. Drugs NG-nitro-L-arginine methyl ester (L-NAME) was obtained from Santa Cruz Biotechnology (Finnell Street, Dallas, Texas, USA.), ramipril was obtained from Sigma (St. Louis, MO, USA). 2.3. Experimental animals Male Wistar rats (150–170 g) were used for the experiment and were obtained from the animal house, University of Ibadan, Oyo State,
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2.6. Biochemical parameters 2.6.1. Blood sample At the third week of the experiment, blood sample was drawn by ocular puncture in conscious rats in the L-NAME and control groups into a plain bottle to assay for serum lipid profile and nitric oxide concentration. This was repeated at the end of the experiment before the animals were sacrificed. The animals were euthanized by overdose of sodium pentobarbitone. The samples were centrifuged at 2500 rpm for 10 min, and the serum was isolated and stored at −20 °C for lipid profile and nitric oxide concentration.
Fig. 2. Serum nitric oxide level. Data are presented as mean ± SEM, (n = 5, p < 0.05), # - compared to C, * - compared to L6. C –control group, L3- L-NAME treated for 3 weeks, L6- L-NAME treated for 6 weeks, LR- L-NAME plus ramipril.
2.6.2. Estimation of serum lipid profile The measurement of serum lipid profile was done according to the methods previously described by Ojiako et al. [14]. Total cholesterol (TC), triglycerides (TAG), and high-density lipoprotein cholesterol (HDL-C) were measured using commercial kits (Fortress Diagnostics Limited, Antrim, UK).
analyze NO plasma concentration [18]. Briefly, Equal volumes of N-(1naphthyl) ethylenediamine (Component A) and sulfanilic acid (Component B) were mixed together to form the Griess Reagent. Sufficient reagent for experiments was prepared immediately (100 μL per spectrophotometer cuvette). The following were mixed in a spectrophotometer cuvette (1 cm light path): 100 μL of Griess Reagent, 300 μL of the nitrite-containing sample and 2.6 mL of deionized water. The mixture was incubated for 30 min at room temperature. A photometric reference sample was prepared by mixing 100 μL of Griess Reagent and 2.9 mL of deionized water. The absorbance of the nitrite-containing sample was measured at 548 nm.
2.6.3. Estimation of low-density lipoprotein cholesterol (LDL-C) Low-density lipoprotein cholesterol (LDL-C) concentration was estimated according to the formula of Friedewald et al. [15]; LDL-C = TC – HDL-C – (TAG/5) 2.6.4. Estimation of very low density lipoprotein cholesterol (VLDL-C) VLDL-C was calculated based on the formula: VLDL-C = TG/5 in mg/dl [16].
2.8. Statistical analysis 2.6.5. Estimation of atherogenic indices All data were expressed as mean ± SEM. Data were analyzed using analysis of variance (ANOVA) using Graphpad prism 7.01 (Graphpad software, Inc., USA). Post-hoc comparison was performed after ANOVA using Turkey’s tests. The level of significance was set at P < 0.05 in all cases.
• The Atherogenic Index of Plasma (AI) was calculated as follows: log (TG/HDL-C) [17].
• Atherogenic Coefficient (AC) was calculated as follows:
3. Results
(TC-HDL-C)/HDL-C. 3.1. Body weight
• Cardiac Risk Ratio (CRR) was calculated as follows:
Fig. 1 shows the mean body weight and weight changes of the studied groups and the control. The initial body weight of all the groups was not significantly different. The progressive weight change of the LNAME group and L-NAME ramipril was not significantly different from that the control. The final body weight recorded in L-NAME group and L-NAME ramipril at sixth week was also not significantly different compared to the control (P < 0.05).
TC/HDL-C. 2.7. Measurement of nitric oxide Nitrite is a principal oxidation product of NO in an aqueous solution, so the nitrite plasma levels (NOx) was determined in order to
3.2. Serum nitric oxide level Fig. 2 shows mean serum nitric oxide concentration. Nitric oxide level decreased significantly in the L-NAME group at the end of induction of reduction in nitric oxide bioavailability compared to control. Table 1 Systolic Blood Pressure (SBP), Diastolic Blood Pressure (DBP), and Mean Arterial Pressure (MAP). Variables SBP DBP MAP
C 87.50 ± 4.92 55.80 ± 7.05 66.30 ± 5.52
L3
L6 #,*
145 ± 3.38 118 ± 3.51#,* 132 ± 5.11#,*
LR #
170 ± 3.74 146 ± 5.51# 154 ± 4.63#
134 ± 2.33#,* 84.7 ± 5.21#,*,a 101 ± 3.01#,*,a
Data are presented as mean ± SEM, (n = 5, p < 0.05). C –control group, L3- L-NAME treated for 3 weeks, L6- L-NAME treated for 6 weeks, LR- L-NAME plus ramipril. # compared to C. * compared to L6. a compared to L3.
Fig. 1. Mean body weight. Data are presented as mean ± SEM, (n = 5, p < 0.05). C –control group, L6- L-NAME group, and LR- L-NAME ramipril group.
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Fig. 5. Serum high density lipoprotein cholesterol (HDL-C). Data are presented as mean ± SEM, (n = 5; p < 0.05). # - compared to C, * - compared to L6, a – compared to L3. C –control group, L3- L-NAME treated for 3 weeks, L6- L-NAME group treated for 6 weeks, LR- L-NAME ramipril group.
Fig. 3. Serum total cholesterol (TC) and low density lipoprotein (LDL-C). Data are presented as mean ± SEM, (n = 5; p < 0.05). #- compared to C, * - compared to L6, a – compared to L3. C –control group, L3- L-NAME treated for 3 weeks, L6- L-NAME group treated for 6 weeks, LR- L-NAME ramipril group.
3.5. Triglyceride (TG) and very low density lipoprotein-cholesterol (VLDLC)
At end of the experiment, nitric oxide level decreased significantly in the L-NAME group and L-NAME ramipril group compared to the control.
At the establishment of reduction in nitric oxide bioavailability, there was a significant increase in TG level in the L-NAME group compared to the control. The study recorded a significant increase in TG level at the end of experiment in L-NAME group compared to the control group and L-NAME group treated with ramipril. There was no significant different in VLDL-C level in all the studied groups compared to the control (Fig. 4).
3.3. Systolic blood pressure, diastolic blood pressure and mean arterial pressure Table 1 shows blood pressure changes. Systolic blood pressure (SBP), diastolic blood pressure (DBP) and mean arterial pressure (MAP) increased significantly in the L-NAME group at the induction of reduction in nitric oxide bioavailability compared to control. At end of the experiment, SBP, DBP and MAP increased significantly in the LNAME group when compared with the control and L-NAME ramipril group. The blood pressure recorded at the induction period was also significantly higher than that of the L-NAME ramipril group and lower than that of L-NAME group at the end of experiment.
3.6. High density lipoprotein-cholesterol (HDL-C) The study recorded a significant reduction in HDL-C level at the induction of reduction in nitric oxide bioavailability in the L-NAME group when compared with the control at the third week. A significant decrease was observed at sixth week in the L-NAME group compared to the control and L-NAME group treated with ramipril. The HDL-C level in the L-NAME ramipril group was also significantly higher than that recorded at the three week of the experiment (Fig. 5).
3.4. Total cholesterol (TC) low density and lipoprotein-cholesterol (LDL-C)
3.7. Atherogenic indices
Fig. 3 shows total cholesterol (TC) and low density lipoprotein (LDLC) levels. The study recorded a significant increase in TC and LDL-C at the third week of the experiment, which marked the induction of reduction nitric oxide bioavailability when compared to the control group. At the end of the experiment, there was a significant increase TC and LDL-C in the L-NAME group compared to the control and the LNAME group treated with ramipril. The L-NAME ramipril group also recorded a significant decrease in LDL-C compared to the level recorded at the third week of the experiment.
There was a significant increase in atherogenic index (AI), atherogenic coefficient (AC) and cardiac risk ratio (CRR) at the third week of the experiment in the L-NAME group compared to control. At the end of the experiment, the study recorded a significant increase in all the atherogenic indices in the L-NAME group compared to the control and the L-NAME group treated with ramipril. The atherogenic indices of the L-NAME group treated with ramipril were also significantly lower than the level recorded at the induction period (Table 2) 4. Discussion The study evaluated the effect ramipril on high blood pressure and high lipid contents induced by NG-nitro-L-arginine methyl ester (LNAME), a non-selective nitric oxide synthase (NOS) inhibitor. L-NAME inhibits NOS by competing with arginine the main substrate of NOS essential for nitric oxide production at the active site. 4.1. Blood pressure Administration of L-NAME for three weeks increased significantly systolic blood pressure, diastolic blood pressure and mean arterial pressure consistent with documented data [19,20]. The use of non-selective nitric oxide synthase inhibitors is a well established and known model for inducing hypertension and its effectiveness in hypertension induction has been reported by several researchers [21]. L-NAME has a
Fig. 4. Serum triglyceride (TG) and very low density lipoprotein-cholesterol (VLDL-C). Data are presented as mean ± SEM, (n = 5; p < 0.05). # - compared to C, * - compared to L6, a – compared to L3. C –control group, L3- L-NAME treated for 3 weeks, L6- L-NAME group treated for 6 weeks, LR- L-NAME ramipril group.
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Table 2 Atherogenic indices (atherogenic index (AI), atherogenic coefficient (AC), and cardiac risk ratio (CRR). Parameters
C
L3
L6
LR
AI AC CRR
−0.02 ± 0.04 2.27 ± 0.39 3.87 ± 0.67
0.25 ± 0.01# 4.23 ± 0.25# 5.23 ± 0.25#,*
0.35 ± 0.03# 5.56 ± 0.47# 7.30 ± 0.82#
−0.08 ± 0.03*,a 2.13 ± 0.27*,a 3.01 ± 0.26*,a
Data are presented as mean ± SEM, (n = 5; p < 0.05). C –control group, L3- L-NAME treated for 3 weeks, L6- L-NAME group treated for 6 weeks, LR- L-NAME ramipril group. # compared to C. * compared to L6. a compared to L3.
Goudarz and co-worker [29]. They reported an increase in total cholesterol, LDL, VLDL, and TG levels, and decreased HDL concentration. However, the present study only observed a marginal increase in VLDLC values. Murugesan and Raja [30] also reported similar results in rats treated with L-NAME. High serum triglycerides (TG), total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) are well known risk factors for cardiovascular disease [31]. Conversely high density lipoprotein (HDL) cholesterol is considered as protective cholesterol. A low serum HDL-cholesterol level is therefore considered as an independent cardiovascular risk factor that leads to the development of atherosclerosis and related cardiovascular events [32]. Interestingly, low levels of triglyceride, total cholesterol, LDL-C and high HDL-C were observed in the L-NAME group treated with ramipril. Similarly, Turhan et al. [33] reported that ACE inhibitor (captopril) decreases triglyceride and cholesterol levels in ovariectomized-aged rats and also another study reported low LDL-C, TC and TG as well as a high HDL-C values in Children with metabolic syndrome treated with ACE inhibitors [34]. The mechanism via which ACE inhibitors modulate lipid metabolism is not well known, however, from this study it is possible that ramipril act via a nitric oxide- independent pathway, since the study recorded a low nitric oxide level in this group. Atherogenic indices are calculated from TC, TG and HDL-C. They reflect the true relationship between protective and atherogenic lipoprotein and are also associated with the size of pro- atherogenic lipoprotein and anti- atherogenic lipoprotein particle [35]. Atherogenic indices have been shown to be strong predictors for metabolic disturbances which include dyslipidemia, atherosclerosis, hypertension and cardiovascular diseases [36]. These indices are atherogenic index of plasma (AIP), atherogenic co-efficient (AC) and cardiac risk ratio (CRR). They give clearer indication of blood cholesterol level that tends towards atherogenic dyslipidemia. Intriguingly, atherogenic indices were significantly low in the L-NAME group treated with ramipril.
progressive time dependent effect on blood pressure, as it was observed in the study that the blood pressure recorded at the sixth week was significantly higher than that of the third week. L-NAME has been reported to have dose and time dependent effect on blood pressure [22,23]. Ramipril cause a reduction in systolic blood pressure, diastolic blood pressure and mean arterial pressure. Ramipril has been reported to alleviate hypertension induced by the administration of L-NAME by inhibiting angiotensin converting enzyme (ACE) [24,25]. ACE is an enzyme that catalyzes the conversion of angiotensin I to angiotensin II, a powerful vasoconstrictor and stimulator of aldosterone release. ACE has also been reported to inhibit kallikrein–kinin system, which is also an important pathway in nitric oxide production [9]. Therefore, inhibiting ACE enhances nitric oxide production in addition to inhibition of angiotensin II production; these two effects consequently reduce blood pressure. 4.2. Serum nitric oxide level Similar to what has been reported, low serum nitric oxide was recorded in the study. In contrast, L-NAME group treated with ramipril recorded a fall in serum nitric oxide. This was similar to the findings of Bernátová et al. [26] who reported that captopril did not affect nitric oxide synthase inhibition; however, the study of Seth et al. [27] reported an increase in nitric oxide in L-NAME rats treated with captopril. The fall in nitric oxide observed in L-NAME rats treated with ramipril actually negate our hypothesis which was based on the kallikrein–kininbradykinin system activated by angiotensin converting enzyme inhibitors. The fall in nitric oxide observed in L-NAME group treated with ramipril indicated that the kallikrein–kinin-bradykinin-nitric oxide pathway might be acting through endothelial nitric oxide synthase (eNOS) dependent pathway, which was actually inhibited in this study. Study has reported that bradykinin upregulated endothelial nitric oxide synthase and neuronal nitric oxide synthase [28].
5. Conclusion In conclusion, ACE inhibitor might be a better therapeutic intervention to curtail both hypertension and hyperlipidemia. Hyperlipidemia and hypertension medications used in combination can effectively lower the possibility of cardiovascular complications in the population with metabolic syndrome. It will be economically and medically prudent to use a therapy that can effectively treat hypertension as well as dyslipidemia concomitantly than using different medications to manage them. The use of ramipril to curtail abnormal high blood pressure and blood lipid content should be recommend as a remedy in medicine. However, further studies are still needed to ascertain the precise mechanism through which ramipril modulates lipid metabolism.
4.3. Lipid profile Studies have suggested the role of nitric oxide in the regulation of lipid metabolism and endothelial and inducible NOS have been shown to be present in adipose tissue of the rat [6], suggesting that adipose tissue may be a potential source of NO production. Nitric oxide (NO) has been suggested to regulate lipid metabolism through the activation of hepatic sterol regulatory element-binding protein (SREBP)-2, a transcriptional factor necessary for cholesterol metabolism and expression of LDL receptor. LDL receptors enhance the uptake of cholesterol into the hepatic cells and thus aid to maintain the blood cholesterol level [7]. Hyperlipidemia is a major risk factor of atherosclerosis and cardiovascular diseases. Nitric oxide has been documented to be a principal factor involved in the anti-atherosclerotic properties of the endothelium [4] and inhibition of NO synthase causes accelerated atherosclerosis in experimental models [5]. The blockade of nitric oxide synthase by L-NAME resulted in high total cholesterol, low density lipoprotein (LDL-C), and a low level of high density lipoprotein (HDL-C); this is similar to the report of
Author’s contributions
• Esther Oluwasola Aluko designed the work and drafted the manuscript. • Temidayo Olutayo Omobowale, Ademola Adetokunbo Oyagbemi, Adejumobi and Temitayo Olabisi Ajibade coordinated and measured
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the blood pressure of the animals.
• Adesoji Adedipe Fasanmade revised and approved the final version
[17]
of the manuscript.
Conflict of interest
[18]
The authors declare no conflict of interest. [19]
Acknowledgment The authors wish to acknowledge Mr. Okon of the Department of Physiology, University of Ibadan for his technical support.
[20]
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Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha
Reduction in nitric oxide bioavailability shifts serum lipid content towards atherogenic lipoprotein in rats
T
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Esther Oluwasola Alukoa, , Temidayo Olutayo Omobowaleb, Ademola Adetokunbo Oyagbemic, Olumuyiwa Abiola Adejumobib, Temitayo Olabisi Ajibadec, Adesoji Adedipe Fasanmaded a
Department of Physiology, Faculty of Basic Medical Sciences, University of Uyo, Uyo, Akwa-Ibom State, Nigeria Department of Veterinary Medicine, Faculty of Veterinary Medicine, University of Ibadan, Nigeria c Department of Veterinary Physiology, Biochemistry and Pharmacology, Faculty of Veterinary Medicine, University of Ibadan, Nigeria d Department of Physiology, Faculty of Basic Medical Sciences, University of Ibadan, Nigeria b
A R T I C LE I N FO
A B S T R A C T
Keywords: Nitric oxide synthase inhibitor Blood pressure Lipid contents ACE inhibitor
Nitric oxide (NO) is major endothelial relaxing factor and reduction in its bioavailabilty has been linked to hypertension. Furthermore, high lipid content is a strong risk factor predisposing to cardiovascular diseases. The principal focus of this study was to investigate the effect of blockade of nitric oxide synthase (NOS) on serum lipid content in rats. Male Wistar rats (150–170 g, n = 15) were randomly divided into two groups designated control (n = 5), and L-Name group (n = 10) and were gavage with distilled water and 60 mg/kg of L-NAME respectively daily for three weeks. After 3 weeks, the L-NAME group was sub-divided into two sub-groups (n = 5 each): L-NAME (60 mg/kg of L-NAME), and L-NAME plus ramipril (LR) (60 mg/kg of L-NAME plus 20 mg/kg of ramipril) and were treated daily for another three weeks. The blood pressure (BP) of the conscious rats was measured by tail-cuff method at the onset, at the third and at the sixth weeks of the experiment; while serum lipid contents and NO were measured at the third and sixth weeks. At the end of the experiment blood sample was drawn by ocular puncture for evaluation of lipid profile and NO, and the animals were later euthanized by overdose of anaesthesia. Data were analyzed using ANOVA at p < 0.05. There was a significant increase in BP, triglyceride, total cholesterol, low density lipoprotein-cholesterol, and atherogenic indices in L-NAME group compared to the control and LR group (p < 0.05); NO and high density lipoprotein-cholesterol was significant lower in the L-NAME group compared to control and LR (p < 0.05). In conclusion, reduction in NO bioavailability alters lipid metabolism, which was rectified by ramipril.
1. Introduction Hypertension and hyperlipidemia are major risk factors for cardiovascular diseases. In addition, the combined occurrence of these two risk factors such as seen in metabolic syndrome augments greatly the risk for cardiovascular complications [1]. Nitric oxide (NO) is a main local mediator released by the endothelium and serves as a key signaling molecule in various physiological processes. It is a major endothelial relaxing factor essential for dilating the blood vessels to facilitate blood flow. Reduction in nitric oxide (NO) bioavailability plays an important role in blood pressure elevation [2] and several diseases associated with vascular dysfunction such as atherosclerosis, diabetes mellitus, hypertension, or preeclampsia are associated with altered NO signaling [3]. Nitric oxide has been documented to be a principal factor involved in the anti-atherosclerotic properties of the endothelium [4] and inhibition of NO synthase causes accelerated atherosclerosis in experimental models [5]. ⁎
Studies have suggested the role of nitric oxide in the regulation of lipid metabolism and endothelial and inducible nitric oxide synthase (NOS) have been shown to be present in adipose tissue of the rat [6], suggesting that adipose tissue may be a potential source of NO production. NO exerts beneficial effects on lipid metabolism through activation of hepatic sterol regulatory element-binding protein (SREBP)-2 [7]. On the contrary, the renin angiotensin aldosterone system (RAAS) plays major role in blood pressure regulation, and excessive activation of this system, is implicated in the pathogenesis of hypertension and end-organ damage associated with hypertension [8]. Furthermore, angiotensin II has been reported to inhibit the endothelium-dependent relaxation by decreasing nitric oxide bioavailability. Angiotensin converting enzyme has also been shown to inhibit kallikrein–kinin-bradykinin system, which is an important system involved in nitric oxide production [9]. The adipose tissue has been shown to express renin-angiotensin
Corresponding author at: Physiology Department, Faculty of Basic Medical Sciences, University of Ibadan, Ibadan, Nigeria. E-mail address: [email protected] (E.O. Aluko).
https://doi.org/10.1016/j.biopha.2018.03.001 Received 29 January 2018; Received in revised form 28 February 2018; Accepted 2 March 2018 0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.
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system components and angiotensin II receptors; moreover, adipocyte lipid metabolism has been reported to be influenced by angiotensin II. Angiotensin II has also been documented to increase inflammatory gene expression and decrease adiponectin expression [10]. Adiponectin is a protein produced by the adipose tissue that has beneficial effects on insulin resistance, atherosclerosis, and inflammation [11]. The use of combination therapies that tackle hyperlipidemia and hypertension has been recommended and predicted to be an effective approach to significantly reduce the occurrence of cardiovascular complications in metabolic syndrome [12]. Therefore, this study is aimed at assessing the effect of ramipril an ACE inhibitor on reduction in nitric oxide bio-availability in rats.
Nigeria. The rats were housed under standard laboratory conditions, fed with normal rat chow and were given access to clean water ad libitum. They were allowed to acclimatize for a period of two (2) weeks before the commencement of the experiment. The experimental protocols were approved by the Animal Ethics Committee of the University of Ibadan, Oyo State, Nigeria.
Diagram illustrating the inter-connections between nitric oxide, renin-angiotensin II-aldosterone system and kallikrein–kinin-bradykinin system. iNOS- inducible nitric oxide synthase and cNOS- constitutive nitric oxide synthase.
in nitric oxide bioavailability was induced by daily oral administration of 60 mg/kg of L-NAME for three (3) weeks. After three weeks, the LNAME group was sub-divided into two sub-groups (n = 5):
2.4. Experimental design Fifteen (15) male Wistar rats (150–170 g) were used for the experiment. These were divided into two groups: the control group (n = 5) and L-NAME group (n = 10). In the L-NAME group, reduction
• L-NAME group: received 60 mg/kg/daily of L-NAME. • L-NAME plus ramipril group: received 60 mg/kg/daily of L-NAME plus 20 mg/kg/daily ramipril. • Administration was done orally for another three (3) weeks.
2. Materials and methods 2.1. List of assay kits used Total cholesterol (TC), Triglycerides (TAG), High-density lipoprotein cholesterol (HDL-C)
2.5. Measurement of blood pressure The blood pressure of the conscious rats was measured non-invasively by using a volume pressure recording sensor through the occlusion tail-cuff method (CODA, Kent Scientific, USA). The rats were placed in a holding device mount on a thermostatically control warming plate with mini-cuffs fix around the tails to detect the artery pulsations. The rats were allowed to acclimate to the cuffs for 10–15 min before the recording session. The data were recorded thrice per session and average values were taken [13]. The blood pressure was measured at the onset, at the third and at the sixth weeks of the experiment.
2.2. Drugs NG-nitro-L-arginine methyl ester (L-NAME) was obtained from Santa Cruz Biotechnology (Finnell Street, Dallas, Texas, USA.), ramipril was obtained from Sigma (St. Louis, MO, USA). 2.3. Experimental animals Male Wistar rats (150–170 g) were used for the experiment and were obtained from the animal house, University of Ibadan, Oyo State,
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2.6. Biochemical parameters 2.6.1. Blood sample At the third week of the experiment, blood sample was drawn by ocular puncture in conscious rats in the L-NAME and control groups into a plain bottle to assay for serum lipid profile and nitric oxide concentration. This was repeated at the end of the experiment before the animals were sacrificed. The animals were euthanized by overdose of sodium pentobarbitone. The samples were centrifuged at 2500 rpm for 10 min, and the serum was isolated and stored at −20 °C for lipid profile and nitric oxide concentration.
Fig. 2. Serum nitric oxide level. Data are presented as mean ± SEM, (n = 5, p < 0.05), # - compared to C, * - compared to L6. C –control group, L3- L-NAME treated for 3 weeks, L6- L-NAME treated for 6 weeks, LR- L-NAME plus ramipril.
2.6.2. Estimation of serum lipid profile The measurement of serum lipid profile was done according to the methods previously described by Ojiako et al. [14]. Total cholesterol (TC), triglycerides (TAG), and high-density lipoprotein cholesterol (HDL-C) were measured using commercial kits (Fortress Diagnostics Limited, Antrim, UK).
analyze NO plasma concentration [18]. Briefly, Equal volumes of N-(1naphthyl) ethylenediamine (Component A) and sulfanilic acid (Component B) were mixed together to form the Griess Reagent. Sufficient reagent for experiments was prepared immediately (100 μL per spectrophotometer cuvette). The following were mixed in a spectrophotometer cuvette (1 cm light path): 100 μL of Griess Reagent, 300 μL of the nitrite-containing sample and 2.6 mL of deionized water. The mixture was incubated for 30 min at room temperature. A photometric reference sample was prepared by mixing 100 μL of Griess Reagent and 2.9 mL of deionized water. The absorbance of the nitrite-containing sample was measured at 548 nm.
2.6.3. Estimation of low-density lipoprotein cholesterol (LDL-C) Low-density lipoprotein cholesterol (LDL-C) concentration was estimated according to the formula of Friedewald et al. [15]; LDL-C = TC – HDL-C – (TAG/5) 2.6.4. Estimation of very low density lipoprotein cholesterol (VLDL-C) VLDL-C was calculated based on the formula: VLDL-C = TG/5 in mg/dl [16].
2.8. Statistical analysis 2.6.5. Estimation of atherogenic indices All data were expressed as mean ± SEM. Data were analyzed using analysis of variance (ANOVA) using Graphpad prism 7.01 (Graphpad software, Inc., USA). Post-hoc comparison was performed after ANOVA using Turkey’s tests. The level of significance was set at P < 0.05 in all cases.
• The Atherogenic Index of Plasma (AI) was calculated as follows: log (TG/HDL-C) [17].
• Atherogenic Coefficient (AC) was calculated as follows:
3. Results
(TC-HDL-C)/HDL-C. 3.1. Body weight
• Cardiac Risk Ratio (CRR) was calculated as follows:
Fig. 1 shows the mean body weight and weight changes of the studied groups and the control. The initial body weight of all the groups was not significantly different. The progressive weight change of the LNAME group and L-NAME ramipril was not significantly different from that the control. The final body weight recorded in L-NAME group and L-NAME ramipril at sixth week was also not significantly different compared to the control (P < 0.05).
TC/HDL-C. 2.7. Measurement of nitric oxide Nitrite is a principal oxidation product of NO in an aqueous solution, so the nitrite plasma levels (NOx) was determined in order to
3.2. Serum nitric oxide level Fig. 2 shows mean serum nitric oxide concentration. Nitric oxide level decreased significantly in the L-NAME group at the end of induction of reduction in nitric oxide bioavailability compared to control. Table 1 Systolic Blood Pressure (SBP), Diastolic Blood Pressure (DBP), and Mean Arterial Pressure (MAP). Variables SBP DBP MAP
C 87.50 ± 4.92 55.80 ± 7.05 66.30 ± 5.52
L3
L6 #,*
145 ± 3.38 118 ± 3.51#,* 132 ± 5.11#,*
LR #
170 ± 3.74 146 ± 5.51# 154 ± 4.63#
134 ± 2.33#,* 84.7 ± 5.21#,*,a 101 ± 3.01#,*,a
Data are presented as mean ± SEM, (n = 5, p < 0.05). C –control group, L3- L-NAME treated for 3 weeks, L6- L-NAME treated for 6 weeks, LR- L-NAME plus ramipril. # compared to C. * compared to L6. a compared to L3.
Fig. 1. Mean body weight. Data are presented as mean ± SEM, (n = 5, p < 0.05). C –control group, L6- L-NAME group, and LR- L-NAME ramipril group.
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Fig. 5. Serum high density lipoprotein cholesterol (HDL-C). Data are presented as mean ± SEM, (n = 5; p < 0.05). # - compared to C, * - compared to L6, a – compared to L3. C –control group, L3- L-NAME treated for 3 weeks, L6- L-NAME group treated for 6 weeks, LR- L-NAME ramipril group.
Fig. 3. Serum total cholesterol (TC) and low density lipoprotein (LDL-C). Data are presented as mean ± SEM, (n = 5; p < 0.05). #- compared to C, * - compared to L6, a – compared to L3. C –control group, L3- L-NAME treated for 3 weeks, L6- L-NAME group treated for 6 weeks, LR- L-NAME ramipril group.
3.5. Triglyceride (TG) and very low density lipoprotein-cholesterol (VLDLC)
At end of the experiment, nitric oxide level decreased significantly in the L-NAME group and L-NAME ramipril group compared to the control.
At the establishment of reduction in nitric oxide bioavailability, there was a significant increase in TG level in the L-NAME group compared to the control. The study recorded a significant increase in TG level at the end of experiment in L-NAME group compared to the control group and L-NAME group treated with ramipril. There was no significant different in VLDL-C level in all the studied groups compared to the control (Fig. 4).
3.3. Systolic blood pressure, diastolic blood pressure and mean arterial pressure Table 1 shows blood pressure changes. Systolic blood pressure (SBP), diastolic blood pressure (DBP) and mean arterial pressure (MAP) increased significantly in the L-NAME group at the induction of reduction in nitric oxide bioavailability compared to control. At end of the experiment, SBP, DBP and MAP increased significantly in the LNAME group when compared with the control and L-NAME ramipril group. The blood pressure recorded at the induction period was also significantly higher than that of the L-NAME ramipril group and lower than that of L-NAME group at the end of experiment.
3.6. High density lipoprotein-cholesterol (HDL-C) The study recorded a significant reduction in HDL-C level at the induction of reduction in nitric oxide bioavailability in the L-NAME group when compared with the control at the third week. A significant decrease was observed at sixth week in the L-NAME group compared to the control and L-NAME group treated with ramipril. The HDL-C level in the L-NAME ramipril group was also significantly higher than that recorded at the three week of the experiment (Fig. 5).
3.4. Total cholesterol (TC) low density and lipoprotein-cholesterol (LDL-C)
3.7. Atherogenic indices
Fig. 3 shows total cholesterol (TC) and low density lipoprotein (LDLC) levels. The study recorded a significant increase in TC and LDL-C at the third week of the experiment, which marked the induction of reduction nitric oxide bioavailability when compared to the control group. At the end of the experiment, there was a significant increase TC and LDL-C in the L-NAME group compared to the control and the LNAME group treated with ramipril. The L-NAME ramipril group also recorded a significant decrease in LDL-C compared to the level recorded at the third week of the experiment.
There was a significant increase in atherogenic index (AI), atherogenic coefficient (AC) and cardiac risk ratio (CRR) at the third week of the experiment in the L-NAME group compared to control. At the end of the experiment, the study recorded a significant increase in all the atherogenic indices in the L-NAME group compared to the control and the L-NAME group treated with ramipril. The atherogenic indices of the L-NAME group treated with ramipril were also significantly lower than the level recorded at the induction period (Table 2) 4. Discussion The study evaluated the effect ramipril on high blood pressure and high lipid contents induced by NG-nitro-L-arginine methyl ester (LNAME), a non-selective nitric oxide synthase (NOS) inhibitor. L-NAME inhibits NOS by competing with arginine the main substrate of NOS essential for nitric oxide production at the active site. 4.1. Blood pressure Administration of L-NAME for three weeks increased significantly systolic blood pressure, diastolic blood pressure and mean arterial pressure consistent with documented data [19,20]. The use of non-selective nitric oxide synthase inhibitors is a well established and known model for inducing hypertension and its effectiveness in hypertension induction has been reported by several researchers [21]. L-NAME has a
Fig. 4. Serum triglyceride (TG) and very low density lipoprotein-cholesterol (VLDL-C). Data are presented as mean ± SEM, (n = 5; p < 0.05). # - compared to C, * - compared to L6, a – compared to L3. C –control group, L3- L-NAME treated for 3 weeks, L6- L-NAME group treated for 6 weeks, LR- L-NAME ramipril group.
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Table 2 Atherogenic indices (atherogenic index (AI), atherogenic coefficient (AC), and cardiac risk ratio (CRR). Parameters
C
L3
L6
LR
AI AC CRR
−0.02 ± 0.04 2.27 ± 0.39 3.87 ± 0.67
0.25 ± 0.01# 4.23 ± 0.25# 5.23 ± 0.25#,*
0.35 ± 0.03# 5.56 ± 0.47# 7.30 ± 0.82#
−0.08 ± 0.03*,a 2.13 ± 0.27*,a 3.01 ± 0.26*,a
Data are presented as mean ± SEM, (n = 5; p < 0.05). C –control group, L3- L-NAME treated for 3 weeks, L6- L-NAME group treated for 6 weeks, LR- L-NAME ramipril group. # compared to C. * compared to L6. a compared to L3.
Goudarz and co-worker [29]. They reported an increase in total cholesterol, LDL, VLDL, and TG levels, and decreased HDL concentration. However, the present study only observed a marginal increase in VLDLC values. Murugesan and Raja [30] also reported similar results in rats treated with L-NAME. High serum triglycerides (TG), total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) are well known risk factors for cardiovascular disease [31]. Conversely high density lipoprotein (HDL) cholesterol is considered as protective cholesterol. A low serum HDL-cholesterol level is therefore considered as an independent cardiovascular risk factor that leads to the development of atherosclerosis and related cardiovascular events [32]. Interestingly, low levels of triglyceride, total cholesterol, LDL-C and high HDL-C were observed in the L-NAME group treated with ramipril. Similarly, Turhan et al. [33] reported that ACE inhibitor (captopril) decreases triglyceride and cholesterol levels in ovariectomized-aged rats and also another study reported low LDL-C, TC and TG as well as a high HDL-C values in Children with metabolic syndrome treated with ACE inhibitors [34]. The mechanism via which ACE inhibitors modulate lipid metabolism is not well known, however, from this study it is possible that ramipril act via a nitric oxide- independent pathway, since the study recorded a low nitric oxide level in this group. Atherogenic indices are calculated from TC, TG and HDL-C. They reflect the true relationship between protective and atherogenic lipoprotein and are also associated with the size of pro- atherogenic lipoprotein and anti- atherogenic lipoprotein particle [35]. Atherogenic indices have been shown to be strong predictors for metabolic disturbances which include dyslipidemia, atherosclerosis, hypertension and cardiovascular diseases [36]. These indices are atherogenic index of plasma (AIP), atherogenic co-efficient (AC) and cardiac risk ratio (CRR). They give clearer indication of blood cholesterol level that tends towards atherogenic dyslipidemia. Intriguingly, atherogenic indices were significantly low in the L-NAME group treated with ramipril.
progressive time dependent effect on blood pressure, as it was observed in the study that the blood pressure recorded at the sixth week was significantly higher than that of the third week. L-NAME has been reported to have dose and time dependent effect on blood pressure [22,23]. Ramipril cause a reduction in systolic blood pressure, diastolic blood pressure and mean arterial pressure. Ramipril has been reported to alleviate hypertension induced by the administration of L-NAME by inhibiting angiotensin converting enzyme (ACE) [24,25]. ACE is an enzyme that catalyzes the conversion of angiotensin I to angiotensin II, a powerful vasoconstrictor and stimulator of aldosterone release. ACE has also been reported to inhibit kallikrein–kinin system, which is also an important pathway in nitric oxide production [9]. Therefore, inhibiting ACE enhances nitric oxide production in addition to inhibition of angiotensin II production; these two effects consequently reduce blood pressure. 4.2. Serum nitric oxide level Similar to what has been reported, low serum nitric oxide was recorded in the study. In contrast, L-NAME group treated with ramipril recorded a fall in serum nitric oxide. This was similar to the findings of Bernátová et al. [26] who reported that captopril did not affect nitric oxide synthase inhibition; however, the study of Seth et al. [27] reported an increase in nitric oxide in L-NAME rats treated with captopril. The fall in nitric oxide observed in L-NAME rats treated with ramipril actually negate our hypothesis which was based on the kallikrein–kininbradykinin system activated by angiotensin converting enzyme inhibitors. The fall in nitric oxide observed in L-NAME group treated with ramipril indicated that the kallikrein–kinin-bradykinin-nitric oxide pathway might be acting through endothelial nitric oxide synthase (eNOS) dependent pathway, which was actually inhibited in this study. Study has reported that bradykinin upregulated endothelial nitric oxide synthase and neuronal nitric oxide synthase [28].
5. Conclusion In conclusion, ACE inhibitor might be a better therapeutic intervention to curtail both hypertension and hyperlipidemia. Hyperlipidemia and hypertension medications used in combination can effectively lower the possibility of cardiovascular complications in the population with metabolic syndrome. It will be economically and medically prudent to use a therapy that can effectively treat hypertension as well as dyslipidemia concomitantly than using different medications to manage them. The use of ramipril to curtail abnormal high blood pressure and blood lipid content should be recommend as a remedy in medicine. However, further studies are still needed to ascertain the precise mechanism through which ramipril modulates lipid metabolism.
4.3. Lipid profile Studies have suggested the role of nitric oxide in the regulation of lipid metabolism and endothelial and inducible NOS have been shown to be present in adipose tissue of the rat [6], suggesting that adipose tissue may be a potential source of NO production. Nitric oxide (NO) has been suggested to regulate lipid metabolism through the activation of hepatic sterol regulatory element-binding protein (SREBP)-2, a transcriptional factor necessary for cholesterol metabolism and expression of LDL receptor. LDL receptors enhance the uptake of cholesterol into the hepatic cells and thus aid to maintain the blood cholesterol level [7]. Hyperlipidemia is a major risk factor of atherosclerosis and cardiovascular diseases. Nitric oxide has been documented to be a principal factor involved in the anti-atherosclerotic properties of the endothelium [4] and inhibition of NO synthase causes accelerated atherosclerosis in experimental models [5]. The blockade of nitric oxide synthase by L-NAME resulted in high total cholesterol, low density lipoprotein (LDL-C), and a low level of high density lipoprotein (HDL-C); this is similar to the report of
Author’s contributions
• Esther Oluwasola Aluko designed the work and drafted the manuscript. • Temidayo Olutayo Omobowale, Ademola Adetokunbo Oyagbemi, Adejumobi and Temitayo Olabisi Ajibade coordinated and measured
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the blood pressure of the animals.
• Adesoji Adedipe Fasanmade revised and approved the final version
[17]
of the manuscript.
Conflict of interest
[18]
The authors declare no conflict of interest. [19]
Acknowledgment The authors wish to acknowledge Mr. Okon of the Department of Physiology, University of Ibadan for his technical support.
[20]
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