Summary
The current study includes the physiological effects of melatonin (MEL) on the vascular response to angiotensin 1-8 octapeptide (Ang 1-8) and angiotensin 1-7 heptapeptide (Ang 1-7). As well as this study includes aortic histological sections and angiotensin type-1 receptor (AT1R), angiotensin-converting enzyme 2 (ACE2), and Mas receptor (MasR) gene expression of male rats’ thoracic aorta in non-diabetic (DM), streptozotocin (STZ) induced type-1 diabetes mellitus (DM), and STZ-induced type-1 DM rats treated with MEL. In the current investigation, 350 male albino rats (Rattus norvegicus) weighing approximately 250-300 gm aged 10-12 weeks were used.
The current study involved two main experiments, along with histological examination and gene expression of the isolated thoracic aorta.
The first experiment of this study was carried out to investigate the effects of MEL on the vascular response to Ang 1-8 with or without MEL, MEL agonist, and ACE2 inhibitor in non-diabetes mellitus intact endothelium (non-DM E+), non-DM denuded endothelium (non-DM E-), and DM intact endothelium (DM E+) aortic rings. The isolated aortic rings were pre-incubated with MEL, ramelteon (RAM), a selective agonist of MEL receptor type 1 and type 2 receptors (MT1R/MT2R), ACE2 inhibitor, MEL+ACE2 inhibitor, MEL+luzindole (LUZ), a non-selective (MT1R/MT2R) antagonist, ACE2 inhibitor+RAM in the DM E+ aortic rings group. Furthermore, this experiment also involved the pre-incubation of MEL, AT1R blocker and Ang 1-8 receptor type-2 (AT2R) blocker including valsartan (VAL), a selective AT1R blocker, PD123319+VAL, PD123319, a selective AT2R blocker, MEL, MEL+VAL, MEL+PD123319+VAL, PD123319, PD123319+VAL, MEL+ PD123319 and MEL+LUZ+ PD123319 in non-DM and DM aortic rings.
The second experiment was designed to investigate the effects of MEL on the vascular response to Ang 1-7. This experiment comprised the pre-incubation of MEL, RAM, LUZ, and MEL with the following blockers and/or inhibitors, including tetraethylammonium (TEA), a nonselective blocker of calcium-activated potassium (KCa) channels, charybdotoxin (ChTX), a big and intermediate conductance calcium-activated potassium (BKca and IKca) channels blocker, clotrimazole (CLT), an intermediate conductance calcium-activated potassium (IKca) channels blocker, glibenclamide (Glib), an adenosine triphosphate-sensitive potassium (KATP) channels blocker, 4- aminopyridine (4-AP), voltage sensitive potassium (KV) channels blocker and barium chloride (BaCl2), a delay inwardly rectifier potassium (Kir) channels blocker in non-DM and DM rats isolated aortic rings. Also, this experiment examined the effect of pre-incubation of MEL, endothelium-derived relaxing factors (EDRFs), inhibitors including L-nitro-arginine methyl ester (L-NAME), an irreversible endothelial nitric oxide synthase (eNOS) inhibitor, indomethacin (IND), a non-selective cyclooxygenase (COX) inhibitor, nifedipine (NIF), the L-type calcium channels blocker, and oxidative stress enzymes inhibitors including RO 31-8220, a protein kinase C (PKC) inhibitor, apocynin (APO), a selective inhibitor of nicotinamide adenine dinucleotide phosphate oxidase (NADPH), rotenone (ROT), a mitochondrial complex I electron transport chain (ETC) inhibitor and oxypurinol (OXY), the xanthine oxidase (XO) inhibitor. Also, this experiment includes the pre-incubation of MasR blocker and phosphoinositide 3 kinase/ protein kinase B/ endothelial nitric oxide synthase (PI3K/Akt/eNOS) signalling pathway inhibitors including PI-3065, a phosphoinositide 3-kinases (PI3K) inhibitor, Ipatasertib (GDC-0068), a selective inhibitor of protein kinase B (AKT) and A779, a selective blocker of MasR, Ang 1-7 receptor in non-DM, DM and DM rats treated with MEL.
In addition, the present study investigated both qualitative and quantitative histological morphometrics of the thoracic aorta. Furthermore, this study involved the examination of gene expression of AT1R, MasR, ACE2, and β-actin (housekeeping gene) with quantitative reverse transcription real time polymerase chain reaction (RT-qPCR) technique.
The present results showed a significant (P < 0.01) increase in the maximum response to MEL in DM aortic rings. The vascular contractility response to Ang 1-8 was also significantly (P < 0.0055) increased in non-DM E- aortic rings group while this response was decreased significantly (P < 0.001) in the DM aortic rings group. Furthermore, both MEL and RAM exhibited dramatic abolishment of the vascular action of Ang 1-8 in both non-DM E+ and non-DM E- aortic rings group. Besides, the ACE2 inhibitor potentiated Ang 1-8 activity in DM aortic rings.
The data obtained revealed novelties, indicating that MT1R is predominantly localised in VSMCs and MT2R in VECs. The current study uncovers a novel localization of carboxypeptidases and their significant function in the metabolism of Ang 1-8 in the rat thoracic aorta. Regarding DM condition, MEL showed a significant role in Ang 1-8 dose response curve (DRC). this pre-incubation of MEL shifted Ang 1-8 DRC to the right side significantly (P < 0.001) while this response did not occur as much as with RAM pre-incubation in DM conditions. On the other hand, ACE2 inhibitor shifted Ang 1-8 DRC to the left side significantly (P < 0.5) in DM aortic rings. Furthermore, the combined pre-incubation of each of MEL+LUZ, MEL+ACE2 inhibitor, MEL+LUZ+ACE2 inhibitor and RAM+ACE2 inhibitor, caused a significant rightward shift. In contrast, pre-incubation of VAL abolished Ang 1-8 DRC completely in non-DM aortic rings, while the pre-incubation VAL+MEL restored Ang 1-8 contractility. On the other hand, the pre-incubation of PD123319 also decreased Ang 1-8 DRC significantly in DM aortic rings, while MEL reversed this effect in non-DM and DM groups as well. In addition, the combined pre-incubation of MEL+LUZ+P123319 decreased Ang 1-8 DRC completely in DM condition.
Therefore, the present study is the first to show that the distribution of vascular AT1R and AT2R is distinct in the thoracic aorta.
The current study reveals a decreased vascular response to Ang 1-7 in DM aortic rings compared to that of non-DM aortic rings. The impaired response promoted a reversed result in the DM aortic ring under MEL, RAM and LUZ, as well. In addition, MEL increased vasodilation with TEA, ChTX and CLT through the Ang 1-7 action. In addition, the present study highlighted the pre-incubation of STZ-induced DM rat aortic rings with the Glib non-significantly shifted the Ang 1-7 DRC vasodilatory response to the right side. Furthermore, the addition of MEL produced a further leftward shift in the STZ-induced DM aortic rings than in non-DM rings, respectively. These outcomes were observed under 4-AP and BaCl2, as well.
The DRC of Ang 1-7 under L-NAME and IND pre-incubation decreased Ang 1-7 potency significantly in DM rings were treated orally with MEL. Furthermore, NIF pre-incubation elevated Emax in DM rats were treated with orally MEL. The present investigation revealed for the first time that MEL administration most likely demonstrated vasodilatory regulation in Ang1-7 through the amplification of Ca2+ vasomotion, but it is still unclear whether IND-induced endothelium-dependent vasodilation may harm or protect the cardiovascular system.
The AUC of Ang 1-7 was decreased significantly (p<0.001), and the Emax was elevated in the same fashion in the DM rats treated with orally MEL. On the other hand, aortic rings incubated with RO-31-8220 showed a significant decrease (p<0.0067) in potency and a significant (p<0.05) decrease in dAUC% in DM rats treated with orally MEL. In addition, the APO pre-incubation produced the same results in DM rats treated with orally MEL, with an elevated vascular response to Ang 1-7 in the DM group. Besides, the ROT and OXY pre-incubation increased the vascular response to Ang 1-7 in the DM aortic rings, also the dAUC% was decreased in the DM rats treated with orally MEL.
The effects of MEL in Ang 1-7 via the PI3K/Akt/eNOS signalling pathway showed a slight effect via PI-3065 and Ipatasertib pre-incubation in the studied groups. In contrast, the pre-incubation of aortic rings with A779 increased the potency significantly (p<0.001) in the non-DM and DM groups. Besides, the pre-incubation in aortic rings with A779 caused an increase of the vascular response to Ang 1-7 in DM rats treated with orally MEL.
The present qualitative and quantitative aortic histological results indicated that MEL administration enhanced the aortic texture through eliminating diabetic degeneration, which included restored aortic layers and smooth muscle cell nuclei count.
By using certain components of the RAS, the molecular roles of MEL that were previously suggested were confirmed. While the DM rats given oral MEL experienced a non-significant decrease in this elevation, the aortic AT1R gene expression level was slightly elevated in the DM group. The DM group experienced a modest drop in the levels of aortic ACE2 and MasR gene expression, while the administration of MEL resulted in non-significant increase in these expressions.