Development of a "turn off" fluorescent molecularly imprinted nanoparticle-based sensor for selective captopril quantification in synthetic urine and wastewater samples.

The combination of silica nanoparticles with fluorescent molecularly imprinted polymers (Si-FMIPs) prepared by a one-pot sol-gel synthesis method to act as chemical sensors for the selective and sensitive determination of captopril is described. Several analytical parameters were optimized, including reagent ratio, solvent, concentration of Si-FMIP solutions, and contact time. Fourier-transform infrared spectroscopy (FT-IR), transmission electron microscopy (TEM), and the ninhydrin assay were used for characterization. The selectivity was evaluated against molecules belonging to other drug classes, such as fluoroquinolones, nonacid nonopioids, benzothiadiazine, alpha amino acids, and nitroimidazoles. Under optimized conditions, the Si-FMIP-based sensor exhibited a working range of 1-15 µM, with a limit of detection (LOD) of 0.7 µM, repeatability of 6.4% (n = 10), and suitable recovery values at three concentration levels (98.5% (1.5 µM), 99.9% (3.5 µM), and 99.2% (7.5 µM)) for wastewater samples. The sensor provided a working range of 0.5-15 µM for synthetic urine samples, with an LOD of 0.4 µM and a repeatability of 7.4% (n = 10) and recovery values of 93.7%, 92.9%, and 98.0% for 1.0 µM, 3.5 µM, and 10 µM, respectively. In conclusion, our single-vessel synthesis approach for Si-FMIPs proved to be highly effective for the selective determination of captopril in wastewater and synthetic urine samples.

Repeated doses of captopril induce airway hyperresponsiveness by modulating the TRPV1 receptor in rats.

Although TRPV1 receptors play an essential role in the adverse effects on the airways following captopril treatment, there is no available evidence of their involvement in treatment regimens involving repeated doses of captopril. Comparing the difference in these two treatment regimens is essential since captopril is a continuous-use medication. Thus, this study explored the role of the transient receptor potential vanilloid 1 (TRPV1) in the effects of captopril on rat airways using two treatment regimens. Airway resistance, bronchoalveolar lavage (BAL), and histological and immunohistochemical analyses were conducted in rats administered with single or repeated doses of captopril. This study showed that the hyperresponsiveness to bradykinin and capsaicin in captopril-treated rats was acute. Treatment with the selective B2 antagonist, HOE140 reduced bradykinin hyperresponsiveness and abolished capsaicin exacerbation in single-dose captopril-treated rats. Likewise, degeneration of TRPV1-positive neurones also reduced hyperresponsiveness to bradykinin. Single-dose captopril treatment increased leukocyte infiltration in the BAL when compared with the vehicle and this increase was reduced by TRPV1-positive neurone degeneration. However, when compared with the vehicle treatment, animals treated with repeated doses of captopril showed an increase in leukocyte influx as early as 1 h after the last captopril treatment, but this effect disappeared after 24 h. Additionally, an increase in TRPV1 expression occurred only in animals who received repeated captopril doses and the degeneration of TRPV1-positive neurones attenuated TRPV1 upregulation. In conclusion, these data strongly indicate that a treatment regimen involving multiple doses of captopril not only enhances sensitisation but also upregulates TRPV1 expression. Consequently, targeting TRPV1 could serve as a promising strategy to reduce the negative impact of captopril on the airways.

Captopril's influence on Danio rerio embryonic development: Unveiling significant toxic outcomes at environmentally relevant concentrations.

Anticipating a global increase in cardiovascular diseases, there is an expected surge in the use of angiotensin-converting enzyme inhibitors, notably captopril (CAP). This heightened usage raises significant environmental apprehensions, mainly due to limited knowledge regarding CAP's toxic effects on aquatic species. In response to these concerns, the current study aimed to tackle this knowledge gap by evaluating the potential influence of nominal concentrations of CAP (0.2-2000 µg/L) on the embryonic development of Danio rerio. The findings revealed that CAP at all concentrations, even at concentrations considered environmentally significant (0.2 and 2 µg/L), induced various malformations in the embryos, ultimately leading to their mortality. Main malformations included pericardial edema, craniofacial malformation, scoliosis, tail deformation, and yolk sac deformation. In addition, CAP significantly altered the antioxidant activity of superoxide dismutase and catalase across all concentrations. Simultaneously, it elevated lipid peroxidation levels, hydroperoxides, and carbonylic proteins in the embryos, eliciting a substantial oxidative stress response. Likewise, CAP, at all concentrations, exerted significant modulatory effects on the expression of genes associated with apoptosis (bax, bcl2, p53, and casp3), organogenesis (tbx2a, tbx2b, and irx3b), and ion exchange (slc12a1 and kcnj1) in Danio rerio embryos. Both augmentation and reduction in the expression levels of these genes characterized this modulation. The Pearson correlation analysis indicated a close association between oxidative damage biomarkers and the expression patterns of all examined genes with the elevated incidence of malformations and mortality in the embryos. In summary, it can be deduced that CAP poses a threat to aquatic species. Nevertheless, further research is imperative to enhance our understanding of the environmental implications of this pharmaceutical compound.

The role of captopril in leukotriene deficient type 1 diabetic mice.

T1D can be associated with metabolic disorders and several impaired pathways, including insulin signaling, and development of insulin resistance through the renin-angiotensin system (RAS). The main precursor of RAS is angiotensinogen (Agt) and this system is often linked to autophagy dysregulation. Dysregulated autophagy has been described in T1D and linked to impairments in both glucose metabolism, and leukotrienes (LTs) production. Here, we have investigated the role of RAS and LTs in both muscle and liver from T1D mice, and its effects on insulin and autophagy pathways. We have chemically induced T1D in 129sve and 129sve 5LO-/- mice (lacking LTs) with streptozotocin (STZ). To further inhibit ACE activity, mice were treated with captopril (Cap). In muscle of T1D mice, treatment with Cap increased the expression of RAS (angiotensinogen and angiotensin II receptor), insulin signaling, and autophagy markers, regardless of the genotype. In the liver of T1D mice, the treatment with Cap increased the expression of RAS and insulin signaling markers, mostly when LTs were absent. 5LO-/- T1D mice showed increased insulin sensitivity, and decreased NEFA, after the Cap treatment. Cap treatment impacted both insulin signaling and autophagy pathways at the mRNA levels in muscle and liver, indicating the potential role of ACE inhibition on insulin sensitivity and autophagy in T1D.

The effect of zofenopril on the cardiovascular system of spontaneously hypertensive rats treated with the ACE2 inhibitor MLN-4760.

BACKGROUND: Angiotensin converting enzyme 2 (ACE2) plays a crucial role in the infection cycle of SARS-CoV-2 responsible for formation of COVID-19 pandemic. In the cardiovascular system, the virus enters the cells by binding to the transmembrane form of ACE2 causing detrimental effects especially in individuals with developed hypertension or heart disease. Zofenopril, a H2S-releasing angiotensin-converting enzyme inhibitor (ACEI), has been shown to be effective in the treatment of patients with essential hypertension; however, in conditions of ACE2 inhibition its potential beneficial effect has not been investigated yet. Therefore, the aim of the study was to determine the effect of zofenopril on the cardiovascular system of spontaneously hypertensive rats, an animal model of human essential hypertension and heart failure, under conditions of ACE2 inhibition induced by the administration of the specific inhibitor MLN-4760 (MLN). RESULTS: Zofenopril reduced MLN-increased visceral fat to body weight ratio although no changes in systolic blood pressure were recorded. Zofenopril administration resulted in a favorable increase in left ventricle ejection fraction and improvement of diastolic function regardless of ACE2 inhibition, which was associated with increased H2S levels in plasma and heart tissue. Similarly, the acute hypotensive responses induced by acetylcholine, L-NAME (NOsynthase inhibitor) and captopril (ACEI) were comparable after zofenopril administration independently from ACE2 inhibition. Although simultaneous treatment with zofenopril and MLN led to increased thoracic aorta vasorelaxation, zofenopril increased the NO component equally regardless of MLN treatment, which was associated with increased NO-synthase activity in aorta and left ventricle. Moreover, unlike in control rats, the endogenous H2S participated in maintaining of aortic endothelial function in MLN-treated rats and the treatment with zofenopril had no impact on this effect. CONCLUSIONS: Zofenopril treatment reduced MLN-induced adiposity and improved cardiac function regardless of ACE2 inhibition. Although the concomitant MLN and zofenopril treatment increased thoracic aorta vasorelaxation capacity, zofenopril increased the participation of H2S and NO in the maintenance of endothelial function independently from ACE2 inhibition. Our results confirmed that the beneficial effects of zofenopril were not affected by ACE2 inhibition, moreover, we assume that ACE2 inhibition itself can lead to the activation of cardiovascular compensatory mechanisms associated with Mas receptor, nitrous and sulfide signaling.

Captopril inhibits thioacetamide-induced chronic liver injury associated with the suppression of inflammation / hypoxia- inducible factor 1-alpha / profibrogenic axis-mediated hepatotoxicity in rats / Captopril inhibe la lesión hepática crónica inducida por tioacetamida asociada con la supresión de la inflamación/factor 1-alfa inducible por hipoxia/hepatotoxicidad mediada por el eje profibrogénico en rata

SUMMARY: Liver transplantation is the only available method to treat liver failure induced by chronic liver injury. We sought to determine whether the angiotensin-converting enzyme inhibitor, captopril, can inhibit the development of chronic liver injury induced by the hepatotoxic agent thioacetamide (TAA) in association with the suppression of inflammation (hsCRP, TNF-α, and IL-6) / hypoxia- inducible factor 1-alpha (HIF-1α) / profibrosis (TIMP-1, MMP-9, and α-SMA) axis that mediates liver injury. Therefore, the model group of rats was injected for eight weeks with 200 mg/kg TAA starting at week two. The protective group was pretreated with 150 mg/ kg captopril daily for two weeks prior to TAA injections and continued receiving both capropril and TAA agents until being humanely scrificed at week 10. We observed a substantial damage to liver tissue in the model group as demonstrated by a significant (p<0.0001) increase in blood and hepatic tissue levels of high sensitivity C-reactive protein (hsCRP), tumor necrosis factor-a (TNF-α), interleukin- 6 (L-6), HIF-1α, tissue inhibitor of metalloproteinases-1 (TIMP-1), matrix metalloproteinase-9 (MMP-9), alpha-smooth muscle actin (α-SMA), alanine aminotransferase (ALT), and aspartate aminotransferase (AST). All these parameters were significantly (p<0.0244) protected by captopril. Also, a significant (p<0.0001) positive correlation was observed between a-SMA (profibrosis) and the serum and tissue levels of hsCRP, TNF-α, HIF-1α, TIMP-1, MMP-9, and ALT. Thus, these findings suggest that the induction of chronic liver injury by the hepatotoxic compound, TAA is associated with the upregulation of inflammation/HIF-1α/profibrosis, with captopril exhibiting beneficial hepatic pleotropic effects.

El trasplante de hígado es el único método disponible para tratar la insuficiencia hepática inducida por una lesión hepática crónica. Buscamos determinar si el inhibidor de la enzima convertidora de angiotensina, captopril, puede inhibir el desarrollo de lesión hepática crónica inducida por el agente hepatotóxico tioacetamida (TAA) en asociación con la supresión de la inflamación (hsCRP, TNF-α e IL-6) / factor inducible por hipoxia 1-alfa (HIF-1α) / profibrosis (TIMP-1, MMP-9 y α- SMA) eje que media la lesión hepática. Por lo tanto, al grupo modelo de ratas se le inyectó durante ocho semanas 200 mg/kg de TAA a partir de la semana dos. El grupo protector fue pretratado con 150 mg/kg de captopril al día durante dos semanas antes de las inyecciones de TAA y continuó recibiendo capropril y agentes TAA hasta que fue sacrificado en la semana 10. Observamos un daño sustancial en el tejido hepático en el grupo modelo, como lo demuestra un aumento significativo (p<0,0001) de los niveles en sangre y tejido hepático de proteína C reactiva de alta sensibilidad (hsCRP), factor de necrosis tumoral-α (TNF-a), interleucina-6 (L-6), HIF-1α, inhibidor tisular de metaloproteinasas-1 (TIMP-1), metaloproteinasa de matriz-9 (MMP-9), actina de músculo liso alfa (α-SMA), alanina aminotransferasa (ALT) y aspartato aminotransferasa (AST). Todos estos parámetros estaban significativamente (p<0,0244) protegidos por captopril. Además, se observó una correlación positiva significativa (p<0,0001) entre α-SMA (profibrosis) y los niveles séricos y tisulares de hsCRP, TNF-α, HIF-1α, TIMP- 1, MMP-9 y ALT. Por lo tanto, estos hallazgos sugieren que la inducción de daño hepático crónico por el compuesto hepatotóxico, TAA, está asociada con la regulación al alza de la inflamación/HIF-1α/profibrosis, con captopril exhibiendo efectos pleotrópicos hepáticos beneficiosos.

COVID-19: Understanding the impact of anti-hypertensive drugs and hydroxychloroquine on the ACE1 and ACE2 in lung and adipose tissue in SHR and WKY rats.

Hypertensive individuals taking anti-hypertensive drugs from renin-angiotensin system inhibitors may exhibit a more severe evolution of the disease when contracting the SARS-CoV-2 virus (COVID-19 disease) due to potential increases in ACE2 expression. The study investigated ACE1 and ACE2 axes and hydroxychloroquine in the lungs and adipose tissue of male and female normotensive Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHRs). SHRs were treated with losartan (10 mg/kg/day) or captopril (10 mg/kg/day) for 14 days or 7 days with hydroxychloroquine (200 mg/kg/day) in drinking water. WKY rats were also treated for 7 days with hydroxychloroquine. Blood pressure (BP), protein, and mRNA expression of ACE1 and ACE2 were analyzed in serum, adipose, and lung tissues. Losartan and captopril reduced BP in both sexes in SHR, whereas hydroxychloroquine increased BP in WKY rats. Losartan reduced ACE2 in serum and lungs in both sexes and in adipose tissue of male SHRs. Captopril decreased ACE2 protein in the lung of females and in adipose tissue in both sexes of SHRs. Hydroxychloroquine decreased ACE1 and ACE2 proteins in the lungs in both sexes and adipose tissue in male SHRs. In female WKY rats, ACE2 protein was lower only in the lungs and adipose tissue. Losartan effectively inhibited ACE2 in male and captopril in female SHRs. Hydroxychloroquine inhibited ACE2 in male SHRs and female WKY rats. These results further our understanding of the ACE2 mechanism in patients under renin-angiotensin anti-hypertensive therapy and in many trials using hydroxychloroquine for COVID-19 treatment and potential sex differences in response to drug treatment.

Angiotensinergic neurotransmission in the bed nucleus of the stria terminalis is involved in cardiovascular responses to acute restraint stress in rats.

The brain angiotensin II acting via AT1 receptors is a prominent mechanism involved in physiological and behavioral responses during aversive situations. The AT2 receptor has also been implicated in stress responses, but its role was less explored. Despite these pieces of evidence, the brain sites related to control of the changes during aversive threats by the brain renin-angiotensin system (RAS) are poorly understood. The bed nucleus of the stria terminalis (BNST) is a limbic structure related to the cardiovascular responses by stress, and components of the RAS system were identified in this forebrain region. Therefore, we investigated the role of angiotensinergic neurotransmission present within the BNST acting via local AT1 and AT2 receptors in cardiovascular responses evoked by an acute session of restraint stress in rats. For this, rats were subjected to bilateral microinjection of either the angiotensin-converting enzyme inhibitor captopril, the selective AT1 receptor antagonist losartan, or the selective AT2 receptor antagonist PD123319 before they underwent the restraint stress session. We observed that BNST treatment with captopril reduced the decrease in tail skin temperature evoked by restraint stress, without affecting the pressor and tachycardic responses. Local AT2 receptor antagonism within the BNST reduced both the tachycardia and the drop in tail skin temperature during restraint. Bilateral microinjection of losartan into the BNST did not affect the restraint-evoked cardiovascular changes. Taken together, these data indicate an involvement of BNST angiotensinergic neurotransmission acting via local AT2 receptors in cardiovascular responses during stressful situations.

Effect of the renin-angiotensin system on the exacerbation of adrenal glucocorticoid steroidogenesis in diabetic mice: Role of angiotensin-II type 2 receptor.

Prior investigation shows an increase in the activity of both hypothalamus-pituitary-adrenal (HPA) axis and the renin-angiotensin system (RAS) in diabetic patients. Moreover, activation of angiotensin-II type 1 receptor (AT1) has been associated with adrenal steroidogenesis. This study investigates the role of RAS on the overproduction of corticosterone in diabetic mice. Diabetes was induced by intravenous injection of alloxan into fasted Swiss-webster mice. Captopril (angiotensin-converting enzyme inhibitor), Olmesartan (AT1 receptor antagonist), CGP42112A (AT2 receptor agonist) or PD123319 (AT2 receptor antagonist) were administered daily for 14 consecutive days, starting 7 days post-alloxan. Plasma corticosterone was evaluated by ELISA, while adrenal gland expressions of AT1 receptor, AT2 receptor, adrenocorticotropic hormone receptor MC2R, pro-steroidogenic enzymes steroidogenic acute regulatory protein (StAR), and 11ß-hydroxysteroid dehydrogenase type 1 (11ßHSD1) were assessed using immunohistochemistry or western blot. Diabetic mice showed adrenal gland overexpression of AT1 receptor, MC2R, StAR, and 11ßHSD1 without altering AT2 receptor levels, all of which were sensitive to Captopril or Olmesartan treatment. In addition, PD123319 blocked the ability of Olmesartan to reduce plasma corticosterone levels in diabetic mice. Furthermore, CGP42112A significantly decreased circulating corticosterone levels in diabetic mice, without altering the overexpression of MC2R and StAR in the adrenal glands. Our findings revealed that inhibition of both angiotensin synthesis and AT1 receptor activity reduced the high production of corticosterone in diabetic mice via the reduction of MC2R signaling expression in the adrenal gland. Furthermore, the protective effect of Olmesartan on the overproduction of corticosterone by adrenals in diabetic mice depends on both AT1 receptor blockade and AT2 receptor activation.

Randomized, double-blinded, controlled clinical trial of the effect of captopril, telmisartan and their combination on systemic inflammation of patients on hemodialysis.

To evaluate individual and combined effect of captopril and telmisartan on systemic inflammation markers of hemodialysis (HD) patients. Randomized, double-blinded, controlled clinical trial. Patients on HD at least 2 months, with arteriovenous fistula, were randomly allocated to groups: (1) captopril/placebo (N 13); (2) telmisartan/placebo (N 13); (3) captopril + telmisartan (N 12); or (4) placebo/placebo (N 12). During 3 months, patients received oral drugs as follows: captopril 50 mg/day, telmisartan 80 mg/day or placebo. Patients excluded if they had conditions or were on drugs potentially influencing on inflammation. Clinical and biochemical evaluations were performed monthly. Serum tumor necrosis factor alpha (TNFα), interleukin 6 (IL-6), and C-reactive protein (CRP) were measured at 0, 1 and 3 months. Baseline, demographic, clinical and biochemical variables were comparable between groups. Baseline versus final inflammatory markers were: captopril/placebo TNFα, 2.47 (0.1-4.5) versus 1.73 (0.3-3.8) pg/ml; IL-6, 17.03 (7.2-23) versus 7.90 (0.7-19) pg/ml; CRP, 4.21 (1.6-18) versus 5.9 (3.0-28) mg/l; telmisartan/placebo TNFα, 3.03 (2.3-4.6) versus 1.70 (1.2-2.0) pg/ml; IL-6, 14.10 (5.5-23) versus 9.85 (6.2-13) pg/ml; CRP, 5.74 (2.1-13) versus 10.60 (1.5-27) mg/l; captopril + telmisartan TNFα, 1.43 (0.7-5.4) versus 0.40 (0.1-2.1) pg/ml; IL-6, 10.05 (4.9-23) versus 4.00 (0.7-7.7) pg/ml (p < 0.05); CRP, 3.26 (0.7-12) versus 2.83 (0.6-6.5) mg/l; placebo/placebo TNFα, 3.13 (1.6-5.6) versus 1.64 (1.6-2.3) pg/ml; IL-6, 8.12 (5.4-16) versus 7.60 (2.4-15) pg/ml; CRP, 5.23 (1.9-16) versus 3.13 (1.5-18) mg/l. Monotherapy with captopril or telmisartan display a trend, but their combined treatment significantly decreased serum levels of IL-6. No remarkable changes on TNFα and CRP were observed.

Captopril oral solution for pediatric use: formulation, stability study and palatability assessment in vivo

Abstract he aim of this work was to develop an oral solution of captopril at 5 mg/mL preservative-free. Two formulations were prepared, one containing sweetener (formulation 1) and the other without this excipient (formulation 2). The results found of validation parameters from analytical method performed by HPLC for captopril were, linearity 0.9998, the limit of detection 15.71 µg/mL, the limit of quantification 47.60 µg/mL, repeatability 1.05%, intermediate precision 2.42%, accuracy intraday 101,53%, accuracy inter-day 99.85%. Moreover, the results found for captopril disulfide were, linearity 0.9999, limit of detection 0.65 µg/mL, limit of quantification 1.96 µg/mL, repeatability 2.28%, intermediate precision 1.51%, accuracy intraday 101.36%, accuracy inter-day 100.29%. The appearance of formulations was clear and colorless, pH measures were 3.12 and 3.04, dosage of captopril and captopril disulfide were 99.45% and 99.82%, 0.24% and 0.12% for formulation 1 and formulation 2, respectively. The stability study demonstrated that the concentration of captopril and captopril disulfide in the formulations was > 90% and below 3%, respectively. The in vivo palatability study in animals and humans showed that Formulation 1 containing the sweetener had better acceptance. Thus, the sweetener was able to improve the unpleasant taste of the formulation

Role of ACE and ACE-2 in abrogated cardioprotective effect of ischemic preconditioning in ovariectomized rat heart

Abstract Ischemic heart disease is the leading cause of death in postmenopausal women. The activity of heart ACE increases whereas the activity of ACE-2 decreases after menopause. The present study was designed to investigate the role of ACE and ACE-2 in the abrogated cardioprotective effect of IPC in OVX rat heart. The heart was isolated from OVX rat and mounted on Langendorff's apparatus for giving intermittent cycles of IPC. The infarct size was estimated using TTC stain, and coronary effluent was analyzed for LDH, CK-MB, and nitrite release. IPC induced cardioprotection was significantly attenuated in the ovariectomized rat heart as compared to the normal rat heart. However, this attenuated cardioprotection was significantly restored by perfusion of DIZE, an ACE-2 activator, and captopril, an ACE inhibitor, alone or in combination noted in terms of decrease in myocardial infarct size, the release of LDH and CK-MB, and also increase in the release of NO as compared to untreated OVX rat heart. Thus, it is suggested that DIZE and captopril, alone or in combination restore the attenuated cardioprotective effect of IPC in OVX rat heart which is due to an increase in ACE-2 activity and decrease in ACE activity after treatment.

High-volume endurance exercise training stimulates hematopoiesis by increasing ACE NH2-terminal activity.

One of the health benefits of endurance exercise training (ET) is the stimulation of hematopoiesis. However, the mechanisms underlying ET-induced hematopoietic adaptations are understudied. N-Acetyl-Seryl-Aspartyl-Lysyl-Proline (Ac-SDKP) inhibits proliferation of early hematopoietic progenitor cells. The angiotensin I-converting enzyme (ACE) NH2-terminal promotes hematopoiesis by inhibiting the anti-hematopoietic effect of Ac-SDKP. Here we demonstrate for the first time the role of ACE NH2-terminal in ET-induced hematopoietic adaptations. Wistar rats were subjected to 10 weeks of moderate-(T1) and high-(T2) volume swimming-training. Although both protocols induced classical ET-associated adaptations, only T2 increased plasma ACE NH2-domain activity (by 40%, P=0.0003) and reduced Ac-SDKP levels (by 50%, P<0.0001). T2 increased the number of hematopoietic stem cells (HSCs; ∼200%, P=0.0008), early erythroid progenitor colonies (∼300%, P<0.0001) and reticulocytes (∼500%, P=0.0007), and reduced erythrocyte lifespan (∼50%, P=0.022). Following, Wistar rats were subjected to T2 or T2 combined with ACE NH2-terminal inhibition (captopril (Cap) treatment: 10 mg.kg-1.day-1). T2 combined with ACE NH2-terminal inhibition prevented Ac-SDKP decrease and attenuated ET-induced hematopoietic adaptations. Altogether, our findings show that ET-induced hematopoiesis was at least partially associated with increased ACE NH2-terminal activity and reduction in the hematopoietic inhibitor Ac-SDKP.

Inhibition of diabetes mellitus-induced knee joint injury in rats by a combination of metformin and captopril / Inhibición de la lesión de la articulación de la rodilla inducida por diabetes mellitus en ratas mediante una combinación de metformina y captopril

SUMMARY: Osteoarthritis (OA) is an inflammatory disease that damages the joints and affects millions of people worldwide. The potential inhibitory effects of the antidiabetic drug metformin combined with captopril, the angiotensin-converting enzyme inhibitor, on diabetes-induced damage to the knee joint articular cartilage associated with the inhibition of glycemia, dyslipidemia, and inflammation has not been investigated before. Therefore, we induced diabetes in rats using high carbohydrate and fat diets and a single injection of streptozotocin (50 mg/kg). The protective group of rats was pre-treated with combined daily doses of metformin (Met; 200 mg/kg body weight) and captopril (Cap; 150 mg/kg body weight) for 14 days before diabetic induction and continued on metformin and resveratrol until the end of the experiment at week 12. Harvested tissues obtained from knee joints were prepared for basic histology staining with haematoxylin and eosin (H&E) and examined under light microscopy. Representative H&E images showed that OA was developed in the diabetic rats as demonstrated by a profound damage to the knee joints such as irregular eroded and a sharp decrease in the thickness of the articular cartilage surface and abnormal remodeling of the subchondral bone that were substantially ameliorated by Met+Cap. Met+Cap also significantly (p< 0.05) reduced blood levels of glucose, glycated hemoglobin (HbA1c), dyslipidemia, and the inflammatory biomarkers, high sensitivity C-reactive protein (hs-CRP), interleukin-6 (IL-6), and tumour necrosis factor-alpha (TNF-α) induced by diabetes. In addition, a significant (p≤ 0.0014) correlation between the articular cartilage thickness and the blood levels of glucose, HbA1c, triglyceride (TG), low density lipoprotein-cholesterol (LDL-C), high density lipoprotein- cholesterol (HDL-C), and hs-CRP were observed. Thus, we demonstrate that Met+Cap effectively protect the knee joint against injuries induced secondary to diabetes in rats, possibly due to the inhibition of glycemia, dyslipidemia, and biomarkers of inflammation.

RESUMEN: La osteoartritis (OA) es una enfermedad inflamatoria que daña las articulaciones y afecta a millones de per- sonas en todo el mundo. No se han investigado los posibles efectos inhibidores del fármaco antidiabético metformina combinado con captopril, el inhibidor de la enzima convertidora de angiotensina, sobre el daño inducido por la diabetes en el cartílago articular de la articulación de la rodilla asociado con la inhibición de la glucemia, dislipidemia e inflamación. En este estudio fue inducida la diabetes en ratas con dietas altas en carbohidratos y grasas y una sola inyección de estreptozotocina (50 mg / kg). El grupo protector de ratas se pretrató con dosis diarias combinadas de metformina (Met; 200 mg / kg de peso corporal) y captopril (Cap; 150 mg / kg de peso corporal) durante 14 días antes de la inducción diabética. El tratamiento se continuó con metformina y resveratrol hasta el final del experimento en la semana 12. Los tejidos obtenidos de las articulaciones de la rodilla se prepararon para la tinción de histología básica con hematoxilina y eosina (H&E) y se examinaron con microscopía óptica. Imágenes representativas de H&E mostraron que la OA se desarrolló en las ratas diabéticas, como lo evidencia un daño profundo en las articulaciones de la rodilla, como la erosión irregular y una fuerte disminución en el grosor de la superficie del cartílago articular y remodelación anor- mal del hueso subcondral que fueron mejorados sustancialmente por Met + Cap. Met + Cap. También redujo significativamente (p <0.05) los niveles sanguíneos de glucosa, hemoglobina glicosilada (HbA1c), dislipidemia y los biomarcadores inflamatorios, proteína C reactiva de alta sensibilidad (hs-CRP), interleucina-6 (IL-6), y factor de necrosis tumoral alfa (TNF-α) inducido por diabetes. Además, una correlación significativa (p≤ 0,0014) entre el grosor del cartílago articular y los niveles sanguíneos de glucosa, HbA1c, triglicéridos (TG), lipoproteínas-colesterol de baja densidad (LDL- C), lipoproteínas de alta densidad-colesterol (HDL-C) ) y hs-CRP. Así, demostramos que Met + Cap protege eficazmente la articulación de la rodilla contra lesiones inducidas por diabetes en ratas, posiblemente debido a la inhibición de la glicemia, dislipidemia y biomarcadores de inflamación.

Effects of physical exercise combined with captopril or losartan on left ventricular hypertrophy of hypertensive rats.

Background: Left ventricular hypertrophy (LVH) is an endpoint of hypertensive cardiac alterations. Renin-angiotensin-aldosterone system (RAAS) blockers are among the most effective on LVH regression. Physical exercise combined to antihypertensive drug contributes to arterial pressure (AP) control and LVH prevention. We evaluated the effects of physical exercise combined to captopril or losartan during eight weeks for spontaneously hypertensive rats (SHR) on some cardiac parameters.Methods: SHR (n=5-6 per group) were sedentary or trained 5 days a week in treadmill during 8 weeks; and they were treated with daily oral captopril (12.5, 25, or 50mg/kg), losartan (2.5, 5, or 10mg/kg), or vehicle. At the end, it was obtained systolic AP (SAP), electrocardiogram (ECG), and hearts metalloproteinase 2 (MMP-2) activity and histology.Results: Captopril 25 and 50 mg/kg, and losartan 10 mg/kg lowered SAP of sedentary and trained SHR. Losartan 5 mg/kg combined with physical exercise also lowered SAP. Combined with exercise, captopril 50 mg/kg lowered 13.6% of QT interval, 14.2% of QTc interval, and 22.8% of Tpeak-Tend compared to sedentary SHR. Losartan 5 and 10mg/kg lowered QT interval of sedentary and trained SHR. Losartan 2.5, 5 and 10mg/kg combined with physical exercise lowered respectively 25.4%, 24.8%, and 31.8% of MMP-2 activity. Losartan (10mg/kg) combined with exercise reduced cardiomyocyte diameter.Conclusion: These data support the hypothesis of physical exercise combined with RAAS blockers could improve the benefits on hypertensive LVH treatment.

Sodium palatability in male spontaneously hypertensive rats.

Spontaneously hypertensive rats (SHRs) ingest more NaCl than normotensive strains. Here we investigated NaCl intake and taste reactivity in adult male SHRs and normotensive Holtzman rats treated or not with AT1 receptor antagonist centrally in euhydrated condition and after fluid depletion. Taste reactivity was measured by the number of orofacial expressions to intra-oral infusions of 0.3 M NaCl. In euhydrated condition, intra-oral infusions of 0.3 M NaCl produced greater number of hedonic responses in SHRs than in normotensive rats, without differences in the number of aversive responses. Compared to euhydrated condition, the treatment with the diuretic furosemide + low dose of captopril (angiotensin converting enzyme blocker) increased the number of hedonic and reduced the number of aversive responses to intra-oral NaCl in normotensive rats, without changing the number of hedonic or aversive responses in SHRs. Losartan (AT1 receptor antagonist, 100 ng/1 µl) injected intracerebroventricularly in SHRs abolished 0.3 M NaCl intake induced by water deprivation + partial rehydration, whereas only transiently (first 30 min of the 60 min test) reduced hedonic responses, without changes in aversive responses to intra-oral NaCl. Losartan intracerebroventricularly also only transiently (first 30 min) reduced the number of hedonic responses to intra-oral NaCl in euhydrated SHRs. The results suggest that NaCl palatability is increased and independent from body fluid balance in SHRs. The results also suggest that central AT1 receptors are part of the mechanisms activated to increase NaCl intake and palatability in SHRs. A partial dissociation between NaCl intake and palatability in SHRs is also suggested.

Inhibitors of angiotensin I converting enzyme potentiate fibromyalgia-like pain symptoms via kinin receptors in mice.

Fibromyalgia is a potentially disabling chronic disease, characterized by widespread pain and a range of comorbidities such as hypertension. Among the mechanisms involved in fibromyalgia-like pain symptoms are kinins and their B1 and B2 receptors. Moreover, angiotensin I converting enzyme (ACE) inhibitors, commonly used as antihypertensive drugs, can enhance pain by blocking the degradation of peptides such as substance P and bradykinin, besides enhancing kinin receptors signalling. We investigated the effect of ACE inhibitors on reserpine-induced fibromyalgia-like pain symptoms and the involvement of kinins in this effect in mice. Nociceptive parameters (mechanical and cold allodynia and overt nociception) were evaluated after ACE inhibitors administration in mice previously treated with reserpine. The role of kinin B1 and B2 receptors was investigated using pharmacological antagonism. Additionally, bradykinin levels, as well as the activity of ACE and kininase I, were measured in the sciatic nerve, spinal cord and cerebral cortex of the mice. The ACE inhibitors enalapril and captopril enhanced reserpine-induced mechanical allodynia, and this increase was prevented by kinin B1 and B2 receptor antagonists. Substance P and bradykinin caused overt nociception and increased mechanical allodynia in animals treated with reserpine. Reserpine plus ACE inhibitors increased bradykinin-related peptide levels and inhibited ACE activity in pain modulation structures. Since hypertension is a frequent comorbidity affecting fibromyalgia patients, hypertension treatment with ACE inhibitors in these patients should be reviewed once this could enhance fibromyalgia-like pain symptoms. Thus, the treatment of hypertensive patients with fibromyalgia could include other classes of antihypertensive drugs, different from ACE inhibitors.

Interference with the renin-angiotensin system reduces the palatability of 0.3 M NaCl in sodium-deplete rats.

The renin-angiotensin system (RAS) controls hypertonic NaCl intake driven by sodium appetite. Here we investigated whether the antagonism of RAS interferes with hedonic and aversive orofacial motor responses, or palatability, to intraoral infusion of 0.3 M NaCl (hNaCl). Adult rats were depleted of sodium by combined sc injection of furosemide and 24 h removal of ambient sodium. In experiment 1, losartan (AT1 angiotensin II receptor antagonist, intracerebroventricular, 200 µg/µl), produced a three-fold increase in aversive orofacial motor responses to hNaCl. Losartan also suppressed hNaCl intake recorded immediately thereafter. In experiment 2, each animal had repeated recordings of hNaCl intake and orofacial responses to hNaCl distributed for 180 min. Paired recordings of intake and orofacial responses occurred within five successive blocks after the recordings of only orofacial responses when the animals were still sodium deplete (block zero). Captopril (angiotensin converting enzyme blocker, intraperitoneal, 30 mg/kg) inhibited by 75% the hedonic orofacial responses to hNaCl in blocks zero and 1. The hedonic responses to captopril remained the same throughout blocks, but became similar to vehicle from blocks 2 to 5. There was no difference in aversive responses to 0.3 M NaCl between captopril and vehicle. Captopril produced a 70-100% inhibition of hNaCl intake in blocks 1 to 5. The results suggest that angiotensin II acts in the brain increasing the palatability of hypertonic sodium during the consummatory phase of sodium appetite.

Blockade of ERK1/2 activation with U0126 or PEP7 reduces sodium appetite and angiotensin II-induced pressor responses in spontaneously hypertensive rats.

Spontaneously hypertensive rats (SHRs) have increased daily or induced sodium intake compared to normotensive rats. In normotensive rats, angiotensin II (ANG II)-induced sodium intake is blocked by the inactivation of p42/44 mitogen-activated protein kinase, also known as extracellular signal-regulated protein kinase1/2 (ERK1/2). Here we investigated if inhibition of ERK1/2 pathway centrally would change sodium appetite and intracerebroventricular (icv) ANG II-induced pressor response in SHRs. SHRs (280-330 g, n = 07-14/group) with stainless steel cannulas implanted in the lateral ventricle (LV) were used. Water and 0.3 M NaCl intake was induced by the treatment with the diuretic furosemide + captopril (angiotensin converting enzyme blocker) subcutaneously or 24 h of water deprivation (WD) followed by 2 h of partial rehydration with only water (PR). The blockade of ERK1/2 activation with icv injections of U0126 (MEK1/2 inhibitor, 2 mM; 2 µl) reduced 0.3 M NaCl intake induced by furosemide + captopril (5.0 ± 1.0, vs. vehicle: 7.3 ± 0.7 mL/120 min) or WD-PR (4.6 ± 1.3, vs. vehicle: 10.3 ± 1.4 mL/120 min). PEP7 (selective inhibitor of AT1 receptor-mediated ERK1/2 activation, 2 nmol/2 µL) icv also reduced WD-PR-induced 0.3 M NaCl (2.8 ± 0.7, vs. vehicle: 6.8 ± 1.4 mL/120 min). WD-PR-induced water intake was also reduced by U0126 or PEP7. In addition, U0126 or PEP7 icv reduced the pressor response to icv ANG II. Therefore, the present results suggest that central AT1 receptor-mediated ERK1/2 activation is part of the mechanisms involved in sodium appetite and ANG II-induced pressor response in SHRs.

Transforming Non-Selective Angiotensin-Converting Enzyme Inhibitors in C- and N-domain Selective Inhibitors by Using Computational Tools.

The two-domain dipeptidylcarboxypeptidase Angiotensin-I-converting enzyme (EC 3.4.15.1; ACE) plays an important physiological role in blood pressure regulation via the reninangiotensin and kallikrein-kinin systems by converting angiotensin I to the potent vasoconstrictor angiotensin II, and by cleaving a number of other substrates including the vasodilator bradykinin and the anti-inflammatory peptide N-acetyl-SDKP. Therefore, the design of ACE inhibitors is within the priorities of modern medical sciences for treating hypertension, heart failures, myocardial infarction, and other related diseases. Despite the success of ACE inhibitors for the treatment of hypertension and congestive heart failure, they have some adverse effects, which could be attenuated by selective domain inhibition. Crystal structures of both ACE domains (nACE and cACE) reported over the last decades could facilitate the rational drug design of selective inhibitors. In this review, we refer to the history of the discovery of ACE inhibitors, which has been strongly related to the development of molecular modeling methods. We stated that the design of novel selective ACE inhibitors is a challenge for current researchers which requires a thorough understanding of the structure of both ACE domains and the help of molecular modeling methodologies. Finally, we performed a theoretical design of potential selective derivatives of trandolaprilat, a drug approved to treat critical conditions of hypertension, to illustrate how to use molecular modeling methods such as de novo design, docking, Molecular Dynamics (MD) simulations, and free energy calculations for creating novel potential drugs with specific interactions inside nACE and cACE binding sites.