Spontaneously Hypertensive Rats Research Articles

Carvacrol Improves Vascular Function in Hypertensive Animals by Modulating Endothelial Progenitor Cells.

Carvacrol, a phenolic monoterpene, has diverse biological activities, highlighting its antioxidant and antihypertensive capacity. However, there is little evidence demonstrating its influence on vascular regeneration. Therefore, we evaluated the modulation of carvacrol on endothelial repair induced by endothelial progenitor cells (EPC) in hypertension. Twelve-week-old spontaneously hypertensive rats (SHR) were treated with a vehicle, carvacrol (50 or 100 mg/kg/day), or resveratrol (10 mg/kg/day) orally for four weeks. Wistar Kyoto (WKY) rats were used as the normotensive controls. Their systolic blood pressure (SBP) was measured weekly through the tail cuff. The EPCs were isolated from the bone marrow and peripherical circulation and were quantified by flow cytometry. The functionality of the EPC was evaluated after cultivation through the quantification of colony-forming units (CFU), evaluation of eNOS, intracellular detection of reactive oxygen species (ROS), and evaluation of senescence. The superior mesenteric artery was isolated to evaluate the quantification of ROS, CD34, and CD31. Treatment with carvacrol induced EPC migration, increased CFU formation and eNOS expression and activity, and reduced ROS and senescence. In addition, carvacrol reduced vascular ROS and increased CD31 and CD34 expression. This study showed that treatment with carvacrol improved the functionality of EPC, contributing to the reduction of endothelial dysfunction.

Epigallocatechin-3-gallate exerts antihypertensive effects and improves endothelial function in spontaneously hypertensive rats

Objective: To investigate the effect of epigallocatechin-3-gallate (EGCG) on endothelial dysfunction in spontaneously hypertensive rats (SHR). Methods: Wistar-Kyoto (WKY) rats and SHR were divided into four groups; WKY control, SHR control and SHR treated with EGCG (50 mg/kg/day) or losartan (10 mg/kg/day). The treatment was given daily for 4 weeks by oral gavage and the blood pressure was monitored by tail-cuff method every 3 days. Acetylcholine-induced endothelium-dependent relaxations were assessed in isolated phenylephrine-precontracted aortic rings at the end of treatment. The vascular levels of reactive oxygen species, nitric oxide, tetrahydrobiopterin, and cyclic guanosine monophosphate were also measured. Moreover, the expression of angiotensin II type 1 (AT1) receptor protein was determined. Results: The systolic blood pressure was significantly decreased in SHR treated with EGCG. The impaired endothelium-dependent relaxation was significantly improved in aortic ring isolated from the EGCG-treated SHR group. EGCG also significantly increased the levels of nitric oxide, tetrahydrobiopterin, and cyclic guanosine monophosphate, while decreasing the level of reactive oxygen species and the protein expression of AT1 receptor in SHR. Conclusions: EGCG attenuates endothelial dysfunction in SHR by decreasing oxidative stress and increasing vascular nitric oxide bioavailability, which may be modulated partly by inhibition of vascular AT1 receptors. An increase in endothelium-dependent relaxation may contribute to a decrease in blood pressure in hypertensive animals.

Content determination of seven active components of Eucommiae Cortex in aortic vascular endothelial cells of spontaneously hypertensive rats by UPLC-MS/MS

In the present study, the contents of seven active components [genipinic acid(GA), protocatechuic acid(PCA), neochlorogenic acid(NCA), chlorogenic acid(CA), cryptochlorogenic acid(CCA),(+)-pinoresinol di-O-β-D-glucopyranosid(PDG), and(+)-pinoresinol 4'-O-β-D-glucopyranoside(PG)] of Eucommiae Cortex in aortic vascular endothelial cells of spontaneously hypertensive rats(SHR) were simultaneously determined by ultra-high liquid chromatography-triple quadrupole mass spectrometry(UPLC-MS/MS). The qualified SHR models were selected. The primary aortic endothelial cells(VECs) of rats were separated and cultured by ligation and adherence, followed by subculture. After successful identification, an UPLC-MS/MS method for simultaneously determining the contents of GA, PCA, NCA, CA, CCA, PDG, PG in seven components of Eucommiae Cortex in VECs was established, including specificity, linearity, matrix effect, recovery, accuracy, precision and stability. The established method had the lo-west limit of quantification of 0.97-4.95 μg·L~(-1), accuracy of 87.26%-109.6%, extraction recovery of 89.23%-105.3%, matrix effect of 85.86%-106.2%, and stability of 86.00%-112.5%. Therefore, the established accurate UPLC-MS/MS method could rapidly and simultaneously determine the contents of the seven active components of Eucommiae Cortex in VECs of SHRs, which provided a refe-rence for the study of cellular pharmacokinetics of active components of Eucommiae Cortex extract.

Weak Hypotensive Effect of Chronic Administration of the Dual FAAH/MAGL Inhibitor JZL195 in Spontaneously Hypertensive Rats as Revealed by Area under the Curve Analysis.

The enhancement of the endocannabinoid tone might have a beneficial influence on hypertension. Polypharmacology proposes multi-target-directed ligands (MTDLs) as potential therapeutic agents for the treatment of complex diseases. In the present paper, we studied JZL195, a dual inhibitor of the two major endocannabinoid-degrading enzymes, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), in spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY). Hemodynamic parameters were assessed in conscious animals via radiotelemetry and tail-cuff methods and then evaluated by the area under the curve (AUC). Single administration of JZL195 induced dose-dependent weak hypotensive and bradycardic responses in SHR but not in WKY. Similarly, its chronic application revealed only a slight hypotensive potential which, however, effectively prevented the progression of hypertension and did not undergo tolerance. In addition, multiple JZL195 administrations slightly decreased heart rate only in WKY and prevented the gradual weight gain in both groups. JZL195 did not affect organ weights, blood glucose level, rectal temperature and plasma oxidative stress markers. In conclusion, chronic dual FAAH/MAGL inhibition prevents the progression of hypertension in SHR without affecting some basal functions of the body. In addition, our study clearly proves the suitability of AUC for the evaluation of weak blood pressure changes.

Acacetin Alleviates Cardiac Fibrosis via TGF-β1/Smad and AKT/mTOR Signal Pathways in Spontaneous Hypertensive Rats

Introduction: Attenuating cardiac fibroblasts activation contributes to reducing excessive extracellular matrix deposition and cardiac structural remodeling in hypertensive hearts. Acacetin plays a protective role in doxorubicin-induced cardiomyopathy and ischemia/reperfusion injury. The aim of this study was to investigate the potential molecular mechanisms underlying the protective role of acacetin on hypertension-induced cardiac fibrosis. Methods: Echocardiography, histopathological methods, and Western blotting techniques were used to evaluate the anti-fibrosis effects in spontaneous hypertensive rat (SHR) which were daily intragastrically administrated with acacetin (10 mg/kg and 20 mg/kg) for 6 weeks. Angiotensin II (Ang II) was used to induce cellular fibrosis in human cardiac fibroblasts (HCFs) in the absence and presence of acacetin treatment for 48 h. Results: Acacetin significantly alleviated hypertension-induced increase in left ventricular (LV) posterior wall thickness and LV mass index in SHR. The expressions of collagen-1, collagen-III, and alpha-smooth muscle actin (α-SMA) were remarkedly decreased after treatment with acacetin (n = 6, p < 0.05). In cultured HCFs, acacetin significantly attenuated Ang II-induced migration and proliferation (n = 6, p < 0.05). Moreover, acacetin substantially inhibited Ang II-induced upregulation of collagen-1 and collagen-III (n = 6, p < 0.05) and downregulated the expression of alpha-SMA in HCFs. Additionally, acacetin decreased the expression of TGF-β1, p-Smad3/Smad3, and p-AKT and p-mTOR but increased the expression of Smad7 (n = 6, p < 0.05). Further studies found that acacetin inhibited TGF-β1 agonist SRI and AKT agonist SC79 caused fibrotic effect. Conclusion: Acacetin inhibits the hypertension-associated cardiac fibrotic processes through regulating TGF-β/Smad3, AKT/mTOR signal transduction pathways.

Identification and characterization of the new generation soluble guanylate cyclase stimulator BAY-747 designed for the treatment of resistant hypertension.

First-generation soluble guanylate cyclase (sGC) stimulators have shown clinical benefit in pulmonary hypertension (riociguat) and chronic heart failure (vericiguat). However, given the broad therapeutic opportunities for sGC stimulators, tailored molecules for distinct indications are required. We report the high-throughput screening (HTS)-based discovery of a second generation of sGC stimulators from a novel imidazo[1,2-a]pyridine lead series. An intense medicinal chemistry programme resulted in the discovery of the sGC stimulator BAY 1165747 (BAY-747). The pharmacokinetic profile of BAY-747 was determined in different species, and it was broadly characterized in pharmacological model systems relevant for vasodilatation and hypertension. BAY-747 is a highly potent sGC stimulator in vitro. In addition, BAY-747 showed an excellent pharmacokinetic profile with long half-life and low peak-to-trough ratio. BAY-747 was investigated in experimental in vivo models of malignant and resistant hypertension (rHT). In spontaneously hypertensive (SH) rats, BAY-747 caused a dose-related and long-lasting decrease in mean arterial blood pressure (MAP). Oral treatment over 12 days resulted in a persistent decrease. BAY-747 provided additional benefit when dosed on top of losartan, amlodipine or spironolactone and even on top of triple combinations of frequently used antihypertensive drugs. In a new canine model of rHT, BAY-747 caused a dose-related and long-lasting (>6 h) MAP decrease. BAY-747 is a potent, orally available sGC stimulator. BAY-747 shows long-acting pharmacodynamic effects with a very low peak-to-trough ratio. BAY-747 could be a treatment alternative for patients with hypertension, especially those not responding to standard-of-care therapy.

Red Quinoa Hydrolysates with Antioxidant Properties Improve Cardiovascular Health in Spontaneously Hypertensive Rats.

In recent years, quinoa has been postulated as an emerging crop for the production of functional foods. Quinoa has been used to obtain plant protein hydrolysates with in vitro biological activity. The aim of the present study was to evaluate the beneficial effect of red quinoa hydrolysate (QrH) on oxidative stress and cardiovascular health in an in vivo experimental model of hypertension (HTN) in the spontaneously hypertensive rat (SHR). The oral administration of QrH at 1000 mg/kg/day (QrHH) showed a significant reduction in SBP from baseline (-9.8 ± 4.5 mm Hg; p < 0.05) in SHR. The mechanical stimulation thresholds did not change during the study QrH groups, whereas in the case of SHR control and SHR vitamin C, a significant reduction was observed (p < 0.05). The SHR QrHH exhibited higher antioxidant capacity in the kidney than the other experimental groups (p < 0.05). The SHR QrHH group showed an increase in reduced glutathione levels in the liver compared to the SHR control group (p < 0.05). In relation to lipid peroxidation, SHR QrHH exhibited a significant decrease in plasma, kidney and heart malondialdehyde (MDA) values compared to the SHR control group (p < 0.05). The results obtained revealed the in vivo antioxidant effect of QrH and its ability to ameliorate HTN and its associated complications.

#4145 ACLARA: AN ON-LINE CALCULATOR TO ESTIMATE CREATININE CLEARANCE IN RATS

Abstract Background and Aims Creatinine clearance (ClCr) is a standard method for the measurement of glomerular filtration rate in rats. Recently, we developed ACLARA, an open and freely available neural network (NN)-based calculator of ClCr in Wistar rats (http://idal.uv.es/aclara). By working solely on plasma creatinine concentration and body weight, ACLARA instantly and easily provides accurate ClCr estimations with an identical concept to that underlying human glomerular filtration rate (GFR)-estimating formulae utility in the clinical setting. In addition to saving costs and time, ACLARA aligns with the 3Rs principles by providing a methodological refinement tool that reduces experimental animal stress derived from avoiding confinement in metabolic cages for individual, 24-hour urine collection. As a potential limitation, ACLARA was developed with data from Wistar rats. Accordingly, the aim of this study is to study ACLARA's accuracy and utility in a different rat strain, namely the spontaneous hypertensive rat (SHR). Method In order to further validate the calculator, we used a dataset with 1,458 entries from unpublished experiments of AKI induced in Wistar and SHR male rats. Measured ClCr (mClCr) was determined with a standard procedure, as we previously described [1], and estimated ClCr (eClCr) was calculated using ACLARA. Calculator performance was assessed with the mean absolute error (MAE) and the Pearson product-moment correlation coefficient calculated as previously described [2]. Results of eCLCr were compared with their corresponding mClCr values in the context of individual experiments. Results Our study reveals that ACLARA performs with reasonably high accuracy at ClCr estimation in both Wistar and SHR rats as indicated by Pearson's product-moment correlation coefficient (Corr) and MAE. The global Corr for the 1,458 measured versus estimated data values reaches 0.9, and a MAE of 0.18 mL/min. In general, estimated data provide identical results to measured data for individual experiments considered in the whole and, if anything, depict more congruent behavior. We interpret this improvement as the consequence of bypassing experimental errors introduced by the well-known discrepancies between the real and measured urinary flow in metabolic cages. Interestingly, ACLARA performs with accuracy through the range of ages and for all experimental conditions tested, including pharmacological treatments. Figure 1 shows a representative experiment in which measured and estimated data are compared holistically. Conclusion ACLARA is an easy-to-use and reliable tool to estimate creatinine clearance in SHR and Wistar rats from plasma creatinine concentration and body weight data. Validation for a second rat strain suggests a potential, wider application of ACLARA through laboratory rats, which needs to be further studied. In this sense, collaboration and use by independent investigators and recruitment of additional data sets from other laboratories is strongly sought to further adjust the calculator in order to enhance its accuracy, extend its applicability and improve its utility to the scientific community. This study was supported by Project PI21/01226, funded by Instituto de Salud Carlos III (ISCIII) and co-funded by the European Union, and a grant from the Consejería de Educación, Junta de Castilla y León (IES160P20), Spain, co-funded by FEDER funds; and a grant from the Valencian Government with reference number CIAICO/2021/184. Noelia Diaz-Morales is recipient of a Juan de la Cierva-Formación postdoctoral contract (FJC2020-043205-I) funded by MCIN/AEI/10.13039/501100011033 and European Union “NextGenerationEU/PRTR”.

#4248 COMBINED EFFECT OF DEHYDRATION, HYPERTENSION, AND AGE ON RENAL FRAILTY

Abstract Background and Aims Renal frailty (RF) is a premorbid and, at least partly, modifiable condition arising from diminished renal functional reserve and defective adaptive response capacity predisposing to acute kidney injury (AKI). RF ensues from subclinical wear or distortion of the renal haemodynamic and tubular homeostatic responses that defend the renal excretory function from supervening circumstances (i.e., stressors such as drugs). In this work, we aimed to study the impact of RF generated by the combined effect of the hydration state, hypertension and ageing on the nephrotoxicity developed by a low dose of cisplatin in the rat. Method Young Wistar rats, young spontaneously hypertensive rats (SHR), and aged Wistar rats were used. Rats in each group were randomly subject to four experimental conditions: - Control: rats with ad libitum water intake, receiving vehicle (0.9% NaCl, i.p). - Cisplatin: rats with ad libitum water intake, treated with 2.5 mg/kg, i.p. cisplatin. - Water deprivation: rats deprived of water for 48 h, receiving vehicle (0.9% NaCl, i.p.). - Water deprivation + cisplatin: rats deprived of water for the 48 h prior to 2.5 mg/kg i.p. cisplatin administration. Tail vein blood and 24-hour urine samples were collected at basal time (B), after water deprivation (D0), and 4 days after cisplatin/vehicle administration (D4, day of maximum kidney damage). Haematocrit was measured and plasma osmolality, plasma creatinine (Jaffe reaction) and urea (Jung method) concentrations were determined. Urine samples were analysed for volume (urinary flow), osmolality, and proteinuria (Bradford method). Body fluid composition was measured by bioimpedance spectroscopy with an ImpediVETTM VetBIS1 device. Results All rats showed clear signs of dehydration after 48 hours of water deprivation: weight loss, increases in plasma osmolality and haematocrit (more pronouncedly in young Wistar and SHR than in aged animals), and reduced flow of a highly concentrated urine (i.e., with elevated osmolality). Bioimpedance analysis revealed a parallel loss of intracellular and extracellular water and net loss of total body water (explaining most of weight loss), with no changes in the liquid distribution between the intracellular and extracellular compartments. Dehydration was reverted after 4 days of rehydration in vehicle-treated rats, and no alteration of renal function was observed due to water deprivation. Administration of cisplatin in young, normohydrated Wistar rats showed minimal loss of renal function that was not worsened by water deprivation. Hypertensive rats showed completely normal renal function when cisplatin was administered under normohydration. Interestingly, however, dehydrated SHR did suffer a cisplatin-induced AKI (significant increases in plasma creatinine, plasma urea and proteinuria). In aged Wistar rats, cisplatin compromised renal function (as supported by increases in plasma creatinine and urea concentration), and this effect was significantly amplified by water deprivation. Conclusion This study suggests that dehydration in fully competent young rats is not in itself a significant risk factor for AKI, while the combination of dehydration and hypertension in young rats significantly increases the risk of AKI. Furthermore, ageing poses animals in a state of frailty that renders their kidneys unable to respond to stressors (such as a low dose of cisplatin). Frailty is boldly worsened in dehydrated aged animals. In perspective, models reproducing conditions of increased vulnerability to AKI provide useful tools to search for biomarkers pre-emptively predicting undesired health outcomes before exposure to stressors, and for developing preventing strategies. This study was supported by Project PI21/01226, funded by Instituto de Salud Carlos III (ISCIII) and co-funded by the European Union, and a grant from the Consejería de Educación, Junta de Castilla y León (IES160P20), Spain, co-funded by FEDER funds. Noelia Diaz-Morales is recipient of a Juan de la Cierva-Formación postdoctoral contract (FJC2020-043205-I) funded by MCIN/AEI/10.13039/501100011033 and European Union “NextGenerationEU/PRTR”.

Rhynchophylline alleviates endothelial injury in spontaneously hypertensive rats by regulating PPARγ mediated iron death

Purpose: To determine the effects of rhynchophylline (Rhy) on the progression of cardiovascular diseases in rats.&#x0D; Methods: Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR, 7 week old, weighing 180 – 200 g) were used in this study. The rats were divided into WKY, SHR, SHR+ Rhy (30 mg/kg), and SHR+ Rhy (50 mg/kg). WKY rats were used as the normal control group which had free access to distilled water. The rats were then orally administered Rhy (30 and 50 mg/kg daily by gavage). For inhibition of PPARr, T0070907 was given at a dose of 2 mg/kg for 24 h. The blood pressure of control, spontaneously hypertensive rats (SHR), and SHR rhynchophylline (Rhy) rats were measured. While PPARr levels in the hypertensive rats were assessed by Western blot. Cell apoptosis in response to SHR and Rhy were evaluated by TdT-mediated dUTP nick-end labeling (TUNEL) and Western blot assays, whereas oxidative stress and inflammatory response in SHR rats treated with Rhy were determined by enzyme linked immunosorbent assay (ELISA) and Immunoblot assays, respectively.&#x0D; Results: Rhy decreased hypertension and up-regulated PPARγ in SHR rats (p &lt; 0.01). In addition, Rhy improved SHR endothelial cell apoptosis by inducing PPARγ, and also produced reduction in proinflammatory factor secretion by inducing PPARγ. Furthermore, Rhy mitigated oxidative stress and iron death by inducing PPARγ (p &lt; 0.001).&#x0D; Conclusion: Rhynchophylline provides a promising method in alleviating endothelial injury in SHR rats by modulating PPARγ-mediated inflammation and oxidative stres. We therefore thought Rhy alleviated endothelial injury and could serve as a promising drug.

Spontaneously hypertensive rats vs. Wistar Kyoto and Wistar rats: An assessment of anxiety, motor activity, memory performance, and seizure susceptibility

Spontaneously hypertensive rats (SHRs) are widely accepted for modeling essential hypertension and Attention deficit hyperactivity disorder (ADHD). However, data concerning central nervous system changes associated with behavioral responses of this strain and usage of Wistar Kyoto (WKY) rats as controls are confounding. The objective of the present study was to assess the impact of anxiety and motor activity on the cognitive responses of SHRs compared to Wistar and WKY rats. In addition, the role of brain-derived neurotrophic factor (BDNF) in the hippocampus on cognitive behavior and seizure susceptibility in the three strains was evaluated. In Experiment#1, SHR demonstrated impulsive responses in the novelty suppression feeding test accompanied by impaired spatial working and associative memory in the Y maze and object recognition test compared with the Wistar rat but not WKY rats. In addition, the WKY rats exhibited diminished activity compared to Wistar rats in an actimeter. In Experiment#2, the seizure susceptibility was assessed by 3-min electroencephalographic (EEG) recording after two consecutive injections of pentylenetetrazol (PTZ) (20+40 mg/kg). The WKY rats were more vulnerable to rhythmic metrazol activity (RMA) than the Wistar rats. In contrast, Wistar rats were more prone to generalized tonic-clonic seizures (GTCS) than WKY rats and SHRs. Control SHR had lower BDNF expression in the hippocampus compared to Wistar rats. However, while the BDNF levels were elevated in the Wistar and WKY rats after PTZ injection, no change in this signaling molecule was observed in the SHR in the seizure condition. The results suggest Wistar rats as a more appropriate control of SHR than WKY rats for studying memory responses mediated by BDNF in the hippocampus. The higher vulnerability to seizures in Wistar and WKY rats compared to SHR might be linked to PTZ-induced decreased expression of BDNF in the hippocampus.

MiR-155-5p Attenuates Vascular Smooth Muscle Cell Oxidative Stress and Migration via Inhibiting BACH1 Expression.

The malfunction of vascular smooth muscle cells (VSMCs) is an initiating factor in the pathogenesis of pathological vascular remodeling, including hypertension-related vascular lesions. MicroRNAs (miRNAs) have been implicated in the pathogenesis of VSMC proliferation and migration in numerous cases of cardiovascular remodeling. The evidence for the regulatory role of miR-155-5p in the development of the cardiovascular system has been emerging. However, it was previously unclear whether miR-155-5p participated in the migration of VSMCs under hypertensive conditions. Thus, we aimed to define the exact role and action of miR-155-5p in VSMC migration by hypertension. Here, we detected that the level of miR-155-5p was lower in primary VSMCs from spontaneously hypertensive rats (SHRs). Its overexpression attenuated, while its depletion accelerated, the migration and oxidative damage of VSMCs from SHRs. Our dual-luciferase reporter assay showed that miRNA-155-5p directly targeted the 3'-untranslated region (3'-UTR) of BTB and CNC homology 1 (BACH1). The miR-155-5p mimic inhibited BACH1 upregulation in SHR VSMCs. By contrast, the deletion of miR-155-5p further elevated the upregulation of BACH1 in SHR-derived VSMCs. Importantly, the overexpression of miR-155-5p and knockdown of BACH1 had synergistic effects on the inhibition of VSMCs in hypertension. Collectively, miR-155-5p attenuates VSMC migration and ameliorates vascular remodeling in SHRs, via suppressing BACH1 expression.

Methyl Palmitate Modulated NMDA-Induced Cerebral Hyperemia in Hypertensive Rats

N-methyl-D-aspartate (NMDA) receptors were found to be dysfunctional in hypertensive rats. Methyl palmitate (MP) has been shown to diminish the nicotine-induced increase in blood flow in the brainstem. The aim of this study was to determine how MP modulated NMDA-induced increased regional cerebral blood flow (rCBF) in normotensive (WKY), spontaneously hypertensive (SHR), and renovascular hypertensive (RHR) rats. The increase in rCBF after the topical application of experimental drugs was measured using laser Doppler flowmetry. Topical NMDA application induced an MK-801-sensitive increase in rCBF in anesthetized WKY rats, which was inhibited by MP pretreatments. This inhibition was prevented by pretreatment with chelerythrine (a PKC inhibitor). The NMDA-induced increase in rCBF was also inhibited by the PKC activator in a concentration-dependent manner. Neither MP nor MK-801 affected the increase in rCBF induced by the topical application of acetylcholine or sodium nitroprusside. Topical application of MP to the parietal cortex of SHRs, on the other hand, increased basal rCBF slightly but significantly. MP enhanced the NMDA-induced increase in rCBF in SHRs and RHRs. These results suggested that MP had a dual effect on the modulation of rCBF. MP appears to play a significant physiological role in CBF regulation.

Network pharmacology-based approach uncovers the pharmacodynamic components and mechanism of Fructus Tribuli for improving endothelial dysfunction in hypertension

Ethnopharmacological relevanceFructus Tribuli (FT), a traditional Chinese medicinal herbal, has been used for the clinical treatment of cardiovascular diseases for many years and affects vascular endothelial dysfunction (ED) in patients with hypertension. Aim of the studyThis study aimed to demonstrate the pharmacodynamic basis and mechanisms of FT for the treatment of ED. Materials and methodsThe present study used ultra-high-performance liquid chromatography coupled with quadruple-time-of-flight mass spectrometry (UHPLC-Q-TOF/MS) to analyze and identify the chemical components of FT. The active components in blood were determined after the oral administration of FT by comparative analysis to blank plasma. Then, based on the active components in vivo, network pharmacology was performed to predict the potential targets of FT in treating ED. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were also performed, and component-target-pathway networks were constructed. Interactions between the major active components and main targets were verified by molecular docking. Moreover, spontaneously hypertensive rats (SHRs) were divided into the normal, model, valsartan, low-dose FT, medium-dose FT, and high-dose FT experimental groups. In pharmacodynamic verification studies, treatment effects on blood pressure, serum markers (nitric oxide [NO], endothelin-1 [ET-1,], and angiotensin Ⅱ [Ang Ⅱ)]) of ED, and endothelial morphology of the thoracic aorta were evaluated and compared between groups. Finally, the PI3K/AKT/eNOS pathway was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot of the thoracic aorta of rats in each group to detect the mRNA expression of PI3K, AKT, and eNOS and the protein expression of PI3K, AKT, p-AKT, eNOS, and p-eNOS. ResultsA total of 51 chemical components were identified in FT, and 49 active components were identified in rat plasma. Thirteen major active components, 22 main targets, and the PI3K/AKT signaling pathway were screened by network pharmacology. The animal experiment results showed that FT reduced systolic blood pressure and ET-1 and Ang Ⅱ levels and increased NO levels in SHRs to varying degrees. The therapeutic effects were positively correlated with the oral dose of FT. Hematoxylin-eosin (HE) staining confirmed that FT could alleviate the pathological damage of the vascular endothelium. qRT-PCR and Western blot analysis confirmed that up-regulated expression of the PI3K/AKT/eNOS signaling pathway could improve ED. ConclusionsIn this study, the material basis of FT was comprehensively identified, and the protective effect on ED was confirmed. FT had a treatment effect on ED through multi-component, multi-target, and multi-pathways. It also played a role by up-regulating the PI3K/AKT/eNOS signaling pathway.

STUDY ON THE MECHANISM OF CARDIAC HYPERTROPHY INDUCED BY TESTOSTERONE THROUGH MTORC2 IN OVARIECTOMIZED FEMALE SPONTANEOUSLY HYPERTENSIVE RATS

Objective: The estrogen level of postmenopausal women decreased sharply, while the androgen level did not decrease significantly, which was characterized by the inversion of estrogen / androgen ratio. Therefore, it could be observed that relative excess androgen in the circulation of postmenopausal women, which may be one of the reasons for the increase of cardiovascular disease in postmenopausal women. In this study, the model of postmenopausal hypertension was established by bilateral ovariectomy in spontaneously hypertensive rats (SHR), and the specific mechanism of testosterone-induced myocardial hypertrophy in ovariectomized SHR was explored. Design and method: Twenty-four SHR were randomly divided into three groups: sham operation group (Sham group, n = 8), bilateral ovariectomized group (OVX group, n = 8) and testosterone propionate intervention group (OVX+T group, 2.85mg/kg Weekly, im for4 weeks, n = 8). The systolic blood pressure, diastolic blood pressure and heart rate of SHR were monitored and recorded twice a week. Western Blot was used to detect the protein expression of mTOR, p-mTOR, mTORC2 and its subunits Rictor and p-Rictor. Moreover, the expression of autophagy-related proteins LC3 and P62/SQSTM1 and Mitofusin2 (MFN2) and Dynamin related protein 1 (DRP1) in cardiac myocytes was observed. Results: 1. Testosterone could activate the expression of mTORC2 in myocardial tissue: testosterone could activate the expression of mTORC2 and the Rictor of mTORC2 in ovariectomized SHR(Figure 1). 2. Testosterone could inhibit cardiomyocyte autophagy: there was no significant difference in the expression of autophagy-related proteins LC3 and P62/SQSTM1 between Sham and OVX groups, while testosterone could decrease the expression of LC3 protein and increase the expression of P62/SQSTM1 protein in OVX+T group (Figure 2). 3. Testosterone could activate the mitochondrial cleavage of cardiomyocytes: the expression of DRP1 was increased in OVX+T group (Figure 3). Conclusions: In bilateral ovariectomized SHR, testosterone may inhibit cardiomyocyte autophagy by activating the expression of mTORC2, which leads to cardiac hypertrophy in SHR. And in cardiomyocyte mitochondria, testosterone can promote mitochondrial cleavage.

Non-Invasive Ultrasound Modulation of Solitary Tract Nucleus Exerts a Sustainable Antihypertensive Effect in Spontaneously Hypertensive Rats.

We applied the method of non-invasive ultrasound (US) neuromodulation to regulate blood pressure (BP) by stimulating the solitary tract nucleus (NTS) of spontaneously hypertensive rats (SHRs). The rats were exposed to US stimulation for 20 mins every day for two months. Morphology and function of the hypertensive target organs (heart and kidney) were then examined by echocardiography and immunohistochemical staining. C-fos immunofluorescence assays were used to evaluate neuronal activity in the US stimulated areas and to explore related neural pathways. Moreover, the effects of US stimulation on biochemical indicators angiotensinII (ANGII), aldosterone (Aldo), endothelin-1 (ET-1), atrial natriuretic factor (ANF), cortisol (Cor) in SHRs were detected. In addition, HE, TUNEL, and Nissl staining were performed to evaluate the safety of long-term transcranial US stimulation. After two months of US stimulation, systolic blood pressure (SBP) decreased from 170 ± 1.1 mmHg to 158 ± 1.8 mmHg, p < 0.01. What's more, US stimulation effectively inhibited the pathological process of target organs from both morphological and functional levels. With US stimulation, neuronal activities were also significantly enhanced in the NTS, ventrolateral periaqueductal gray (vlPAG), and the caudal ventrolateral medulla (CVLM) region. And US stimulation did not cause brain tissue damage. Meanwhile, the plasma levels of ANF, ANGII, Aldo, and Cor content were inhibited. US stimulation of the NTS could significantly lower BP in SHRs. Non-invasive transcranial US stimulation acting on the NTS might be a potential therapeutic intervention due to its efficacy and safety.

Effect of combination of active ingredients of Salvia miltiorrhiza on vascular inflammation in spontaneously hypertensive rat

This study explored the effect of combination of active ingredients of Salvia miltiorrhiza (SM) on vascular inflammation in spontaneously hypertensive rats (SHRs). SHRs were treated with optimal compatibility of SM active ingredients (SABP), SM lyophilized powder (SMLP), and Perindopril (PD), followed by analysis of arterial histology by HE staining, blood pressure, inflammatory factors and MCP-1 by ELISA, cell migration by Transwell assay, MCP-1 and CCR2 level by Reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Compared to normal group, arterial wall of rats in model group was significantly thickened with rough intima, without obvious divisions. Some blood vessels were enlarged at the 6th and 8th week, and arterial vessel wall of the SMLP and PD groups was thinned. SABP treatment significantly alleviated vascular inflammation, where blood vessels basically returned to normal at 6th week. The blood pressure of rats in the 1st, 6th and 12th weeks decreased, especially in SABP group (P &lt;0.05). Nevertheless, blood pressure of rats in model group kept rising while levels of various inflammatory factors increased and decreased in the three experimental groups, with SABP group having the lowest levels. SABP, PD, and SMLP treatments significantly upregulated MCP-1 and CCR2 when decreasing the mRNA expressions, with more significant changes in the SABP group. The SAMP exhibited obvious antihypertensive effect with highest compatibility activity of combining SM’s active ingredients, reducing inflammatory factors and MCP-1 in the rats, thereby improving vascular inflammatory response.

DISTINCT CARDIAC CONNEXIN-43 EXPRESSION IN HEMODYNAMICALLY UPLOADED AND UNLOADED HEART MAY IMPACT VULNERABILITY TO MALIGNANT ARRHYTHMIAS

Objective: Experimental and clinical studies suggest that myocardial hypertrophy in response to hypertension and hyperthyroidism increases propensity to cardiac arrhythmias. While these are rare in conditions resulting in myocardial atrophy, such as hypothyroidism or type-1 diabetes mellitus (T1DM). One of the crucial factors impacting the susceptibility of the heart to life-threatening arrhythmias is gap junction channel protein connexin-43 (Cx43), which ensure cardiac cell-to-cell coupling for electrical signal propagation that induce contraction. We aimed to explore abundance of Cx43 protein and its cardiomyocyte topology in hemodynamically uploaded hypertrophied versus unloaded hypotrophic cardiac phenotype. Design and method: Analysis were performed in left ventricular tissue of adult male rats of following groups: 1. spontaneously hypertensive rat (SHR); 2. normotensive Wistar Kyoto rats (WKY) treated for 8-weeks with L-thyroxine (0.15 μg/kg, i.v.) to induce hyperthyroid status; 3. WKY rats treated with methimazol (0.05% solution, p.o.) to induce hypothyroid status; 4. WKY rats treated with strepotozotocin (50mg/kg, i.p.) to induce T1DM. Animals were compared with non-treated normotensive control rats. Results: Comparing to healthy non-treated rats there was a decrease of total myocardial Cx43 protein and its variant phosphorylated at serine368 in SHR and hyperthyroid rats. Besides, enhanced abnormal localisation of Cx43 was demonstrated on lateral sides of hypertrophied cardiomyocytes. In contrast, total Cx43 protein and its serine368 variant were increased in atrophied left ventricle of hypothyroid and T1DM rats. It was associated with less pronounced alterations in Cx43 topology. In parallel, the abundance of PKC epsilon, which phosphorylates Cx43 at serine368 that stabilize Cx43 function and distribution was reduced in hypertrophied heart while enhanced in atrophied once. Conclusions: Findings suggest that differences in the abundance of cardiac Cx43, its variant phosphorylated at serine368 and Cx43 topology may explain, in part, distinct propensity of uploaded and unloaded heart to malignant arrhythmias. It suggests that hemodynamically overloaded heart might benefit from interventions promoting unloading. This research was supported by VEGA grants 2/0002/20, 2/0006/23, Slovak Research and Development Agency under the Contract no. 21-0410

Comparative Analysis of Hypertensive Tubulopathy in Animal Models of Hypertension and Its Relevance to Human Pathology.

Assessment of hypertensive tubulopathy for more than fifty animal models of hypertension in experimental pathology employs criteria that do not correspond to lesional descriptors for tubular lesions in clinical pathology. We provide a critical appraisal of experimental hypertension with the same approach used to estimate hypertensive renal tubulopathy in humans. Four models with different pathogenesis of hypertension were analyzed-chronic angiotensin (Ang) II-infused and renin-overexpressing (TTRhRen) mice, spontaneously hypertensive (SHR), and Goldblatt two-kidney one-clip (2K1C) rats. Mouse models, SHR, and the nonclipped kidney in 2K1C rats had no regular signs of hypertensive tubulopathy. Histopathology in animals was mild and limited to variations in the volume density of tubular lumen and epithelium, interstitial space, and interstitial collagen. Affected kidneys in animals demonstrated lesion values that are significantly different compared with healthy controls but correspond to mild damage if compared with hypertensive humans. The most substantial human-like hypertensive tubulopathy was detected in the clipped kidney of 2K1C rats. For the first time, our study demonstrated the regular presence of chronic progressive nephropathy (CPN) in relatively young mice and rats with induced hypertension. Because CPN may confound the assessment of rodent models of hypertension, proliferative markers should be used to verify nonhypertensive tubulopathy.

APELIN AS A POTENTIAL DOWNSTREAM MEDIATOR OF THE IGF-1 CARDIOPROTECTIVE PATHWAY IN THE HYPERTROPHIED MYOCARDIUM OF SPONTANEOUSLY HYPERTENSIVE RATS

Objective: Hypertension is a complex cardiovascular disease and contributes to worldwide morbidity and mortality associated to the development of hypertensive cardiac hypertrophy and the progression of heart failure. Hypertensive myocardium is characterized by mitochondrial dysfunction and redox disbalance. On the other hand, aerobic exercise upregulates several humoral factors such as insulin like growth factor-1 (IGF-1) and apelin, involved in cardioprotective effects. Particularly, whether apelin mediates IGF-1 cardioprotective effects by an autocrine paracrine mechanism is not clear yet. Objective. To determine the acute effects of IGF-1/apelin on oxidative stress and mitochondrial status, as well as the possible crosstalk between the signaling pathways triggered by them in spontaneously hypertensive rats (SHR) myocardium. Design and method: Adult SHR isolated hearts or cardiomyocytes were perfused during 10 min in the presence or absence of IGF-1 or apelin, and the specific antagonist of their receptors (AG1024 and ML221 for IGF1R and APJ, respectively). We explored reactive oxygen species (ROS) production, SOD activity, APJ cellular distribution, mitochondrial membrane potential (↖ ⇘) and permeability transition pore opening through the calcium retention capacity (CRC). Results: IGF-1 exerted an antioxidant effect evidenced by an increase in SOD activity (U/mg, IGF-1: 59.8±5.2, vs Control: 41.6±3) and decrease in ROS production (%, IGF-1: 66±12, vs Control: 100±8). In mitochondria, IGF-1 prevented loss induced by H2O2 (IGF-1: 0.934±0.016, vs Control: 0.827±0.027) and increased CRC (IGF-1: 251±34, vs Control: 135±23). Interestingly, IGF-1 upregulated APJ expression (IGF-1: 175.8±27.7 vs Control: 100±16 % mRNA) while favoring its translocation to the sarcolemma (Pearson coefficient: IGF-1: 0.880±0.011 vs Control: 0.758±0.031) in isolated cardiomyocytes. Moreover, exogenous apelin was able to emulate IGF-1 antioxidant and mitochondrial actions (SOD: 64±5.3 U/mg; ROS: 64± 7 %; ↖ ⇘ loss induced by H2O2: 0.981±0.025 and CRC 264±31). These effects were prevented by inhibition of APJ (Apelin+ML221: SOD: 33±4.4, CRC: 84±17, 0.808±0.042) but not by AG1024 (Apelin+AG: 0.985±0.068, CRC: 245±47). Conclusions: In conclusion, our data suggest that IGF-1 by sequentially activating its own receptor and the apelin/APJ pathway is responsible for cardioprotection. Therefore, apelin emerges as a downstream effector of the cardiac IGF-1 signaling pathway.