Wistar-Kyoto Groups Research Articles

Renal denervation alleviates vascular remodeling in spontaneously hypertensive rats by regulating perivascular adipose tissue.

Vascular remodeling is the main pathological process that causes the damage of the target organ of hypertension. Perivascular adipose tissue (PVAT) surrounds blood vessels and plays a key role in the pathogenesis of various cardiovascular diseases. This study aimed to investigate the effects of renal denervation (RDN) on hypertensive vascular remodeling and to elucidate the role of PVAT in this process. Male spontaneously hypertensive rat (SHR) and Wistar-Kyoto (WKY) rat were selected. Aortic vascular remodeling was evaluated using hematoxylin and eosin (H&E) staining and Masson's trichrome staining. Morphological changes in the PVAT were observed through H&E and Oil Red O staining. Dihydroethidium was used to measure oxidative stress levels in PVAT, while western blot analysis was used to determine the expression levels of proteins associated with vascular remodeling. The results showed that the aortic medial thickness, media thickness/lumen diameter, collagen volume fraction, and reactive oxygen species (ROS) level in PVAT were significantly higher in the SHR group than in the WKY group. The indexes mentioned above were lower in the SHR-RDN group than in the SHR group. H&E staining revealed that fat droplets in PVAT in the SHR-RDN group became smaller and browning occurred. Moreover, the protein expression of uncoupling protein-1 (UCP-1) and neuregulin 4 (Nrg4) was significantly increased in the SHR-RDN group. In addition, the expression of adiponectin increased and the expression of leptin decreased in the SHR-RDN group compared to the SHR group. In conclusion, RDN can relieve hypertensive vascular remodeling, which may be associated with PVAT.

Effects and mechanisms of negative air ions on blood pressure, oxidative stress and inflammation levels in SHR rats

To investigate the effects and possible mechanisms of negative air ions(NAIs) on blood pressure, oxidative stress, and inflammatory status in spontaneous hypertension rats(SHR). A total of 60 SHR(half male and half female) were randomly divided into one-month and three-month groups, 30 rats per groups, based on the duration of the intervention. Each group was further randomized into three groups based on the daily intervention time: SHR control group, 2 h NAIs-SHR group, and 6 h NAIs-SHR group, 10 rats per groups. In addition, 20 Wistar Kyoto(WKY)(half male and half female), were randomized into one-month WKY group and three-month WKY group, 10 rats per groups, based on the intervention time. The 2 h NAIs-SHR group and 6 h NAIs-SHR group were exposed to an environment with NAIs concentrations of 4.5×10~4-5×10~4 cm~3 per day for 2 h and 6 h. The WKY group and SHR group were exposed to normal air on a daily basis. Blood pressure of rats in each group was measured every three days, while weight was measured once a week. After sacrificing the rats in the first month and the third month of rearing, wet weight of the organs was weighed. The enzyme linked immunosorbent assay(ELISA) was used to detect 8-hydroxylated deoxyguanosine(8-OHdG), interleukin-6(IL-6), interleukin-8(IL-8), tumor necrosis factor-α(TNF-α), nitric oxide(NO) and endothelin-1(ET-1) levels. Reactive oxygen species(ROS) detection kit was used to detect ROS level. Malondialdehyde(MDA) and superoxide dismutase(SOD), glutathione(GSH) and glutathione disulfide(GSSG) were measured by colorimetric analysis. HE staining was conducted to observe the histopathological morphological changes of the thoracic aorta in each group, and Western blot was conducted to detect the thoracic aortap38 mitogen-activated protein kinase(p38 MAPK), extracellular signal-regulated kinases(ERK), c-Jun n-terminal kinase(JNK), c-fos proteins, c-jun proteins and their phosphorylated proteins level. The weight of WKY male mice in the same week age group was higher than that of SHR control group, and there was no significant difference in the weight between the other groups. The coefficient of heart in SHR control group(4.66±0.48) was higher than that in WKY group(3.73±0.15)(P<0.05), while there were no significant differences in the coefficients of brain, kidney, liver and spleen among the groups. Blood pressure in WKY group at the same age was lower than that in SHR group, and blood pressure in SHR control group at 2-5 and 8-11 weeks was higher than that in 2 h NAIs-SHR and 6 h NAIs-SHR groups(P<0.05). HE staining showed that the internal, middle and external membranes of thoracic aorta in 2 h NAIs-SHR group and 6 h NAIs-SHR group were improved to varying degrees compared with those in SHR control group, including disordered internal membrane structure, thickened middle membrane and broken external membrane. In terms of oxidative stress levels, compared with the SHR control group, the ROS(0.66%±0.17%, 0.49%±0.32%) and 8-OHdG((48.29±8.00) ng/mL, (33.13±14.67)ng/mL) levels were lower in the 6 h NAIs-SHR group(P<0.05), while the GSH/GSSG ratio was higher in the one-month 6 h NAIs-SHR group(10.08±4.93). Compared with the 2 h NAIs-SHR group, the ROS level(0.99%±0.19%) was lower in the 6 h NAIs-SHR group(P<0.05). In terms of inflammatory factor levels, compared with the SHR control group, the IL-8 levels((160.44±56.54) ng/L, (145.77±38.39) ng/L) were lower in the 6 h NAIs-SHR group(P<0.05), while the ET-1 level((249.55±16.98) ng/L) was higher in the one-month WKY group. There was no significant difference in NO levels among the groups. The relative expression of p-p38 protein in the thoracic aorta of rats in the one-month SHR control group was lower than that in the WKY group(P<0.05). The relative expression of p-p38 and p-c-fos proteins in the thoracic aorta of rats at three-months was higher in the SHR control group than in the 2 h NAIs-SHR and 6 h NAIs-SHR groups(P<0.05). The intervention of NAIs at a concentration of 4.5×10~4-5×10~4/cm~3 may regulate the partial oxidation and inflammatory state of SHR rats through the ROS/MAPK/AP1 signaling pathway, thereby reducing their blood pressure level.

Patchouli alcohol against renal fibrosis of spontaneously hypertensive rats via Ras/Raf-1/ERK1/2 signalling pathway.

The present study was designed to obverse the protection of patchouli alcohol (PA) ameliorates hypertensive nephropathy in spontaneously hypertensive rats (SHR) and reveals potential mechanism. Briefly, the adult spontaneously hypertensive rats (SHR) or Wistar-Kyoto (WKY) rats (half male and half female) were intragastric gavaged or not with PA (80, 40 and 20 mg/kg) for 8 weeks. Body weight, blood pressure (BP), renal weight, renal function and renal morphology were measured. Further, western blotting and immunohistochemical analysis were used to study the underlying mechanism. Compared with the WKY group, plasmatic levels of renin, angiotensin II (Ang-II), transforming growth factor beta 1(TGF-β1), plasminogen activator inhibitor-1(PAI-1), creatinine (Cr), blood urea nitrogen (BUN), renal index, mRNA levels of ERK1/2 and α-SMA were significantly increased in SHR. Histology results showed that renal tubular injury and tubulointerstitial fibrosis occurred in SHR. After administration, SBP of captopril group decreased at each week after administration, especially at 3, 5, 6 7 and 8 weeks (P < 0.05 or P < 0.01). There is no significant effect was assessed in the olive oil group. Decreased plasma Cr, Renin, Ang-II, TGF-β1, PAI-1, SCFAs and Renin, TGF-β1, PAI-1 in renal tissues were observed significantly in captopril (P <0.05 or P < 0.01). Plasma BUN, Ang-II, TGF-β1 and PAI-1 in renal tissues decreased in the olive oil group significantly (P <0.05 or P < 0.01). PA (80, 40 and 20 mg/kg) lowered BP and plasmatic levels of Renin, Ang-II, TGF-β1 and PAI-1. Treatment with PA (40, 20 mg/kg) decreased levels of Cr, BUN and suppressed of activation of pro-fibrosis cytokines including TGF-β1 in kidney. There is no ameliorative change in the olive oil group and the captopril group (P > 0.05) while PA treatment alleviated renal tubular injury and produced dramatic collagen fibre area reductions in mesangial membrane, basement membrane, and renal interstitium obviously (P < 0.05 or P < 0.01). Treatment of SHR with PA-inhibited MFB activation and downregulated mRNA of α-SMA. Treatment with PA suppressed excessive production of the extracellular matrix (ECM) via decreasing Col I, III and FN, downregulating mRNA of tissue inhibitor of TIMP-1 along with upregulating mRNA of MMP-9. The expression of Col III and MMP-9 mRNA-reduced in the captopril group (P < 0.05). In addition, the expression of ERK1/2 and pERK1/2 also reduced in the captopril group significantly (P < 0.05 or P < 0.01). Treatment with PA (20 mg/kg) downregulated proteins expression of Raf-1, ERK1/2 and pERK1/2 and mRNA expression of Ras, Raf-1 and ERK1/2. Overall, PA restored normal BP, alleviated renal dysfunction and renal fibrosis, possibly by suppressing Ang II and TGF-β1-mediated Ras/Raf-1/ERK1/2 signalling pathway.

Gastrodin attenuates renal injury and collagen deposition via suppression of the TGF-β1/Smad2/3 signaling pathway based on network pharmacology analysis.

Background: Gastrodin has been widely used clinically in China as an antihypertensive drug. However, its effect on hypertensive renal injury is yet to be elucidated. The current study aimed to investigate the effects of gastrodin on hypertensive renal injury and its underlying mechanisms by network pharmacology analysis and validation in vivo and in vitro. Methods: A total of 10 spontaneously hypertensive rats (SHRs) were randomly categorized into the following two groups: SHR and SHR + Gastrodin groups. Wistar Kyoto (WKY) rats were used as the control group (n = 5). The SHR + Gastrodin group was intragastrically administered gastrodin (3.5mg/kg/day), and the rats in both WKY and SHR groups were intragastrically administered an equal amount of double-distilled water for 10 weeks. Hematoxylin-eosin, Masson's trichrome, and Sirius red staining were used to detect the pathological changes and collagen content in the renal tissues. Network pharmacology analysis was performed to explore its potential targets and related pathways. In vitro, the CCK-8 assay was used to determine the cell viability. Immunohistochemistry and western-blotting analyses were employed to assess the protein expression associated with renal fibrosis and transforming growth factor-β1 (TGF-β1) pathway-related proteins in the renal tissues or in TGF-β1-stimulated rat kidney fibroblast cell lines (NRK-49F). Results: Gastrodin treatment attenuates renal injury and pathological alterations in SHRs, including glomerular sclerosis and atrophy, epithelial cell atrophy, and tubular dilation. Gastrodin also reduced the accumulation of collagen in the renal tissues of SHRs, which were confirmed by downregulation of α-SMA, collagen I, collagen III protein expression. Network pharmacology analysis identified TGFB1 and SMAD2 as two of lead candidate targets of gastrodin on against hypertensive renal injury. Consistently, gastrodin treatment downregulated the increase of the protein expression of TGF-β1, and ratios of both p-Smad2/Smad2 and p-Samd3/Smad3 in renal tissues of SHRs. In vitro, gastrodin (25-100μM) treatment significantly reversed the upregulation of α-SMA, fibronectin, collagen I, as well as p-Smad2 and p-Smad3 protein expressions without affecting the cell viability of TGF-β1 stimulated NRK-49F cells. Conclusion: Gastrodin treatment significantly attenuates hypertensive renal injury and renal fibrosis and suppresses TGF-β1/Smad2/3 signaling in vivo and in vitro.

Cardioprotective effect of NRG-4 gene expression on spontaneous hypertension rats and its mechanism through mediating the activation of ErbB signaling pathway

To explore the myocardial protective effect of Neuregulin-4 (NRG-4) gene expression on spontaneous hypertension (SHR) rats and its mechanism through mediating the activation of the ErbB signaling pathway, this study was conducted. For this purpose, forty 24-week-old male SPF SHR rats were selected as the experimental group, and 10 age and sex-matched Wistar Kyoto (WKY) rats were selected as the control group. Cardiac tissues were collected for hematoxylin-eosin (HE) staining, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining, Masson staining, and immunohistochemical staining. Following tail vein injection of recombinant lentiviral vectors, the experimental groups were constructed as the control group (SHR rats without any treatment), Empty vector group (Empty Vector transfection), shRNA negative control (NC) group (LV-shRNA-NRG-4 NC transfection to silence the expression of NRG-4), shRNA group (LV-shRNA-NRG-4 transfection), pcDNA3.1(-) NC group (pcDNA3.1(-)-NRG-4 empty vector transfection) and pcDNA3.1(-) group (pcDNA3.1(-)-NRG-4 transfection to overexpress NRG-4). Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were used to detect the expression levels In addition, methyl thiazolyl tetrazolium salt (MTT) assay and flow cytometry were performed to detect the proliferation and apoptosis of cardiomyocytes, respectively. Results showed that in the SHR group, the cardiomyocytes showed hypertrophy, disordered arrangement, hyperchromatic nucleus, irregular shape, obvious rupture of myocardial fibers, and obvious proliferation of fibrous stroma; obvious myocardial fibrosis, and there were more blue collagen fibers around cardiomyocytes and myocardial arterioles; cardiomyocytes were swollen, muscle fibers arranged disorderly, collagen around the coronary artery and myocardial interstitium increased significantly with a cross-linking appearance; besides, compared with WKY group, the apoptosis index of cardiomyocytes in SHR group was significantly increased (P&lt;0.05). The expression of NRG-4 protein was decreased in the SHR group compared with the WKY group (P&lt;0.05). In vitro, there was no difference in the mRNA expression of NRG-4, ErbB2 and ErbB4, MMP2, TGFβ1 and α-SMA, as well as Caspase3, Bax and Bcl-2 among the control group, Empty vector group, shRNA NC group and pcDNA3.1(-) NC group (P&gt;0.05). While shRNA group showed decreased expressions of NRG-4, ErbB2, ErbB4, MMP2, TGFβ1, α-SMA and Bcl-2, while increased Caspase3 and Bax expressions, as well as promoted cell proliferation and cell apoptosis when compared with the shRNA NC group (P&lt;0.05); while compared with pcDNA3.1(-) NC group, pcDNA3.1(-) group had highly increased expressions of NRG-4, ErbB2, ErbB4, MMP2, TGFβ1, α-SMA and Bcl-2, while decreased Caspase3 and Bax expressions, inhibited cell proliferation and cell apoptosis (all P&lt;0.05). It is concluded Upregulation of NRG-4 gene expression can promote the activation of the ErbB signaling pathway, thus inhibiting the proliferation and apoptosis of cardiomyocytes in SHR rats, reversing myocardial fibrosis, and playing its cardioprotective role.

In vivo assessment of hypertensive nephrosclerosis using ultrasound localization microscopy.

As a typical chronic kidney disease (CKD), hypertensive nephrosclerosis (HN) is a common syndrome of hypertension, characterized by chronic kidney microvascular damage. Early diagnosis of microvascular damage using conventional ultrasound imaging encounters challenges in sensitivity and specificity owing to the inherent diffraction limit. Ultrasound localization microscopy (ULM) has been developed to obtain microvasculature and microvascular hemodynamics within the kidney, and would be a promising tool for the early diagnosis of CKD. In this study, the advantage of quantitative indexes obtained by using ULM (mean arterial blood flow speeds of different segments of interlobular arteries) over indexes obtained using conventional clinical serum (β2-microglobulin, serum urea nitrogen, and creatinine) and urine (24-h urine volume and urine protein) tests and ultrasound Doppler imaging (peak systolic velocity [PSV], end-diastolic velocity [EDV], and resistance index [RI]) and contrast-enhanced ultrasound imaging (CEUS; rise time [RT], peak intensity [IMAX], mean transit time [mTT], and area under the time-intensity curve [AUC]) for early diagnosis of HN were investigated. Examinations were carried out on six spontaneously hypertensive rats (SHR) and five normal Wistar-Kyoto (WKY) rats at the age of 10weeks. The experimental results show that the indicators derived from conventional clinical inspections (serum and urine tests) and ultrasound imaging (PSV, EDV, RI, RT, IMAX, mTT, and AUC) do not show significant difference between hypertensive and healthy rats (p>0.05), while the TTP of the SHR group (28.52 ± 5.52 s) derived from CEUS is significantly higher than that of the WKY group (18.68 ± 7.32 s; p<0.05). The mean blood flow speed in interlobular artery of SHR (12.47 ± 1.06mm/s) derived from ULM is significantly higher than that of WKY rats (10.13 ± 1.17mm/s; p<0.01). The advantages of ULM over conventional clinical inspections and ultrasound imaging methods for early diagnosis of HN were validated. The quantitative results show that ULM can effectively diagnose HN at the early stage by detecting the blood flow speed changes of interlobular arteries. ULM may promise a reliable technique for early diagnosis of HN in the future.

Qingda granules mitigate cardiac inflammation in spontaneously hypertensive rats via the MCP-1/CCR2 signaling pathway

ObjectiveThis study investigated effects and underlying mechanisms of Qingda granules (QDG) on cardiac inflammation in spontaneously hypertensive rats (SHRs). MethodsTwelve SHRs (17 weeks old) were randomly divided into SHR and SHR + QDG groups, with six rats in each group. We also used six 17-week-old Wistar Kyoto (WKY) rats as the WKY group. While rats in the SHR + QDG group were administered QDG (0.8 g/kg/d) for eight weeks, those in the WKY and SHR groups were administered an equal volume of normal saline. Blood pressure was then monitored weekly. Subsequently, hematoxylin and eosin (HE) staining was used to detect pathological changes in the cardiac tissue. Besides, enzyme-linked immunosorbent assay (ELISA) was used to detect the serum content of inflammatory cytokines. Subsequently, real-time quantitative polymerase chain reaction and immunohistochemical (IHC) staining were conducted to determine the expression of inflammatory cytokines and inflammatory cell infiltration, including the activation of the MCP-1/CCR2 signaling pathway. ResultsAs observed, QDG inhibited the elevation of systolic blood pressure, diastolic blood pressure, and mean arterial pressure in the understudied SHRs. HE staining showed that this drug attenuated pathological changes and inflammatory cell infiltration of cardiac tissue samples in the SHRs. However, ELISA and IHC confirmed a reduction in the expression of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the serum and cardiac tissue of SHRs with QDG treatment. Additionally, QDG treatment significantly attenuated protein levels of interferon-γ, CD3, and Mac-2 in the cardiac tissue samples of SHRs. Moreover, mRNA and protein expressions of monocyte chemoattractant protein 1 (MCP-1) and chemotactic cytokine receptor 2 (CCR2), which were upregulated in cardiac tissue samples of SHRs, became downregulated through treatment with QDG. ConclusionTherefore, by decreasing the levels of inflammatory cytokines and inflammatory cell infiltration through suppression of the MCP-1/CCR2 signaling pathway, QDG treatment attenuated blood pressure and cardiac inflammatory changes in SHRs.

Reduced Relaxant Response to Adenine in the Superior Mesenteric Artery of Spontaneously Hypertensive Rats.

We investigated the vascular response to nucleobase adenine using freshly isolated superior mesenteric arteries of spontaneously hypertensive rats (SHR) and its control, Wistar Kyoto (WKY) rats. Endothelium-dependent and endothelium-independent relaxations were assessed in isolated segments in an organ bath. The releases of the metabolites of thromboxane A2 and prostaglandin I2 were also detected. Adenine induced vasorelaxation in both the endothelium-intact and endothelium-denuded arteries in a concentration-dependent manner. In the SHR group, the adenine-induced relaxation was slightly but significantly reduced in the endothelium-intact rings when compared with that in the WKY group. However, the relaxation in the endothelium-denuded rings were similar between the two groups. The difference in the adenine-mediated relaxation in the superior mesenteric arteries between the SHR and WKY groups was eliminated by endothelial denudation and a nitric oxide (NO) synthase inhibitor. In the absence and presence of adenine, SHR tended to have higher levels of metabolites of thromboxane A2 and prostaglandin I2 compared with WKY. However, adenine did not induce the release of these substances in the arteries in both the SHR and WKY groups. These results suggest that the reduced adenine-mediated relaxation in the superior mesenteric arteries in SHR is due to a lack of contribution from the endothelium-derived NO and not from the release of prostanoids.

P–073 High-intensity interval training modulates the effects of hypertension on inflammatory mediators in testis in adult spontaneously hypertensive rats

Abstract Study question Is the high-intensity interval training (HIIT) able to prevent the increase of the inflammatory proteins in the testis of spontaneously hypertensive rats (SHR)? Summary answer HIIT for 8 weeks inhibits the increase of the tumor necrosis factor alpha (TNFα) and the interleukin 6 (IL6) in the testis of SHR. What is known already Hypertension increases the inflammatory cytokines of the cardiovascular system, causing damage in the microcirculation and in the testes. Hypertension is a cause of low fertility in men and exercises are indicated to decrease blood pressure and improve overall health. HIIT is characterized by short periods of exercise, with an intensity equal to or greater than the anaerobic threshold, separated by recovery periods. HIIT can be indicated for cardiac patients, as this type of training improves the cardiac autonomic nervous system and lipemic control in both hypertensive and normotensive individuals. Study design, size, duration Male Wistar-Kyoto rats without hypertension and SHRs were divided into 3 groups (n = 4): WKY (Wistar-Kyoto); SHR; and SHR-HIIT (SHRs that performed HIIT). The HIIT was realized in treadmill for 5 days/week for 8 weeks. HIIT started with 5 times of 4 minutes of 100% of the maximum exhaust speed, with active rest intervals of 3 minutes at 60%. There was an increase of once every week until reaching 7 times each session. Participants/materials, setting, methods Samples from testicles were used for immunostainig IL6 and TNFα. After the blocking, the sections were subjected to reaction of specific antibodies IL6 (1:50, E–4, sc–28343) and TNFα (1:50, 52B83, sc–257) at 4ºC overnight, and with secondary antibodies m-IgGK (1:200, IgG-HRP, sc–516) at room temperature for 2 hours. Diaminobenzidine (1:50) was used against Harris stained with Hematoxylin and evaluated in the photomicroscope. Data were analyzed by One-Way ANOVA followed by Tukey post test (P &amp;lt; 0.05). Main results and the role of chance The immunostainig of IL6 was higher in SHR (10.23 ± 0.47) than in SHR-HIIT (8.69 ± .44) group (P = 0,0237). From the same perspective, immunoexpression of TNFα was higher in SHR (10.10 ± 0.42) than WKY (8.24 ± 0.24) and SHR-HIIT (7.82 ± 0.39) groups (P = 0.0018). Limitations, reasons for caution As a limitation of the study, we have no measurement of fertility parameters to affirm that the HIIT improve the fertility because of the reduction of inflammatory mediators. Wider implications of the findings: The hypertension drugs have a negative effect on fertility. HIIT can be suggested as a treatment for hypertension as an alternative to medication, since HIIT can inhibit the increase of the inflammatory mediators in testis and its consequences to the reproduction. Financial support by São Paulo Research foundation, FAPESP (2018/22682–0). Trial registration number 2018/22682–0

WHAT IS THE MAJOR FACTOR ON THE KIDNEY INJURY INDUCED BY HYPERTENSION OR HYPERHOMOCYSTEINEMIA

Objective: As we all know kidney injury could be induced by hypertension or hyperhomocysteinemia (HHcy). What is the major factor? HHcy plays a synergistic role with hypertension in vascular injury; however, the relationship between HHcy and hypertension in renal injury remains unclear. Here, we sought to evaluate the relationship between HHcy and hypertension in the context of renal injury and to elucidate the mechanism of action underlying this relationship. Design and method: Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats were randomized into WKY, WKY+HHcy, SHR, and SHR+HHcy groups. Blood pressure, plasma homocysteine, creatinine, serum malondialdehyde (MDA), serum superoxide dismutase (SOD), and urinary albumin creatinine ratio (UACR) were measured. mRNA and protein expression levels of NOX2, NOX4, and nephrin in the kidneys were also examined. Results: The WKY+HHcy group exhibited lower serum SOD levels, relative to the WKY group, along with higher levels of both serum MDA and UACR. Similar effects were observed in the SHR+HHcy group, relative to the SHR group. Moreover, compared with the levels in the WKY+HHcy group, mean SOD levels in the SHR+HHcy group were lower, while mean MDA and UACR levels were higher. Higher mRNA and protein expression levels of NOX2 and NOX4, along with lower mRNA and protein expression levels of nephrin, were observed in the kidneys of WKY+HHcy and SHR+HHcy rats, relative to SHR and WKY controls, respectively. Furthermore, the mRNA and protein levels of NOX2 and NOX4 were higher and the mRNA and protein levels of nephrin were lower in the SHR+HHcy group than in the WKY+HHcy group. In addition, compared with WKY+HHcy group, the UACR level of SHR group was higher. Conclusions: HHcy appears to synergistically increase hypertensive renal damage by enhancing oxidative stress. However, hypertension remains a major risk factor for renal impairment compared to HHcy.

Pingyang Jiangya Fang pretreatment reduces the blood pressure of spontaneously hypertensive rats with Liver-Yang hyperactivity syndrome via ROS/Akt oxidative stress pathway.

ROS/Akt pathway oxidative stress refers to a procedural response that activates various stress-sensitive signaling pathways in the cell thereby inducing insulin resistance (IR), and is closely related to high blood pressure. This study aims to investigate the effects of Pingyang Jiangya Fang (PYJYF) on the ROS/Akt pathway oxidative stress response in spontaneously hypertensive rats (SHRs) with Liver-Yang hyperactivity syndrome. Except for the Wistar-Kyoto (WKY) and SHR groups, the other groups' rats were administered with Fuzi Decoction, to duplicated the model of hyperactivity of Liver-Yang. These rat's scleral color of deepens and become red, and rat's degree of irritability reached to above II degree. Compared with control group, rat's water consumption increased significantly, rotation tolerance time reduced significantly, pain threshold reduced significantly, rat tail vein systolic blood pressure (SBP) increased significantly. It shows that the model rats with hyperactivity of Liver-Yang have successfully replicated. Then, the corresponding drugs in the positive medicine group (candesartan cilexetil tablets) and the high, middle, and low dose groups of PYJYF (PYJYF-H, PYJYF-M, PYJYF-L) were administered. This work detected and analyzed behavior, SBP, renin-angiotensin system (RASS), expression of skeletal muscle angiotensin II type 1 receptor/angiotensin II type 2 receptor (AT1R/AT2R), IR index, renal/renal vascular/skeletal muscle tissue damage in each group. Moreover, we identified the target effect of point composition in the ROS/Akt signaling pathway. Unlike the WKY and SHR groups, the model group exhibited the phenotype of Liver-Yang hyperactivity syndrome, markedly increased SBP, hyper-RASS system, increased urine N-acetyl-β-Dglucosidase (NAG) and microalbuminuria (m-ALB), increased AT1R/AT2R ratio of skeletal muscle, IR, concomitant renal and skeletal muscle damage, as well as significant upregulation in the expression of ROS/ Akt signaling factor. PYJYF-H and PYJYF-M improved Liver-Yang hyperactivity syndrome, reduced SBP, the RASS system, urine NAG and m-ALB, AT1R/AT2R ratio, IR index, repair renal, renal vascular, and skeletal muscle tissue injury, as well as downregulated the expression of ROS/Akt signal factor. PYJYF reduces blood pressure by inhibiting ROS/Akt pathway oxidative stress and reducing IR.

Increased Notch2/NF-κB Signaling May Mediate the Depression Susceptibility: Evidence from Chronic Social Defeat Stress Mice and WKY Rats

The susceptibility to depression has been attributed to the chronic stress and genetic factors but still fails to identify definite biomarkers. The present study aimed to investigate the role of disrupted Notch signaling in the medial prefrontal cortex of the chronic social defeat stress (CSDS) mice and Wistar Kyoto (WKY) rats. RNA-sequencing and quantitative real-time PCR analyses evidenced the involvement of Notch signaling pathway in depression. Western blotting reported an increased level of Notch2 and NF-κB and a decreased level of Hes1 and Bcl2/Bax ratio both in the susceptible mice when compared with the control or resilient ones and in the depression WKY rats when compared with the Wistar or non-depression WKY groups. Further analysis showed that the above-mentioned changes were significantly correlated with the depression-like behaviors and that the elicited Notch2 strongly correlated with the upregulated NF-κB, not with the downregulated Hes1 or Bcl2/Bax ratio. In conclusion, the increased Notch2/NF-κB signaling in the medial prefrontal cortex may mediate depression susceptibility, providing a potential diagnostic biomarker or therapeutic target for treating major depressive disorder.

Hyperhomocysteinemia-Induced Oxidative Stress Aggravates Renal Damage in Hypertensive Rats.

Hyperhomocysteinemia (HHcy) plays a synergistic role with hypertension in vascular injury; however, the relationship between HHcy and hypertension in renal injury remains unclear. Here, we sought to evaluate the relationship between HHcy and hypertension in the context of renal injury and to elucidate the mechanism of action underlying this relationship. Wistar Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) were randomized into WKY, WKY + HHcy, SHR, and SHR + HHcy groups. Blood pressure, plasma homocysteine, serum malondialdehyde (MDA), serum superoxide dismutase (SOD), urinary albumin creatinine ratio (UACR), and glomerular filtration rate (GFR) were measured. Renal histopathology and expression levels of NOX2, NOX4, and nephrin in the kidneys were examined. The WKY + HHcy and SHR groups exhibited lower serum SOD and GFR levels, relative to the WKY group, along with higher levels of both serum MDA and UACR. Higher mRNA and protein expression levels of NOX2 and NOX4, along with lower expression levels of nephrin, were observed in the kidneys of WKY + HHcy and SHR rats, relative to WKY controls, respectively. Similar effects were observed in the SHR + HHcy group, relative to the SHR group and WKY + HHcy group, respectively. Periodic acid-Schiff staining showed an increase in the glomerular extracellular matrix in the WKY + HHcy and SHR + HHcy groups compared with their respective controls. HHcy appears to synergistically increase hypertensive renal damage by enhancing oxidative stress.

Metabolomics study of the prefrontal cortex in a rat model of attention deficit hyperactivity disorder reveals the association between cholesterol metabolism disorder and hyperactive behavior

Attention deficit hyperactivity disorder (ADHD) is a neurodevelopmental disease for which specific biomarkers and pathological mechanisms have yet to be identified. Methylphenidate (MPH) is commonly used to treat ADHD, but its therapeutic mechanisms and its impact on brain metabolites remain unclear. Metabolomics can help to discover biomarkers and identify pathophysiological mechanisms. We adopted an untargeted metabolomics approach based on gas chromatography-mass spectrometry to investigate the potential biomarkers and pathogenesis of ADHD. Ten Wistar-Kyoto (WKY) rats were chosen as healthy controls (vehicle, i.g.). Twenty young spontaneously hypertensive rats (SHR) were randomly allocated to the SHR group (vehicle, i.g.) and MPH group (2 mg/kg/day, i.g.). We identified 103 metabolites from the prefrontal cortex (PFC). Orthogonal partial least square-discriminate analysis showed the differential expression of these metabolites between the groups. Multivariate and univariate statistical analyses isolated 12 metabolites that differed significantly between the WKY and SHR groups: 3-hydroxymethylglutaric acid, 3-phosphoglyceric acid, adenosine monophosphate, cholesterol, lanosterol, and o-phosphoethanolamine; 3-hydroxymethylglutaric acid and cholesterol were reversed with MPH treatment. Pathway and enrichment analyses revealed that the altered metabolites belonged to the cholesterol metabolism pathways. ELISA and western blotting showed that the activity of 3-hydroxy-3-methyl-glutaryl-CoA reductase and the expression of sterol regulatory element-binding protein-2 and ATP-binding cassette transporter A1 were reduced in the PFC of the SHR; the latter two proteins were upregulated by MPH. In conclusion, metabolomics analysis identified potential biomarkers that influence cholesterol metabolism and may be implicated in the development of ADHD-like behavior. MPH can regulate cholesterol metabolism in the PFC of ADHD models. This study uncovered potential biomarkers and pathways involved in ADHD, providing new insight into its pathogenesis.

Validation of chronic mild stress in the Wistar-Kyoto rat as an animal model of treatment-resistant depression.

A recent review proposed four criteria for an animal model of treatment-resistant depression (TRD): a phenotypic resemblance to a risk factor for depression; enhanced response to stress; nonresponse to antidepressant drugs and response to treatments effective in TRD, such as deep brain stimulation (DBS) of the prefrontal cortex or ketamine. Chronic mild stress (CMS) provides a valid model of depression; the Wistar-Kyoto (WKY) rat is considered to be nonresponsive to antidepressant drugs. Here, we applied CMS to WKY rats. WKY and Wistar rats were exposed to CMS, then treated with saline, imipramine, citalopram or venlafaxine. After 5 weeks of CMS and 3 weeks of drug treatment, all WKY groups were implanted unilaterally with DBS electrodes in the prefrontal cortex, and examined in sucrose intake, elevated plus maze (EPM; decreased entries and time in the open arms) and novel object recognition (decreased exploration) tests, following 2×2 h of DBS. CMS decreased sucrose intake, open arm entries on the EPM, and object recognition. Relative to Wistars, WKY rats showed evidence of increased emotionality in the EPM and novel object recognition tests, and a greater impact of CMS on body weight gain and open arm entries. Wistars responded to drug treatment with an increase in sucrose intake but WKY were nonresponsive to drug treatment on all three behavioural tests. With one exception, DBS reversed the anhedonic, anxiogenic and dyscognitive effects of CMS in all groups of WKY rats. In a further experiment, subacute ketamine (10 mg/kg) also normalized behaviour on all three tests. We conclude that WKY rats subjected to CMS meet all four criteria for a valid model of TRD, and provide a basis for studying the mechanism of action of DBS.

Changes in cerebral glucose metabolism after acupuncture at KI3 in spontaneously hypertensive rats: a positron emission tomography study.

The aim of this study was to explore the effect of acupuncture stimulation at KI3 on brain glucose metabolism in spontaneously hypertensive rats (SHRs). Brain glucose metabolism in SHRs after acupuncture stimulation at KI3 was detected using 18F-2-fluorodeoxy-D-glucose positron emission tomography (18F-FDG-PET). SHRs were randomly divided into three groups: no treatment (SHR group); acupuncture at KI3 (KI3 group); and sham acupuncture (Sham group). Wistar Kyoto (WKY) rats were used as a normal blood pressure (BP) control group. Rats were subjected to 10 min of acupuncture once a day for 7 days. BP and positron emission tomography-computed tomography (PET-CT) were measured after the first acupuncture session and after 7 days of treatment. The results showed that BP was lower in the KI3 group than in the SHR group, both 30-60 min after the first acupuncture session and 24-48 h after the 7-day treatment. Compared with the WKY group, the SHR group had lower glucose metabolism in the motor cortex, sensory cortex, basal ganglia, corpus callosum, caudate putamen, and visual cortex. Compared with the untreated/sham-treated SHR control groups, cerebral glucose metabolism was lower in the medulla oblongata, thalamus, dorsal thalamus, orbital cortex, and hypothalamus after acupuncture at KI3, while it was higher in the olfactory cortex and inferior phrenic muscle. Our results show that, in SHRs, needling at KI3 reduces high BP, most likely by altering the activation of cerebral regions.

Effect of glucocorticoid receptor function on the behavior of rats with attention deficit hyperactivity disorder

To investigate the ideal animal models for attention deficit hyperactivity disorder (ADHD) subtypes and the effect of glucocorticoid receptor (GR) function on the behavior of ADHD rats by comparing behavioral differences between spontaneously hypertensive rats (SHRs), Wistar Kyoto (WKY) rats, and Sprague-Dawley (SD) rats. A total of 24 male SHRs aged 21 days were randomly divided into GR agonist group, GR inhibitor group, and SHR group, with 8 rats in each group. Eight male WKY rats and 8 male SD rats, also aged 21 days, were enrolled as WKY group and SD group respectively. The GR agonist group was treated with intraperitoneal injection of dexamethasone (0.5 mg/kg daily); the GR inhibitor group was treated with intraperitoneal injection of mifepristone (RU486) (54 mg/kg daily); the SHR, WKY, and SD groups were treated with intraperitoneal injection of normal saline (0.5 mL/kg daily). The course of treatment was 14 days for all groups. The open field test and Lat maze test were used to evaluate spontaneous activity and non-selective attention. The open field test showed that before drug intervention the SHR group had significantly higher numbers of line crossings and rearings than the WKY and SD groups (P<0.05); the WKY group had a significantly higher number of line crossings than the SD group (P<0.05); the SD group had a significantly higher number of groomings than the WKY group (P<0.05). After drug intervention, the GR agonist group had significantly lower numbers of line crossings and groomings than the SHR group (P<0.05). The Lat maze test indicated that before drug intervention the SHR group had significantly higher numbers of corner crossings and rearings than the WKY and SD groups (P<0.05); the WKY group had significantly higher numbers of rearings and leanings than the SD group (P<0.05). After drug intervention, the GR agonist group had significantly lower numbers of corner crossings and rearings than the SHR group (P<0.05); the GR inhibitor group had a significantly higher number of rearings than the SHR group (P<0.05); the WKY group had significantly higher numbers of rearings and leanings than the SD group (P<0.05). SHR is an ideal animal model for mixed subtype ADHD, and further studies are needed to determine whether WKY rats can be used as an animal model for attention-deficit subtype ADHD. GR agonist can effectively improve spontaneous activity and non-selective attention in SHRs.

Chronic, low-dose TMAO treatment reduces diastolic dysfunction and heart fibrosis in hypertensive rats.

Several studies have suggested negative effects of trimethylamine oxide (TMAO) on the circulatory system. However, a number of studies have shown protective functions of TMAO, a piezolyte and osmolyte, in animals exposed to high hydrostatic and/or osmotic stress. We evaluated the effects of TMAO treatment on the development of hypertension and its complications in male spontaneously hypertensive rats (SHRs) maintained on water (SHR-Water) and SHRs drinking TMAO water solution from weaning (SHR-TMAO). Wistar-Kyoto (WKY) rats were used as normotensive controls to discriminate between age-dependent and hypertension-dependent changes. Telemetry measurements of blood pressure were performed in rats between the 7th and 16th weeks of life. Anesthetized rats underwent echocardiographic, electrocardiographic, and direct left ventricular end-diastolic pressure (LVEDP) measurements. Hematoxylin and eosin as well as van Gieson staining for histopathological evaluation were performed. Plasma TMAO measured by chromatography coupled with mass spectrometry was significantly higher in the SHR-Water group compared with the WKY group (~20%). TMAO treatment increased plasma TMAO by four- to fivefold and did not affect the development of hypertension in SHRs. Sixteen-week-old rats in the SHR-Water and SHR-TMAO groups (12-wk TMAO treatment) showed similar blood pressures, angiopathy, and cardiac hypertrophy. However, the SHR-TMAO group had lower plasma NH2-terminal pro-B-type natriuretic peptide, LVEDP, and cardiac fibrosis. In contrast to age-matched WKY rats, 60-wk-old SHRs showed hypertensive angiopathy and heart failure with preserved ejection fraction. Compared with the SHR-Water group, the SHR-TMAO group (56-wk TMAO treatment) showed significantly lower plasma NH2-terminal pro-B-type natriuretic peptide and vasopressin, significantly lower LVEDP, and cardiac fibrosis. In conclusion, a four- to fivefold increase in plasma TMAO does not exert negative effects on the circulatory system. In contrast, increased dietary TMAO seems to reduce diastolic dysfunction in pressure-overloaded hearts in rats. NEW & NOTEWORTHY Chronic, low-dose trimethylamine oxide (TMAO) treatment that increases plasma TMAO by four- to fivefold reduces plasma NH2-terminal pro-B-type natriuretic peptide and vasopressin, left ventricular end-diastolic pressure, and cardiac fibrosis in pressure-overloaded hearts in hypertensive rats. Our study provides evidence that a moderate increase in plasma TMAO does not have a negative effect on the circulatory system. In contrast, increased dietary TMAO seems to reduce diastolic dysfunction in the pressure-overloaded heart.

Neuropeptidase activity in the frontal cortex of Wistar-Kyoto and spontaneously hypertensive rats treated with vasoactive drugs: a bilateral study.

Background and objective:Hypertension can lead to mood disorders that may worsen or ameliorate depending on the type of antihypertensive prescribed. Depression is associated with modifications in basal brain asymmetry particularly that of the frontal cortex, which is involved in blood pressure control. Furthermore, different vasoactive drugs may change the brain's asymmetry in a manner that contributes to cognition status. We studied the bilateral activity of several neuropeptidases in frontal cortex as a reflect of the functional status of certain neuropeptides involved in mood.Methods:Using arylamide derivatives as substrates, we fluorometrically analysed the activity of these enzymes in the left and right frontal cortex of control untreated Wistar–Kyoto (WKY) and spontaneously hypertensive rats (SHRs) and compared their activities with WKY or SHR treated with the antihypertensive drugs captopril (CAP) and propranolol (PRO) or with the hypertensive N (G)-nitro-l-arginine methyl ester. SBP was also measured in all WKY and SHR groups.Results:Untreated WKY, WKY treated with CAP or PRO and SHR treated with CAP exhibited normotensive values of SBP. However, WKY treated with N (G)-nitro-l-arginine methyl ester as well as untreated SHR and SHR treated with PRO and N(G)-nitro-l-arginine methyl ester demonstrated hypertensive values of SBP. Changes in the bilateral distribution of neuropeptidases were depending on the strain, the enzyme analysed and the drug used. Normotensive WKY groups (WKY, CAP, PRO) revealed intrahemispheric correlations mainly in the left hemisphere. In contrast, WKY treated with N(G)-nitro-l-arginine methyl ester and SHR groups demonstrated intrahemispheric correlations mainly in the right hemisphere. Interhemispheric correlations were mostly observed in WKY as well as in SHR groups with antihypertensive treatments (CAP, PRO).Conclusion:Our results suggest specific brain bilateral patterns of neuropeptidase activities in WKY that change in SHR. This observation may be related to the cognitive disorders that have been described in these animals and that change under antihypertensive or hypertensive drug's treatments.

Aerobic exercise enhanced endothelium-dependent vasorelaxation in mesenteric arteries in spontaneously hypertensive rats: the role of melatonin.

Melatonin, a neuroendocrine hormone synthesized primarily by the pineal gland, provides various cardiovascular benefits. Regular physical activity is an effective non-pharmacological therapy for the prevention and control of hypertension. In the present study, we hypothesized that melatonin plays an important role in the aerobic exercise-induced increase of endothelium-dependent vasorelaxation in the mesenteric arteries (MAs) of spontaneously hypertensive rats (SHRs) in a melatonergic receptor-dependent manner. To test this hypothesis, we evaluated the vascular mechanical and functional properties in normotensive Wistar Kyoto (WKY), SHRs, and SHRs that were trained on a treadmill (SHR-EX) for 8 weeks. Exercise training produced a significant reduction in blood pressure and heart rate in SHR, which was significantly attenuated by the intraperitoneal administration of luzindole, a non-selective melatonin receptor (MT1/MT2) antagonist. Serum melatonin levels in the SHR group were significantly lower than those in the WKY group at 8:00-9:00 and 21:00-22:00, while exercise training reduced this difference. Endothelium-dependent vessel relaxation induced by acetylcholine was significantly blunted in SHR compared with age-matched WKY. Both exercise training and luzindole ameliorated this endothelium-dependent impairment of relaxation in hypertension. Immunohistochemistry and Western blotting showed that the protein expression of the MT2 receptor and eNOS, as well as their colocalization in the endothelial cell layer in SHRs, was significantly decreased; as exercise training suppressed this reduction. These results provide evidence that regular exercise has a beneficial effect on improving endothelium-dependent vasorelaxation in MAs, in which melatonin plays a critical role by acting on MT2 receptors to increase NO production and/or NO bioavailability.