Jian-Guang Yu

KETANSERIN‐INDUCED BAROREFLEX ENHANCEMENT IN SPONTANEOUSLY HYPERTENSIVE RATS DEPENDS ON CENTRAL 5‐HT2A RECEPTORS

1 Ketanserin may influence baroreflex function by blocking 5‐HT2A receptors and/or α1‐adrenoceptors through central and/or peripheral mechanisms. 2 In the present study, we tested the hypothesis that the baroreflex sensitivity (BRS)‐enhancing effects of ketanserin are mediated by central 5‐HT2A receptors in spontaneously hypertensive rats (SHR). 3 Using a conjugate of a monoclonal antibody to the serotonin reuptake transporter (SERT) and the toxin saporin (anti‐SERT‐SAP), which specifically eliminates the neurons that express SERT, the effects of ketanserin (0.3 and 3.0 mg/kg, i.g.) on BRS, blood pressure (BP), heart period (HP) and blood pressure variability (BPV) were compared between conscious intact SHR and SHR pretreated with anti‐SERT‐SAP. 4 Immunochemistry showed that, 2 weeks after intracerebroventricular injection of the toxin, 5‐HT expression was strikingly attenuated in the brain, whereas values of BRS, BPV and BP were similar to those in the sham group. In intact SHR, 0.3 mg/kg ketanserin significantly improved BRS (191% control) and reduced BPV without affecting BP; at 3.0 mg/kg, ketanserin significantly increased BRS (197% control) and decreased BPV and BP. In toxin‐pretreated SHR, only the high dose of ketanserin improved BRS (132% control), neither of the ketanserin doses reduced BPV, but both significantly decreased BP. 5 We conclude that the BRS‐enhancing effects of ketanserin are mediated largely by central 5‐HT2A receptors, whereas the antihypertensive effect of ketanserin persists even after destruction of serotonergic neurons in the central nervous system.

MicroRNA-124 negatively regulates LPS-induced TNF-α production in mouse macrophages by decreasing protein stability

Aim:MicroRNAs play pivotal roles in regulation of both innate and adaptive immune responses. In the present study, we investigated the effects of microRNA-124 (miR-124) on production of the pro-inflammatory cytokine TNF-α in lipopolysaccharide (LPS)-treated mouse macrophages.Methods:Mouse macrophage cell line RAW264.7 was stimulated with LPS (100 ng/mL). The levels of miR-124 and TNF-α mRNA were evaluated using q-PCR. ELISA and Western blotting were used to detect TNF-α protein level in cell supernatants and cells, respectively. 3′-UTR luciferase reporter assays were used to analyze the targets of miR-124. For in vivo experiments, mice were injected with LPS (30 mg/kg, ip).Results:LPS stimulation significantly increased the mRNA level of miR-124 in RAW264.7 macrophages in vitro and mice in vivo. In RAW264.7 macrophages, knockdown of miR-124 with miR-124 inhibitor dose-dependently increased LPS-stimulated production of TNF-α protein and prolonged the half-life of TNF-α protein, but did not change TNF-α mRNA levels, whereas overexpression of miR-124 with miR-124 mimic produced the opposite effects. Furthermore, miR-124 was found to directly target two components of deubiquitinating enzymes: ubiquitin-specific proteases (USP) 2 and 14. Knockdown of USP2 or USP14 accelerated protein degradation of TNF-α, and abolished the effect of miR-124 on TNF-α protein stability.Conclusion:miR-124, targeting USP2 and USP14, negatively regulates LPS-induced TNF-α production in mouse macrophages, suggesting miR-124 as a new therapeutic target in inflammation-related diseases.

Ketanserin improves cardiac performance after myocardial infarction in spontaneously hypertensive rats partially through restoration of baroreflex function

Aim:Baroreflex dysfunction is associated with a higher rate of sudden death after myocardial infarction (MI). Ketanserin enhances baroreflex function in rats. The present work was designed to examine whether ketanserin improves the post-MI cardiac function and to explore the possible mechanism involved.Methods:Spontaneously hypertensive rats (SHR) were treated with ketanserin (0.3 mg·kg−1·d−1). Two weeks later, blood pressure and baroreflex function were measured, followed by a ligation of the left coronary artery. The expressions of vesicular acetylcholine transporter (VAChT) and α7 nicotinic acetylcholine receptor (α7-nAChR) in ischemic myocardium, angiogenesis, cardiac function, and left ventricular (LV) remodeling were evaluated subsequently.Results:Ketanserin significantly improved baroreflex sensitivity (0.62±0.21 vs 0.34±0.12 ms/mmHg, P<0.01) and vagal tonic activity (heart rate changes in response to atropine, 54.8±16.2 vs 37.6±13.4 bpm, P<0.01) without affecting the blood pressure or basic heart rate in SHR. Treatment of SHR with ketanserin prominently improved cardiac function and alleviated LV remodeling, as reflected by increases in the ejection fraction, fractional shortening, and LV systolic pressure as well as decreases in LV internal diameter and LV relative weight. The capillary density, vascular endothelial growth factor expression, and blood flow in the ischemic myocardium were significantly higher in the ketanserin-treated group. In addition, ketanserin markedly increased the expression of VAChT and α7-nAChR in ischemic myocardium.Conclusion:Ketanserin improved post-MI cardiac function and angiogenesis in ischemic myocardium. The findings provide a mechanistic basis for restoring baroreflex function using ketanserin in the treatment of MI.

Activation of α7 Nicotinic Acetylcholine Receptor Decreases On-site Mortality in Crush Syndrome through Insulin Signaling-Na/K-ATPase Pathway

On-site mortality in crush syndrome remains high due to lack of effective drugs based on definite diagnosis. Anisodamine (Ani) is widely used in China for treatment of shock, and activation of α7 nicotinic acetylcholine receptor (α7nAChR) mediates such antishock effect. The present work was designed to test whether activation of α7nAChR with Ani decreased mortality in crush syndrome shortly after decompression. Sprague-Dawley rats and C57BL/6 mice with crush syndrome were injected with Ani (20 mg/kg and 28 mg/kg respectively, i.p.) 30 min before decompression. Survival time, serum potassium, insulin, and glucose levels were observed shortly after decompression. Involvement of α7nAChR was verified with methyllycaconitine (selective α7nAChR antagonist) and PNU282987 (selective α7nAChR agonist), or in α7nAChR knockout mice. Effect of Ani was also appraised in C2C12 myotubes. Ani reduced mortality and serum potassium and enhanced insulin sensitivity shortly after decompression in animals with crush syndrome, and PNU282987 exerted similar effects. Such effects were counteracted by methyllycaconitine or in α7nAChR knockout mice. Mortality and serum potassium in rats with hyperkalemia were also reduced by Ani. Phosphorylation of Na/K-ATPase was enhanced by Ani in C2C12 myotubes. Inhibition of tyrosine kinase on insulin receptor, phosphoinositide 3-kinase, mammalian target of rapamycin, signal transducer and activator of transcription 3, and Na/K-ATPase counteracted the effect of Ani on extracellular potassium. These findings demonstrated that activation of α7nAChR could decrease on-site mortality in crush syndrome, at least in part based on the decline of serum potassium through insulin signaling-Na/K-ATPase pathway.

Synergic Effects of Levamlodipine and Bisoprolol on Blood Pressure Reduction and Organ Protection in Spontaneously Hypertensive Rats

Aims: Stroke is a major cause of disability and death worldwide. Hypertension is one of the most important risk factors for stroke. The objective of this work was to study the synergic effects of levamlodipine and bisoprolol on blood pressure reduction and organ protection in spontaneously hypertensive rats (SHR). Methods: Blood pressure was continuously monitored in conscious SHR. For acute study, a single dose of drugs was administrated via an intragastric catheter. For chronic study (4 months), drugs were delivered via rat chow. Results: A single dose of levamlodipine (from 1 mg/kg), bisoprolol (from 0.125 mg/kg), and their combinations significantly decreased blood pressure. The levamlodipine‐induced tachycardia and the bisoprolol‐induced bradycardia were temporized by the combination of these two drugs. Upon chronic treatment, this combination also decreased blood pressure variability and reduced organ damage. Conclusion: Levamlodipine and bisoprolol produce synergic effects on blood pressure reduction and organ protection in SHR.

Role of Intracranial Microaneurysm in the Pathogenesis of Stroke in SHR-SP

Stroke-prone spontaneously hypertensive rats (SHR-SP) is a substrain of spontaneously hypertensive rats (SHR) with much more vulnerability to stroke. Cerebral artery remodeling, for example, increased vascular wall thickness and decreased lumen diameter, represents a key pathological feature that underlies such a difference. Cerebral vascular malformation, for example, microaneurysm, is also an important factor in the pathogenesis of stroke in human. About 15% of all strokes are secondary to ruptured aneurysms [1]. Microaneurysm may rupture upon sudden increase of blood pressure [BP; Ref. 2]. Ischemic stroke, accounting for ∼70% of all stroke events in China, can be a presenting clinical feature of intracranial aneurysms [3]. This work examined a potential role of intracranial microaneurysms in the pathogenesis of stroke in SHR-SP and the underlying mechanisms. The experiments included 26 SHR-SP (male 13, female 13) and 25 SHR (male 13, female 12). BP and baroreflex sensitivity (BRS) were determined at the age of 28 weeks in conscious state, as previously described [4]. Serial transverse paraffin brain sections (5 μm thick, at 100 μm interval) were then prepared using routine techniques. Ten slides were stained with hematoxylin–eosin and observed under a light microscope. Microaneurysms were calculated with an image analysis system (SDK-2000, 10moons Technology Development, China). Data are expressed as mean ± SD. P < 0.05 was considered statistically significant. Intracranial microaneurysms occurred in 25 of the 26 SHR-SP (incidence: 96.15%; 2.96 ± 1.51 microaneurysms per slide), and none of the 25 SHR. In SHR-SP, the number of microaneurysms was positively correlated with systolic BP (SBP; Figure 1A; r = 0.60, P < 0.05), and negatively correlated with BRS (Figure 1B; r = 0.54, P < 0.05). The enzyme 5-lipoxygenase is a key enzyme in the biosynthesis of proinflammatory leukotriene lipid mediators and Intracranial microaneurysm predicts stroke in SHR-SP is believed to be responsible for the occurrence of aneurysm [5,6]. In this study, western blot analysis showed that the expression of 5-lipoxygenase in the brain tissue was significantly higher in SHRSP (Figure 1C; P < 0.01 vs. SHR). A separate group of 40 male SHR-SP (age: 14 weeks) were randomly divided into two groups and received either the 5-lipoxygenase inhibitor zileuton (0.8 g/kg/day, delivered in food, n = 20) or the vehicle (n = 20). The rats were sacrificed when death was deemed inevitable after stroke. The brains were harvested and stained with hematoxylin–eosin. Zileuton treatment significantly decreased the occurrence of intracranial microaneurysms (Figure 2A; P < 0.01 vs. control). Kaplan–Meier survival analysis revealed a significantly longer survival time in SHRSP who received zileuton than the vehicle control (Figure 2B; P = 0.019). Intracranial microaneurysm is an important pathological basis for stroke, especially for spontaneous cerebral hemorrhage. Results from this study showed a much higher incidence of intracranial microaneurysms in SHR-SP compared with SHR. Furthermore, the number of microaneurysms was positively correlated with SBP and negatively correlated with BRS. The enzyme 5-lipoxygenase, an essential factor for the occurrence of aneurysm, was higher in SHR-SP. Inhibiting 5-lipoxygenase with zileuton decreased the occurrence of intracranial microaneurysms and prolonged the lifespan of SHR-SP. In conclusion, intracranial microaneurysm may play an important role in the pathogenesis of stroke in SHR-SP; inhibiting 5-lipoxygenase could suppress the development of intracranial microaneurysms.

Protective cerebrovascular effects of hydroxysafflor yellow A (HSYA) on ischemic stroke

Abstract The purpose of the present work was designed to explore protective cerebrovascular effects of hydroxysafflor yellow A (HSYA), and provide preclinical efficacy and mechanism data for its possible application in patients with cerebral ischemia. The protective effect of HSYA on ischemic stroke was evaluated by infarct sizes and neurological scores in Sprague‐Dawley (SD) rats with middle cerebral artery occlusion (MCAO). Cerebrovascular permeability was detected by Evans blue dye leakage in MCAO rats. Cerebral blood flow, as well as blood pressure and heart rate were monitored using flow probes in Beagle dogs. Basilar artery tension isolated from Beagle dogs was evaluated with an MPA 2000 data‐acquisition system. Coagulation‐related function was also judged, including rabbit platelet aggregation by adenosine diphosphate (ADP) and platelet‐aggregating factor (PAF), rabbit blood viscosity by a blood viscometer, and thrombus formation by rat arterial‐venous shunts. Results showed that HSYA treatment significantly decreased the infarct sizes, neurological scores and cerebrovascular permeability in rats with MCAO. However, cerebral blood flow, blood pressure and heart rate were not affected by HSYA. In vitro, HSYA had a strong effect on cerebrovascular vasodilatation, and significantly decreased platelet aggregation, blood viscosity, and thrombogenesis. Besides well‐known anti‐coagulation effects, HSYA protects against ischemic stroke by dilating cerebral vessels and improving cerebrovascular permeability.

Estrogen weakens muscle endurance via estrogen receptor-p38 MAPK-mediated orosomucoid (ORM) suppression.

Gender differences in fatigue manifest as females being more prone to feel exhaustion and having lower muscle endurance. However, the mechanisms of these effects remain unclear. We investigated whether orosomucoid, an endogenous anti-fatigue protein that enhances muscle endurance, is involved in this regulation. Female rats exhibited lower muscle endurance, and this gender difference disappeared in orosomucoid-1-deficient mice. Female rats also exhibited weaker orosomucoid induction in serum, liver and muscle in response to fatigue compared with male rats. Ovariectomy elevated orosomucoid levels and increased swimming time, and estrogen replenishment reversed these effects. Exogenous estrogen treatment in male and female mice produced opposite effects. Estrogen decreased orosomucoid expression and its promoter activity in C2C12 muscle and Chang liver cells in vitro, and estrogen receptor or p38 mitogen-activated protein kinase blockade abolished this effect. Therefore, estrogen negatively regulates orosomucoid expression that is responsible for the weaker muscle endurance in females.

Diurnal Variation of the Peripheral Cholinergic Antiinflammatory Function in Mice.

Cholinergic antiinflammatory (CAI) pathway functions importantly in inflammation via α7 nicotinic acetylcholine receptors (α7nAChR). The present work tested circadian rhythm in peripheral CAI activity and validities of CAI activity and glucocorticoids in chronotherapy for lipopolysaccharide (LPS)‐induced shock.

Establishment of depression behavioral criteria in cynomolgus monkeys.

Depression is a common mental disorder with relatively high lifetime prevalence and substantial disability, especially in women [1–3]. Clinical manifestations of depression include loss of interest or pleasure, disturbed sleep or appetite, poor concentration, etc., worst of which is suicide. To solve such an important global public health issue, it is essential to establish ideal animal models for study and treatment of depression. Cynomolgus monkeys have been proved to be advantageous for modeling human depression through mother–infant separation [4] and social subordination stress [5]. Behavioral tests have been performed in animals to test severity of depression and successfulness of modeling, for example, forced swim test and tail suspension test for rodents [6,7] and sucrose intake test for nonhuman primates [8]. In this study, we attempted to establish several behavioral criteria that can be easily measured to distinguish depression in cynomolgus monkeys. After a fourteen-month observation in groups, six socially subordinate female cynomolgus monkeys and six socially dominant counterparts were chosen as depressed models and alert controls, respectively, based on their social status hierarchy (Figure 1). The subordinate and dominant monkeys came from six and four groups, respectively, and each group consisted of 14–26 monkeys. The monkeys were 6–10 years of age and housed alone in adjacent cages separated with transparent plexiglass. All the monkeys were provided by Suzhou Xishan Zhongke Lab Animal Ltd (Suzhou, China) and received humane care. In this study, body weight, food consumption, preference for sucrose water, attempts for apple, and time of depressed posture were measured. Data are expressed as mean SEM and analyzed with unpaired t-test. P < 0.05 was considered statistically significant. The body weight of depressed monkeys was significantly lower than that of nondepressed monkeys (3.03 0.07 vs. 3.57 0.13 kg, P < 0.01; Figure 2A). Food consumption relative to body weight was calculated as the mean of 3-day measurements. Our results showed that depressed monkeys ate more food than the nondepressed (40.76 2.76 vs. 18.25 0.86 g/kg, P < 0.01; Figure 2B). The monkeys were deprived of water for 23 h and obliged to drink from two bottles containing pure water at the 24th h. After a 3-day adaptation, pure water was changed to 1.5% sucrose water in one bottle and then consumption of both kinds of water was recorded for 3 days. Preference of sucrose water was calculated as the percentage of sucrose water intake to total water intake. Depressed monkeys exhibited less preference for sucrose as compared to nondepressed monkeys (51.5 12.6% vs. 81.0 2.9%, P < 0.05; Figure 2C). Apples were given to monkeys in the cages for 3 days and then put out of the cages. Distance between the wall of the cage and the apple was a bit longer than the length of the monkeys arm. Monkeys could stretch their arms through the meshes of the cage wall. Times of reaching for apple were counted for 15 min on 3 days. Attempts for apple were calculated as times of reaching for apple in 15 min. Depressed monkeys showed more attempts for apple than nondepressed monkeys, however, without a statistical significance (Figure 2D). Depressed posture was defined as a slumped or collapsed body posture [9,10]. Behavior was recorded for 15 min during 14:00–16:00 on 3 days, avoiding feeding period. Time of depressed posture was calculated as the percentage of time keeping slumped or collapsed body posture to 15 min. Depressed monkeys spent more time in depressed posture than nondepressed monkeys (57.3 10.1% vs. 19.4 10.2%, P < 0.05; Figure 2E). Most of our results were consistent with results of the previous study [5,8]. Interestingly, food consumption and attempts for