Guo-Jun Cai

Antishock effect of anisodamine involves a novel pathway for activating alpha7 nicotinic acetylcholine receptor.

Objective:Vagus nerve stimulation inhibits proinflammatory cytokine production by signaling through the α7 nicotinic acetylcholine receptor (α7nAChR). Anisodamine, a muscarinic acetylcholine receptor antagonist, has been used clinically in China for treatment of various shocks, but the mechanism was poorly understood. Here, we tested the hypothesis whether anisodamine attained its antishock effect through activation of α7nAChR. Design:Randomized and controlled in vitro and in vivo study. Settings:Research laboratory and animal facility rooms. Subjects:Sprague-Dawley rats, Kunming mice, α7nAChR-deficient mice, and RAW264.7 cells. Interventions:Sprague-Dawley rats were injected with lipopolysaccharide (LPS) (15 mg/kg, intravenous) to induce septic shock. Methyllycaconitine, a selective α7nAChR antagonist, was administered (10 mg/kg, intraperitoneal) 10 minutes before anisodamine (10 mg/kg, intravenous). Mean arterial pressure was monitored and cytokines were analyzed 2 hours after the onset of LPS. In vagotomized mice and α7nAChR-deficient mice, the antishock effect of anisodamine was appraised, respectively. RAW264.7 cells were stained by fluorescein isothiocyanate- labeled-α-bungarotoxin and the fluorescence intensity was observed. Mice peritoneal macrophages were pretreated and stimulated with LPS, and tumor necrosis factor (TNF)-α in the supernatant was measured by enzyme-linked immunosorbent assay. Measurements and Main Results:Methyllycaconitine significantly antagonized the beneficial effect of anisodamine on mean arterial pressure and TNF-α, interleukin-1β expression in response to LPS. The antishock effects of anisodamine were markedly attenuated in vagotomized mice and α7nAChR-deficient mice. In vitro, anisodamine significantly augmented the effect of acetylcholine on fluorescence intensity stained with fluorescein isothiocyanate-labeled-α-bungarotoxin and TNF-α production stimulated with LPS. Conclusion:These findings demonstrate that the antishock effect of anisodamine is intimately linked to α7nAChR-dependent anti-inflammatory pathway.

Blood pressure variability, cardiac baroreflex sensitivity and organ damage in experimentally hypertensive rats.

1. The present study was designed to investigate the haemodynamic features and morphological changes in experimentally hypertensive rat models.

Effects of combination therapy with atenolol and amlodipine on blood pressure control and stroke prevention in stroke-prone spontaneously hypertensive rats

AbstractAim:To test the effects of atenolol and amlodipine, either alone or in combination, on blood pressure, blood pressure variability (BPV), baroreflex sensitivity (BRS), and the prevalence of stroke in stroke-prone spontaneously hypertensive rats (SHR-SP).Methods:In the first set of the study, 24 8-month-old, female SHR-SP rats were randomly divided into 3 groups. Blood pressure, heart period, and BRS were determined before and after the intragastric administration of atenolol (10 mg/kg) and amlodipine (1.0 mg/kg), either alone or in combination. In the second set of the study, 40 male and 40 female rats were randomly assigned to 1 of the following 4 groups: control, atenolol (10 mg·kg−1·d−1), amlodipine (1.0 mg·kg−1·d−1), and both (10 male and 10 female in each group). The stroke incident and survival time were recorded.Results:Atenolol and amlodipine, either alone or in combination, significantly decreased blood pressure, with the exception of the amlodipine-induced effect on diastolic blood pressure. Meanwhile, only the combination treatment significantly decreased the BPV levels for the same period. The q-values calculated by the probability sum analysis were 1.17 and 2.67 for systolic and diastolic blood pressure, respectively, and were 2.48 and 2.10 for systolic and diastolic BPV, respectively, following administration. Neither drug exhibited any significant effect on BRS. Atenolol and amlodipine, either alone or in combination, significantly increased the lifespan of SHR-SP, with the best effect elicited by the combination therapy.Conclusion:A significant synergism exists between atenolol and amlodipine in lowering and stabilizing blood pressure in SHR-SP. Combination therapy may be an optimal strategy for the prevention of stroke in hypertension.

Morphological evidence of reinnervation of the baroreceptive regions in sinoaortic-denervated rats

1. The arterial baroreflex (ABR) plays an important role in the maintenance of the stability of blood pressure. Sinoaortic denervation (SAD) destroys the integrity of the reflex arc and produces severe organ damage in rats. However, partial recovery of ABR function has been observed following chronic denervation. The aim of the present study was to determine whether there was morphological evidence of reinnervation of the aortic arch and carotid sinus following SAD.

The protective action of ketanserin against lipopolysaccharide-induced shock in mice is mediated by inhibiting inducible NO synthase expression via the MEK/ERK pathway

Nitric oxide (NO) plays an important role in the pathogenesis of endotoxic shock. This work tested the hypothesis that ketanserin could attenuate endotoxic shock by inhibiting the expression of inducible NO synthase (iNOS). The results demonstrated that ketanserin could inhibit iNOS expression in the heart, lungs, liver, and kidneys and nitrate production in the serum upon endotoxic shock in mice. In RAW264.7 cells, ketanserin significantly inhibited the expression of iNOS and decreased the production of NO, TNFα, IL-6, and reactive oxygen species upon lipopolysaccharide (LPS) challenge. Ketanserin also increased the level of ATP and mitochondrial membrane potential in RAW264.7 cells upon LPS exposure. LPS-induced iNOS expression was inhibited by the 5-HT2A receptor antagonist ritanserin and not the α1 receptor antagonist prazosin. Knockdown of 5-HT2A receptor by siRNA abolished the inhibitory effect of ketanserin on the expression of iNOS. These results indicated that the inhibitory effect of ketanserin on the expression of iNOS is mediated by blocking the 5-HT2A receptor. Furthermore, ketanserin significantly inhibited the activation of ERK1/2 and NF-κB signal. Pretreatment with PD184352, a specific inhibitor of ERK1/2, blocked the inhibitory effect of ketanserin on the expression of iNOS and NO production, indicating a critical role for the MEK/ERK1/2 signaling pathway. Collectively, our findings indicate that inhibition of the expression of iNOS via the MEK/ERK pathway mediates the protective effects of ketanserin against LPS-induced shock in mice.

Effects of Ketanserin on Endotoxic Shock and Baroreflex Function in Rodents

BACKGROUND Ketanserin, a 5-hydroxytryptamine receptor antagonist, is clinically used as an antihypertensive agent and could enhance baroreflex function. The present work tested the hypothesis that restoration of baroreflex function is an effective treatment for lipopolysaccharide (LPS)-induced shock. METHODS Kunming mice were injected with LPS (30 mg/kg; intraperitoneal) to induce endotoxic shock. Ketanserin (0.3, 1, 3, or 10 mg/kg; intraperitoneal) was administered immediately after LPS injection. Survival time was monitored, and serum cytokines were analyzed after the onset of LPS. Effects of ketanserin were also examined in IL-10-deficient mice and mice with sinoaortic denervation. Finally, effects of ketanserin on blood pressure, heart rate, and baroreflex sensitivity were examined in Wistar-Kyoto (WKY) rats with endotoxic shock. RESULTS Ketanserin significantly increased survival time and decreased serum levels of tumor necrosis factor α and interleukin (IL) 1β in mice with endotoxic shock. At a dose of 10 mg/kg, ketanserin also significantly increased serum IL-10 concentration. The antishock effect of ketanserin was also apparent in IL-10-knockout mice. In mice with sinoaortic denervation, however, ketanserin had little antishock effects. In WKY rats, ketanserin significantly prevented the baroreflex impairment induced by LPS and prolonged the survival time. CONCLUSIONS Ketanserin could ameliorate endotoxic shock by restoring baroreflex function.

GREATER HYPERTROPHY IN RIGHT THAN LEFT VENTRICLES IS ASSOCIATED WITH PULMONARY VASCULOPATHY IN SINOAORTIC‐DENERVATED WISTAR‐KYOTO RATS

1. Biventricular hypertrophy has been described in a high blood pressure variability (BPV) model of sinoaortic‐denervated (SAD) rats without systemic hypertension. To explore the possible involvement of the lung in SAD‐induced right ventricular hypertrophy (RVH), we examined lung morphology, in addition to systemic haemodynamics and ventricle morphology, in Wistar‐Kyoto rats 32 weeks after SAD.

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.

Baroreflex deficiency aggravates atherosclerosis via α7 nicotinic acetylcholine receptor in mice

OBJECTIVE Inflammation and oxidative stress play a key role in the initiation, propagation, and development of atherosclerosis. Arterial baroreflex (ABR) dysfunction induced by sinoaortic denervation (SAD) promoted the development of atherosclerosis in ApoE-/- mice. The present work was designed to examine whether ABR deficiency affected inflammation and oxidative stress via α7 nicotinic acetylcholine receptor (α7nAChR) leading to the aggravation of atherosclerosis in mice. METHODS AND RESULTS ApoE-/- mice were fed with a high-cholesterol diet for 6weeks and half of the mice received sinoaortic denervation that destroyed ABR. We studied the expression of vesicular acetylcholine transporter (VAChT), α7nAChR and levels of inflammatory response and oxidative stress. The results showed that baroreflex dysfunction could promote atherosclerosis, meanwhile, decrease the expression of VAChT and α7nAChR and significantly increase the levels of oxidative stress and inflammation in SAD mice. After treated with PNU-282987 (a selective α7nAChR agonist, 0.53mg/kg/day) for 6weeks in SAD and Sham mice, we found that PNU-282987 could attenuate atherosclerosis and significantly decreased oxidative stress and inflammation after SAD. In addition, α7nAChR+/+ and α7nAChR-/- mice fed with a high-cholesterol diet for 8weeks were co-treated with ketanserin (0.6mg/kg/day), a drug that can enhance baroreflex sensitivity (BRS). Ketanserin could alleviate atherosclerosis and markedly decrease oxidative stress and inflammation in α7nAChR+/+ mice. But there were no effects in α7nAChR knockout mice. CONCLUSIONS Our results demonstrate that ABR dysfunction aggravates atherosclerosis in mice via the vagus-ACh-α7nAChR-inflammation and oxidative stress pathway.

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.