Shu-Wei Song

Dysfunction of the Cholinergic Anti-Inflammatory Pathway Mediates Organ Damage in Hypertension

Inflammatory responses are associated with the genesis and progression of end-organ damage (EOD) in hypertension. A role for the &agr;7 nicotinic acetylcholine receptor (&agr;7nAChR) in inflammation has recently been identified. We tested the hypothesis that &agr;7nAChR dysfunction contributes to hypertensive EOD. In both spontaneously hypertensive rats (SHRs) and rats with abdominal aorta coarctation–induced hypertension, atropine-induced tachycardia was blunted compared with normotensive controls. Both models of hypertension were associated with deficits in expression of the vesicular acetylcholine transporter and the &agr;7nAChR in cardiovascular tissues. In hypertension induced by abdominal aorta coarctation, deficits in aortic vesicular acetylcholine transporter and &agr;7nAChR were present both above and below the coarctation site, indicating that they were independent of the level of arterial pressure itself. Hypertension in 40-week-old SHRs was associated with cardiac and aortic hypertrophy. Morphological abnormalities consistent with EOD, along with elevated tissue levels of proinflammatory cytokines (tumor necrosis factor-&agr;, interleukin-1&bgr;, and interleukin-6) were observed in the heart, kidney, and aorta. Chronic treatment of SHRs with the &agr;7nAChR agonist PNU-282987 relieved EOD and inhibited tissue levels of proinflammatory cytokines and activation of nuclear factor &kgr;B. Greater serum levels of proinflammatory cytokines and more severe damage in the heart, aorta, and kidney were seen in &agr;7nAChR−/− mice subjected to 2-kidney-1-clip surgery than in wild-type mice. A deficit in the cholinergic anti-inflammatory pathway appears to contribute to the pathogenesis of EOD in models of hypertension of varying etiology. This pathway may provide a new target for preventing cardiovascular disease resulting from hypertension.

Baroreflex deficiency hampers angiogenesis after myocardial infarction via acetylcholine-α7-nicotinic ACh receptor in rats

AIMS Angiogenesis is critical for re-establishing blood supply to ischaemic myocardium after myocardial infarction (MI). Human studies have associated arterial baroreflex (ABR) deficiency with higher rate of sudden death after MI. The present work was designed to examine whether ABR deficiency affects angiogenesis in MI rats. METHODS AND RESULTS Baroreflex sensitivity (BRS) was determined in conscious rats at 1 month after occlusion of the left anterior descending coronary artery. The survival time was significantly shorter in Sprague-Dawley rats with BRS <0.60 ms/mmHg vs. those with BRS ≥0.60 ms/mmHg. Sinoaortic denervation destroyed ABR, and decreased capillary density, regional blood flow and vascular endothelial growth factor (VEGF) concentration after MI. Ketanserin (0.6 mg/kg/day) enhanced BRS, and increased capillary density, regional blood flow, and VEGF. Sinoaortic denervation also reduced the expression of vesicular acetylcholine (ACh) transporter and α7-nicotinic ACh receptor (α7-nAChR). Angiogenesis after MI was significantly attenuated in α7-nAChR knockout mice. In contrast, increase in endogenous ACh with cholinesterase inhibitor pyridostigmine (30 mg/kg/day) increased angiogenesis after MI. In cultured cardiac microvascular endothelial cells, ACh stimulated the expression of VEGF, phosphorylation of VEGF receptor 2, and tube formation in a manner dependent upon α7-nAChR. CONCLUSION Our results demonstrated that ABR deficiency could attenuate angiogenesis in ischaemic myocardium. These findings provide further mechanistic basis for enhancing baroreflex function in the treatment of MI.

Magnesium Lithospermate B Protects Cardiomyocytes from Ischemic Injury Via Inhibition of TAB1–p38 Apoptosis Signaling

Danshen has been used in traditional Chinese medicine for hundreds of years to treat cardiovascular diseases. However, its precise cardioprotective components and the underlying mechanism are still unclear. In the present study, we demonstrated that in a rat model of acute myocardial infarction, the treatment with magnesium lithospermate B (MLB), the representative component of phenolic acids in Danshen, significantly reduced the infarct size and the blood lactate dehydrogenase level. In contrast, tanshinone IIA, the representative component of lipophilic tanshinones in Danshen, had no such protective effects. Moreover, in the simulated ischemia cell model, MLB treatment considerably increased the cell viability and reduced the sub-G1 population and the apoptotic nuclei, indicating its anti-apoptotic effect. Further mechanism study revealed that the ischemia-induced p38 phosphorylation was abolished by MLB treatment. Interestingly, MLB specifically inhibited the TGFβ-activated protein kinase 1-binding protein 1 (TAB1) mediated p38 phosphorylation through disrupting the interaction between TAB1 and p38, but it did not affect the mitogen-activated protein kinase 3/6 mediated p38 phosphorylation. In conclusion, the present study identifies MLB as an active component of Danshen in protecting cardiomyocytes from ischemic injury through specific inhibition of TAB1–p38 apoptosis signaling. These results indicate TAB1–p38 interaction as a putative drug target in treating ischemic heart diseases.

Blood Pressure Reduction Combining Baroreflex Restoration for Stroke Prevention in Hypertension in Rats

Blood pressure reduction is an important and effective strategy in stroke prevention in hypertensives. Recently, we found that baroreflex restoration was also crucial in stroke prevention. The present work was designed to test the hypothesis that a combination of blood pressure reduction and baroreflex restoration may be a new strategy for stroke prevention. In Experiment 1, the effects of ketanserin (0.3, 1, 3, 10 mg/kg), amlodipine (0.3, 1, 2, 3 mg/kg) and their combination (1 + 0.3, 1 + 1, 1 + 2, 1 + 3 mg/kg) on blood pressure and baroreflex sensitivity (BRS) of stroke-prone spontaneously hypertensive rats (SHR-SP) were determined under conscious state. It was found that both amlodipine and ketanserin decreased blood pressure dose-dependently. Ketanserin enfanced BRS from a very small dose but amlodipine enfanced BRS only at largest dose used. At the dose of 1 + 2 mg/kg (ketanserin + amlodipine), the combination possessed the largest synergism on blood pressure reduction. In Experiments 2 and 3, SHR-SP and two-kidney, two-clip (2K2C) renovascular hypertensive rats received life-long treatments with ketanserin (1 mg/kg) and amlodipine (2 mg/kg) or their combination (0.5 + 1, 1 + 2, 2 + 4 mg/kg). The survival time was recorded and the brain lesion was examined. It was found that all kinds of treatments prolonged the survival time of SHR-SP and 2K2C rats. The combination possessed a significantly better effect on stroke prevention than mono-therapies. In conclusion, combination of blood pressure reduction and baroreflex restoration may be a new strategy for the prevention of stroke in hypertension.

Synergism of telmisartan and amlodipine on blood pressure reduction and cardiorenal protection in hypertensive rats.

Aim: This study was designed to investigate the effects of telmisartan and amlodipine on reduction of blood pressure (BP), myocardial hypertrophy, and renal injury in hypertensive rats. Method: In acute experiments, the BP was measured in conscious freely moving rats. Spontaneously hypertensive rats were treated with intragastric administration of amlodipine (1, 2, 4 mg/kg), telmisartan (4, 8, 12, 16, 20 mg/kg), and their different combinations (4 + 4, 2 + 4, 4 + 8, 4 + 12, 1 + 4, 2 + 8, 4 + 16, 2 + 12, 1 + 8, 2 + 16, 2 + 20, 1 + 12, 1 + 16, 1 + 20 mg/kg). The probability sum test (q test) was used to evaluate the synergistic action on BP reduction. In two-kidney, one-clip rats, the effects of amlodipine (1 mg/kg), telmisartan (6 mg/kg) and their combination on BP reduction were observed. In the chronic study, spontaneously hypertensive rats were treated with amlodipine (1 mg/kg), telmisartan (6 mg/kg), and their combination for 4 months. Histopathologic examinations were performed after the determination of BP and BP variability. Results: There is a synergistic interaction between amlodipine and telmisartan on BP reduction. The optimal dose ratio was found at 1:6. The synergistic effect of this dose ratio (1:6) was also seen in two-kidney, one-clip rats. Long-term treatment with this combination results in a beneficial effect on the reduction of BP and BP variability. The end-organ damage, including myocardial hypertrophy, glomerular atrophy, and fibrosis, was significantly attenuated by this combination. Conclusion: The optimal dose ratio of amlodipine and telmisartan on BP was 1:6. This combination is beneficial for the BP and BP variability reduction and end-organ damage prevention.

The features of reserpine-induced gastric mucosal lesions

AbstractAim:To reinvestigate the characteristics of reserpine-induced gastric mucosal lesions (GMLs).Methods:The GML-inducing effect of reserpine and the time-course of recovery from reserpine-induced GMLs were examined in Sprague-Dawley (SD) rats. The GML-inducing and blood pressure-decreasing effects of Compound Hypotensive Tablets (CHTs) were investigated in spontaneously hypertensive rats (SHRs). Intracerebroventricular (icv) injection and vagotomy were performed to verify the central vagal mechanism in reserpine-induced GMLs.Results:Single intraperitoneal (ip) injections of reserpine (0.25, 0.5, 1, 2, 4, and 6 mg/kg) dose-dependently induced GMLs in SD rats. Both single and repeated (2 weeks) oral administrations of reserpine led to slight GMLs at doses of 24 mg/kg and 10 mg/kg, respectively. Blood pressure was significantly decreased in SHRs after 2 months of CHT administration (0.01 and 0.03 mg/kg; doses were expressed as the amount of reserpine in the CHT). CHT doses of 0.3 mg/kg induced GMLs, but 0.1 mg/kg did not. Examining the time course of recovery from GMLs, severe GMLs occurred 18 h after ip reserpine (4 mg/kg), obviously lessened at 1 week and healed spontaneously at 3 weeks. Intracerebroventricular injections of reserpine caused GMLs at much lower doses (0.08 and 0.4 mg/kg), and reserpine-induced GMLs were greatly inhibited by vagotomy, suggesting the involvement of a central vagal mechanism.Conclusion:Reserpine-induced GMLs were dose-dependent, and the lesions healed spontaneously within 3 weeks. Long-term treatment with CHT at doses adequate to decrease blood pressure will not induce GMLs. A central vagal mechanism was involved in reserpine-induced GMLs.

Arterial Baroreflex Dysfunction Impairs Ischemia-Induced Angiogenesis

Background Endothelium‐derived acetylcholine (eACh) plays an important role in the regulation of vascular actions in response to hypoxia, whereas arterial baroreflex (ABR) dysfunction impairs the eACh system. We investigated the effects of ABR dysfunction on ischemia‐induced angiogenesis in animal models of hindlimb ischemia with a special focus on eACh/nicotinic ACh receptor (nAChR) signaling activation. Methods and Results Male Sprague‐Dawley rats were randomly assigned to 1 of 3 groups that received (1) sham operation (control group), (2) sinoaortic denervation (SAD)‐induced ABR dysfunction (SAD group), or (3) SAD rats on diet with an acetylcholinesterase inhibitor pyridostigmine (30 mg/kg per day, SAD+Pyr group). After 4 weeks of the SAD intervention, unilateral limb ischemia was surgically induced in all animals. At postoperative day 14, SAD rats exhibited impaired angiogenic action (skin temperature and capillary density) and decreased angiogenic factor expressions (vascular endothelial growth factor [VEGF] and hypoxic inducible factor [HIF]‐1α) in ischemic muscles. These changes were restored by acetylcholinesterase inhibition. Rats with ABR dysfunction had lower eACh levels than did control rats, and this effect was recovered in SAD+Pyr rats. In α7‐nAChR knockout mice, pyridostigmine improved ischemia‐induced angiogenic responses and increased the levels of VEGF and HIF‐1α. Moreover, nicotinic receptor blocker inhibited VEGF expression and VEGF receptor 2 phosphorylation (p‐VEGFR2) induced by ACh analog. Conclusions Thus, ABR dysfunction appears to impair ischemia‐induced angiogenesis through the reduction of eACh/α7‐nAChR‐dependent and ‐independent HIF‐1α/VEGF‐VEGFR2 signaling activation.

Risperidone Enhances the Vulnerability to Stroke in Hypertensive Rats

Background: Stroke is the second most common cause of death and a major cause of disability worldwide. Risperidone is an atypical antipsychotic drug that may increase the risk of stroke. The present work examined whether risperidone enhances the vulnerability to stroke in hypertensive rats and the potential mechanisms underlying such action. Methods: Experiment 1: Wistar‐Kyoto (WKY) rats, spontaneously hypertensive rats (SHRs) and stroke‐prone SHRs (SHR‐SPs) were treated with risperidone (0.8 and 2.4 mg/kg/d) or vehicle for 30 consecutive days. Tissue damage in response to middle cerebral artery occlusion (MCAO) was measured microscopically. The activity of superoxide dismutase, glutathione peroxidase, the levels of malondialdehyde were also determined. Experiment 2: Survival data were recorded in SHR‐SPs that received daily risperidone perpetually. Experiment 3: Effect of risperidone on interleukin‐6 and tumor necrosis factor‐α was examined in quiescent or LPS‐activated cortical microglias from WKY rats. Experiment 4: Potential damage of risperidone exposure to neurons was examined in primary neuronal culture obtained from WKY rats, SHRs, and SHR‐SPs. Results: Risperidone increased infarct areas upon MCAO in SHR‐SPs and SHRs, but not in WKY rats. Survival time in SHR‐SPs was shortened by risperidone. Apoptosis was augmented by risperidone through enhanced Bax. Risperidone also increased endothelial injury. Conclusions: Risperidone enhances the vulnerability to stroke in hypertensive rats through increasing neuronal apoptosis and endothelial injury.

The Effects of Anakinra on Focal Cerebral Ischemic Injury in Rats

The inflammatory immune response plays an important role in the pathophysiological process of cerebral ischemic injury, especially during the enlargement of damage and the secondary damage after cerebral ischemia. There are high cytokine expression and proinflammatory infiltration in damage area after ischemic cerebral injury, which aggravates brain injury especially in reperfusion period [1,2]. Interleukin-1b (IL-1b) is an inflammatory factor regulating the complex pathologic changes in acute ischemic brain injury. Increase in IL-1b mRNA and protein level was observed after cerebral ischemia–reperfusion. IL-1b plays an important role in inflammation of focal cerebral ischemia. IL-1b contributes to brain tissue injury following cerebral ischemia–reperfusion [3,4]. Anakinra, a recombinant, nonglycosylated form of human interleukin-1 receptor antagonist (rhIL-1ra), is identical to a native IL-1 receptor antagonist except for a methionine residue that has been added to the beginning of the amino acid sequence [5]. Intracerebroventricular injection of IL-1ra (10 mg) significantly reduced the lesion volume after middle cerebral artery occlusion (MCAO) in rats [6]. There are no articles about animal studies of anakinra being administered i.v. and therapeutic time window in cerebral ischemia. This study was designed to document the therapeutic effect and therapeutic time window of anakinra after transient focal MCA occlusion and its mechanism at molecular level. Male Wistar rats (250–350 g) were divided into 18 groups, normal groups, sham groups, control groups, and anakinra groups (5, 10, or 20 mg/kg, i.v.) at 3, 6, or 12 h after ischemia, respectively. Twenty-four hours after cerebral ischemia, rats were euthanized and decapitated. The brains were removed, and then, RNA was extracted. The mRNA primer of intercellular adhesion molecule 1 (ICAM-1), inducible nitric oxide synthase (iNOS), IL-1b, and tumor necrosis factor-a (TNF-a) were synthesized. Anakinra decreased infarct percentage and improved neurological deficits induced by MCAO in male Wistar rats dose dependently and time dependently in Figure 1A–C. Significant difference was found between anakinra 5 mg/kg i.v. at 3 or 6 h after ischemia and the control group. Similar findings were observed in anakinra 10 or 20 mg/kg i.v. at 3, 6, or 12 h. The effect of anakinra on ICAM-1 mRNA, iNOS mRNA, IL-1b mRNA, and TNF-a mRNA was assessed by semiquantitative reverse transcription PCR [7]. The integral gray value of normal group was set as 1, and the values of other groups were expressed as a ratio versus normal group. As shown in Figure 2A–D, ischemia–reperfusion upregulated ICAM-1, iNOS, IL-1b, and TNF-a mRNA level. Anakinra 5~20 mg/kg i.v. at 3, 6, or 12 h after ischemia could significantly downregulate their mRNA levels (P < 0.01 or P < 0.05, vs. control group). Results have demonstrated that anakinra has an inhibitory effect on these overexpression cytokine. Anakinra is a kind of biomacromolecule, which is composed of 153 amino acids, and it cannot cross blood–brain barrier by i.v. administration in normal animals. It has been reported that natural IL-1ra could improve brain injury resulting from focal cerebral ischemia through cerebral ventricle injection [8]. In this study,

Donepezil Protects Endothelial Cells against Hydrogen Peroxide-Induced Cell Injury

Vascular endothelial cells serve as a barrier between tissue and blood stream, which plays an important role in maintaining vascular homeostasis. Oxidative stress has been implicated as a major cause of endothelial injuries in a variety of clinical abnormalities including artherosclerosis, ischemia reperfusion injury, and diabetes [1]. Donepezil, a reversible noncompetitive acetylcholinesterase inhibitor, used to treat Alzheimer’s disease, has been reported that it could protect neuronal cells against cell injury [2]. In this study, the protective effect of donepezil against hydrogen peroxide (H2O2)-induced cell injury in vitro was determined. Human umbilical vein endothelial cell (HUVEC) line obtained from Department of Pharmacology, College of Pharmacy, Second Military Medical University, was cultured in RPMI-1640 medium (Gibco, NY, USA), containing 10% fetal bovine serum (Gibco, NY, USA), 100 U/mL penicillin (Invitrogen, NY, USA), and 100 μg/mL streptomycin (Invitrogen, NY, USA). Cells were grown in a 37◦C, 5% CO2 incubator with media replenishment every 3 days. Before experimental intervention, confluent cultured cells were preincubated for 12 h in starved medium. The starved cells were then divided into four experimental groups: (1) control; (2) cells incubated with H2O2 (100 μM) (Sinopharm Chemical Reagent Co. Ltd, China) 1.5 h alone; (3) cells pretreated with donepezil (Sigma, MO, USA) for 1 h before coincubated with H2O2; (4) cells incubated with donepezil alone. To evaluate HUVEC cell viability, MTT assay was conducted. After being treated with different medium conditions, the hypoxia-inducible factor alpha (HIF1α) and manganese superoxide dismutase (MnSOD) expression in HUVECs were analyzed by Western blot. Levels of basic fibroblast growth factor (bFGF) and stromal-derived factor-1 (SDF-1) in cellular supernatant of each group were determined by ELISA kits (R&D Systems, MN, USA). All data are presented as mean ± SD. Comparisons of the groups were evaluated by using the t-test with one-way analysis of variance (ANOVA). Statistical significance was set at P < 0.05.