Chin J Integr Med 2011 Feb;17(2):134-140
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ORIGINAL ARTICLE Therapeutic Benefit of Yangxue Qingnao Granule (养血清脑颗粒) on Cognitive Impairment Induced by Chronic Cerebral Hypoperfusion in Rats XIONG Li (熊 ABSTRACT
丽), ZHANG Jun-jian (章军建), SUN Dong (孙
冬), and LIU Hui (刘
晖)
Objective: To observe the therapeutic effect of Yangxue Qingnao Granule (养血清脑颗粒,
YXQNG) on cognitive impairment induced by chronic cerebral hypoperfusion and to investigate its impact on oxidative stress, apoptosis, and the cholinergic system. Methods: Adult male Wistar rats were subjected to chronic cerebral hypoperfusion by permanent occlusion of bilateral common carotid arteries (2-VO). Thirty rats were randomly assigned to one of the five treatment groups in a 1:1:1:1:1 ratio: sham operation plus normal saline treatment, 2-VO plus normal saline treatment, 2-VO plus YXQNG at a dose of 2 g·kg-1·d-1 or 4 g·kg-1·d-1, or 2-VO plus rivastigmine 2 mg·kg-1·d-1. The Morris water maze test was used to assess the spatial memory retrieval. Apoptosis, total antioxide capacity (T-AOC), acetylcholine esterase (AchE) and choline acetyl transferase (ChAT) activities in the hippocampus and the cortex were investigated. Results: In the chronic cerebral hypoperfusion model, the 2-VO plus saline treatment resulted in impaired special learning as shown by the significantly prolonged escape latency and shorter swim time in the first quadrant as compared to the sham operation. The impairment was associated with apoptosis and significant decreases in T-AOC, AchE and ChAT activities in the hippocampus and the cortex. Treatment with YXQNG at either 2 g·kg-1·d-1 or 4 g·kg-1·d-1 dose, or rivastigmine resulted in significantly shorter escape latencies and longer swim time in the first quadrant. YXQNG at both doses, but not rivastigmine, had significant reduction in apoptosis, and significant increases in T-AOC and ChAT activity in both the hippocampus and the cortex. Unlike rivastigmine, neither dose of YXQNG showed significant reduction in AchE activity. Conclusions: YXQNG ameliorated cognitive impairment induced by chronic cerebral hypoperfusion. The protective effect may be mediated through its regulation of apoptosis and activities of T-AOC and ChAT in the hippocampus and cortex of the rats in the chronic cerebral hypoperfusion model, a mechanism that is different from rivastigmine. KEYWORDS Yangxue Qingnao Granule, chronic cerebral hypoperfusion, apoptosis, total antioxide capacity, acetylcholine esterase, choline acetyl transferase
Increasing evidence from epidemiological studies suggests that there are vascular risk factors for cognitive impairment and dementia, not only in vascular dementia (VD) but also in Alzheimer's disease (AD)(1). Chronic reduction of cerebral blood flow in patients with arteriovenous malformations, carotid stenosis/occlusion, or cerebral small vessel diseases has been shown to be associated with cognitive impairment, and researches have been focused on understanding the pathophysiologic changes associated with cognitive function decline due to chronic cerebral hypoperfusion and on developing new therapeutics that may intervene in VD and AD or slow down the pathological processes(2,3). Chronic cerebral hypoperfusion can be induced by the permanent occlusion of bilateral common carotid arteries (2-vessel occlusion, 2-VO) in rats.
This procedure is applied to establish a rat model of chronic cerebral hypoperfusion and such a model is commonly used to evaluate the relationship between chronic cerebral hypoperfusion and cognition (4). Yangxue Qingnao Granule (养血清脑 颗粒, YXQNG) is a compound drug extracted from multiple components of Chinese medicines, and has been shown to be effective in treating chronic cerebrovascular insufficiency in clinical practice (5). However, the underlying molecular mechanism remains to be elucidated. The purposes of this study
©The Chinese Journal of Integrated Traditional and Western Medicine Press and Springer-Verlag Berlin Heidelberg 2011 Department of Neurology, Zhongnan Hospital of Wuhan University, Wuhan (430071), China Correspondence to: Prof. ZHANG Jun-jian, Tel: 86-27-67812885, E-mail:
[email protected] DOI: 10.1007/s11655-011-0643-0
Chin J Integr Med 2011 Feb;17(2):134-140
are to determine the therapeutic benefit of YXQNG in cognitive impairment and to investigate its regulatory effects on apoptosis, total antioxidative capacity (T-AOC), and the activities of acetylcholinesterase (AchE) and choline acetyltransferase (ChAT) in the rat model of chronic cerebral hypoperfusion. Rivastigmine is a cholinesterase inhibitor for the treatment of mild to moderate dementia of the Alzheimer's type and dementia due to Parkinson's disease. It was used in the current study as a positive control agent for the treatment of cognitive impairment and its mechanism of action was compared with YXQNG.
METHODS Drugs and Reagents YXQNG, containing the extract from Radix Angelica sinensis , rhizome chuanxiong, Radix Paeoniae Alba , rehmannia praeparata , concha margaritifera usta and Rhizoma corydalis tuber, etc., was purchased from Tianjin Tianshili Pharmaceutical Co., Ltd (State Drug Approval Document No. Z10960082). The control drug rivastigmine, a cholinesterase inhibitor, was purchased from Novartis (State Drug Approval Document No. X20000170). Fluorescein isothiocyanate-conjugated Annexin Ⅴ (annexin Ⅴ-FITC) and propidium iodide (PI) were from Jingmei Biological Company (China). Kits for quantifying T-AOC, AchE and ChAT activities were from the Nanjingjiancheng Biological Company (China).
Animals and Surgical Procedure Specific pathogen free (SPF) adult male Wistar rats were provided by the Laboratory Animal Central of Hubei province [License No. SCXK (Hubei) 2008-0005]. All animals (approximately 10 weeks old, 275±25 g) were group-housed in the laboratory animal room maintained at 25±1 ℃ with food and water supply, with a regular 12-h light-dark cycle. All animals were treated in accordance with the National Institutes of Health Guidance for the Care and Use of Laboratory Animals, and their use was approved by the Animal Ethics Committee of the Medical School of Wuhan University. All animals were housed for at least one week prior to any experimental procedures. As described previously, 2-VO was used to induce chronic cerebral hypoperfusion(6). Briefly, rats were anesthetized with 10% chloral hydrate (350 mg/kg) by intraperitoneal injection and allowed to breathe
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spontaneously throughout the surgical procedure. Both common carotid arteries were exposed via a midline cervical incision and were doubly ligated with silk sutures. Animals serving as sham-operated controls received the same surgical operation without ligation of the carotid arteries. Rectal temperature was monitored and maintained between 36.0 ℃ and 37.0 ℃ during the surgical procedure. All animals underwent a three-week recuperation and drug treatments began on week 4.
Grouping and Drug Treatments Rats were randomly assigned to one of the five treatment groups in a 1:1:1:1:1 ratio: sham operation plus normal saline; 2-VO plus normal saline; 2-VO plus YXQNG at a dose of 2 g・kg -1・d -1; 2-VO plus YXQNG at a dose of 4 g・kg -1・d -1 ; or 2-VO plus rivastigmine at a dose of 2 mg・kg-1・d-1. All treatments were administered orally once per day for four weeks.
The Morris Water Maze Test The Morris water maze test consists of a circular pool (diameter: 150 cm; height: 60 cm; depth of water: 32 cm) with a featureless inner surface and a hidden escape platform (diameter: 10 cm) submerged 2 cm below the surface of the water and invisible from the surface of the water. The water was darkened with 50 mL of prepared Chinese ink and maintained at 22±1 ℃. The animals were trained to locate the hidden platform. The test was given starting on the first day at the completion of the 4-week treatment. All animals received four trials per day for five consecutive days with a constant interval of one hour between trials. The probe trials were performed on the 6th day(7). Swimming pathways were analyzed by a computerized video imaging analysis system (Institute of Materia Medica, Chinese Academy of Medical Sciences, China). Time spent in each quadrant was analyzed and escape latency recorded. Longer escape latency and shorter time spent in quadrant 1 indicated impaired spatial leaning ability.
Tissue Collection and Apoptosis Determination After the completion of the Morris water maze test, all animals were anesthetized with 10% chloral hydrate (350 mg/kg, intraperitoneal injection). Their brains were quickly removed, and the frontal lobe cortex and whole hippocampus were carefully isolated. The tissues collected from the left hemisphere were snap frozen in liquid nitrogen
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Biochemical Examinations The frozen tissues were weighed and homogenized in ice-cold normal saline that was nine times the volume of the tissue. The homogenate was centrifuged at 2 500 r/min for 10 min at 4 ℃. The supernatant was maintained on ice and was used for the succeeding measurements. Protein content was determined by the Coomassie blue protein binding method using bovine serum albumin as standard. ChAT and AchE activities were determined per instruction in the kits by spectrometry. The level of T-AOC was assessed according to the instruction provided by the Nanjing Jiancheng Bioengineering Institute, China.
Statistical Analysis All data were analyzed using GraphPad Prism 4.0 software. Group differences in escape latency in the Morris water maze test were analyzed using a twoway ANOVA followed by Bonferroni multiple group comparisons. Biochemical data and apoptosis were analyzed by a one-way ANOVA followed by a post Dunnett's test. Statistical significance was defined as P
