Aβ诱导AD大鼠的炎症机制及乙酰葛根素和壳聚糖磷脂酰胆碱的干预作用
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摘要
研究背景和意义
阿尔茨海默病(Alzheimer disease, AD)是一种起病隐匿的进行性发展的中枢神经系统退行性疾病,临床上主要表现为进行性记忆减退和认知障碍。AD可能是一组异质性疾病,病因尚未十分明确,存在多种因素,包括生物因素和社会心理因素。近年来大量研究证实,AD脑(尤其在海马)内持续存在着慢性进展性的炎性反应,认为小胶质细胞活化及炎症介导的神经毒性在神经退行性疾病的发病机制中起了决定性作用,炎性反应是AD出现认知和记忆障碍的重要机制。其中,有证据表明,p淀粉样蛋白(amyloid-beta, Aβ)沉积激活小胶质细胞引起的炎症反应是AD的发病核心,Aβ诱导引起的神经炎症是AD进展的关键事件之一,其中小胶质细胞是主要涉及的细胞,因此对小胶质细胞的激活进行干预可能会减缓疾病的进程,从而对中枢神经系统起保护作用,因而,以小胶质细胞为靶目标的研究有望成为新型抗AD药物研发的新方向。另外,目前公认的是胆碱能假说,认为老年认知障碍的临床症状是由于患者脑内胆碱酯能神经元损伤引起乙酰胆碱生成、释放减少所致。AD患者乙酰胆碱递质减少,因此,可通过增加乙酰胆碱的含量来治疗AD。
目前有关AD抗炎方面药物的研究主要集中在诸如非类固醇抗炎药(NSAIDS)、雌激素等,其防治AD的共同通路可能都是通过抑制AD的始动环节,即抑制患者脑内Ap沉积和老年斑的形成以及由此而引发的脑内炎症反应。但NSAIDs的长期应用可导致严重的胃粘膜损伤和肾功能障碍等,长期使用雌激素可能诱发乳腺或子宫内膜的癌变；NSAIDs和雌激素的毒副作用,限制了它们在临床上的使用。
葛根素(Puerarin)是从中药葛根中提取的异黄酮类化合物,具有阻断p受体、增加脑血流和脑代谢、清除氧自由基、促进免疫、改善学习记忆、抑制Ap激发的大鼠海马炎性反应等功效,是临床上治疗冠心病、脑血栓的常用和有效药物,但由于其脂溶性差,血脑屏障透过率低,因此在治疗脑部疾病时不能充分发挥其药理活性,很大程度上限制了其临床疗效的发挥。乙酰葛根素(六乙酰葛根素,化学名8-C-β-D-2",3”,4”,6”-四乙酰基吡喃葡萄糖-7,4’-二乙酰基异黄酮)是由葛根素经用醋酐溶解并酰化得到的葛根素衍生物,在大鼠体内代谢为葛根素,口服后在大鼠体内的暴露水平得到显著提高,更容易通过血脑屏障发挥作用,有更大的临床应用价值和前景。以往研究显示乙酰葛根素对脑缺血再灌注的保护作用,其机制可能通过作用于NMDA受体、抗氧自由基、抗脂质过氧化、抑制C-fos和ICAM-1、诱导VEGF等。但关于乙酰葛根素用于AD抗炎作用的研究文献报道较少。
壳聚糖(Chitosan)是甲壳素脱乙酰化的产物,为天然阳离子聚合物,无毒,安全可靠,呈碱性,且具有很好的生物相容性。国外研究表明,壳聚糖能调节细胞分化、增殖及细胞因子的产生,清除自由基,中和体内毒素,保护脑神经细胞和神经胶质细胞膜。有研究发现,高分子量水溶性壳聚糖能预防神经细胞凋亡。Mi-Sun Kim等体外研究显示水溶性壳聚糖的抗炎作用,可降低并延迟AD病理改变。卵磷脂是生物细胞膜的主要构成成份,且具有抗炎、增强免疫作用,所含胆碱可作为神经递质乙酰胆碱的前体物质,是神经元之间依靠化学物质传递信息的一种最主要的“神经递质”,一旦与壳聚糖形成复合物,可作为载体携带壳聚糖通过血脑屏障。本课题组前期应用溶剂分散法和湿态研磨法制备壳聚糖磷脂复合物,并进行了其防治老年性痴呆的基础和临床研究,从组织形态学角度研究发现壳聚糖磷脂胆碱的抗氧化、保护神经细胞作用,显示壳聚糖磷脂胆碱可提高痴呆大鼠和老年性痴呆病人的记忆能力,修复痴呆大鼠的神经细胞膜损伤,增加脑乙酰胆碱含量,清除自由基,改善脑功能。但壳聚糖磷脂胆碱用于对AD的抗炎作用及机制研究,文献中尚未见报道。
本研究分两部分：
第一部分Aβ诱导AD大鼠的炎症机制及乙酰葛根素的干预作用
第二部分壳聚糖磷脂酰胆碱对Aβ诱导AD大鼠的学习记忆作用及抗炎机制
第一部分Ap诱导AD大鼠的炎症机制及乙酰葛根素的干预作用
目的
本研究采用体内实验,从整体、组织、细胞、分子水平,利用双侧海马注射Aβ1-42所致AD模型大鼠为研究对象,利用行为学、免疫组织化学、分子生物学等技术方法,观察乙酰葛根素对Aβ诱导大鼠学习记忆的作用及抗炎机制,为AD治疗提供有效的策略方法和理论依据。
方法
采用双侧海马注射Aβl-42法所致AD模型大鼠为研究对象。实验动物分组：将40只wistar大鼠随机分为空白对照组、Aβ模型组、乙酰葛根素低剂量组和高剂量组,每组各10只。术后14天,药物低剂量组和高剂量组分别腹腔注射给予乙酰葛根素100mg/kg和200mg/kg,共12天。对照组和模型组给予腹腔注射相同容量生理盐水,共12天。应用Morris水迷宫试验观察乙酰葛根素对Aβ1-42诱导模型大鼠的学习记忆功能,包括观察记录大鼠的逃逸时间及在原平台象限停留时间。应用免疫组织化学法、透射电镜技术,观察乙酰葛根素对Ap1.-42模型大鼠大脑皮层及海马小胶质细胞的影响。并应用免疫组织化学法检测海马PKC-δ、IKK-β、iNOS阳性表达及定位。应用Western-blot法检测Aβ1-42大鼠海马PKC-δ、IKK-β及IL-1β的蛋白质表达水平。应用ELISA法检测大鼠血清中IL-6含量的变化。
全部数据分析均采用SPSS17.0软件进行。实验数据以均数±标准差表示,组间比较采用方差分析及t检验,P<0.05为具有统计学意义。
结果
1.Aβ1-42诱导对大鼠学习记忆能力的影响及乙酰葛根素的作用
Morris水迷宫定位航行实验显示,模型制备前,4组大鼠到达平台的逃避潜伏期大致相同(P>0.05)；术后14天,模型组、药物低剂量组、高剂量组大鼠到达平台的潜伏期明显长于空白对照组(P<0.01)；术后26天,即乙酰葛根素治疗12天后,空白组、药物低剂量、高剂量组大鼠到达平台的潜伏期明显短于模型组(乙酰葛根素低剂量组,P<0.05；乙酰葛根素高剂量组,P<0.01)。空间探索实验显示：模型制备前,4组大鼠在原平台象限停留时间大致相同(P>0.05)；术后14天,模型组、药物低剂量组、高剂量组大鼠在原平台象限停留时间减少,明显少于空白对照组(P<0.01)；乙酰葛根素治疗12天后,空白对照组、药物低剂量组、高剂量组大鼠在原平台象限停留时间明显高于模型组(乙酰葛根素低剂量组,P<0.05；乙酰葛根素高剂量组,P<0.01)。
2.Aβ1-42诱导对大鼠大脑皮层及海马小胶质细胞的影响及乙酰葛根素的作用
大脑皮层Ibal免疫组织化学染色结果：模型组小胶质细胞(Microglia,MG)数目最多,胞体增大增粗,黄染,着色深；乙酰葛根素低剂量组、高剂量组MG数目较少,胞体变小,淡染,着色浅；对照组MG数目最少,淡染。Ibal阳性细胞计数结果显示,模型组与对照组比较,MG数目明显增多(P<0.05),而乙酰葛根素低剂量组和高剂量组与模型组比较,前额皮层小胶质细胞数目明显减少(乙酰葛根素低剂量组,P<0.05；乙酰葛根素高剂量组,P<0.01)。
透射电镜结果显示：对照组MG较小,可见清晰的粗面内质网和线粒体,核仁清楚,异染色质明显,电子密度高。模型组MG有较多突起,异染色质减少和松散,胞浆丰富,核糖体数量增加,并可见溶酶体颗粒。乙酰葛根素低剂量组：胞浆电子密度低,可见破碎的线粒体嵴。乙酰葛根素高剂量组：细胞核形状规则,可见高尔基复合体。
3.Aβ1-42诱导对大鼠海马组织中PKC-δ及IKK-β的影响及乙酰葛根素的作用
PKC-δ、IKK-β被选为本研究与NF-κB通路相关的指标。免疫组织化学法检测了每组大鼠海马组织PKC-δ和IKK-β的免疫染色反应。PKC-δ染色结果：其免疫反应阳性物质呈棕黄色,定位于胞膜和胞浆。AD模型组PKC-δ的免疫反应阳性细胞最多,着色最深,细胞内的阳性颗粒最多,主要位于胞浆内；对照组和药物组阳性细胞较少,神经元胞浆着色浅。定量分析结果显示：模型PKC-δ海马阳性细胞的数量高于对照组、乙酰葛根素低剂量组和乙酰葛根素高剂量组,差异有统计学意义(P<0.05),并呈剂量依赖性。
在IKK-β免疫反应染色也观察到了类似的结果：模型组海马区IKK-β阳性细胞最多并成深棕色；对照组IKK-β阳性细胞较少,基本没有棕黄色颗粒表达的IKK-β,偶见神经元胞浆浅着色；乙酰葛根素组可见到少量IKK-β免疫反应阳性细胞且着色浅。定量分析结果显示：模型IKK-β海马阳性细胞的数量高于对照组、乙酰葛根素低剂量组和乙酰葛根素高剂量组,差异有统计学意义,(P<0.05),并呈剂量依赖性。
Western blot法观测了大鼠海马组织PKC-δ和IKK-β的表达,结果显示：双侧海马注射Aβ1-42能显著增加PKC-δ和IKK-β的表达,模型组约为正常对照组的2倍。乙酰葛根素治疗能显著减低Aβ1-42引起PKC-δ和IKK-β的表达,并呈剂量依赖性。
4.Ap1-42诱导对大鼠海马组织中iNOS、IL-1β及血清IL-6的影响及乙酰葛根素的作用
iNOS、IL-1β及IL-6被选为本研究炎症反应的标志物。免疫组织化学法检测了海马组织iNOS的表达。iNOS染色结果：其免疫反应阳性物质呈棕黄色,定位于细胞的胞膜和胞浆。其中,AD模型组iNOS的免疫反应阳性细胞最多,着色最深,细胞内的阳性颗粒最多,主要位于胞浆内；对照组和药物组阳性细胞较少,神经元胞浆着色浅。定量分析结果显示：模型iNOS海马阳性细胞的数量高于对照组、乙酰葛根素低剂量组和乙酰葛根素高剂量组,差异有统计学意义(乙酰葛根素低剂量组P<0.05；乙酰葛根素高剂量组P<0.01),并呈剂量依赖性。
Western blot分析表明,双侧海马注射Aβ1-42能显著增加IL-1p的表达,模型组约为正常对照组的2倍。
Elisa实验结果显示,治疗后模型组血清中IL-6的含量显著高于空白对照组、乙酰葛根素低剂量组、高剂量组,差异具有统计学意义(P<0.05)。
结论
1.Ap1-42双侧海马注射能明显降低大鼠学习记忆能力,成功模拟炎性AD模型,可以用于检测药物的抗炎作用。
2.乙酰葛根素对Aβ1-42所致AD模型大鼠学习记忆障碍具有明显的改善作用；由此推断出,乙酰葛根素能够成为防治AD发生发展的新型药物。
3.乙酰葛根素的作用机制可能是抑制了Ap沉积所引起的小胶质细胞激活,下调脑组织PKC-δ和IKK-β的表达,从而抑制NF-κB信号通路的激活,减少了炎性因子iNOS、IL-1β、 IL-6的分泌,减轻大脑皮层、海马神经元及小胶质细胞的炎性反应,从而发挥其神经保护作用。
第二部分壳聚糖磷脂酰胆碱对Aβ诱导AD大鼠的学习记忆作用及抗炎机制
目的
本研究采用体内实验,从整体、组织水平,利用双侧海马注射Aβ25-35所致AD模型大鼠为研究对象,应用行为学、免疫组织化学技术观察壳聚糖磷脂酰胆碱对Ap诱导大鼠的作用及抗炎机制,为AD治疗提供有效的策略方法。
方法
采用喷雾干燥器制备壳聚糖磷脂酰胆碱复合物微球,用分光光度法分别测定壳聚糖、卵磷脂的含量。采用双侧海马注射Aβ25-35法所致AD模型大鼠为研究对象。实验动物分组：将50只Wistar大鼠随机分为对照组、Aβ模型组、壳聚糖磷脂酰胆碱低剂量组、中剂量组和高剂量组,每组各10只。术后第7天开始,壳聚糖磷脂酰胆碱低剂量组、中剂量组、高剂量组分别给予0.2/(k·d)、0.4/(kg·d)和1.0/(kg·d)灌胃,共30天。对照组和模型组给予相当剂量生理盐水灌胃,共30天。应用Morris水迷宫试验,观察壳聚糖磷脂酰胆碱对Aβ25-35诱导模型大鼠的学习记忆功能,包括观察记录大鼠的逃逸时间、穿越平台次数及在原平台象限停留时间。应用免疫组织化学技术,观察壳聚糖磷脂酰胆碱对Aβ25-35AD模型大鼠大脑皮层小胶质细胞的影响,并应用免疫组织化学法检测海马PKC-δ、iNOS及IL-1β的阳性表达及定位。
全部数据分析均采用SPSS17.0软件进行。实验数据以均数±标准差表示,组间比较采用方差分析及t检验,P<0.05为具有统计学意义。
结果
1.壳聚糖磷脂酰胆碱对AD模型大鼠学习记忆能力的影响
AD动物模型制备前,各组大鼠学习记忆水平比较,差异没有统计学意义(P>0.05)。造模14d后,模型组和壳聚糖磷脂酰胆碱组大鼠的学习记忆水平明显低于对照组(P<0.01)。药物干预30d后,各壳聚糖磷脂酰胆碱组及对照组大鼠平均潜伏期(average escape latency, AEL)明显缩短,且单位时间内跨越原平台次数及在原平台象限停留时间增加,与模型组比较差异有统计学意义(P<0.01)。
2.A β25-35诱导对大鼠大脑皮层MC的影响及壳聚糖磷脂酰胆碱的作用
大脑皮层Ibal免疫组织化学染色结果：模型组MG数目多,胞体增大增粗,黄染,着色深；壳聚糖磷脂酰胆碱低剂量组、中剂量组、高剂量组MG数目减少,胞体变小,淡染,着色浅；对照组MG数目最少,淡染。定量分析结果显示：模型组Ibal反应强度明显高于正常组(P<0.05),壳聚糖磷脂酰胆碱低剂量组、中剂量组、高剂量组阳性表达依然强于正常组,但明显弱于模型组P<0.05)。
3.壳聚糖磷脂酰胆碱对Aβ25-35诱导Wistar大鼠海马PKC-δ的影响
PKC-δ在大鼠海马区的免疫反应程度,依次为模型组、壳聚糖磷脂酰胆碱低剂量组、中剂量组、高剂量组、对照组。PKC-δ免疫反应阳性物质呈棕黄色,定位于细胞的胞膜和胞浆。其中,模型组PKC-δ阳性细胞最多并着色深,细胞内的阳性颗粒最多,主要位于胞浆内；对照组PKC-δ阳性细胞最少,偶见神经元胞浆浅着色。定量分析结果显示：模型PKC-δ海马阳性细胞的数量高于对照组、壳聚糖磷脂酰胆碱低剂量组、中剂量组、高剂量组,差异有统计学意义(P<0.05),并呈剂量依赖性。
4.壳聚糖磷脂酰胆碱对Aβ25-35诱导Wistar大鼠海马iNOS、IL-1β的影响
Ap模型组iNOS和IL-1β阳性细胞表达明显高于对照组,壳聚糖磷脂酰胆碱低剂量组、中剂量组、高剂量组。壳聚糖磷脂酰胆碱药物组和模型组相比,炎性因子iNOS和IL-1p的阳性细胞数显著下降,差异有统计学意义(P<0.01),壳聚糖磷脂酰胆碱药物组和对照组相比,阳性细胞表达数差异无统计学意义(P>0.05)。提示Aβ25-35诱导可激发大鼠海马组织iNOS和IL-1p的表达,而壳聚糖磷脂酰胆碱可降低iNOS和IL-1β的表达。
结论
1.以壳聚糖和大豆卵磷脂为原料经喷雾干燥法可以合成壳聚糖磷脂酰胆碱复合物微球。这一方面提高壳聚糖的脂溶性,使壳聚糖更有效地发挥作用；另一方面可以更好地发挥卵磷脂供给胆碱及保护神经元细胞膜的作用。
2.壳聚糖磷脂酰胆碱对Aβ25-35所致AD模型大鼠的学习记忆障碍有明显的改善作用；
3.壳聚糖磷脂酰胆碱可能是通过抑制小胶质细胞增生及激活,进而减少细胞因子iNOS、IL-1β的产生,从而发挥其神经保护作用。
4.壳聚糖磷脂酰胆碱可能是通过下调脑组织PKC-δ的表达,进而抑制NF-kB通路激活,从而降低炎性因子的分泌。
Background and significance
Alzheimer disease (AD) is one of neuro-degenerative diseases of central nervous system, with the main clinical manifestations of memory and cognitive impairment. In recent years, a large number of studies have confirmed that there continuously exists chronic progressive inflammatory reaction in the brain of AD, especially in the hippocampus, and microglia activation and inflammation mediated neurotoxicity played a decisive role in the pathogenesis of neurodegenerative diseases, inflammatory reaction is an important mechanism of AD cognition and memory impairment. In which, more and more evidence shows that inflammatory reaction of activated microglia induced by amyloid beta (Aβ) deposition is the core reason of AD, and the role of neural inflammation and microglia activation in the pathogenesis of AD can not be ignored. The intervention to the activation of microglia may slow the disease process, so as to protect the central nervous system, therefore, research targeting on microglial cells is expected to become the new direction of anti AD drugs. In addition, the currently accepted is the cholinergic hypothesis, that clinical symptoms of senile cognitive impairment is due to the reduction of acetylcholine formation and release caused by cholinergic neurons damage. Acetylcholine neurotransmitter reduction plays an important role in the disease, increase the content of acetylcholine can be used to treatment AD.
At present, drug studies related to AD anti-inflammatory aspects mainly focused on non-steroidal anti-inflammatory drugs (NSAIDS), estrogen, etc, the common pathway of prevention and control of AD may inhibit the initial step of AD, and inhibition of Aβ deposition and formation of senile plaques and the inflammatory reaction in the brain. But the long-term application of NSAIDs can lead to severe gastric mucosal injury and renal dysfunction, long-term use of estrogen may induce breast or endometrial cancer. The side effects of NSAIDs and estrogen limit their use in the clinic. Therefore, to find a new anti-inflammatory drugs to reduce inflammation in the brain of AD, so to delay the progression of the disease will become a new starting point in the AD treatment.
Puerarin is isoflavone compounds extracted from Radix Puerariae, with the effects of blocking β receptor, increase cerebral blood flow and cerebral metabolism, scavenging oxygen free radicals, promoting immunity, and improving the learning and memory function. Wang Wensheng's study reported that puerarin could inhibit amyloid β-stimulated inflammatory reaction of rat hippocampi, but puerarin is not easy to pass through blood-brain barrier(BBB), its application is limited in the diseases of central nervous system. Acetylpuerarin is a modified form of puerarin. It is more lipid-soluble than puerarin and can successfully cross the blood-brain barrier, with great clinical application value. Previous research has shown that acetylpuerarin's protective effect on cerebral ischemia reperfusion, its mechanism may be mediated by NMDA receptors, anti oxygen free radical, lipid peroxidation, inhibition of C-fos and ICAM-1, Induction of VEGF. But the study regarding whether acetylpuerarin can reduce the inflammatory effects of AD is few.
Chitosan is a deacetylated product of chitin, a kind of cationic polymer, natural alkaline, non-toxic, safe and reliable, and has good biocompatibility. Foreign studies showed that chitosan can regulate cell differentiation, proliferation and cytokine production, scavenging free radicals, and the toxin in the body, protecting the brain nerve cells and glial cell membrane. A study found that high molecular weight water-soluble chitosan can prevent nerve cell apoptosis. Another study on anti-inflammatory effects of water-soluble chitosan suggested that it can reduce and delay the pathological changes of AD. Lecithin is the main component of membrane of cells and has the effect of anti-inflammation and enhances immune function, the choline in it can be used as the precursor of acetylcholine. Once the complex of lecithin with chitosan is formed, it can be used as carrier and carry chitosan through BBB. We used the method of solvent dispersion and wet grinding for the preparation of chitosan phospholipid complex before, and undertook the basic and clinical research on the treatment of senile dementia, and found that chitosan phospholipid choline can repair nerve cell membrane damage of dementia rats, improve memory capacity of dementia rats and patients with senile dementia, increase the brain acetylcholine content, scavenge free radicals, improve brain function. But the antiinflammatory effect and mechanism of the chitosan phospholipid choline on AD has not been eported in the literature.
This study inclueds the following two parts:
1. Mechanisms of inflammation in AD rats induced by Aβ and the intervention effect of Acetylpuerarin.
2. Effects and its mechanisms of Chitosan Phosphatidylcholine on learning and memory and inflammation in AD rats induced by Aβ.
Part I Mechanisms of Inflammation in AD Rats Induced by Aβ and the Intervention Effect of Acetylpuerarin
Objective
This study used in vivo experiments, from the whole, tissue, cell, molecular levels to observe the learning and memory function and the antiinflammatory mechanism of acetylpuerarin on AD model rats. The bilateral hippocampal injection of Aβ1-42induced AD model Wistar rats was used as subjects, and behavior, immunohistochemistry and molecular biology techniques were used to explore the effect and mechanism of acetylpuerarin on AD rat model, aimed to explain the effect of acetylpuerarin on AD and antiinflammatory mechanisms, so to provide strategy and theory basis for AD treatment.
Methods
The bilateral intrahippocampal Aβ1-42injection induced AD model rats were used as the research subjects.40Wistar rats were randomly divided into blank control group, model group (Aβ only group), acetylpuerarinin low dose group and acetylpuerarinin high dose group, with10rats in each group. After14days, rats of acetylpuerarin low dose group and high dose group were given intraperitoneal injection of acetylpuerarin100mg/kg and200mg/kg respectively, a total of12days. The control group and model group were given intraperitoneal injection of normal saline, a total of12days. Morris water maze test was used to observe the learning and memory function of acetylpuerarin on Aβ1-42induced rats from the overall level, including the escape time(average escape latency, AEL), and time in the original platform quadrant explorating the platform. Immunohistochemistry and Transmission Electron Microscopic examination methods were used to observe the effects of acetylpuerarin on microglia of cortex and hippocampus. And immunohistochemical staining method was applied to observe the expression of PKC-δ, IKK-β, iNOS in hippocampus. Western-blot method was used to observe the protein expression of PKC-δ, IKK-β and IL-1β in rat hippocampus. Elisa method was applied to detect the changes of IL-6in serum of rats.Values are given as mean±standard deviation, and analysis of variance and t-tests were performed with SPSS17.0software. P<0.05was considered to be statistically significant.
Results
1. Influence of Aβ1-42to learning and memory of Rats and the effects of Acetylpuerarin
We tested escape latency and exploring time in target area in the Morris water maze (MWM) before surgery,2weeks after surgery, and26days after surgery (after treatment). We found that the Aβ1-42treated rats were significantly impaired2weeks after surgery, compared with the control group, their mean escape latency was significantly increased in the place navigation test (P<0.01), and mean exploring time in the target area was significantly decreased in the spatial probe test (P<0.05for Acetylpuerarin low dose group; P<0.01for Acetylpuerarin high dose group). After12days of acetylpuerarin treatment, we found that both doses significantly attenuated Aβ1-42induced learning and memory deficits, as evidenced by the decreased escape latency in the place navigation test (P<0.01), and longer mean exploring time in the target area in the spatial probe test (P<0.05for Acetylpuerarin low dose group;P<0.01for Acetylpuerarin high dose group).
We did not observe any changes in locomotion during the MWM, suggesting that the increased time was due to impaired learning rather than difficulty swimming.
2. Influence of Aβ1-42to Microglial of rats and the effects of Acetylpuerarin
Cerebral cortex Ibal immunohistochemical staining results:in the model group, there were more MG cells, with thick body, yellow dye, color depth; in Acetylpuerarin low dose group and high dose group, there were less number of MG, with smaller size, light color; in control group, theere were the least MG cells, with pale staining. Ibal positive cell counting analysis showed that microglial cell number increased significantly (P<0.05) in model group compared with the control group, while compared with the model group, the number of microglial cells in Acetylpuerarin low dose group and high dose group was significantly reduced (Acetylpuerarin low dose group, P<0.05; acetyl puerarin in high dose group, P<0.01).
Transmission electron microscope showed:microglia of the control group is small, with clear rough endoplasmic reticulum and mitochondria, clear nucleolus, obvious heterochromatin, high electron density. The model group microglial cell has many neurites, less heterochromatin and loose, rich cytoplasm, and the number of ribosomes increased, with visible lysosomal granules. Low doses Acetylpuerarin group:low cytoplasmic electron density, with visible broken mitochondrial crista. High doses Acetylpuerarin group:big nuclear chromatin, with visible Golgi complex.
3. Influence of Aβ1-42to PKC-δ and IKK-β in hippocampal tissue and the effects of Acetylpuerarin
PKC-δ and IKK-β were selected as markers related to NF-κB pathway in this study. Immunohistochemistry method was used to detect the PKC-δ and IKK-β staining reaction of hippocampal tissue. PKC-δ staining results:the immunoreactive positive substance was brown, localized in cell membrane and cytoplasmic. PKC-8immunoreactive cells was the most in AD model group, with the deepest coloring, most positive granular cells, mainly located in the cytoplasm; there were less positive cells in control group and drug groups, and the cytoplasm of neurons was light coloured. Quantitative analysis showed:the number of PKC-8positive cells in model group was higher than that of control group, Acetylpuerarin low dose group and Acetylpuerarin high dose group, with significant statistical difference (P<0.05), and in a dose-dependent manner.
Similar result was also observed in IKK-β immunoreactive staining:in model group, with the most IKK-P positive cells and a dark brown color. There were Less IKK-β positive cells in the control group,basically no expression of brown granules of IKK-β, occasionally the cytoplasm of neurons light colored. There was a small amount of IKK-P immune immunoreactive cells in Acetylpuerarin groups, with shallow coloring. Quantitative analysis showed:the number of IKK-P hippocampal positive cells in model group was more than that in the control group, Acetylpuerarin low dose group and Acetylpuerarin high dose group, with significant statistical difference (P<0.05), and in a dose-dependent manner.
Western blot method was used to observe the expression of PKC-δ and IKK-β in the hippocampus of rats, and the results showed:bilateral hippocampal injection of Aβ1-42can significantly increase the expression of PKC-δ and IKK-β, approximately2times as much as normal control group. Acetylpuerarin therapy can significantly reduce the expression of PKC-δ and IKK-β induced by Aβ1-42,with significant statistical difference(P<0.05), and in a dose-dependent manner.
4. Influence of Aβ1-42to the expression of hippocampal iNOS、IL-1β and serum IL-6and the effects of Acetylpuerarin
iNOS, IL-1β and IL-6were chosen as the markers of inflammation for this study. Immunohistochemistry method was used to detect the iNOS expression in hippocampal tissue. The staining result of iNOS showed:the positive material of immunoreactivity was brown, localized in cell membrane and cytoplasm. Among them, there were the most immunoreactive iNOS positive cells in model group, with the deepest coloring, and the most positive granular. The immunoreaction positive cells in control group and drug groups were less, with lightly coloured cytoplasm of neurons. Quantitative analysis showed:the number of iNOS hippocampal positive cells in model group was more than that in the control group, Acetylpuerarin low dose group and Acetylpuerarin high dose group, with significant statistical difference (P<0.05), and in a dose-dependent manner.
Western blot method was used to detect the quantitative expression of IL-1β in hippocampal tissue. The results showed that bilateral hippocampal injection of Aβ1-42can significantly increase the expression of IL-1β, about3times as much as normal control group. Acetylpuerarin therapy can significantly reduce the expression of IL-1β induced by Aβ1-42, with significant statistical difference (P<0.05), and in a dose-dependent manner.
Elisa method was used to detect the serum IL-6level of rats.Results showed after Acetylpuerarin treatment, the level of serum IL-6of control group, Acetylpuerarin low dose group and Acetylpuerarin high dose group was significantly lower than that in the model group respectively, with significant statistical difference (P<0.05).
Conclusions
1. Aβ1-42bilateral intrahippocampal injection method can successfully simulate the inflammatory AD model rats, which can be used to test the effects of drugs.
2. Acetylpuerarin has obvious effect on learning and memory disorder in AD model rats induced by Aβ1-42.Thus infer, acetylpuerarin can become a new drug for prevention of the occurrence and development of AD.
3.The mechanism of Acetylpuerarin may possibly through inhibit microglia proliferation and activation, down-regulate the expression of brain PKC-δ, IKK-β, thus inhibit the activation of NF-κB signal channel, down-regulate the secretion of iNOS, IL-1β and IL-6,decrease the inflammatory reaction of brain cortex and hippocampus neurons, which play a role in brain protection and anti dementia.
Part Ⅱ Effects and its Mechanisms of Chitosan Phosphatidylcholine on Learning and Memory and Inflammation in AD Rats Induced by Aβ
Objective
This study used in vivo experiments, from the whole and tissue levels to observe the learning and memory function and the anti-inflammatory mechanism of Chitosan Phosphatidylcholine on AD rats. The bilateral hippocampal injection of Aβ25-35induced AD model Wistar rats were used as subjects. We used behaviorial and immunohistochemistry techniques to observe the effect and anti inflammation mechanism of chitosan phosphatidyl choline induced by Aβ in rats, aims to explain the effect of Chitosan Phosphatidylcholine on AD and anti-inflammatory mechanisms, so to provide strategy and theory basis for AD treatment.
Methods
The bilateral intrahippocampal Aβ25-35injection induced AD model rats were used as the research subjects.50rats were randomly divided into5groups:blank control group, model group (AP only group), Chitosan Phosphatidylcholine low dose group, midian dose group and high dose group, with10rats in each group. After7days, rats of Chitosan Phosphatidylcholine low dose group, midian dose group and high dose group were given Chitosan Phosphatidylcholine of0.2、0.6and1.0g/(kg·d) respectively by the way of intragastrition, a total of30days. Rats of the control group and model group were given equal dose of normal saline intragastrically, a total of30days. Morris water maze test was used to observe the learning and memory function of Chitosan Phosphatidylcholine on Aβ25-35induced rats, including the escape time(average escape latency, AEL), times crossing the postion of platform, and time staying in the original platform quadrant spending in explorating the platform. Immunohistochemical method was used to detect the expression of Ibal in cerebral cortex, and PKC-8, IL-1β, iNOS expression in hippocampus. Values are given as mean±standard deviation, and analysis of variance and t-tests were performed with SPSS13.0software. P<0.05was considered to be statistically significant.
Results
1. Chitosan Phosphatidylcholine Improved Learning and Memory
We tested escape latency, times crossing the position of platform, and target area exploring time in the Morris water maze (MWM) before surgery,14days after surgery, and30days after surgery. We found that the learning and memory ability of Aβ25-35treated rats was significantly impaired2weeks after surgery, compared with the control group, their mean escape latency was significantly increased in the place navigation test (P<0.01), with fewer times crossing the postion of platform (P<0.01), and mean exploring time in the target area was significantly decreased in the spatial probe test (P<0.01). After30days of Chitosan Phosphatidylcholine treatment, we found that Chitosan Phosphatidylcholine significantly attenuated Aβ25-35induced learning and memory deficits, as evidenced by the decreased escape latency in the place navigation test (P<0.01), more times crossing the postion of platform, and longer mean exploring time in the target area in the spatial probe test (P<001).
2. Chitosan Phosphatidylcholine Reduced Aβ25-35induced Microglial Activation
Cerebral cortex Ibal immunohistochemical staining results:in the model group, there was more MG cells, with thick cell body, yellow and deep color; in the chitosan phosphatidylcholine low dose group, midian dose group and high dose group, the number of MG cells decreased, the cell body became small, light color; in the control group, there was the least MG cells, with pale staining. Quantitative analysis showed: the number of Ibal positives cells in model group was significantly higher than that in normal group (P<0.05), the Number of Ibal positives cells in chitosan phosphatidylcholine low dose group, midian dose group and high dose group was significantly higher than that in normal group, but was lower than that in the model group P<05).
3. Chitosan Phosphatidylcholine Reduced Aβ25-35induced PKC-δ expression in rat hippocampus
The immune response level of PKC-δ in the rat hippocampus in model group was significantly higher than the control group, the chitosan phosphatidylcholine low dose group, median dose group, high dose group. The PKC-δ immunoreactive material was brown, localized in cell membrane and cytoplasm. PKC-δ immunoreactive cells was the most in AD model group, with the deepest coloring, most positive granular cells, mainly located in the cytoplasm; there were the least positive cells in control group and drug group, and the cytoplasm of neurons was light coloured. Quantitative analysis showed: the number of PKC-δ positive cells in model group was higher than that of control group, chitosan phosphatidylcholine low dose group, median dose group, high dose group, with significant statistical difference (P<005), and in a dose-dependent manner.
4. Chitosan Phosphatidylcholine reduced Aβ25-35induced iNOS and IL-1β expression in rat hippocampus
The immune response level of iNOS and IL-1β in the rat hippocampus in model group was significantly higher than the control group, the chitosan phosphatidylcholine low dose group, median dose group, and high dose group. The number of iNOS and IL-1β positive cells in chitosan phosphatidylcholine groups was lower than that in Aβ model group respectively, and the difference was statistically significant (P<0.05).Compared with the control group, the number of iNOS and IL-1β positive cells in Chitosan phosphatidylcholine low dose group, median dose group and high dose group had no significant statistical difference (P>0.05). Indicating that Aβ25-35could stimulate the expression of iNOS and IL-1β in rat hippocampuss, and chitosan phosphatidylcholine can reduce the expression of iNOS and IL-1β.
Conclusions
1.Using chitosan and soy lecithin can synthesize chitosan phosphatidylcholine compound microspheres by the method of spray drying.On one hand, it can improve chitosan's lipid solubility, be more effective for chitosan's action; on the other hand,it can play a better role for the supply of choline and protect neuron membrane for lecithin.
2. Chitosan phosphatidylcholine has obvious effect on Aβ25-35induced learning and memory impairment in AD rat model.
3. Chitosan phosphatidylcholine may be through the suppression of microglia in brain cortex and hippocampus, thereby reducing the expression of inflammation factors, such as iNOS and IL-1β, which exerts its neuroprotective effect.
4. Chitosan phosphatidylcholine may be through the downregulation of PKC-8in brain tissue, and inhibit the NF-kB pathway, thereby reducing the expression of inflammation factors.
阿尔茨海默病(Alzheimer disease, AD)是一种起病隐匿的进行性发展的中枢神经系统退行性疾病,临床上主要表现为进行性记忆减退和认知障碍。AD可能是一组异质性疾病,病因尚未十分明确,存在多种因素,包括生物因素和社会心理因素。近年来大量研究证实,AD脑(尤其在海马)内持续存在着慢性进展性的炎性反应,认为小胶质细胞活化及炎症介导的神经毒性在神经退行性疾病的发病机制中起了决定性作用,炎性反应是AD出现认知和记忆障碍的重要机制。其中,有证据表明,p淀粉样蛋白(amyloid-beta, Aβ)沉积激活小胶质细胞引起的炎症反应是AD的发病核心,Aβ诱导引起的神经炎症是AD进展的关键事件之一,其中小胶质细胞是主要涉及的细胞,因此对小胶质细胞的激活进行干预可能会减缓疾病的进程,从而对中枢神经系统起保护作用,因而,以小胶质细胞为靶目标的研究有望成为新型抗AD药物研发的新方向。另外,目前公认的是胆碱能假说,认为老年认知障碍的临床症状是由于患者脑内胆碱酯能神经元损伤引起乙酰胆碱生成、释放减少所致。AD患者乙酰胆碱递质减少,因此,可通过增加乙酰胆碱的含量来治疗AD。
目前有关AD抗炎方面药物的研究主要集中在诸如非类固醇抗炎药(NSAIDS)、雌激素等,其防治AD的共同通路可能都是通过抑制AD的始动环节,即抑制患者脑内Ap沉积和老年斑的形成以及由此而引发的脑内炎症反应。但NSAIDs的长期应用可导致严重的胃粘膜损伤和肾功能障碍等,长期使用雌激素可能诱发乳腺或子宫内膜的癌变；NSAIDs和雌激素的毒副作用,限制了它们在临床上的使用。
葛根素(Puerarin)是从中药葛根中提取的异黄酮类化合物,具有阻断p受体、增加脑血流和脑代谢、清除氧自由基、促进免疫、改善学习记忆、抑制Ap激发的大鼠海马炎性反应等功效,是临床上治疗冠心病、脑血栓的常用和有效药物,但由于其脂溶性差,血脑屏障透过率低,因此在治疗脑部疾病时不能充分发挥其药理活性,很大程度上限制了其临床疗效的发挥。乙酰葛根素(六乙酰葛根素,化学名8-C-β-D-2",3”,4”,6”-四乙酰基吡喃葡萄糖-7,4’-二乙酰基异黄酮)是由葛根素经用醋酐溶解并酰化得到的葛根素衍生物,在大鼠体内代谢为葛根素,口服后在大鼠体内的暴露水平得到显著提高,更容易通过血脑屏障发挥作用,有更大的临床应用价值和前景。以往研究显示乙酰葛根素对脑缺血再灌注的保护作用,其机制可能通过作用于NMDA受体、抗氧自由基、抗脂质过氧化、抑制C-fos和ICAM-1、诱导VEGF等。但关于乙酰葛根素用于AD抗炎作用的研究文献报道较少。
壳聚糖(Chitosan)是甲壳素脱乙酰化的产物,为天然阳离子聚合物,无毒,安全可靠,呈碱性,且具有很好的生物相容性。国外研究表明,壳聚糖能调节细胞分化、增殖及细胞因子的产生,清除自由基,中和体内毒素,保护脑神经细胞和神经胶质细胞膜。有研究发现,高分子量水溶性壳聚糖能预防神经细胞凋亡。Mi-Sun Kim等体外研究显示水溶性壳聚糖的抗炎作用,可降低并延迟AD病理改变。卵磷脂是生物细胞膜的主要构成成份,且具有抗炎、增强免疫作用,所含胆碱可作为神经递质乙酰胆碱的前体物质,是神经元之间依靠化学物质传递信息的一种最主要的“神经递质”,一旦与壳聚糖形成复合物,可作为载体携带壳聚糖通过血脑屏障。本课题组前期应用溶剂分散法和湿态研磨法制备壳聚糖磷脂复合物,并进行了其防治老年性痴呆的基础和临床研究,从组织形态学角度研究发现壳聚糖磷脂胆碱的抗氧化、保护神经细胞作用,显示壳聚糖磷脂胆碱可提高痴呆大鼠和老年性痴呆病人的记忆能力,修复痴呆大鼠的神经细胞膜损伤,增加脑乙酰胆碱含量,清除自由基,改善脑功能。但壳聚糖磷脂胆碱用于对AD的抗炎作用及机制研究,文献中尚未见报道。
本研究分两部分：
第一部分Aβ诱导AD大鼠的炎症机制及乙酰葛根素的干预作用
第二部分壳聚糖磷脂酰胆碱对Aβ诱导AD大鼠的学习记忆作用及抗炎机制
第一部分Ap诱导AD大鼠的炎症机制及乙酰葛根素的干预作用
目的
本研究采用体内实验,从整体、组织、细胞、分子水平,利用双侧海马注射Aβ1-42所致AD模型大鼠为研究对象,利用行为学、免疫组织化学、分子生物学等技术方法,观察乙酰葛根素对Aβ诱导大鼠学习记忆的作用及抗炎机制,为AD治疗提供有效的策略方法和理论依据。
方法
采用双侧海马注射Aβl-42法所致AD模型大鼠为研究对象。实验动物分组：将40只wistar大鼠随机分为空白对照组、Aβ模型组、乙酰葛根素低剂量组和高剂量组,每组各10只。术后14天,药物低剂量组和高剂量组分别腹腔注射给予乙酰葛根素100mg/kg和200mg/kg,共12天。对照组和模型组给予腹腔注射相同容量生理盐水,共12天。应用Morris水迷宫试验观察乙酰葛根素对Aβ1-42诱导模型大鼠的学习记忆功能,包括观察记录大鼠的逃逸时间及在原平台象限停留时间。应用免疫组织化学法、透射电镜技术,观察乙酰葛根素对Ap1.-42模型大鼠大脑皮层及海马小胶质细胞的影响。并应用免疫组织化学法检测海马PKC-δ、IKK-β、iNOS阳性表达及定位。应用Western-blot法检测Aβ1-42大鼠海马PKC-δ、IKK-β及IL-1β的蛋白质表达水平。应用ELISA法检测大鼠血清中IL-6含量的变化。
全部数据分析均采用SPSS17.0软件进行。实验数据以均数±标准差表示,组间比较采用方差分析及t检验,P<0.05为具有统计学意义。
结果
1.Aβ1-42诱导对大鼠学习记忆能力的影响及乙酰葛根素的作用
Morris水迷宫定位航行实验显示,模型制备前,4组大鼠到达平台的逃避潜伏期大致相同(P>0.05)；术后14天,模型组、药物低剂量组、高剂量组大鼠到达平台的潜伏期明显长于空白对照组(P<0.01)；术后26天,即乙酰葛根素治疗12天后,空白组、药物低剂量、高剂量组大鼠到达平台的潜伏期明显短于模型组(乙酰葛根素低剂量组,P<0.05；乙酰葛根素高剂量组,P<0.01)。空间探索实验显示：模型制备前,4组大鼠在原平台象限停留时间大致相同(P>0.05)；术后14天,模型组、药物低剂量组、高剂量组大鼠在原平台象限停留时间减少,明显少于空白对照组(P<0.01)；乙酰葛根素治疗12天后,空白对照组、药物低剂量组、高剂量组大鼠在原平台象限停留时间明显高于模型组(乙酰葛根素低剂量组,P<0.05；乙酰葛根素高剂量组,P<0.01)。
2.Aβ1-42诱导对大鼠大脑皮层及海马小胶质细胞的影响及乙酰葛根素的作用
大脑皮层Ibal免疫组织化学染色结果：模型组小胶质细胞(Microglia,MG)数目最多,胞体增大增粗,黄染,着色深；乙酰葛根素低剂量组、高剂量组MG数目较少,胞体变小,淡染,着色浅；对照组MG数目最少,淡染。Ibal阳性细胞计数结果显示,模型组与对照组比较,MG数目明显增多(P<0.05),而乙酰葛根素低剂量组和高剂量组与模型组比较,前额皮层小胶质细胞数目明显减少(乙酰葛根素低剂量组,P<0.05；乙酰葛根素高剂量组,P<0.01)。
透射电镜结果显示：对照组MG较小,可见清晰的粗面内质网和线粒体,核仁清楚,异染色质明显,电子密度高。模型组MG有较多突起,异染色质减少和松散,胞浆丰富,核糖体数量增加,并可见溶酶体颗粒。乙酰葛根素低剂量组：胞浆电子密度低,可见破碎的线粒体嵴。乙酰葛根素高剂量组：细胞核形状规则,可见高尔基复合体。
3.Aβ1-42诱导对大鼠海马组织中PKC-δ及IKK-β的影响及乙酰葛根素的作用
PKC-δ、IKK-β被选为本研究与NF-κB通路相关的指标。免疫组织化学法检测了每组大鼠海马组织PKC-δ和IKK-β的免疫染色反应。PKC-δ染色结果：其免疫反应阳性物质呈棕黄色,定位于胞膜和胞浆。AD模型组PKC-δ的免疫反应阳性细胞最多,着色最深,细胞内的阳性颗粒最多,主要位于胞浆内；对照组和药物组阳性细胞较少,神经元胞浆着色浅。定量分析结果显示：模型PKC-δ海马阳性细胞的数量高于对照组、乙酰葛根素低剂量组和乙酰葛根素高剂量组,差异有统计学意义(P<0.05),并呈剂量依赖性。
在IKK-β免疫反应染色也观察到了类似的结果：模型组海马区IKK-β阳性细胞最多并成深棕色；对照组IKK-β阳性细胞较少,基本没有棕黄色颗粒表达的IKK-β,偶见神经元胞浆浅着色；乙酰葛根素组可见到少量IKK-β免疫反应阳性细胞且着色浅。定量分析结果显示：模型IKK-β海马阳性细胞的数量高于对照组、乙酰葛根素低剂量组和乙酰葛根素高剂量组,差异有统计学意义,(P<0.05),并呈剂量依赖性。
Western blot法观测了大鼠海马组织PKC-δ和IKK-β的表达,结果显示：双侧海马注射Aβ1-42能显著增加PKC-δ和IKK-β的表达,模型组约为正常对照组的2倍。乙酰葛根素治疗能显著减低Aβ1-42引起PKC-δ和IKK-β的表达,并呈剂量依赖性。
4.Ap1-42诱导对大鼠海马组织中iNOS、IL-1β及血清IL-6的影响及乙酰葛根素的作用
iNOS、IL-1β及IL-6被选为本研究炎症反应的标志物。免疫组织化学法检测了海马组织iNOS的表达。iNOS染色结果：其免疫反应阳性物质呈棕黄色,定位于细胞的胞膜和胞浆。其中,AD模型组iNOS的免疫反应阳性细胞最多,着色最深,细胞内的阳性颗粒最多,主要位于胞浆内；对照组和药物组阳性细胞较少,神经元胞浆着色浅。定量分析结果显示：模型iNOS海马阳性细胞的数量高于对照组、乙酰葛根素低剂量组和乙酰葛根素高剂量组,差异有统计学意义(乙酰葛根素低剂量组P<0.05；乙酰葛根素高剂量组P<0.01),并呈剂量依赖性。
Western blot分析表明,双侧海马注射Aβ1-42能显著增加IL-1p的表达,模型组约为正常对照组的2倍。
Elisa实验结果显示,治疗后模型组血清中IL-6的含量显著高于空白对照组、乙酰葛根素低剂量组、高剂量组,差异具有统计学意义(P<0.05)。
结论
1.Ap1-42双侧海马注射能明显降低大鼠学习记忆能力,成功模拟炎性AD模型,可以用于检测药物的抗炎作用。
2.乙酰葛根素对Aβ1-42所致AD模型大鼠学习记忆障碍具有明显的改善作用；由此推断出,乙酰葛根素能够成为防治AD发生发展的新型药物。
3.乙酰葛根素的作用机制可能是抑制了Ap沉积所引起的小胶质细胞激活,下调脑组织PKC-δ和IKK-β的表达,从而抑制NF-κB信号通路的激活,减少了炎性因子iNOS、IL-1β、 IL-6的分泌,减轻大脑皮层、海马神经元及小胶质细胞的炎性反应,从而发挥其神经保护作用。
第二部分壳聚糖磷脂酰胆碱对Aβ诱导AD大鼠的学习记忆作用及抗炎机制
目的
本研究采用体内实验,从整体、组织水平,利用双侧海马注射Aβ25-35所致AD模型大鼠为研究对象,应用行为学、免疫组织化学技术观察壳聚糖磷脂酰胆碱对Ap诱导大鼠的作用及抗炎机制,为AD治疗提供有效的策略方法。
方法
采用喷雾干燥器制备壳聚糖磷脂酰胆碱复合物微球,用分光光度法分别测定壳聚糖、卵磷脂的含量。采用双侧海马注射Aβ25-35法所致AD模型大鼠为研究对象。实验动物分组：将50只Wistar大鼠随机分为对照组、Aβ模型组、壳聚糖磷脂酰胆碱低剂量组、中剂量组和高剂量组,每组各10只。术后第7天开始,壳聚糖磷脂酰胆碱低剂量组、中剂量组、高剂量组分别给予0.2/(k·d)、0.4/(kg·d)和1.0/(kg·d)灌胃,共30天。对照组和模型组给予相当剂量生理盐水灌胃,共30天。应用Morris水迷宫试验,观察壳聚糖磷脂酰胆碱对Aβ25-35诱导模型大鼠的学习记忆功能,包括观察记录大鼠的逃逸时间、穿越平台次数及在原平台象限停留时间。应用免疫组织化学技术,观察壳聚糖磷脂酰胆碱对Aβ25-35AD模型大鼠大脑皮层小胶质细胞的影响,并应用免疫组织化学法检测海马PKC-δ、iNOS及IL-1β的阳性表达及定位。
全部数据分析均采用SPSS17.0软件进行。实验数据以均数±标准差表示,组间比较采用方差分析及t检验,P<0.05为具有统计学意义。
结果
1.壳聚糖磷脂酰胆碱对AD模型大鼠学习记忆能力的影响
AD动物模型制备前,各组大鼠学习记忆水平比较,差异没有统计学意义(P>0.05)。造模14d后,模型组和壳聚糖磷脂酰胆碱组大鼠的学习记忆水平明显低于对照组(P<0.01)。药物干预30d后,各壳聚糖磷脂酰胆碱组及对照组大鼠平均潜伏期(average escape latency, AEL)明显缩短,且单位时间内跨越原平台次数及在原平台象限停留时间增加,与模型组比较差异有统计学意义(P<0.01)。
2.A β25-35诱导对大鼠大脑皮层MC的影响及壳聚糖磷脂酰胆碱的作用
大脑皮层Ibal免疫组织化学染色结果：模型组MG数目多,胞体增大增粗,黄染,着色深；壳聚糖磷脂酰胆碱低剂量组、中剂量组、高剂量组MG数目减少,胞体变小,淡染,着色浅；对照组MG数目最少,淡染。定量分析结果显示：模型组Ibal反应强度明显高于正常组(P<0.05),壳聚糖磷脂酰胆碱低剂量组、中剂量组、高剂量组阳性表达依然强于正常组,但明显弱于模型组P<0.05)。
3.壳聚糖磷脂酰胆碱对Aβ25-35诱导Wistar大鼠海马PKC-δ的影响
PKC-δ在大鼠海马区的免疫反应程度,依次为模型组、壳聚糖磷脂酰胆碱低剂量组、中剂量组、高剂量组、对照组。PKC-δ免疫反应阳性物质呈棕黄色,定位于细胞的胞膜和胞浆。其中,模型组PKC-δ阳性细胞最多并着色深,细胞内的阳性颗粒最多,主要位于胞浆内；对照组PKC-δ阳性细胞最少,偶见神经元胞浆浅着色。定量分析结果显示：模型PKC-δ海马阳性细胞的数量高于对照组、壳聚糖磷脂酰胆碱低剂量组、中剂量组、高剂量组,差异有统计学意义(P<0.05),并呈剂量依赖性。
4.壳聚糖磷脂酰胆碱对Aβ25-35诱导Wistar大鼠海马iNOS、IL-1β的影响
Ap模型组iNOS和IL-1β阳性细胞表达明显高于对照组,壳聚糖磷脂酰胆碱低剂量组、中剂量组、高剂量组。壳聚糖磷脂酰胆碱药物组和模型组相比,炎性因子iNOS和IL-1p的阳性细胞数显著下降,差异有统计学意义(P<0.01),壳聚糖磷脂酰胆碱药物组和对照组相比,阳性细胞表达数差异无统计学意义(P>0.05)。提示Aβ25-35诱导可激发大鼠海马组织iNOS和IL-1p的表达,而壳聚糖磷脂酰胆碱可降低iNOS和IL-1β的表达。
结论
1.以壳聚糖和大豆卵磷脂为原料经喷雾干燥法可以合成壳聚糖磷脂酰胆碱复合物微球。这一方面提高壳聚糖的脂溶性,使壳聚糖更有效地发挥作用；另一方面可以更好地发挥卵磷脂供给胆碱及保护神经元细胞膜的作用。
2.壳聚糖磷脂酰胆碱对Aβ25-35所致AD模型大鼠的学习记忆障碍有明显的改善作用；
3.壳聚糖磷脂酰胆碱可能是通过抑制小胶质细胞增生及激活,进而减少细胞因子iNOS、IL-1β的产生,从而发挥其神经保护作用。
4.壳聚糖磷脂酰胆碱可能是通过下调脑组织PKC-δ的表达,进而抑制NF-kB通路激活,从而降低炎性因子的分泌。
Background and significance
Alzheimer disease (AD) is one of neuro-degenerative diseases of central nervous system, with the main clinical manifestations of memory and cognitive impairment. In recent years, a large number of studies have confirmed that there continuously exists chronic progressive inflammatory reaction in the brain of AD, especially in the hippocampus, and microglia activation and inflammation mediated neurotoxicity played a decisive role in the pathogenesis of neurodegenerative diseases, inflammatory reaction is an important mechanism of AD cognition and memory impairment. In which, more and more evidence shows that inflammatory reaction of activated microglia induced by amyloid beta (Aβ) deposition is the core reason of AD, and the role of neural inflammation and microglia activation in the pathogenesis of AD can not be ignored. The intervention to the activation of microglia may slow the disease process, so as to protect the central nervous system, therefore, research targeting on microglial cells is expected to become the new direction of anti AD drugs. In addition, the currently accepted is the cholinergic hypothesis, that clinical symptoms of senile cognitive impairment is due to the reduction of acetylcholine formation and release caused by cholinergic neurons damage. Acetylcholine neurotransmitter reduction plays an important role in the disease, increase the content of acetylcholine can be used to treatment AD.
At present, drug studies related to AD anti-inflammatory aspects mainly focused on non-steroidal anti-inflammatory drugs (NSAIDS), estrogen, etc, the common pathway of prevention and control of AD may inhibit the initial step of AD, and inhibition of Aβ deposition and formation of senile plaques and the inflammatory reaction in the brain. But the long-term application of NSAIDs can lead to severe gastric mucosal injury and renal dysfunction, long-term use of estrogen may induce breast or endometrial cancer. The side effects of NSAIDs and estrogen limit their use in the clinic. Therefore, to find a new anti-inflammatory drugs to reduce inflammation in the brain of AD, so to delay the progression of the disease will become a new starting point in the AD treatment.
Puerarin is isoflavone compounds extracted from Radix Puerariae, with the effects of blocking β receptor, increase cerebral blood flow and cerebral metabolism, scavenging oxygen free radicals, promoting immunity, and improving the learning and memory function. Wang Wensheng's study reported that puerarin could inhibit amyloid β-stimulated inflammatory reaction of rat hippocampi, but puerarin is not easy to pass through blood-brain barrier(BBB), its application is limited in the diseases of central nervous system. Acetylpuerarin is a modified form of puerarin. It is more lipid-soluble than puerarin and can successfully cross the blood-brain barrier, with great clinical application value. Previous research has shown that acetylpuerarin's protective effect on cerebral ischemia reperfusion, its mechanism may be mediated by NMDA receptors, anti oxygen free radical, lipid peroxidation, inhibition of C-fos and ICAM-1, Induction of VEGF. But the study regarding whether acetylpuerarin can reduce the inflammatory effects of AD is few.
Chitosan is a deacetylated product of chitin, a kind of cationic polymer, natural alkaline, non-toxic, safe and reliable, and has good biocompatibility. Foreign studies showed that chitosan can regulate cell differentiation, proliferation and cytokine production, scavenging free radicals, and the toxin in the body, protecting the brain nerve cells and glial cell membrane. A study found that high molecular weight water-soluble chitosan can prevent nerve cell apoptosis. Another study on anti-inflammatory effects of water-soluble chitosan suggested that it can reduce and delay the pathological changes of AD. Lecithin is the main component of membrane of cells and has the effect of anti-inflammation and enhances immune function, the choline in it can be used as the precursor of acetylcholine. Once the complex of lecithin with chitosan is formed, it can be used as carrier and carry chitosan through BBB. We used the method of solvent dispersion and wet grinding for the preparation of chitosan phospholipid complex before, and undertook the basic and clinical research on the treatment of senile dementia, and found that chitosan phospholipid choline can repair nerve cell membrane damage of dementia rats, improve memory capacity of dementia rats and patients with senile dementia, increase the brain acetylcholine content, scavenge free radicals, improve brain function. But the antiinflammatory effect and mechanism of the chitosan phospholipid choline on AD has not been eported in the literature.
This study inclueds the following two parts:
1. Mechanisms of inflammation in AD rats induced by Aβ and the intervention effect of Acetylpuerarin.
2. Effects and its mechanisms of Chitosan Phosphatidylcholine on learning and memory and inflammation in AD rats induced by Aβ.
Part I Mechanisms of Inflammation in AD Rats Induced by Aβ and the Intervention Effect of Acetylpuerarin
Objective
This study used in vivo experiments, from the whole, tissue, cell, molecular levels to observe the learning and memory function and the antiinflammatory mechanism of acetylpuerarin on AD model rats. The bilateral hippocampal injection of Aβ1-42induced AD model Wistar rats was used as subjects, and behavior, immunohistochemistry and molecular biology techniques were used to explore the effect and mechanism of acetylpuerarin on AD rat model, aimed to explain the effect of acetylpuerarin on AD and antiinflammatory mechanisms, so to provide strategy and theory basis for AD treatment.
Methods
The bilateral intrahippocampal Aβ1-42injection induced AD model rats were used as the research subjects.40Wistar rats were randomly divided into blank control group, model group (Aβ only group), acetylpuerarinin low dose group and acetylpuerarinin high dose group, with10rats in each group. After14days, rats of acetylpuerarin low dose group and high dose group were given intraperitoneal injection of acetylpuerarin100mg/kg and200mg/kg respectively, a total of12days. The control group and model group were given intraperitoneal injection of normal saline, a total of12days. Morris water maze test was used to observe the learning and memory function of acetylpuerarin on Aβ1-42induced rats from the overall level, including the escape time(average escape latency, AEL), and time in the original platform quadrant explorating the platform. Immunohistochemistry and Transmission Electron Microscopic examination methods were used to observe the effects of acetylpuerarin on microglia of cortex and hippocampus. And immunohistochemical staining method was applied to observe the expression of PKC-δ, IKK-β, iNOS in hippocampus. Western-blot method was used to observe the protein expression of PKC-δ, IKK-β and IL-1β in rat hippocampus. Elisa method was applied to detect the changes of IL-6in serum of rats.Values are given as mean±standard deviation, and analysis of variance and t-tests were performed with SPSS17.0software. P<0.05was considered to be statistically significant.
Results
1. Influence of Aβ1-42to learning and memory of Rats and the effects of Acetylpuerarin
We tested escape latency and exploring time in target area in the Morris water maze (MWM) before surgery,2weeks after surgery, and26days after surgery (after treatment). We found that the Aβ1-42treated rats were significantly impaired2weeks after surgery, compared with the control group, their mean escape latency was significantly increased in the place navigation test (P<0.01), and mean exploring time in the target area was significantly decreased in the spatial probe test (P<0.05for Acetylpuerarin low dose group; P<0.01for Acetylpuerarin high dose group). After12days of acetylpuerarin treatment, we found that both doses significantly attenuated Aβ1-42induced learning and memory deficits, as evidenced by the decreased escape latency in the place navigation test (P<0.01), and longer mean exploring time in the target area in the spatial probe test (P<0.05for Acetylpuerarin low dose group;P<0.01for Acetylpuerarin high dose group).
We did not observe any changes in locomotion during the MWM, suggesting that the increased time was due to impaired learning rather than difficulty swimming.
2. Influence of Aβ1-42to Microglial of rats and the effects of Acetylpuerarin
Cerebral cortex Ibal immunohistochemical staining results:in the model group, there were more MG cells, with thick body, yellow dye, color depth; in Acetylpuerarin low dose group and high dose group, there were less number of MG, with smaller size, light color; in control group, theere were the least MG cells, with pale staining. Ibal positive cell counting analysis showed that microglial cell number increased significantly (P<0.05) in model group compared with the control group, while compared with the model group, the number of microglial cells in Acetylpuerarin low dose group and high dose group was significantly reduced (Acetylpuerarin low dose group, P<0.05; acetyl puerarin in high dose group, P<0.01).
Transmission electron microscope showed:microglia of the control group is small, with clear rough endoplasmic reticulum and mitochondria, clear nucleolus, obvious heterochromatin, high electron density. The model group microglial cell has many neurites, less heterochromatin and loose, rich cytoplasm, and the number of ribosomes increased, with visible lysosomal granules. Low doses Acetylpuerarin group:low cytoplasmic electron density, with visible broken mitochondrial crista. High doses Acetylpuerarin group:big nuclear chromatin, with visible Golgi complex.
3. Influence of Aβ1-42to PKC-δ and IKK-β in hippocampal tissue and the effects of Acetylpuerarin
PKC-δ and IKK-β were selected as markers related to NF-κB pathway in this study. Immunohistochemistry method was used to detect the PKC-δ and IKK-β staining reaction of hippocampal tissue. PKC-δ staining results:the immunoreactive positive substance was brown, localized in cell membrane and cytoplasmic. PKC-8immunoreactive cells was the most in AD model group, with the deepest coloring, most positive granular cells, mainly located in the cytoplasm; there were less positive cells in control group and drug groups, and the cytoplasm of neurons was light coloured. Quantitative analysis showed:the number of PKC-8positive cells in model group was higher than that of control group, Acetylpuerarin low dose group and Acetylpuerarin high dose group, with significant statistical difference (P<0.05), and in a dose-dependent manner.
Similar result was also observed in IKK-β immunoreactive staining:in model group, with the most IKK-P positive cells and a dark brown color. There were Less IKK-β positive cells in the control group,basically no expression of brown granules of IKK-β, occasionally the cytoplasm of neurons light colored. There was a small amount of IKK-P immune immunoreactive cells in Acetylpuerarin groups, with shallow coloring. Quantitative analysis showed:the number of IKK-P hippocampal positive cells in model group was more than that in the control group, Acetylpuerarin low dose group and Acetylpuerarin high dose group, with significant statistical difference (P<0.05), and in a dose-dependent manner.
Western blot method was used to observe the expression of PKC-δ and IKK-β in the hippocampus of rats, and the results showed:bilateral hippocampal injection of Aβ1-42can significantly increase the expression of PKC-δ and IKK-β, approximately2times as much as normal control group. Acetylpuerarin therapy can significantly reduce the expression of PKC-δ and IKK-β induced by Aβ1-42,with significant statistical difference(P<0.05), and in a dose-dependent manner.
4. Influence of Aβ1-42to the expression of hippocampal iNOS、IL-1β and serum IL-6and the effects of Acetylpuerarin
iNOS, IL-1β and IL-6were chosen as the markers of inflammation for this study. Immunohistochemistry method was used to detect the iNOS expression in hippocampal tissue. The staining result of iNOS showed:the positive material of immunoreactivity was brown, localized in cell membrane and cytoplasm. Among them, there were the most immunoreactive iNOS positive cells in model group, with the deepest coloring, and the most positive granular. The immunoreaction positive cells in control group and drug groups were less, with lightly coloured cytoplasm of neurons. Quantitative analysis showed:the number of iNOS hippocampal positive cells in model group was more than that in the control group, Acetylpuerarin low dose group and Acetylpuerarin high dose group, with significant statistical difference (P<0.05), and in a dose-dependent manner.
Western blot method was used to detect the quantitative expression of IL-1β in hippocampal tissue. The results showed that bilateral hippocampal injection of Aβ1-42can significantly increase the expression of IL-1β, about3times as much as normal control group. Acetylpuerarin therapy can significantly reduce the expression of IL-1β induced by Aβ1-42, with significant statistical difference (P<0.05), and in a dose-dependent manner.
Elisa method was used to detect the serum IL-6level of rats.Results showed after Acetylpuerarin treatment, the level of serum IL-6of control group, Acetylpuerarin low dose group and Acetylpuerarin high dose group was significantly lower than that in the model group respectively, with significant statistical difference (P<0.05).
Conclusions
1. Aβ1-42bilateral intrahippocampal injection method can successfully simulate the inflammatory AD model rats, which can be used to test the effects of drugs.
2. Acetylpuerarin has obvious effect on learning and memory disorder in AD model rats induced by Aβ1-42.Thus infer, acetylpuerarin can become a new drug for prevention of the occurrence and development of AD.
3.The mechanism of Acetylpuerarin may possibly through inhibit microglia proliferation and activation, down-regulate the expression of brain PKC-δ, IKK-β, thus inhibit the activation of NF-κB signal channel, down-regulate the secretion of iNOS, IL-1β and IL-6,decrease the inflammatory reaction of brain cortex and hippocampus neurons, which play a role in brain protection and anti dementia.
Part Ⅱ Effects and its Mechanisms of Chitosan Phosphatidylcholine on Learning and Memory and Inflammation in AD Rats Induced by Aβ
Objective
This study used in vivo experiments, from the whole and tissue levels to observe the learning and memory function and the anti-inflammatory mechanism of Chitosan Phosphatidylcholine on AD rats. The bilateral hippocampal injection of Aβ25-35induced AD model Wistar rats were used as subjects. We used behaviorial and immunohistochemistry techniques to observe the effect and anti inflammation mechanism of chitosan phosphatidyl choline induced by Aβ in rats, aims to explain the effect of Chitosan Phosphatidylcholine on AD and anti-inflammatory mechanisms, so to provide strategy and theory basis for AD treatment.
Methods
The bilateral intrahippocampal Aβ25-35injection induced AD model rats were used as the research subjects.50rats were randomly divided into5groups:blank control group, model group (AP only group), Chitosan Phosphatidylcholine low dose group, midian dose group and high dose group, with10rats in each group. After7days, rats of Chitosan Phosphatidylcholine low dose group, midian dose group and high dose group were given Chitosan Phosphatidylcholine of0.2、0.6and1.0g/(kg·d) respectively by the way of intragastrition, a total of30days. Rats of the control group and model group were given equal dose of normal saline intragastrically, a total of30days. Morris water maze test was used to observe the learning and memory function of Chitosan Phosphatidylcholine on Aβ25-35induced rats, including the escape time(average escape latency, AEL), times crossing the postion of platform, and time staying in the original platform quadrant spending in explorating the platform. Immunohistochemical method was used to detect the expression of Ibal in cerebral cortex, and PKC-8, IL-1β, iNOS expression in hippocampus. Values are given as mean±standard deviation, and analysis of variance and t-tests were performed with SPSS13.0software. P<0.05was considered to be statistically significant.
Results
1. Chitosan Phosphatidylcholine Improved Learning and Memory
We tested escape latency, times crossing the position of platform, and target area exploring time in the Morris water maze (MWM) before surgery,14days after surgery, and30days after surgery. We found that the learning and memory ability of Aβ25-35treated rats was significantly impaired2weeks after surgery, compared with the control group, their mean escape latency was significantly increased in the place navigation test (P<0.01), with fewer times crossing the postion of platform (P<0.01), and mean exploring time in the target area was significantly decreased in the spatial probe test (P<0.01). After30days of Chitosan Phosphatidylcholine treatment, we found that Chitosan Phosphatidylcholine significantly attenuated Aβ25-35induced learning and memory deficits, as evidenced by the decreased escape latency in the place navigation test (P<0.01), more times crossing the postion of platform, and longer mean exploring time in the target area in the spatial probe test (P<001).
2. Chitosan Phosphatidylcholine Reduced Aβ25-35induced Microglial Activation
Cerebral cortex Ibal immunohistochemical staining results:in the model group, there was more MG cells, with thick cell body, yellow and deep color; in the chitosan phosphatidylcholine low dose group, midian dose group and high dose group, the number of MG cells decreased, the cell body became small, light color; in the control group, there was the least MG cells, with pale staining. Quantitative analysis showed: the number of Ibal positives cells in model group was significantly higher than that in normal group (P<0.05), the Number of Ibal positives cells in chitosan phosphatidylcholine low dose group, midian dose group and high dose group was significantly higher than that in normal group, but was lower than that in the model group P<05).
3. Chitosan Phosphatidylcholine Reduced Aβ25-35induced PKC-δ expression in rat hippocampus
The immune response level of PKC-δ in the rat hippocampus in model group was significantly higher than the control group, the chitosan phosphatidylcholine low dose group, median dose group, high dose group. The PKC-δ immunoreactive material was brown, localized in cell membrane and cytoplasm. PKC-δ immunoreactive cells was the most in AD model group, with the deepest coloring, most positive granular cells, mainly located in the cytoplasm; there were the least positive cells in control group and drug group, and the cytoplasm of neurons was light coloured. Quantitative analysis showed: the number of PKC-δ positive cells in model group was higher than that of control group, chitosan phosphatidylcholine low dose group, median dose group, high dose group, with significant statistical difference (P<005), and in a dose-dependent manner.
4. Chitosan Phosphatidylcholine reduced Aβ25-35induced iNOS and IL-1β expression in rat hippocampus
The immune response level of iNOS and IL-1β in the rat hippocampus in model group was significantly higher than the control group, the chitosan phosphatidylcholine low dose group, median dose group, and high dose group. The number of iNOS and IL-1β positive cells in chitosan phosphatidylcholine groups was lower than that in Aβ model group respectively, and the difference was statistically significant (P<0.05).Compared with the control group, the number of iNOS and IL-1β positive cells in Chitosan phosphatidylcholine low dose group, median dose group and high dose group had no significant statistical difference (P>0.05). Indicating that Aβ25-35could stimulate the expression of iNOS and IL-1β in rat hippocampuss, and chitosan phosphatidylcholine can reduce the expression of iNOS and IL-1β.
Conclusions
1.Using chitosan and soy lecithin can synthesize chitosan phosphatidylcholine compound microspheres by the method of spray drying.On one hand, it can improve chitosan's lipid solubility, be more effective for chitosan's action; on the other hand,it can play a better role for the supply of choline and protect neuron membrane for lecithin.
2. Chitosan phosphatidylcholine has obvious effect on Aβ25-35induced learning and memory impairment in AD rat model.
3. Chitosan phosphatidylcholine may be through the suppression of microglia in brain cortex and hippocampus, thereby reducing the expression of inflammation factors, such as iNOS and IL-1β, which exerts its neuroprotective effect.
4. Chitosan phosphatidylcholine may be through the downregulation of PKC-8in brain tissue, and inhibit the NF-kB pathway, thereby reducing the expression of inflammation factors.
引文
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