Effect of Oral Administration of Pheretima Aspergillum (Earthworm) in Rats with Cerebral Infarction Induced by Middle-Cerebral Artery Occlusion

We investigated the curative effect of Pheretima aspergillum (earthworm, PA) on rats with middle cerebral artery occlusion (MCAo). The MCAo-induced cerebral infarction was established and its underlying mechanisms by counting the infarction areas and evaluating the rats'' neurological status. Immunostaining was used to test the expression of NeuN, and glial fibrillary acidic (GFAP), S100B, and brain-derived neurotrophic factor (BDNF) proteins. Our results showed that oral administration of PA for two weeks to rats with MCAo successfully reduced cerebral infarction areas in the cortex and striatum, and also reduced scores of neurological deficit. The PA-treated MCAo rats showed greatly decreased neuronal death, glial proliferation, and S100B proteins in the penumbra area of the cortex and in the ischemic core area of the cortex, but BDNF did not changed. These results demonstrated novel and detailed cellular mechanisms underlying the neuroprotective effects of PA in MCAo rats     

Improvement and Application of Acute Blood Stasis Rat Model Aligned with the 3Rs (Reduction, Refinement and Replacement) of Humane Animal Experimentation

Objective: To establish a novel cardiocentesis method for withdrawing venous blood from the right atrium, and to improve an acute blood stasis rat model using an ice bath and epinephrine hydrochloride(Epi) while considering the 3 Rs(reduction, refinement, and replacement) of humane animal experimentation. Methods: An acute blood stasis model was established in male Sprague-Dawley rats by subcutaneous injection(s.c.) Epi(1.2 mg/kg) administration at 0 h, followed by a 5-min exposure to an ice-bath at 2 h and s.c. Epi administration at 4 h. Control rats received physiological saline. Rats were fasted overnight and treated with Angelicae Sinensis Lateralis Radix(ASLR) and Pheretima the following day. Venous blood was collected using our novel cardiocentesis method and used to test whole blood viscosity(WBV), prothrombin time(PT), activated partial thromboplastin time(APTT), and fibrinogen(FIB) content. Results: The rats survived the novel cardiocentesis technique; WBV value returned to normal while hematological parameters such as hemoglobin level and red blood cell count were restored to 94% of the corresponding values in normal rats following a 14-day recovery. Epi(1.2 mg/kg, s.c.) combined with a 5-min exposure to the ice bath replicated the acute blood stasis rat model and was associated with the highest WBV value. In rats showing acute blood stasis, ASLR treatment [4 g/(kg·d) for 8 days] decreased WBV by 9.98%, 11.09%, 9.34%, 9.00%, 7.66%, and 7.03%(P0.05), while Pheretima treatment [2.6 g/(kg·d), for 8 days] decreased WBV by 25.49%, 25.94%, 16.28%, 17.76%, 11.07%, and 7.89%(P0.01) at shear rates of 1, 3, 10, 30, 100, and 180 s-1, respectively. Furthermore, Pheretima treatment increased APTT significantly(P0.01). Conclusions: We presented a stable, reproducible, and improved acute blood stasis rat model, which could be applied to screen drugs for promoting blood circulation and eliminating blood stasis.     

HPLC法测定地龙注射液中主要氨基酸的含量

目的：建立测定地龙注射液中主要氨基酸含量的方法。方法：以6-氨基喹啉-N-羟基琥珀酰亚胺基氨基甲酸酯（AQC）为衍生剂,与地龙注射液中的游离氨基酸柱前定量衍生,用HPLC法测定衍生物含量。结果：丙氨酸、缬氨酸、盐酸赖氨酸、异亮氨酸和亮氨酸在各自浓度范围内线性关系良好（r≥0．9998）,平均回收率102．6％～104．9％,RSD0．3％～0．9％。结论：本法简便、准确,可用于地龙注射液的生产和质量控制。     

一种新型蚯蚓纤溶酶的研究

采用加提取剂的匀浆、盐析、超滤及层析方法从一种环毛蚓中获得一种新型纤溶酶。此酶分子量为22000,为单链蛋白。它不仅有强烈的直接溶解血栓及人纤维蛋白活性,而且还有纤溶酶原激活作用;动物试验表明此酶的毒副作用很小。本方法获得的酶的活性、纯度及得率都很高。     

修饰蚯蚓纤溶酶的药代动力学研究

环毛蚓纤溶酶经聚乙二醇（PEG）和β-环糊精（β-CD）修饰后，分别静脉注射到猪体内和兔体内进行药代动力学研究。结果表明PEG-EFE在猪体内的半衰期为0.96h，而原酶在猪体内的半衰期为0.59h，PEG-EFE较原酶提高了1.62倍。β-CD-EFE注射于兔体内的药代动力学证明，β-CD-EFE在兔体内的分布相半衰期为1.00h，而原酶仅为0.65h，β-CD-EFE较原酶的体内半衰期提高了1.52倍。说明2种修饰方法都是有效的。     

地龙提取物的抗凝血作用

目的研究地龙提取物的抗凝血作用。方法观察地龙提取物对小鼠凝血、出血时间和家兔离体血浆复钙时间的影响,测定提取物中游离氨基酸的量。结果地龙提取物能延长小鼠凝血、出血时间和家兔离体血浆复钙时间。游离氨基酸测定结果表明,提取物中含有丰富的氨基酸。结论地龙提取物所含的游离氨基酸可能是其抗凝血作用的有效成分。     

广地龙饮片炮制工艺研究

目的:优选广地龙饮片最佳炮制工艺。方法:采用正交试验法,以次黄嘌呤的含量为考察指标,对加水量,淘洗时间,干燥方式3个因素进行研究。结果:广地龙饮片的最佳炮制工艺为加5倍量的水,淘洗3 min,40℃烘干。淘洗时间对次黄嘌呤的含量有显著性差异,加水量和干燥方式对次黄嘌呤的含量影响不显著。结论:本研究对规范广地龙饮片炮制工艺具有一定意义。     

黄精地龙提取液对老年痴呆大鼠脑内烟碱型乙酰胆碱受体的影响

目的:探讨黄精地龙提取液(EPP)对老年痴呆大鼠脑内α3和α7烟碱型乙酰胆碱受体(α3,α7-nAChR)阳性神经元数量、蛋白及mRNA表达的影响。方法:SD大鼠40只,随机均分为正常组,模型组,脑复康(阳性药物,0.6 g·kg~(-1)·d~(-1))组,EPP(相当生药量4 g·kg~(-1)·d~(-1))组。给大鼠颈背部皮下注射1%D-半乳糖(5 m L·kg~(-1)·d~(-1),连续3周)后,腹腔注射东莨菪碱(2 mg·kg~(-1)·d~(-1),连续后2周)复制大鼠老年痴呆模型;造模1周后同时对脑复康组及EPP组大鼠每天灌胃给相应实验药物,正常组及模型组大鼠灌胃给生理盐水1 mL。实验结束,处死各组大鼠,取出脑组织,一半置于10%中性甲醛液浸泡固定待组织切片检查,另一半置于冰上匀浆,进行mRNA及蛋白提取;运用尼氏染色、免疫组化观察各大鼠大脑皮层第一躯体感觉区(S1Tr)和海马CA1与CA3区的阳性神经元数量;实时荧光定量聚合酶链式反应(Real-time PCR),蛋白质免疫印迹(Western blot)检测各大鼠大脑内的α3和α7-nAChR mRNA及蛋白的表达。结果:与正常组比较,模型组大鼠大脑S1Tr区和海马CA1及CA3区神经元数量显著减少,大脑内α3和α7-n AChR mRNA和蛋白表达水平均降低(P0.01);EPP能增加痴呆大鼠大脑S1Tr区和海马CA1及CA3区神经元数量,能升高痴呆大鼠大脑内α3和α7-n AChR mRNA和蛋白的表达水平,与模型组比较差异有统计学意义(P0.05,P0.01)。结论:EPP能保护大脑认知学习记忆有关重要区的神经细胞,同时触发α3和α7-n AChR受体效应,提高中枢乙酰胆碱(ACh)含量,进而升高老年痴呆大鼠中枢胆碱能系统活性。     

地龙的多重位点特异性PCR鉴别

目的:近年来,中药材地龙的商品价格不断上涨,市场的混伪品随之增加。其主要鉴别特征在药材中大部分丢失,难以区分。因此,急需建立一种准确、稳定的中药材地龙基原鉴别方法。方法:根据地龙及其混伪品的COI基因序列差异找到变异位点,从而设计参环毛蚓、通俗环毛蚓、威廉环毛蚓及栉盲环毛蚓的特异性鉴别引物,优化反应体系并进行耐受性和适用性的考察和验证。在此基础上将特异性引物组合,构建多重聚合酶链式反应(PCR)体系,从而建立了一种高效准确、操作简便、专属性强、重复性好的中药材地龙及其常见混伪品的多重位点PCR鉴别方法。结果:在该方法下广地龙基原参环毛蚓可得到366 bp片段,沪地龙基原通俗环毛蚓和威廉环毛蚓可得到487 bp片段,栉盲环毛蚓可得到475 bp片段,其他混伪品均无条带。将之组合形成的多重PCR可在一次鉴定操作中根据条带位置同时鉴别沪地龙和广地龙。结论:该文所建立的多重位点特异性PCR鉴别方法可准确鉴别中药材地龙真伪。     

正交设计优选地龙的提取工艺

目的优选地龙的最佳提取工艺。方法通过琼脂纤维蛋白平板法测量溶圈面积,优选出用水匀浆法作为地龙的提取方法;综合考虑得率和溶圈面积这两个指标,采用正交实验对地龙的水匀浆工艺条件进行优化。结果最佳工艺参数为:即加入5倍量水,浸渍2h,以10000r/min转速在40℃下匀浆6h。结论优选出的工艺稳定可行。