摘要
一、目的与意义
颈椎病又称颈椎综合症(Cervical syndrome),是颈部椎间盘、骨骼、软骨、韧带发生退行性变,继发性刺激或压迫周围或临近的脊髓、神经根、血管、软组织、交感神经等组织结构,并由此而引起各种不适的一组症候群。流行病学资料显示：2002年广东省佛山市大学教职工及其家属调查显示：患病率10.78%,2008年兰州市居民患病率13.70%。2002年湘雅二院体检病人中各职业颈椎病发病率分别为干部、技术员、财会人员发病率分别高达78.83%、74.21%、58.70%,其中工作紧张、长期伏案者占59.75%；2005年上海陆家嘴地区金融从业人员的调查显示：患病率17.53%；2008年武汉市中小学生中有颈椎病症状的高达30%。
随着工作、生活方式的改变,如长期伏案工作、电脑和空调的广泛使用、私家车的普及等,人们屈颈和遭受风寒湿的机会不断增加,造成颈椎病的患病率不断上升,且发病年龄有年轻化的趋势。
颈椎间盘退变被认为是颈椎病发生和发展的根本原因。退变的椎间盘出现含水能力降低,蛋白多糖含量下降,胶原类型发生转换,炎症介质大量释放。椎间盘的主要生物力学功能是维持椎间隙高度,对抗压缩力。颈椎病患者的影像学检查可见椎间隙变窄的改变。多种因素参与了椎间盘退变的病理过程。其中,椎间盘细胞营养供给的减少是椎间盘退变的基本因素。Nerlich等认为椎间盘退变的最关键因素是营养供应减少。
血管内皮细胞生长因子(VEGF)是在胚胎发生和创伤愈合过程中启动血管形成的一个高度特异的有丝分裂原,也是一种有效的血管通透性诱导因子,VEGF的表达与组织中微血管的密度及新生血管的数量密切相关。VEGF可由许多正常细胞产生和分泌,血管内皮细胞在正常情况下不表达VEGF,但当内皮细胞被置于缺氧环境时,细胞内VEGF的mRNA水平明显增加,表明VEGF不仅通过旁分泌途径作用于内皮细胞,而且有自分泌机制参与。退变椎间盘血管内瘀血导致压力增加,血管受压,循环受阻,局部组织缺氧,从而诱导VEGF的表达。
水通道蛋白的主要功能是转运水。根据AQPs对水和其它溶质的通透性,Ishibashi将AQPs家族分为两个亚族:AQPO, AQP1, AQP2, AQP4, AQP5, AQP6, AQP8为一亚族,AQP3, AQP7, AQP9、10属于另一亚族。椎间盘是一个富含水分的器官,其中髓核的含水量约为80%。而退变椎间盘的一个特征性表现即为含水量的下降,在MRI表现为T2加权信号减弱。Miyanoto等将髓核皱缩或消失作为椎间盘退变的病理指标。所以水的代谢在维持椎间盘的正常生理功能中具有重要的作用。水作为主要的营养成分之一,其在渗透压的作用下,进出椎间盘组织,并保持软骨终板的水化状态。营养物的渗透率与纤维环与终板的水化程度密切相关,含水越多,渗透率越高,渗透速度越快。水作为一种营养物质,对其它营养物质的吸收有重要的作用。故研究水的代谢对了解椎间盘退变的始动因素有着重要的意义。
课题组根据长期临床实践,以传统中医理论自拟“颈椎舒”方,临床运用近20年,疗效肯定。前期的临床研究和动物实验提示,“颈椎舒”方能改善微循环、降低血小板聚集率、降低血粘度、增加血管壁弹性、扩张血管,能显著改善神经根型颈椎病患者疼痛、麻木、僵硬、寒冷、肌肉萎缩无力等症状。君药葛根和川芎提取物能修复受损神经根,提高神经传导速度、升高波幅,对神经根型颈椎病根性痛有明显的止痛作用。
本研究通过测量椎间盘高度变化与椎间盘含水量之间的关系,明确颈椎舒颗粒治疗椎间盘退变的途径。通过比较治疗前后椎间盘退变程度、椎间盘高度、椎间盘含水量与水通风道蛋白1、3及血管内皮生长因子的变化关系,进一步明确颈椎舒颗粒预防椎间盘退变的机理。本文进一步研究了颈椎舒颗粒中的主要有效成分葛根素对水通道蛋白1、3的调节作用,探讨复方有效成分对椎间盘退变的作用。
二、方法与内容
1大鼠颈椎间盘退变模型的建立
健康清洁级SD雄性大鼠,体重200-220g,购自南方医科大学实验动物中心(动物质量合格证号：SCXK粤2006-0015)。
将非假手术组大鼠颈后部剪毛和清洁后,用10%水合氯醛按3.5m1.kg1体重腹腔内注射,麻醉成功后,颈后部剪毛,碘伏消毒,取颈背部正中纵向切口,长约2-2.5cm,切开皮肤后,充分游离各层肌肉,横向切断深群颈夹肌和头、颈、寰最长肌,完全切除颈髂肋肌与头半棘肌,然后再依次切断C2-C7棘上和棘间韧带,彻底止血后缝合皮肤。术后3天给予青霉素肌注,8万μ/天。伤口感染水肿较严重的可适当延长注射时间或增大注射剂量。假手术组的大鼠切开皮肤后就彻底止血后缝合皮肤。术后12周后造模完成。
2分组与药物干预
2.1颈椎舒颗粒的干预作用
将SD大鼠140只,适应性喂养一周后,按上述方法建立动物模型,其中手术组大鼠124只,假手术中16只。假手术中16只全部造模成功。手术组在手术中及术后共死亡14只,随机抽取80只分成5组分别为：颈椎舒颗粒高剂量组、颈椎舒颗粒中剂量组、颈椎舒颗粒低剂量组、模型组、颈复康组,每组16只。上述6组在干预4周、8周后各处死8只大鼠。
折算各组药物的大鼠灌胃量：人的每日剂量折算成大鼠的中等剂量。颈椎舒颗粒的高、中、低剂量分别设置为2.8g·kg-1.·d-1、1.4g·kg-1·d-1、0.7g·kg-1·d-1。颈复康颗粒根据推荐剂量确定为0.7g·kg-1·d-1。模型组和假手术组给予等容积生理盐水。上述药物配制成相同体积不同浓度的药液。
2.2葛根素对退变椎间盘中AQP1、3的影响
将SD大鼠90只,适应性喂养一周后,按前述方法建立椎间盘退变大鼠模型。其中非手术组大鼠80只,假手术中8只。假手术中8只全部造模成功。手术中及术后共死亡9只。随机抽取40只分成5组分别为：葛根素高剂量组、葛根素中剂量组、葛根素低剂量组、模型组、西乐葆组。上述6组在干预8周后处死。颈椎舒颗粒中葛根素的含量约为1.2%,确定葛根素高、中、低的剂量分别为80mgkg·kg-1·d-1、40mg·kg-1·d-1、20mg·kg-1·d-1,将葛根素配成等体积溶液,西药组给予西乐葆胶囊18mg·kg-1·d-1腹腔注射。假手术组、模型组注射同等体积的生理盐水。
3 X片观察：
分别在灌胃4周和8周时,3%戊巴比妥钠40mg·kg-1腹腔内注射,麻醉后拍摄大鼠颈椎的正侧位片,严格掌握麻醉药剂量以及麻醉后至X光片拍摄的时间,以控制肌肉紧张程度对椎间盘高度的影响。计算C4-5椎间盘高度指数(DHI)。
4病理切片观察：
分别在给药4周及8周后用急性失血法处死大鼠,立即取大鼠整段颈椎,范围自枕骨基底部到第二胸椎,每只大鼠的C5-6椎间盘及相邻椎体分别放在10%福尔马林溶液固定,EDTA脱钙2周,正中矢状位切开颈椎,用酒精梯度脱水,二甲苯透明,石蜡块包埋,切片机切片,苏木素-伊红染色,显微镜下观察髓核、纤维环、终板及椎体退变情况。
Miyamoto等的分级标准对椎间盘评分：
1级：正常椎间盘,纤维环与髓核排列规则,软骨终板分为生长软骨和关节软骨层,潮标清晰。(1分)
2级：纤维环板层结构消失,软骨终板增生。(2分)
3级：髓核皱缩或消失。(3分)
4级：纤维环有裂隙。(4分)
5级：椎间盘突出或骨赘形成。(5分)
5椎间盘含水量的变化：
采用真空冻干法测定椎间盘水含量。将大各组鼠处死后,其中C4-5椎间盘用于水含量测定。取完整椎间盘组织,剥除周围软骨组织,以电子天平称重记录(湿重)。椎间盘组织置于-80℃真空冻干机内冷冻干燥24小时后称重(干重)。椎间盘水含量记算公式如下：椎间盘含水量=湿重—干重。根据上述公式计算各组动物椎间盘水含量。
6 Western blotting法检测椎间盘水通道蛋白AQP1、3的变化
将冻干称重后的C4-5椎间盘用细胞裂解液裂解组织,BCA定量后上样。在有足量电泳缓冲液的恒压电泳仪进行电泳(5%积层胶80V,10%分离胶120V)。电泳结束后,转膜、封闭,然后依次加入一抗、二抗,各步骤之间用TBST充分洗涤。最后用ECL显色液浸湿后显像。同上步骤测各组β-actin的蛋白表达。用AQP1/β-actin的平均灰度值表示AQP1的相对蛋白表达量；用AQP3/β-actin的平均灰度值表示AQP3的相对蛋白表达量。
7 Western blotting法检测椎间盘VEGF的变化
将冻干称重后的C4-5椎间盘用细胞裂解液裂解组织,BCA定量后上样。在有足量电泳缓冲液的恒压电泳仪进行电泳(5%积层胶80V,10%分离胶120V)。电泳结束后,转膜、封闭,然后依次加入一抗、二抗,各步骤之间用TBST充分洗涤。最后用ECL显色液浸湿后显像。同上步骤测各组β-actin的蛋白表达。用VEGF/β-actin的平均灰度值表示VEGF的相对蛋白表达量。
8数据处理
数据用SPSS16.0软件进行析因设计的方差分析,计量资料用均数±标准差(x±s)表示。各组大鼠治疗4周和8周椎间盘含水量、椎间盘高度指数、AQP1、AQP3和VEGF的变化比较,采用两独立样本t检验(Two Independent-Samples t Test)进行统计分析,不同用药组间比较采用单因素方差分析(One-way ANOVA)进行统计分析,方差齐时多重比较采用LSD-t法检验；方差不齐时,用近似方差分析的Welch法,多重比较采用Dunnett T3法检验。各组大鼠退变等级采用多组等级资料,对等级资料采用频数进行统计描述,采用多个独立样本非参数检验Kruskal-Wallis法,P<0.05为有统计学意义。
三、结果
所有大鼠无一脱失,均进入实验结果分析,无明显的体重减轻。伤口愈合良好,无自噬及舔咬,无脓性分泌物。
1颈椎舒颗粒对大鼠退变颈椎间盘高度指数变化的影响
4周组与8周组大鼠颈椎间盘高度指数差异有显著性意义(F=5.52,P=0.026),各组之间差异有统计学意义(F=60.28,P=0.000),各时间点与分组间有交互效应(F=4.45,P=0.001),说明组间差异在4周和8周不一样。
4周组与8周组两独立样本t检验显示,各组除假手术组差异无显著性意义(t=0.79,P=0.454)外,其余各组用药4周、用药8周大鼠椎间盘高度指数差异均有显著性意义(分别为t=10.78,t=7.64,t=11.53,t=15.28,t=9.75；P<0.01)。
组间多重比较,用药4周后,与模型组相比,各组均能增加椎间盘高度指数,差异有显著性意义(P<0.05或P<0.01)；高剂量组与中剂量组、颈复康组比较差异无显著性意义(P>0.05)；中剂量组与低剂量组比较差异有显著性意义(P<0.05)。各药物干预组,以高剂量组疗效最佳,高剂量组椎间盘高度指数最高,模型组最低。用药8周后,与模型组相比,各组组均能增加椎间盘的高度指数,差异有显著性意义(P<0.05或P<0.01)；高剂量组与中剂量组、颈复康组比较差异无显著性意义(P>0.05)；中剂量组与低剂量组比较差异有显著性意义(P<0.05)。各药物干预组,以高剂量组疗效最佳,高剂量组椎间盘高度指数最高,模型组最低。
由上说明,与模型组相比,各药物干预组均能增加椎间盘的高度指数,并且存在时间和剂量依赖性,其中颈椎舒颗粒高剂量组疗效最好。
2颈椎舒颗粒对大鼠退变颈椎间盘病理切片变化的影响
由病理切片可见,假手术组大鼠颈椎间盘潮标清晰可见,纤维环结构清晰,髓核组织未见明显的皱缩,钙化的关节软骨较薄,钙化的软骨下分布有大的血管,4周组与8周组差异不明显。模型组大鼠颈椎间盘出现退行性改变,纤维环板层结构紊乱,排列轻度不规则,髓核组织皱缩或变小,少数髓核轻度突出,关节软骨钙化层及非钙化层均增厚。8周组较4周组变化明显。各药物干预组：大鼠椎间盘形态较模型组有明显改善。颈椎舒颗粒各剂量组大鼠椎间盘形态接近对照组,光镜下观察形态结构较完整,纤维环排列比较规则,髓核比较饱满,潮标可见,终板内血管充血情况改善,血管芽多于模型组,其中8周组较4周组变化明显。
治疗4周后,6组之间差异有统计学意义(X2=11.43,P=0.043)；椎间盘退变级别秩次分别为20.88、26.12、30.25、33.56、22.81,13.38根据平均秩次推断,以颈椎舒高剂量组退变级别最少,颈复康组次之,模型组最多。治疗8周后,6组之间差异有显著性意义(X2=12.06,P=0.034)；颈椎舒高、中、低剂量组,模型组,颈复康组,假手术组发作级别秩次分别为20.12、25.00、29.12、36.06、21.50、15.19,根据平均秩次推断,高剂量组退变级别最轻,颈复康组次之,模型组最多。
3颈椎舒颗粒对大鼠退变颈椎间盘含水量变化的影响
4周组与8周组大鼠颈椎间盘的含水量差异有显著性意义(F=10.52,P=0.002),各组之间差异有统计学意义(F=220.78,P=0.000),各时间点与分组间有交互效应(F=6.69,P=0.000),说明组间差异在4周和8周不一样。
4周组与8周组两独立样本t检验显示,各组除假手术组差异无显著性意义(t=1.23,P=0.241)外,其余各组用药4周、用药8周大鼠椎间盘含水量差异均有显著性意义(分别为t=3.34,P=0.005；t=2.30,P=0.037；t=2.75,P=0.016；t=5.89,P=0.000；t=4.02,P=0.001)。
组间多重比较,用药4周后,与模型组相比,各组组均能增加椎间盘的含水量,差异有显著性意义(P<0.05或P<0.01)；高剂量组与中剂量组、颈复康组比较差异无显著性意义(P>0.05)；中剂量组与低剂量组比较差异有显著性意义(P<0.05)。各药物干预组,以高剂量组疗效最佳,高剂量组含水量最高,模型组最低。用药8周后,与模型组相比,各组组均能增加椎间盘的含水量,差异有显著性意义(P<0.05或P<0.01)；高剂量组与中剂量组、颈复康组比较差异无显著性意义(P>0.05)；中剂量组与低剂量组比较差异有显著性意义(P<0.05)。各药物干预组,以高剂量组疗效最佳,高剂量组含水量最高,模型组最低。
由上说明,与模型组相比,各药物干预组均能增加椎间盘的含水量,并且存在时间和剂量依赖性,其中颈椎舒颗粒高剂量组疗效最好。
4颈椎舒颗粒对大鼠退变颈椎间盘AQP1变化的影响
4周组与8周组大鼠颈椎间盘水通道蛋白1的含量差异有显著性意义(F=70.65,P=0.000),各组之间差异有统计学意义(F=102.60,P=0.000),各时间点与分组间有交互效应(F=476.09,P=0.000),说明组间差异在4周和8周不一样。
4周组与8周组两独立样本t检验显示,各组除假手术组差异无显著性意义(t=1.60,P=0.184)外,用药4周、用药8周大鼠颈椎间盘水通道蛋白1的含量差异均有显著性意义(分别为t=7.50,P=0.000；t=12.51,P=0.000；t=6.15,P=0.004；t=9.40,P=0.001；t=9.23,P=0.001)。
组间多重比较,用药4周后,与模型组相比,各组均能增加颈椎间盘水通道蛋白1的含量,差异有显著性意义(P<0.05或P<0.01)；高剂量组与中剂量组、颈复康组比较差异无显著性意义(P>0.05)；中剂量组与低剂量组比较差异有显著性意义(P<0.05)。各药物干预组,以高剂量组疗效最佳,高剂量组颈椎间盘水通道蛋白1的含量最高,模型组最低。用药8周后,与模型组相比,各组组均能增加颈椎间盘水通道蛋白1的含量,差异有显著性意义(P<0.05或P<0.01)；高剂量组与中剂量组、颈复康组比较差异无显著性意义(P>0.05)；中剂量组与低剂量组比较差异有显著性意义(P<0.05)。各药物干预组,以高剂量组疗效最佳,高剂量组水通道蛋白1最高,模型组最低。
由上说明,与模型组相比,各药物干预组均能增加颈椎间盘水通道蛋白1的含量,并且存在时间和剂量依赖性,其中颈椎舒颗粒高剂量组疗效最好。
5颈椎舒颗粒对大鼠退变颈椎间盘AQP3变化的影响
4周组与8周组大鼠颈椎间盘水通道蛋白3的含量差异有显著性意义(F=25.43,P=0.000),各组之间差异有统计学意义(F=665.59,P=0.000),各时间点与分组间有交互效应(F=26.07,P=0.000),说明组间差异在4周和8周不一样。
4周组与8周组两独立样本t检验显示,各组除假手术组差异无显著性意义(t=2.44,P=0.720)外,用药4周、用药8周大鼠颈椎间盘水通道蛋白3的含量差异均有显著性意义(分别为t=7.00,P=0.002；t=10.39,P=0.000；t=13.79,P=0.000；t=5.16,P=0.007；t=9.31,P=0.001)。
组间多重比较,用药4周后,与模型组相比,各组均能增加颈椎间盘水通道蛋白3的含量,差异有显著性意义(F=566.36,P=0.000)。各药物干预组,以高剂量组疗效最佳,高剂量组颈椎间盘水通道蛋白3的含量最高,模型组最低。用药8周后,与模型组相比,各组组均能增加颈椎间盘水通道蛋白3的含量,差异有显著性意义(P<0.05或P<0.01)；高剂量组与中剂量组、颈复康组比较差异无显著性意义(P>0.05)；中剂量组与低剂量组比较差异有显著性意义(P<0.051。各药物干预组,以高剂量组疗效最佳,高剂量组水通道蛋白3量最高,模型组最低。
由上说明,与模型组相比,各药物干预组均能增加颈椎间盘水通道蛋白3的含量,并且存在时间和剂量依赖性,其中颈椎舒颗粒高剂量组疗效最好。
6颈椎舒颗粒对大鼠退变颈椎间盘VEGF变化的影响
4周组与8周组大鼠椎间盘VEGF的含量差异有显著性意义(F=18.345,P=0.000),各组之间差异有统计学意义(F=590.71,P=0.000),各时间点与分组间有交互效应(F=29.85,P=0.000),说明组间差异在4周和8周不一样。
4周组与8周组两独立样本t检验显示,各组除假手术组差异无显著性意义(t=2.92,P=0.056)外,用药4周、用药8周大鼠椎间盘VEGF的含量差异均有显著性意义(分别为t=12.81,P=0.000；t=4.66,P=0.010；t=6.39,P=0.003；t=7.95,P=0.001：t=5.44,P=0-006)。组间多重比较,用药4周后,与模型组相比,各组均能增加椎间盘VEGF的含量,差异有显著性意义(P<0.05或P<0.01)；高剂量组与中剂量组、颈复康组比较差异无显著性意义(P>0.05)；中剂量组与低剂量组比较差异有显著性意义(P<0.05)。各药物干预组,以高剂量组疗效最佳,高剂量组椎间盘VEGF的含量最高,模型组最低。用药8周后,与模型组相比,各组组均能增加颈椎间盘VEGF的含量,差异有显著性意义(P<0.05或P<0.01)；高剂量组与中剂量组、颈复康组比较差异无显著性意义(P>0.05)；中剂量组与低剂量组比较差异有显著性意义(P<0.05)。各药物干预组,以高剂量组疗效最佳,高剂量组VEGF最高,模型组最低。
由上说明,与模型组相比,各药物干预组均能增加椎间盘VEGF的含量,并且存在时间和剂量依赖性,其中颈椎舒颗粒高剂量组疗效最好。
7葛根素对大鼠退变颈椎间盘含水量的影响
水含量检测表明,葛根素高剂量组、中剂量组、低剂量组,模型组,西乐葆组,假手术组,各组水含量差异有显著性意义(F=321.26,P=0.000)。组间多重比较,葛根素高剂量组、中剂量组、低剂量组,西乐葆组,含水量高于模型组,差异有显著性意义(P值分别为0.000、0.000、0.013、0.003)。葛根素高剂量组、中剂量组含水量高于西乐葆组,差异有显著性意义(P值分别为0.000、0.011)。葛根素高剂量组、中剂量组与低剂量组相比差异有显著性意义(P值分别为0.000、0.013)。葛根素低剂量组与西乐葆组差异无显著性意义(P=0.998)。
由上说明,与模型组相比,各药物干预组均能增加椎间盘含水量,并且存在一定的量效关系,其中葛根素高剂量组疗效最好。与西乐葆相比除葛根素低剂量组疗效相当外,葛根素高剂量、中剂量组疗效均优于西乐葆组。
8葛根素对大鼠退变颈椎间盘水通道蛋白1的影响
水通道蛋白1检测表明,葛根素高剂量组、中剂量组、低剂量组,模型组,西乐葆组,假手术组,各组水通道蛋白1的含量差异有显著性意义(F=123.21,P=0.000)。组间多重比较,葛根素高剂量组、中剂量组、低剂量组,西乐葆组,水通道蛋白1的含量高于模型组,差异有显著性意义(P值分别为0.001、0.001、0.015、0.026)。葛根素高剂量组、中剂量组水通道蛋白1的含量高于西乐葆组,差异有显著性意义(P值分别为0.004、0.007)。葛根素高剂量组、中剂量组与低剂量组相比差异有显著性意义(P值分别为0.001、0.019)。葛根素低剂量组与西乐葆组差异无显著性意义(P=0.166)。
由上说明,与模型组相比,各药物干预组均能增加椎间盘水通道蛋白1的表达,并且存在一定的量效关系,其中葛根素高剂量组疗效最好。与西乐葆组相比除葛根素低剂量组疗效相当外,葛根素高剂量、中剂量组疗效均优于西乐葆组。
9葛根素对大鼠退变颈椎间盘水通道蛋白3的影响
水通道蛋白3检测表明,葛根素高剂量组、中剂量组、低剂量组,模型组,西乐葆组,假手术组,各组水通道蛋白3的含量差异有显著性意义(F=548.96,P=0.000)。组间多重比较,葛根素高剂量组、中剂量组、低剂量组,西乐葆组,水通道蛋白3的含量高于模型组,差异有显著性意义(P值分别为0.000、0.000、0.005、0.046)。葛根素高剂量组、中剂量组水通道蛋白3的含量高于西乐葆组,差异有显著性意义(P值分别为0.000、0.000)。葛根素高剂量组、中剂量组与低剂量组相比差异有显著性意义(P值分别为0.000、0.001)。葛根素低剂量组与西乐葆组差异无显著性意义(P=0.233)。
由上说明,与模型组相比,各药物干预组均能增加椎间盘水通道蛋白3的表达,并且存在一定的量效关系,其中葛根素高剂量组疗效最好。与西乐葆组相比除葛根素低剂量组疗效相当外,葛根素高剂量、中剂量组疗效均优于西乐葆组。
四、结论
1、颈椎舒颗粒能明显延缓大鼠颈椎间盘的退变级别,这一趋势存在一定的量效和时效关系；2、颈椎舒颗粒能从阻止椎间盘高度降低和减缓水含量减少的角度延缓大鼠颈椎间盘退变,并且这一趋势存在一定的量效和时效关系；3、颈椎舒颗粒能调节水通道蛋白1、3及血管内皮生长因子的表达,这可能是延缓椎间盘退变的原因之一；4、葛根素能调节水通道蛋白1、3的表达,并且存在一定的量效关系。可能是颈椎舒颗粒治疗椎间盘退变的机制之一。
Purpose and significance
Cervical spondylosis, is neck intervertebral discs, bone and cartilage and ligament degenerative change happened, this change stimulation or oppression of the spinal cord, around nerve root, the blood vessel, soft tissue, sympathetic nerve, etc, this organization caused a group of syndrome of discomfort.。Epidemiological data shows:Shanghai, foshan, lanzhou, the incidence of cervical spondylosis, respectively; 13.70% 10.78%,17.35%, Cadres, technicians, accounting personnel 78.83%, respectively 74.21% incidence 58.70%, including work, nervous and long-term volt case 59.75% 14.9% of; Wuhan have symptoms of cervical spondylosis of primary and middle school students as much as 30%. Along with the work and life style changes, such as long-term working at his desk, computer and the widespread use of air conditioning, the popularity of private cars, the people and suffering from the wind cold dampness neck flexion, causing the opportunity to increase the prevalence of cervical spondylosis rising, and onset ages have younger trend.
Cervical disc degeneration is considered the emergence and development of cervical spondylosis basic reasons. Degenerative disc appear capacity reduction, proteoglycan aquifer down the content, collagen type conversions, a large release inflammatory mediators. The main biomechanics function disc height, is to maintain the vertebral clearance against compression force. Cervical spondylosis patients imaging examination showed the vertebral clearance narrowed changes. Many factors involved in the disc degeneration pathological process. Among them, the disc cell nutrition decrease supply is the basic factors of cervical disc degeneration. Nerlich think disc degeneration of the most key factor is the nutrition supply reduced. VEGF is a highly specific mitotic original, it in embiyogeny and wound healing process start the formation of blood vessels. VEGF also is a kind of effective vascular permeability inducing factors, VEGF expression and organization of density and capillaries is closely related to the number of new blood vessels. VEGF by many kinds of normal cells can secrete. Vascular endothelial cells in does not normally express VEGF, but when the endothelial cells, when placed in hypoxic conditions inside cells mRNA VEGF was increased, indicating the level obviously not only through the paracrine way to remove VEGF in the role of endothelial cells, and there are secreted oneself mechanism involved. Degenerative disc of intravascular blood stasis caused pressure increases, vascular compression, circulation suffocate, local hypoxia, thus inducing VEGF expression.
The water channel protein main function is to transport water. According to AQPs for water and other solute permeability, Ishibashi is divide AQPs family into two and family AQP0, AQP1, AQP2:AQP4, AQP5, AQP6, AQP8 for a family, AQP7 AQP3, Abigail, AQP9,10, belong to other. Disc is a rich moisture organs, including the moisture content of nucleus pulposus about 80%. And a degenerative disc for water content characteristic expression that drop in MPI, T2 weighted signal weakening for performance. Miyanoto think the nucleus pulposus shrinks or disappear is disc degeneration pathological index. Water metabolism in maintaining the normal physiology function discs plays an important role. Water as the main nutrients in one of the role of osmotic pressure, the next, access, and maintain discs organization of cartilage endplate hydration state. Nutrients and the permeability of fiber ring and endplate is closely related to the hydration degree, the more the higher permeability aquifer, permeate the faster. Water, as a kind of nutrients to other nutrient absorption have important role. We investigated water metabolism for understanding the disc degeneration of tumor-initiating factors have important meaning.
Professor Chan famous Chinese medicine compound created a "Jingzhui Shu", through 20 years of clinical use was satisfied with the effect. Through early animal experiments and clinical trials, our team found that "Jingzhui shu" to improve microcirculation and reduce platelet aggregation, reduce blood viscosity, increased vascular wall flexibility, expansion vessel, can significantly improve nerve root cervical spondylosis Pain, numbness, stiffness, chills, muscle atrophy unable symptoms.
We compared the intervertebral disc height and water content, so that the relationship between the treatment of cervical disc degeneration shu particles of the way. Through comparing the before and after the treatment of cervical disc degeneration degree, disc height, disc moisture content and water 1,3 and ventilation protein vascular endothelial growth factor, further clarify the changing relations between the jingzhuishu Granule prevent cervical disc degeneration mechanism. We studied the regulation effects of puerarin AQP1、3, further discussing compound effective composition on disc degeneration role.
Method and content
1、Rats of cervical disc degeneration was established
3-month-old male SPF SD rat (200-220)g were used, in which conersion rat was in anesthesia through 10% Chloral hydrate(3.5ml/kg) was given by intraperitoneal injection.Then shearing rats'sheep, iodophors disinfect aseptic technique. These rats were given a median longitudinal incision about 2-2.5cm long on the back of neck. When the skin is incised, separating each layer muscle sufficiently, severing splenius cervicis, muscle longissimuscapitis, longissimus cervicis and longissimus atlantis at the transverse, excising costocervicalis and trachelo-occipitalis completely. Then excising ligamenta supraspinale and ligamenta interspinalia of C2-C7 in proper order, suturing peau after arresting bleeding completely. For 3 days after operation penicillin was given by intramuscular injection, inject 8ten thousand u a day. False operation group were given a median incision about 2-2.5 cm long on the back of neck, suturing peau after arresting bleeding, and the stitches shed off of itself. After operation, taking these rats into cage and let them play by themselves. The models complete after 12 weeks.
2、X-ray observations
Respectively in 4 weeks and 8 weeks,3% of pentobarbital sodium 40mg·kg-1 intra-abdominal injection, rats after anesthesia was filmed cervical lateral a piece, strictly mastering the drug dose and anesthesia to X-ray after filming the time to control muscle tension the disc height influence degree. Calculation C5-6 disc height index (DHI).
3、Pathological observation
Killing the rats by excessive loss of blood in a short temper after 4 and 8 weeks respectively, taking the whole cervical vertebra range from basilar part of occipital bone to Second dorsal vertebra immediately. Putting the intervertebral disc and interfacing centrums of C5-6 of each rat and fixing them in 10% liquor formalin, to detect morphology and observe their degeneration situation.
Specimens for light microscopy in 10% neutral formaldehyde,15% EDTA decalcified for 2 weeks, With the median sagittal cervical incision, Dehydrated with alcohol gradients, Xylene, Embedded with paraffin blocks, Slices with a slicer, HE staining, Observed under the microscope, Observed nucleus, annulus, endplate and vertebral degeneration. Scores on the disc with Grading standards of Miyamoto:
1 Level:Normal disc, annulus and nucleus arrangement rules, the growth of cartilage., endplate cartilage and articular cartilage is divided into layers, tidemark clear.
2Level:Anular lamellar structure disappeared,proliferation ofcartilage endplate.
3Level:Nucleus shrink or disappear.
4Level:Fissures of the Anular.
5Level:Herniated Disc or Osteophyte formation.
4、Disc moisture detection
Apply the vacuum freeze-drying water content determination of intervertebral disc. Will large rats were controlled, including C4-death after five vertebral asked plate used in water content determination. Take full disc tissue; divest cartilage tissue around, electronic scales weighing record (wet weight). Disc in Vacuum dryer 80 degrees below zero freeze-drying 24 hours after weighing (dry weight),1 week weighing record again. Disc water content recorded calculate formula is as follows: disc moisture content=wet weight-dry weight. According to the above formula animal intervertebral discs groups water content.
5、Western blotting detection disc AQP1,3 expression
We put the C4-5 disc cracking. The expression of AQP1、AQP3 andβ-actin was detected by way of Western blotting. We evaluated the expression of protein by analyzing the gray scale, and calculated the ratio of the value of gray scale of the protein with the value of gray scale ofβ-actin.
6、Western blotting detection disc VEGF expression
We put the C4-5 disc cracking. The expression of VEGF andβ-actin was detected by way of Western blotting. We evaluated the expression of protein by analyzing the gray scale, and calculated the ratio of the value of gray scale of the protein with the value of gray scale ofβ-actin.
7、Statistical methods
Apply the statistical soft system of Spss 16.0 for windows to analyze the materials, the measurement data indicated by the mean value and standard deviation. Each rat treatment 4 weeks and eight weeks disc water content, disc height index, AQP1、AQP3 and VEGF, using Independent sample t-test, different between treatment group compares the single factor analysis of variance (ANOVA), variance when using multiple comparison together-t method inspection; LSD Variance not neat, the use of approximate variance analysis, multiple comparison Welch method using Dunnett T3 method inspection. Each rat degeneration level by level on the material level of a statistical description by frequency data, using multiple independent sample non-parameter test Kruskal-Wallis method, P< 0.05 for with a statistical significance.
Result
All rats without a loss and the experimental results are incorporated into the analysis, no significant weight loss. Wound healing well, no autophagy bite and lick, no purulent secretions.
1. The influence of jingzhuishu Granule in disc high index
4 weeks group compared with 8 weeks, disc height index difference was significant (F=5.518, P=0.026), A statistically significant differences between groups (F=60.276, P=0.000), Groups with the interaction between before and after treatment effect (F=4.445, P=0.001)。
Two independent sample t-test shows, Each groups disc height index are significant differences (t=10.787, P=0.000; t=7.640, P=0.000; t=11.532, P=0.000; t=15.278, P=0.000; t=9.751, P=0.000), except false group (t=0.793, P=0.454)
Multiple comparisons between groups, Drug use 4 weeks later, compared with model group, each group is can increase disc height index significance(F=130.722, P=0.000). The results showed that the best curative effect of high dose, model group curative effect is the worst. Drug use 8 weeks later, compared with model group, each group is can increase disc height index significance(F=121.730, P=0.000). The results showed that the best curative effect of high dose, model group curative effect is the worst.
Hence, Compared with model group, each drug intervention group all can increase the height of the disc, and exist time and index of dose-response, including cervical curative effect of particle high doses is best.
2. The influence of jingzhuishu Granule in pathological section
By pathological visible, false group cervical intervertebral tide mark clearly visible, fiber ring structure is clear, not seen nucleus pulposus obviously shrinks, calcification articular cartilage is thinner, calcification subchondral distribution has large vessels,4 weeks group and eight weeks group no obvious difference. Model rats, cervical intervertebral appear degenerative change, fiber ring board layer structure disorder, arrangement, mild irregular corrugation of nucleus pulposus or smaller, a few prominent, the nucleus pulposus mild calcified layer and the articular cartilage were thickening calcified layer. Eight weeks than 4 weeks group of a marked change. Each drug intervention group:rat disc form a model group got obvious improvement. Puerarin extract each dose rats, disc form close to the control group, light-microscopy morphology intact, fiber circle permutation nucleus pulposus comparative rules, comparative full, tide mark visible, endplate congestion conditions improve within blood vessels, vascular shoots more than model group, including eight weeks than 4 weeks group of a marked change.
4 weeks after treatment, degenerative level difference between groups was statistically significant (χ2=11.781, P=0.038); Disc degeneration level respectively 15.00.25.94.28.69.35.62.22.56.19.19, according to the average rank times we concluded, the high dose degeneration is lightest, jingfuk group is secondly serious, model group is the most serious.
8 weeks after treatment, degenerative level difference between groups was statistically significant (χ2=11.992, P=0.035); Disc degeneration level respectively 13.25.26.94.29.76.34.06,22.44.20.56, according to the average rank times we concluded, the high dose degeneration is lightest, jingfuk group is secondly serious, model group is the most serious.
3. The influence of jingzhuishu Granule in Disc water content
4 weeks group compared with 8 weeks, Disc water content difference was significant (F=10.518, P=0.002), A statistically significant differences between groups (F=220.777, P=0.000), Groups with the interaction between before and after treatment effect (F=6.694, P=0.000)。
Two independent sample t-test shows, Each groups Disc water content is significant differences (t=3.342, P=0.005; t=2.300, P=0.037; t=2.750, P=0.016; t=5.889, P=0.000; t=4.022, P=0.001), except false group (t=1.226, P=0.241)
Multiple comparisons between groups, Drug use 4 weeks later, compared with model group, each group is can increase Disc water content significance(F=113.590, P=0.000). The results showed that the best curative effect of high dose, model group curative effect is the worst. Drug use 8 weeks later, compared with model group, each group is can increase Disc water content significance(F=113.119, P=0.000). The results showed that the best curative effect of high dose, model group curative effect is the worst. Hence, Compared with model group, each drug intervention group all can increase the Disc water content, and exist time and index of dose-response, including cervical curative effect of particle high doses is best.
4. The influence of jingzhuishu Granule in AQP1
4 weeks group compared with 8 weeks, AQP1 difference was significant (F=70.653, P=0.000), A statistically significant differences between groups (F=102.600, P=0.000), Groups with the interaction between before and after treatment effect (F-476.093, P=0.000)。
Two independent sample t-test shows, Each groups AQP1 is significant differences (t=7.499, P=0.000; t=12.506, P=0.000; t=6.154, P=0.004; t=9.396, P=0.001; t=9.232, P=0.001), except false group (t=1.603, P=0.184)
Multiple comparisons between groups, Drug use 4 weeks later, compared with model group, each group is can increase AQP1 significance(F=366.466, P=0.000). The results showed that the best curative effect of high dose, model group curative effect is the worst. Drug use 8 weeks later, compared with model group, each group is can increase AQP1 significance(F=358.012, P=0.000). The results showed that the best curative effect of high dose, model group curative effect is the worst. Hence, Compared with model group, each drug intervention group all can increase the AQP1, and exist time and index of dose-response, including cervical curative effect of particle high doses is best.
5. The influence of jingzhuishu Granule in AQP3
4 weeks group compared with 8 weeks, AQP3 difference was significant (F=25.427, P=0.000), A statistically significant differences between groups (F=665.591, P=0.000), Groups with the interaction between before and after treatment effect (F=26.073, P=0.000)。
Two independent sample t-test shows, Each groups AQP3 is significant differences (t=6.997, P=0.002; t=10.394, P=0.000; t=13.794, P=0.000; t=5.155, P=0.007; t=9.307, P=0.001), except false group (t=2.435, P=0.720)
Multiple comparisons between groups, Drug use 4 weeks later, compared with model group, each group is can increase AQP3 significance(F=566.358, P=0.000). The results showed that the best curative effect of high dose, model group curative effect is the worst. Drug use 8 weeks later, compared with model group, each group is can increase AQP3 significance(F=495.975, P=0.000). The results showed that the best curative effect of high dose, model group curative effect is the worst. Hence, Compared with model group, each drug intervention group all can increase the AQP3, and exist time and index of dose-response, including cervical curative effect of particle high doses is best.
6. The influence of jingzhuishu Granule in VEGF
4 weeks group compared with 8 weeks, VEGF difference was significant (F=18.345, P=0.000), A statistically significant differences between groups (F=590.710, P=0.000), Groups with the interaction between before and after treatment effect (F=29.853, P=0.000)。
Two independent sample t-test shows, Each groups VEGF is significant differences (t=O12.811, P=0.000; t=4.657, P=0.010; t=6.388, P=0.003; t=7.953, P=0.001; t=5.443, P=0.006), except false group (t=2.923, P=0.056)
Multiple comparisons between groups, Drug use 4 weeks later, compared with model group, each group is can increase VEGF significance(F=143.668, P=0.000). The results showed that the best curative effect of high dose, model group curative effect is the worst. Drug use 8 weeks later, compared with model group, each group is can increase AQP3 significance(F=374.502, P=0.000). The results showed that the best curative effect of high dose, model group curative effect is the worst. Hence, Compared with model group, each drug intervention group all can increase the VEGF, and exist time and index of dose-response, including cervical curative effect of particle high doses is best.
7. The influence of puerarin in Disc water content
One-way ANOVA shows, Each groups Disc water content is significant differences (F=321.260, P=0.000).Multiple comparisons between groups, Drug use 8 weeks later, compared with model group, each group is can increase Disc water content significance(P=0.000、0.000、0.013、0.003). high and middle puerarin group compared with celebrex group show the significant differences(P=0.000、0.011).high and middle puerarin group compared with low group show the significant differences (P=0.000、0.013). low puerarin group compared with celebrex group show no significant differences (P=0.998)
Hence, Compared with model group, each drug intervention group all can increase the Disc water content, and exist dose-response, including cervical curative effect of particle high doses is best,.high and middle puerarin group more effect than celebrex group.
8. The influence of puerarin in AQP1
One-way ANOVA shows, Each groups AQP1 is significant differences (F=123.205, P=0.000).Multiple comparisons between groups, Drug use 8 weeks later, compared with model group, each group is can increase AQP1 significance(P=0.001、0.001、0.015、0.026). high and middle puerarin group compared with celebrex group show the significant differences (P=0.004、0.007).high and middle puerarin group compared with low group show the significant differences (P=0.001、0.019). low puerarin group compared with celebrex group show no significant differences (P=0.166)
Hence, Compared with model group, each drug intervention group all can increase the AQP1, and exist dose-response, including cervical curative effect of particle high doses is best,.high and middle puerarin group more effect than celebrex group.
9. The influence of puerarin in AQP3
One-way ANOVA shows, Each groups AQP3 is significant differences (F=548.956, P=0.000).Multiple comparisons between groups, Drug use 8 weeks later, compared with model group, each group is can increase AQP3 significance(P=0.000、0.000、0.005、0.046). high and middle puerarin group compared with celebrex group show the significant differences (P=0.000、0.000).high and middle puerarin group compared with low group show the significant differences (P=0.000、0.001). low puerarin group compared with celebrex group show no significant differences (P=0.233)
Hence, Compared with model group, each drug intervention group all can increase the AQP3, and exist dose-response, including cervical curative effect of particle high doses is best,.high and middle puerarin group more effect than celebrex group.
Conclusion
1. jingzhuishu Granule can significantly delay cervical rats of the cervical intervertebral disc degeneration level, this trend has certain concentration-response and aging relations;
2. jingzhuishu Granule can prevent cervical disc height lower water content and slow, and then delay reduced the cervical intervertebral disc degeneration in rats, and this trend has certain concentration-response and aging relations;
3. jingzhuishu Granule can adjust cervical AQP1,3 and VEGF expression, this may be slow disc degeneration of one reason;
4. puerarin can adjust cervical AQP1,3 expression, this may be the mechanism of jingzhuishu Granule treatment cervical disc degeneration.
引文
[1]Crusher HE, Hanley EN. Analysis of aging and degeneration of the human intervertebral disc. Comparison of surgical specimens with normal controls [J].Spine,1998; 23:751-757
[2]Nachemson A, Lew in T, Marouds A, et al.In vitro diffusion of dye through the endplates and the annulus fibrosus of human lumber intervertebral disc[J].Acts Orthop Scand,1970; 41:589-607
[3]Nerlich AG, Schleicher ED, Boos N.Immunohistologic markers for age-related changes of human lumbar intervertehral discs [J].Spine,1997; 22:2781-2795
[4]吕刚,芮钢,王星铎,等.椎体软骨终板血管芽的形态学实验研究[J].中华骨科杂志,1998,18(12)：722-724
[5]Roberts S, Menage J, Urban JPG. Biochemical and structural properties of the cartilage end- plate and its relation to the intervertebral disc [J]. Spine,1989,14 (2):166-174
[6]Hassler O. The human intervertebral disc:a microangiographical study on the vascular supply of vaious ages [J]. Acta Orthop Scand,1970,40:765-781
[7]Ogata K, W h iteside LA. Nutritional pathways of the intervertebral disc:An experimental study using hydrogen washout technique [J]. Spine,1981,6: 211-216
[8]Cinotti G, Della-Rocca C, Romeo S, et al. Degenerative changes of porcine intervertebral disc induced by vertebral endplate injuries [J].Spine,2005, 30(2):174-180
[9]Benneker LM, Heini PF, Alini M, Anderson SE, and Ito K.2004 Young. Investigator Award Winner:vertebral endplate marrow contact channel occlusions and intervertebral disc degeneration [J]. Spine 2005; 30:167-73
[10]Tolonen J, Gronblad M, Virri J, et al. Basic fibroblast growth factor immunoreactivity in blood vessels and cells of disc herniations[J]. Spine,1995, 20 (3):271-276
[11]涂兵,金立人.血管内皮细胞生长因子及有关肢体缺血基因治疗的研究进展[J].中国普外基础与临床杂志,2000；7(3)：202-204
[12]王拥军,施杞,周重建,等.益气化瘀方对大鼠颈椎间盘软骨终板内血管的影响[J].中国中医骨伤科杂志,2002；10(4)：1-4
[13]Ishibashi K, Kuwahara M, Gu Y, et al. Aquaporin(water channels):role in vasopresin-activated water transport [J]. Biochem Biophy Res Comma,1998; 244:268-274
[14]Humzah MD, Soames RW. Human intervertebral disc-Structure and function [J]. Anat Rec,1988; 220:337-356
[15]Iatridis JC, MacLean JJ, O'Brien M et al. Measurements of proteoglycan and water content distribution in human lumbar intervertebral discs [J]. Spine,2007; 32:1493-1497
[16]尚铁松,王云钊.腰椎间盘退变的X线、MRI表现与病理对照[J].中华放射学杂志,2002；36(9)：828-832
[17]Miyanoto S, Yonenobu K, Ono K. Experimental cervical spondylosis in the mouse [J]. Spine,1991; 16(10 Suppl):495-500
[18]曹国永,周跃,滕海军等.水通道蛋白在正常大鼠椎间盘组织中的表达与分布[J].中国矫形外科杂志,2005,13(13)：996-999
[19]曹国永,曹兴,周跃等.椎间盘退变时水通道蛋白1表达变化研究[J].颈腰痛杂志,2008；29(4)：300-303
[20]S. M. Richardson R. Knowles D. Marples et al. Aquaporin expression in the human intervertebral disc [J]. J Mol Hist,2008; 39:303-309
[21]孔小宝,黄敏.内外合治治疗颈椎病240例[J].湖南中医药导 报,1997,3(23)：31-32
[22]刘英如,刘玫.贾福奎主任医师辨治颈椎病经验[J].新中医,2001；33(7)：10
[23]陈建鸿.中药内服配合手法治疗椎动脉型颈椎病疗效观察[J].中国中医骨伤科杂志,2003；11(6)：12-14
[24]施杞,王拥军,沈培芝等.益气化瘀法延缓颈椎间盘退变机制研究[J].BullMed Res,2003；32(6)：26-27
[25]刘立,陈宝田,张云昆.颈椎舒冲剂对椎动脉型颈椎病患者椎—基底动脉血流的影响[J].第一军医大学学报,1995,15(4)：292.
[26]刘立,陈宝田,童丽.颈椎舒冲剂对颈椎病患者血液流变学的影响[J].解放军医学杂志,1995,20(5)382-383.
[27]刘立,陈宝田.颈椎舒冲剂治疗椎动脉型颈椎病的临床与实验研究[J].中医杂志,1995,36(6)：351-353.
[28]刘立,陈宝田,薛秀琼.颈椎舒冲剂对颈椎病甲襞微循环的影响[J].人民军医,1997,40(4)：234-235.
[29]谢炜,陈宝田.颈椎舒冲剂治疗颈肩痛37例[J].辽宁中医学院学报,1999,1(1)：49.
[30]谢炜,于林,赵伟宏,等.葛根素两种提取工艺的比较研究[J].安徽中医学院学报,2008,27(2)：44-46.
[31]谢炜,赵伟宏,于林,等.川芎提取物对神经根型颈椎病大鼠根性疼痛的保护作用研究[J].广东药学院学报,2008,24(5)：496-499.
[32]杨军.颈椎病常用内服中药选配规律[J].辽宁中医杂志,2004；31(7)：559
[33]Kim KW, Lim TH, Kim JG,, et al. The origin of chondrocytes in the nucleus pulposus and histologic findings associated with the transition of a notochordal nucleus pulposus to a fibrocartilaginous nucleus pulposus in intact rabbit intervertebral discs[J]. Spine,2003; 28(10):982-990
[34]Holm S, Maroudas A, Urban JP, et al. Nutrition of the intervertebral disc:solute transport and metabolism [J].Connect Tissue Res,1981; 8(2):101-119
[35]Elliott DM, Sarver JJ. Young investigator award winner:volition of the mouse and rat disc as disc asmechanicalmodels of the human lumbar disc[J]-Spine, 2004; 29 (7):713
[36]O'Connell GD, Vresilovic EJ, Elliott DM, et al. Comparison of animals used in disc research to human lumbar disc geometry[J]·Spine,2007; 32 (3):328
[37]王拥军,施杞,沈培芝,等.动静力失衡性大鼠颈椎间盘退变模型的动态观察[J].中国中西医结合杂志,2001；20(3)：199
[38]刘斌,瞿东滨,金大地,等.NO含量和NOS活性在兔软骨终板退变过程中的变化[J].第一军医大学学报,2004；24(3)：278
[39]Lindblom K. Imervertebral disc degeneration considered as a pressure atrophy[J]·J Bone Joint Surg Am,1957; 39 (4):933
[40]HiguchiM,Abe K,Kaneda K-Changes in the nucleus pulposus of the intervertebraldisc in bipedalmice-A lightand electron microscopic study[J]-Clin Onhop RelatRes,1983,175(3):251·
[41]MacLean JJ, Lee CR, Grad S, et al. Effects of immobilization and dynamic compression on intervertebral disc cell gene expression in vivo [J]-Spine 2003; 29(10):973
[42]Demers CN, Antoniou J, Mwale F. Value and limitations of using the bovine tail as a model for the human lumbar spine [J]. Spine,2004; 29(24):2793-2799
[43]Sobaima S, Shimer AL, Chadderdon RC, et al. Quantitative analysis of gene expression in a rabbitmodel of intervertebral disc degeneration by real-time polymerase chain reaction [J]·Spine,2005; 5(1):14
[44]Melrose J, Roberts S, Smith S, et al. Increased nerve and blood vessel in growth associated with proteoglyean depletion in an ovine anular lesion model of experimental disc degeneration [J]. Spine,2002; 27(12):1278
[45]Holm S, Holm AK, Ekstrm L, et al. Experimental disc degeneration due to endplate injury[J]. J Spinal Disord Tech,2004; 17(1):64
[46]Sakuma M, Fujii N, Takahashi T, et al. Effect of chondroitinase ABC on matrix metalloproteinases and inflammatory mediators produced by intervertebral disc of rabbit in vitro[J]. Spine,2002; 27(6):576-580
[47]Norcross JP, Lester GE, Weinhold P, et al. An in vivo model of degenerative disc disease [J]. J Orthop Res,2003; 21(1):183-188
[48]Benneker LM, Heini PF, Alini M, et al.2004 Young Investigator Award Winner: vertebral endplate marrow contact channel occlusions and intervertebral disc degeneration [J]. Spine,2005; 30(2):167-173
[49]Hutton WC, Murakami H, Li J, et al. The effect of blocking a nutritional pathway to the intervertebral disc in the dog model [J]. J Spinal Disord Tech.2004; 17(1):53-63
[50]Krebs J, Ferguson SJ, Goss BG, et al. Degenerative changes of intervertebral discs after vertebroplasty. In:Proceedings of the 33rd annual meeting of the international society for the study of the lumbar spine [J].Bergen,2006; 31.
[51].Lu DS, Shono Y, Oda I, et al. Effects of chondroitinase ABCand chymopapain on spinal motion segment biomechanics. An in vivo biomechanical, radiologic, and histologic canine study [JJ].Spine,1997; 22(16):1828-1834
[52]Kaapa E, Holm S, Inkinen R, et al. Proteoglycan chemistry in experimentally injuried porcine intervertebral disc[J].J Spinal Disord,1994;7(4):296-306
[53]Buckwalter JA. Aging and degeneration of the human intervertebral Disc [J].Spine,1995; 20(11):1307-1314
[54]Agre P, Kozono D. Aquaporin water channels:molecula rmechanisms for human diseases. FEBS Letters [J],2003; 555(1):72
[55]Castle NA. Aquaporins as targets for drug discovery [J]. Drug Discovery Today, 2005; 10(7):485
[56]Jung JS, Preston GM, Smith BL, et al. Molecular structure of the water channel through aquaporin CHIP. The hour glass mode [J].J Biol Chem,1994; 269(20):14648-14654
[57]WangY, Tajkhorshid E. Molecular mechanisms of conduction and selectivity in aquaporin water channels [J]. J Nutr,2007,137(6 Suppll):1509-1515
[58]Mobasheri A, Trujilo E, Bell S, et al. Aquaporin water channels AQP1 and AQP3 are expressed in equine articular chondrocytes [J]. Vet J,2004; 168:143-150
[59]Trujillo E, Gonzalez T, Marin R, et al. Human articular chondrocytes, synoviocytes and synovialmicrovessels express aquaporin water channels; upregulation ofAQPl in rheumatoid arthritis[J]. Histol Histopatho,2004; 19:435-444
[60]Meng Juan-hong, MaXu-chen, Li Zhi-min, et al. Aquaporin-1 and aquaporin-3 expressions in the temporomandibular joint condylar cartilage after an experimentally induced osteoarthritis [J]. Chinese Medical Journal,2007; 120(24):2191-2194
[61]曹国永,周跃,滕海军,等.水通道蛋白在正常大鼠椎间盘组织中的表达与分布.中国矫形外科杂志,2005；13：996-999
[62]Ogata K, Whiteside LA. Nutritional pathways of the intervertebral disc:An experimental study using hydrogen washout technique. Spine,1981; 6:211-216
[63]曹国永,曹兴,周跃,等.椎间盘退变时水通道蛋白1表达变化研究.颈腰痛杂志,2008；29(4)：300-303
[64]Hara M, Verkman AS. Glycerol replacement corrects defective skin hydration, elasticity, and barrier function in aquaporin-3-deficient mice [J].Proc Natl Acad Sci USA,2003; 100(12):7360-7365
[65]Trujillo E, Gonzalez T, Marin R, et al. Human articular chondrocytes, synoviocytes and synovial microvessels express aquaporin water channels; up regulation of AQP1 in rheumatoid arthritis [J].Histol Histopathol,2004; 19(2): 435-444
[66]曹国永,周跃,滕海军,等.水通道蛋白在正常大鼠椎间盘组织中的表达与分布[J].中国矫形外科杂志,2005；13：996-999
[67]Richardson SM, Knowles R, Marples D, et al. Aquaporin expression in the human intervertebral disc[J]. J Mol Histol,2008; 39(3):303-309
[68]Zheng X, Bollinger-Bollag W. Aquaporin 3 colocates with phospholipase d2 in caveolin-rich membrane microdomains and is downregulated upon keratinocyte differentiation [J].J Invest Dermatol,2003;121(6):1487-1495
[69]Towne JE, Krane CM, Bachurski CJ, et al. Tumor necrosis factor-alpha inhibits aquaporin 5 expression in mouse lung epithelial cells [J]. J Biol Chem,2001; 276 (22):18657-18664
[70]Zelenina M, Bondar AA, Zelenin S, et al. Nickel and extra-cellular acidification inhibit the water permeability of human aquaporin 3 in lung epithelial cells [J].J Biol Chem,2003; 278(32):30037-30043
[71]Nerlich AG, Schaaf R, Wlchli B, Boos N, et al. Temporospatial distribution of blood vessels in human lumbar intervertebral discs [J].Eur Spine J,2007; 16(4):547-555
[72]Roberts S, Evans H, Trivedi J, Menage J, et al. Histology and pathology of the human intervertebral disc [J].J Bone Joint Surg Am,2006; 88(2):10-14
[73]葛鸿庆,林定坤,邓晋丰,等.补肾活血方对脊髓型颈椎病动物模型VEGF及其mRNA表达的影响[J].新中医,2009,41(2)：107-108
[74]崔学军,施杞,王拥军,等.颈复康颗粒对颈椎动静力失衡大鼠颈椎间盘形态学改变的影响[J].中医杂志,2009,50(12)：1121-1124
[75]Johnson WE, Evans H. Immunohistochemical detection of Schwann cells in innervated and vascularised human intervertebral discs[J].Spine,2001;26: 2550-2557
[76]Yasuma T, Araik K. The histology of lumbar intervertebral dischemiation[J]. Spine,1993; 18:1761-1765
[77]Marker K, Larsson K. Tumor necrosis factor and nucleus pulposus induced nerve root injury [J]. Spine,1998; 23(23):2538-2544
[78]王世军,史仁华,姬广臣.葛根素对家兔软脑膜微循环的影响[J].微循环杂志,1999：9(4)：19-21
[79]段重高,李宏伟,徐理纳,等.葛根素对金黄地鼠脑微循环的影响[J].中华医学杂志,1991；71(9)：516-517
[80]姜秀莲,徐理纳.葛根素对小鼠实验性微循环障碍的改善作用[J].药学学报,1989：24(4)：251-254
[81]刘振威,潘爱美,李公宝,等.磷脂对葛根素改善家兔血液流变学和微循环作用的影响[J].潍坊医学院学报,1998；20(4)：250-252
[82]高尔,王金红,李红军,等.乳化葛根素对高脂血症家兔血清NO及PGI2的影响[J].潍坊医学院学报,2001；23(1)：1-3
[83]禹志领,张广钦,赵红旗,等.葛根总黄酮对血液粘度、血栓形成及血小板功能的影响[J].中药材,1997；20(9)：468-470
[84]邓娟,周华东,陈曼娥.葛根素注射液对急性脑梗塞患者微循环保护机制的临床研究[J].微循环学杂志,2001,11(1)：14-15
[85]金惠铭,卢建,殷莲华.细胞分子病理生理学[M].郑州大学出版社,2002；602-604
[86]董丽萍,王天佑.葛根素抗谷氨酸对小鼠神经细胞兴奋毒的作用[J].中国药理学报,1998；19(4)：339-342
[87]桑韩飞,张英民,徐礼鲜,等.葛根素对兔脊髓缺血再灌注损伤的保护作用[J].第四军医大学学报,2001；22(5)：414-417
[88]曹建中,刘书山,杨光田,等.鼠急性全脑缺血再灌注后葛根素对海马CA1区BCl-2、Bax表达的影响[J].中国药理学通报,2003；19(11)：1281-1283
[89]潘洪平,杨嘉玲,莫祥兰,等.葛根素注射液对急性脑缺血模型大鼠脑细胞损伤的保护作用[J].中国中药杂志,2005；6(3)：457-459
[90]闫福岭,鲁国,王雅琼,等.葛根素对AD大鼠脑内Aβ1-40和Bax表达的影响[J].中华神经医学杂志,2006；6(1)：6-9
[91]吕俊华,张世平,沈飞海,等.葛根素对D-半乳糖诱导糖基化大鼠脑损害的干预作用[J].中国中药杂志,2006；31(14)：1184-1187
[92]杜德极,聂梅,杨薇,等.葛根提取物体外对TNF、IL-1及IL-2产生的影响[J].中草药,1997；28(3)：160-162
[93]周军,方素萍,齐云,等.葛根汤对佐剂性关节炎大鼠关节液炎症介质的影响[J].中国实验方剂学杂志,2001；7(3)：29-31
[94]周军,方素萍,霍海如,等.葛根汤对退变颈椎间盘组织磷脂酶A2的影响[J].中国中医骨伤科杂志,2002；10(4)：12-14
[95]周军,方素萍,霍海如,等.葛根汤对退变颈椎间盘组织前列腺素E2及环氧合酶的影响[J].中国骨伤,2002；15(12)：724-726
[96]王拥军、施杞,周重建,等.葛根汤与桂枝汤对兔颈椎间盘组织IL-1β、iNOS、 TNF-α、TGF-βmRNA表达的调节作用[J].中西医结合学报,2003；1(4)：273-276
[97]施杞、王拥军、周重建,等.葛根汤、桂枝汤对家兔颈椎间盘组织白介素1p、诱导型一氧化氮合酶mRNA表达的调节作用[J].中医杂志,2004；45(10)：777-779
[98]刘梅,王拥军,施杞,等.葛根汤和桂枝汤调节椎间盘组织Fas、bcl-2蛋白表达的实验研究[J].中国骨伤,2004；17(4)：198-200
[99]Boue SM, Wiese TE, Nehls S, et al. Evaluation of the estrogenic effects of legume extracts containing phytoestrogens. J Agric Food Chem,2003; 51(8): 2193-9
[100]Chen G, Zhang J, jiannong Y. Determination of puerarin, daidzein and rutin in Pueraria lobata (Wild.) Ohwi by capillary electrophoresis with electrochemical detection. J Chromatogr A 2001; 923 (1-2):255-62
[101]郑高利,张信岳,郑经伟,等.葛根素和葛根总黄酮的雌激素样活性[J].中药材,2002；25(8)：566-568
[102]郑高利,张信岳,孟倩超,等.葛根异黄酮对地塞米松致大鼠骨质疏松症的保护作用[J].中药材,2002；25(9)：643-645
[103]周强,付庭斌.葛根素对去卵巢大鼠骨质疏松性骨折愈合的促进作用[J].中国临床康复,2006；10(27)：45-47
[104]田泉,汤旭磊,白孟海,等.葛根素对去卵巢大鼠骨质疏松和血脂的作用[J].中国老年医学杂志,2006：25(7)：543-545
[105]徐芾,金邦荃,武卫平,等.植物异黄酮对更年期妇女骨密度和骨代谢血清生化指标的影响[J].中国妇幼保健,2007；22(11)：1517-1519
[106]郭辉、刘奕琛.葛根素对大鼠成骨细胞代谢调控机制的实验研究[J].现代中医药,2008；28(1)：48-50
[107]S Theoleyre, Y Witt rant, S Couillaud, et al. Cellular activity and signaling induced by osteoprotegerin in osteoclasts:involvement of receptor activator of nuclear factor nB ligand and MAPK [J]. Biochimica et Biophysica Acta,2004; 1644 (1):1-7
[108]吴俊兰,李琰,伏静瑗,等.葛根素治疗冠心病心律失常的效果[J].心脏杂志,2000,12(4)：275-277
[109]钟萍.葛根素对老年高血压病患者血浆内皮素的影响[J].四川医学,2002；23(12)：1318-1319
[110]汪进益,肖少军,候绪伟.葛根素注射液对原发性高血压患者血管内皮细胞损伤的影响[J].井冈山医专报,2001；8(3)：10-11
[111]孙纪荣,卢顺麟,等.葛根素对不稳定型心绞痛患者QT离散度的影响[J].黑龙江医学,2004；28(12)：894-895
[112]徐平湘,孙薇,黑爱莲,等.葛根素对异丙肾上腺素诱导人鼠心肌损伤的保护作用[J].首都医科大学学报,2003；24(3)：251.
[113]刘素蓉,吕敏贤,狄灵.中西药治疗对椎基底动脉供血不足性眩晕患者血液流变学的影响[J].中国临床康复,2004；8(34)：7637-7637
[114]冼冰.葛根素注射液治疗椎-基底动脉供血不足胜眩晕临床观察[J].海南医学,2003；14(4)：33-35
[115]朱庆磊,何爱霞,吕欣然.葛根素对氧自由基的清除和抗氧化损伤作用[J].解放军药学学报,2001；17(1)：1-3
[116]王福文,朱燕,胡志力,等.葛根素对体内血栓形成及血液流变学的影响[J].山东医药工业,2003；22(2)：52-54
[117]茅彩萍,顾振纶.葛根素对糖尿病人鼠主动脉糖基化终产物的形成及其受体表达的影响[J].中国药理通报,2004；20(4)：393-395
[118]王金红,高尔,孙善明,等.乳化葛根素对高脂血症家兔模型调血脂和抗氧化作用[J].潍坊医学院学报,2001；23(1)：6-9
[119]金惠铭,卢建,殷莲华.细胞分子病理生理学[M].郑州大学出版社,2002；602-604
[120]董丽萍,王天佑.葛根素抗谷氨酸对小鼠神经细胞兴奋毒的作用[J].中国药理学报,1998；19(4)：339-342
[121]曹建中,刘书山,杨光田,等.鼠急性全脑缺血再灌注后葛根素对海马CAl区BCl-2、Bax表达的影响[J].中国药理学通报,2003；19(11)：1281-1283
[122]潘洪平,杨嘉玲,莫祥兰,等.葛根素注射液对急性脑缺血模型大鼠脑细胞损伤的保护作用[J].中国中药杂志,2005；6(3)：457-459
[123]闫福岭,鲁国,王雅琼,等.葛根素对AD大鼠脑内Aβ1-40和Bax表达的影响[J].中华神经医学杂志,2006；6(1)：6-9
[124]吕俊华,张世平,沈飞海,等.葛根素对D-半乳糖诱导糖基化大鼠脑损害的干预作用[J].中国中药杂志,2006；31(14)：1184-1187
[125]李斌斌,于世凤.葛根素调控骨代谢的体外实验[J].北京大学学报·医学版,2003：35(1)：74-77
[126]郑高利,张信岳,郑经伟,等.葛根素和葛根总黄酮的雌激素样活性[J].中药材,2002；25(8)：566-568
