【作者】 于林；

【导师】 陈宝田；

【作者基本信息】 南方医科大学 ， 中西医结合临床医学， 2011， 博士

【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。更多还原

【Abstract】 Purpose and significanceCervical 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 content1、Rats of cervical disc degeneration was established3-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 observationsRespectively 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 observationKilling 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 detectionApply 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 expressionWe 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 expressionWe 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 methodsApply 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.ResultAll 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 index4 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 sectionBy 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 content4 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 AQP14 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 AQP34 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 VEGF4 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 AQP1One-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.Conclusion1. 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.更多还原