葛根提取物对神经根型颈椎病大鼠根性疼痛的作用及机制研究
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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。流行病学调查显示,颈椎病在我国成人中的患病率大约为3.8%～17.6%。按现有人口保守推算,至少有5000万至1亿人正在受到颈椎病的折磨。神经根型颈椎病是颈椎病中发病率最高的一种类型,约占其总发病率的60%～70%。神经根型颈椎病(cervical spondylotic radiculopathy,CSR)是神经根受各种因素侵袭损伤而导致其支配区域感觉、运动及反射障碍而引发的以疼痛为主要临床表现的一种疾病。医学界最早关于颈椎病的概念,大多源于神经根型。神经根型颈椎病的主要症状为根性神经痛(Radicular Neuralgia,RN)。目前对神经根型颈椎病根性痛的确切机制尚不完全清楚,但机械压迫因素和生物化学因素在发病机制中的作用得到越来越多的认同。椎间盘退变引起的骨赘增生压迫神经根,引起无神经外膜保护的神经根产生以神经内水肿和缺血为主的病理改变。大量文献表明,退变的椎间盘及损伤的神经根存在多种炎性递质,如细胞因子、白三烯(LT)、肿瘤坏死因子(TNF)、免疫球蛋白和白介素(IL)等,机械性压迫与炎性刺激的协同作用造成神经根更严重的损伤。因此,保护和修复受损的神经根成为防治神经根型颈椎病根性痛的有效方法。目前对于神经根型颈椎病根性痛尚无有效的治疗药物,多采用牵引、针灸、手法推拿按摩、理疗等综合治疗,但只能暂时缓解疼痛,病人依从性差。对于一些病情严重者可选择外科手术治疗,但受到适应症的限制,风险大,费用昂贵,还存在复发的可能。
如何发挥中医药治疗神经根型颈椎病就成了摆在我们面前的课题。本课题组根据神经根型颈椎病“多风、多寒、多湿、多瘀、多虚”的特点,自拟“颈椎舒”方临床运用多年取得满意的疗效。动物实验和临床研究提示,“颈椎舒”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。葛根为方中君药之一,葛根素为葛根的主要有效成分,其化学名8-β-D-葡萄吡喃糖-4',7-二羟基异黄酮。葛根素治疗神经根型颈椎病已有报道,研究证明,葛根素具有调节骨代谢、改善血液循环、降低心肌耗氧量、降低血糖、防治高血压及动脉硬化、保护神经细胞、抗肝脏毒性、抗炎、祛痰、解热、提高机体免疫力、抗菌、抗病毒等多种药理作用。
为了研究葛根素治疗神经根型颈椎病根性痛的可能机制,探讨葛根素对受损的颈神经根的修复作用作用,本课题组从神经行为学、神经形态学和神经电生理学角度进行研究,既为了探讨葛根对神经根型颈椎病根性痛及其受损神经根修复的作用,也为明确中药复方“颈椎舒”的疗效机制提供实验依据。
二、方法与内容
1.葛根药材的鉴定
薄层色谱鉴定照薄层色谱法(附录VIB)试验,吸取上述两种溶液各10μl,分别点于同—以羧甲基纤维素钠为黏合剂的硅胶H薄层板上,使成条状,以三氯甲烷-甲醇-水(7:2.5:0.25)为展开剂,展开,取出,晾干,置紫外光灯(365nm)下检视。供试品色谱中,在与对照品色谱图相应的位置上,显示相同颜色的荧光斑点。
葛根中葛根素含量的测定根据2005版《中国药典(一部)》中规定,按照高效液相色谱法测定,葛根干燥品中葛根素含量不低于2.4%。
2.葛根素最佳提取工艺的确定及质量控制标准的建立
2.1最佳提取工艺的确定
以葛根素为指标,以料液比(14:1、16:1、18:1)g、提取时间(1、1.5、2)h、提取次数(1、2、3)次为考察因素,按L_9(3~4)正交表进行正交试验,比较水提取法和乙醇提取法,确定葛根提取的最佳工艺及质量控制标准
2.2质量控制标准的建立
按照最佳提取工艺做三批验证试验求得RSD值,计算平均浓度。
3.模型的建立及干预
所有大鼠在造模术前行左侧SLSEP检测,获取参考值。仿照文献施行椎管插线造模手术,略作改进:以0.8cm长、直径0.55 mm尼龙渔线制备成插线,C7椎板开窗后,分别沿脊髓纵轴插入至C7-T1,及C5-C7神经根处,部分重叠,牢固嵌入骨性椎管内。术中及术后死亡7只。余53只大鼠在术后第5天接受评价,筛选符合以下条件:①造模前后术侧E'-C6 Lat的增长为0.2-0.4ms;②步态评分为2-3分,无脊髓压迫症状,共47只大鼠入组。随机编号,使用excel均衡生成5个处理组,每组8只:生理盐水组、葛根提取物低、中、高剂量组、颈复康组。颈复康组用颈复康颗粒配制成密度为1:1溶液,所有处理组给药均定容到3ml灌胃。治疗期间对大鼠进行行为学观察记录自发疼痛行为和步态异常评分,治疗10天后行正中神经短潜伏期体感诱发电位(SLSEP)测定和压线段脊髓及神经根病理切片的制作与观察。
4.葛根提取物对受损神经根修复作用的评价
行为学观察实验开始及结束时分别测量大鼠体重;每日在9:00-10:30间将大鼠放置在观察箱中30min后观察:有无自噬、舔咬、嘶叫等;记录5min(300s)内患肢离地的累积时间,取三次的平均值;结合Kawakami与Dubuisson法对其由疼痛挛缩造成的步态障碍进行评估:1分=正常步态、足无畸形;2分=受损伤足轻触玻璃台面,不持重或轻微持重,走时跛行;足内收蜷缩畸形;3分=受损足抬起,走动时不着台面;伴明显足内收蜷缩畸形。
正中神经短潜伏期体感诱发电位(SLSEP)测定方法如文献,刺激和记录均使用2402号针灸针,参考电极置于附近1cm处皮下,刺激正中神经。记录并计算术侧Erb's点与C6椎板上点潜伏期的差值(E'-C6 Lat)、C6椎板上点波幅(C6Amp),Erb's点与C6椎板测量点距离,计算体感诱发电位传导速度(MCV)。
病理切片的制作与观察大鼠各项指标检测完成后,大剂量麻醉致死,迅速取出压线段脊髓及神经根,固定、石蜡包埋,进行HE染色,光镜下观察轴索、髓鞘和神经元的形态及神经内膜及间质内血管的变化、是否有异常物质的沉积和炎症细胞的浸润等来明确病变性质;分别统计各组:神经细胞空泡样变、坏死灶、轴突扭曲髓鞘脱失呈鱼骨状等阳性病变的病例数。
5.数据处理
提取工艺的优化使用正交设计(Orthogonal design);使用SPSS11.5行单因素重复测量数据的方差分析(Repeated Measures),Mauchly球形检验结果,若P＞0.05,满足球形假设,无需校正;若P≤0.05,则不满足球形假设,用ε校正系数为Greenhouse-Geisser校正的结果。计量数据用均数±标准差((?)±s)描述,治疗前后用配对t检验,各组间比较用单向方差分析(One-Way ANOVA)。
三、结果
实验大鼠均无明显的体重减轻,伤口愈合良好,无自噬及舔咬,无脓性分泌物。
1.葛根提取物对大鼠颈神经根性疼痛行为的影响
在术后5天时,分别评价各组自发疼痛行为和步态异常评分差异无显著性意义(P＞0.05)。实验结束时,生理盐水组的大鼠上肢仍弯曲紧贴胸口,其前肢避免触地,触地部位为肘部,其5分钟内前肢离地时间值无明显变化,步态蹒跚,躯体倾斜;EP高、中、低剂量治疗组的大鼠疼痛相关行为明显减轻,四肢趋向于平等支持体重,腕部或爪背部触地,呈稍不平衡步态。
2.葛根提取物对大鼠受损神经根修复的影响
生理盐水组大鼠的组织病理切片形态学观察见神经元坏死,假单极神经细胞结构消失,形成卫星细胞环绕的空洞;后根神经轴突及膜细胞结构不清,雪旺细胞核堆积,轴突有断裂,周围髓鞘消失,形成空洞;脊髓白质的后索、外侧索呈空泡样变性,脊髓灰质、白质处均有胶质细胞增生。葛根提取物高、中、低剂量组和颈复康组神经损伤程度较轻,出现节细胞空泡变性,坏死及髓鞘鱼骨变的例数较少,提示葛根提取物对大鼠受损神经有修复和保护作用。
3.葛根提取物对大鼠受损神经根电生理的影响
3.1对C6椎板上点波幅(C6 Amp)的影响不同组之间评分结果无显著性差异(F=6.878,P＜0.001),治疗前后评分结果有显著差异(F=1394.719,P＜0.001);在不同分组之间,除生理盐水组(t=0.890,P=0.403)无显著性差异,其余各组经配对t检验,结果均有显著差异,t值分别为28.714、17.540、12.529和21.616,均为P＜0.001。从不同组来看,治疗前差异没有统计学意义(F=0.822,P=0.520),治疗后评分结果差异有显著差异(F=14.432,P＜0.001),其中以颈复康组疗效最显著。各组与治疗前后之间存在交互效应(F=93.059,P＜0.001),由表可知,治疗后波幅的最大值在颈复康组,最小值在治疗后生理盐水组;
3.2对潜伏期的差值(E'-C6Lat)的影响不同组之间评分结果无显著性差异(F=20.025,P＜0.001),治疗前后评分结果有显著差异(F=384.062,P＜0.001);在不同分组之间,除生理盐水组(t=0.314,P=0.763)无显著性差异,其余各组经配对t检验,结果均有显著差异,t值分别为11.081、9.178、16.024和10.299,均为P＜0.001。从不同组来看,治疗前差异没有统计学意义(F=0.368,P=0.830),治疗后评分结果差异有显著差异(F=39.430,P＜0.001),其中以颈复康组疗效最显著。各组与治疗前后之间存在交互效应(F=27.133,P＜0.001),由表可知,治疗后潜伏期差值的最大值在生理盐水组,最小值在治疗后颈复康组;
3.3对体感诱发电位传导速度(MCV)的影响不同组之间评分结果有显著性差异(F=3.183,P=0.025),治疗前后评分结果有显著差异(F=187.437,P＜0.001);在不同分组之间,生理盐水组(t=0.708,P=0.502)无显著性差异,高剂量组(t=9.642,P＜0.001)、中剂量组(t=5.170,P=0.001)和低剂量组(t=14.803,P＜0.001)结果均有显著性差异。从不同组来看,治疗前差异没有统计学意义(F=0.394,P=0.812),治疗后评分结果差异有显著差异(F=9.327,P＜0.001),其中以高剂量组疗效最显著。各组与治疗前后之间存在交互效应(F=16.372,P＜0.001),由表可知,治疗后传导速度最大值在颈复康组,最小值在治疗后生理盐水组;
四、结论
1.葛根提取物对神经根型颈椎病神经根性疼痛大鼠有较好的止痛作用,当葛根提取物中含葛根素剂量为266.1、133.0、66.5mg·kg~(-1)·d~(-1)时可以显著减轻大鼠疼痛行为,并且存在一定的量效关系;
2.葛根提取物各剂量组可以改善根性痛大鼠受损神经根形态结构,减少出现节细胞空泡变性、坏死及髓鞘鱼骨变的例数,存在一定的量效关系;
3.对神经电生理的影响研究中发现,葛根提取物各剂量组能升高波幅、缩短潜伏期及加快体感诱发电位传导速度,并且提示存在一定的量效关系。
Objective and significance
Cervical disc disease is the neck,bone,cartilage,ligaments became degeneration, the secondary stimulation or around the organizational structure of oppression such as the spinal cord and nerve roots,blood vessels,soft tissue,sympathetic and so on, caused a group of sick syndrome eventually.Epidemiological investigations revealed that the adults in our country in the rate of cervical syndrome approximately 3.8%to 17.6%.
Conservative projections based on the current population,there are at least 50 to 100 million people are afflicted by cervical syndrome.Cervical spondylotic radiculopathy is the highest incidence of the cervical syndrome,about the total incidence of 60%to 70%.CSR is the nerve root injury by various factors which led invasion and its dominant regional sensory,motor and reflex obstacles for the pain caused to the main clinical manifestations of a disease.In the medical field on cervical syndrome understanding of the concept of the earliest,most from cervical spondylotic radiculopathy.CSR's main symptoms of nerve root pain.
At present,the type of cervical nerve root pain of the illness the exact mechanism is not yet entirely clear,but more and more people think that oppression mechanical and biochemical factors in the pathogenesis of factors play an important role.Disc degeneration caused Osteophyte proliferation,proliferation of osteophyte nerve root compression caused nerve membrane without the protection of the nerve root to produce nerve edema and ischemia-based pathological changes.
Lots of documents indicate that the disc degeneration of the nerve root injury and multiple inflammatory neurotransmitters,such as cytokines,leukotrienes,and tumor necrosis factor,immunoglobulin and interleukin and etc.However,machinery of oppression and stimulate synergies inflammatory nerve root caused more serious damage.Therefore,the prevention and treatment of cervical nerve root pain of the illness is the most effective way to protect and repair damaged nerve root.At present, there is no effective drug treatment of cervical nerve root pain of the illness;doctors use the combination therapy treatments such as traction,acupuncture,massage manipulation,physical therapy,etc.The severity of the patients may choose surgical treatment,but indications are the restrictions,and surgery risky,costly,and there still exist the possibility of recurrence.
How to play in medical treatment of cervical nerve root disease on before us has become the subject.According to the nerve root of the characteristics of cervical disease,we developed a "JingZhuiShu" clinical use for many years to achieve a satisfactory effect.Animal experiments and clinical studies suggest that "JingZhuiShu " to improve microcirculation and reduce platelet aggregation,lower blood viscosity, increased flexibility vessel wall,expanding blood vessels,can significantly improve the nerve root in patients with cervical pain,numbness,stiffness,cold,The inability of the symptoms of muscular dystrophy.Puerarin the main contain isoflavones, triterpene saponins and alkaloids,etc.Of these components,puerarin(Pur) as its main active ingredient,the chemical name is 8 -β-D-glucopyranosyl-4',7 - dihydroxy Isoflavones.Studies have shown that puerarin regulate bone metabolism and improve the body blood circulation,reducing myocardial oxygen consumption,lower blood sugar,prevent hypertension and arteriosclerosis,protect nerve cells,anti-liver drug, anti-inflammatory,expectorant,antipyretic and improve body immunity,antibacterial, antiviral and other pharmacological effect.
In order to study the puerarin treatment of cervical nerve root pain Binggen of the possible mechanisms of puerarin on the cervical nerve root damage to repair,we learn from the behavior of nerve morphological,physiological and neurological perspective.Our aim is not only to explore both the puerarin cervical nerve root of the root of pain and damage repair of nerve root,but also to clear Chinese herbal compound,"JingZhuiShu" of the effect of mechanisms to provide experimental basis.
Method and content
1.Puerarin Lobata(Willd.) Ohwi Identification.
TLC Identification According to TLC(AppendixⅥB) demands of a test solution of the lessons of these two 10μl,respectively,at the same point in sodium carboxymethyl cellulose for the silicone adhesive H plate,into strips,chloroform -methanol - water(7:2.5:0.25) for the launch of start,removed,dried,home UV light (365 nm) under review.In the chromatogram of the corresponding position for the test and reference substance two chromatograms of the same color have shown that the fluorescence spots.According to the 2005 edition of "Chinese Pharmacopoeia (1)",Puerarin in dry Puerarin Lobata(Willd.) Ohwi content of not less than 2.4%.
2.Puerarin optimum extraction of the establishment and the establishment of standards for quality control.
Puerarin to target for inspection,the proportion of raw materials solution (14:1,16:1,18:1),the extraction time(1,1.5,2) h,extraction times(1,2,3) for the inspection,by L9(34) orthogonal table orthogonal test,compare water extraction and ethanol extraction method to determine the best Puerarin extraction technology and quality control standards.Extraction Process in accordance with the best verification test done three batches obtained RSD value,calculating the average concentration.
3.Model and intervention.
All rats in the left-SLSEP preoperative detection,detection of the normal access. According to the literature Oxfam spinal surgery modeling interpolation line,slightly improved:to 0.8 cm in length,has a diameter of 0.55 mm nylon fishing line interpolation of a line,C7 laminectomy fenestration,were inserted along the longitudinal axis to the spinal cord C7-T1 and C5-C7 nerve roots,some overlapping, firmly embedded in the bone of the spinal canal.A total of seven rats in surgery or died after surgery.More than 53 rats in the first five days after receiving evaluation, selection meet the following conditions:①model around the side of E'-C6 Lat for the growth of 0.2-0.4 ms;②gait score of 2-3 points,no symptoms of spinal cord compression,a total of 47 rats into the group.Random numbers,use excel balanced generate five treatment groups,each with eight:saline group,puerarin extract of low, medium and high-dose group,control group.Control group with "Jingfukang" of particle density of 1:1 made solution,all treatment groups will be concentrated liquid capacity to 1 ml,by gavages' method of administration.
4.The evaluation of the extraction of Puerarin lobata(Willd.) Ohwi to restoration of the injured nerve behavior observed in rats.
Measurement of body weight in the experimental rats at the beginning and end; 9-10.30 everyday observed in rats placed in the box observation after 30 min:See whether there are rats autophagy,licking bite,scream and recorded within 5 min of the affected limbs from the cumulative time,the calculation of the average of three times;Use the method of Dubuisson and Kawakami and evaluate pain from the contraction of gait disturbance:1 = normal gait,no foot deformities;= 2 damage feet touch the glass surface,walking limp;foot or huddling malformation;3 = damaged foot lift,walking cannot touch the table,foot deformity or crouch obviously.Median nerve short-latency somatosensory evoked potentials(SLSEP) Determination.
Use acupuncture needles stimulate and record the reference electrode placed 1 cm subcutaneous rats and stimulating the median nerve.Recorded and calculated operated side Erb's point C6 laminectomy,and the latency point margin(E'-C6 Lat), the C6 laminectomy point amplitude(C6 Amp),and C6 Erb's point distance measurement points laminectomy,somatosensory evoked potentials calculated conduction velocity(MCV).
Detection of indicators after the completion of large doses of narcotic death, pressure line quickly removed spinal cord and nerve roots,fixed,paraffin-embedded, HE staining,was observed under light microscope axonal,and the myelin sheath of the neurons and neural patterns and endometrial stromal vascular changes,whether there are abnormal substances deposition and the infiltration of inflammatory cells, such as to the nature of specific diseases;each group respectively statistics:nerve cells vacuolar changes,necrotic foci showed loss of myelin axons distorted fish bone-shaped lesions,such as the number of cases positive.
5.The statistical analysis of experimental data.
Extraction of optimizing the use of orthogonal design,SPSS 11.5 single factor to the use of repeated measurement data analysis of variance(Repeated Measures), Mauchly spherical test results if P＞0.05 meets spherical assumption,without correction;if P≤0.05,spherical not meet the assumptions used s correction coefficient of Greenhouse-Geisser correction results.Measurement data with mean±standard deviation((?)±s) described before and after treatment using paired t test, each group compared with one-way analysis of variance(One-Way ANOVA).
Result
The rats had no significant weight loss,wound healing well,no self-licking macrophages and bite,no Nongxing secretions.
1.The EP on the treatment of pain in rats.
After 5 days,the evaluations of each group were spontaneous acts of pain and abnormal gait score difference was not significant(P＞0.05).The end of the experiment,the rats saline group still bending close upper chest,to avoid conflict with its foreleg,the touchdown site for the elbow,five minutes from the time percentile forelimb no significant changes in gait stumble,body tilt;EP high,medium and low dose treatment group pain-related behavior in rats reduced significantly limbs tend to support equal weight,or claw back contravention of the wrist,a slightly uneven gait;
2.The EP on rat nerve root injury treatment.
Saline group of rats observed histologically visible morphological neuronal death,false unipolar nerve cell structure disappeared,a satellite cells surrounded by empty;axons after nerve cell membrane structure and unclear,Schwann cell accumulation shaft a sudden rupture,the surrounding myelin disappeared,a hollow; spinal cord white matter after the cable,a cable lateral vacuolar degeneration of spinal cord gray matter,white matter gliosis were.Puerarin extract high,medium and low dose group and neck nerve injury rehabilitation lesser extent,there ganglion cell degeneration and necrosis and myelin fish bone changes were fewer,suggesting that the extract of Pueraria damage in rats nerve restoration and protection.
3.The EP on the nerve root injury Electroneurophysiological therapeutic effect.
On the group on rat(C6 Amp) impact.Score results among different groups had no significant difference(F = 6.878,P＜0.001),and score the results before and after treatment were significantly different(F = 1394.719,P＜0.001) between different groups,in addition to the normal saline group(t = 0.890,P = 0.403) with no significant difference in the rest of the group,paired t test,the results were significantly different,t = 28.714,17.540,12.529 and 21.616,respectively,P＜0.001. From different groups,the treatment did not statistically significant difference(F = 0.822,P = 0.520),post-treatment score difference in the results were significantly different(F = 14.432,P＜0.001),in which the carotid rehabilitation of the most significant group.Groups with the interaction between before and after treatment effect(F = 93.059,P＜0.001),by the table,we can see that after treatment the maximum amplitude in the neck rehabilitation group,the minimum in the saline group after treatment;
On the group on rat(E'-C6Lat) impact.Score results among different groups had no significant difference(F = 20.025,P＜0.001),and score the results before and after treatment were significantly different(F = 384.062,P＜0.001) between different groups,in addition to the normal saline group(t = 0.314,P = 0.763) with no significant difference in the rest of the group,paired t test,the results were significantly different,t = 11.081,9.178,16.024 and 10.299,respectively,P＜0.001. From different groups,the treatment did not statistically significant difference(F = 0.368,P = 0.830),post-treatment score difference in the results were significantly different(F = 39.430,P＜0.001),in which the carotid rehabilitation of the most significant group.Groups with the interaction between before and after treatment effect(F = 27.133,P＜0.001),by the table,we can see that the maximum incubation period after treatment in the saline group,the minimum rehabilitation in the treatment of persistent group;
On the group on rat(MCV) impact.The results among different groups score There was a significant difference(F = 3.183,P = 0.025),the score results before and after treatment were significantly different(F = 187.437,P＜0.001) between different groups,and normal saline group(t = 0.708,P = 0.502) with no significant difference between the high dose group(t = 9.642,P＜0.001) in the dose group(t = 5.170,P = 0.001) and low dose group(t = 14.803,P＜0.001) results there was significant difference.From different groups,the treatment did not statistically significant difference(F=0.394,P = 0.812),post-treatment score difference in the results were significantly different(F = 9.327,P＜0.001),with the high dose group of the most notable.Groups with the interaction between before and after treatment effect(F = 16.372,P＜0.001),by the table,we can see that the maximum rate after treatment in the neck rehabilitation group,the minimum in the saline group after treatment;
Conclusions
1.Extraction of Puerarin lobata(Willd.) Ohwi have better analgesic effect on nerve root type of cervical nerve root pain.When the Puerarin of Puerarin lobata(Willd.) Ohwi in the concentration of 266.1,133.0,66.5 mg ~ kg"l ~ dl,can significantly reduce pain in rats,and there are certain dose-effect relationship;
2.EP each dose group can significantly improve the structure of nerve damage, reduced ganglion cell degeneration and necrosis,and the myelin sheath of fish bone changes;
3.In the impact of electrophysiology study,EP-dose groups can increase the volatility,reduce the incubation period and accelerate somatosensory evoked potential conduction velocity,and prompts a certain dose-effect relationship.
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。流行病学调查显示,颈椎病在我国成人中的患病率大约为3.8%～17.6%。按现有人口保守推算,至少有5000万至1亿人正在受到颈椎病的折磨。神经根型颈椎病是颈椎病中发病率最高的一种类型,约占其总发病率的60%～70%。神经根型颈椎病(cervical spondylotic radiculopathy,CSR)是神经根受各种因素侵袭损伤而导致其支配区域感觉、运动及反射障碍而引发的以疼痛为主要临床表现的一种疾病。医学界最早关于颈椎病的概念,大多源于神经根型。神经根型颈椎病的主要症状为根性神经痛(Radicular Neuralgia,RN)。目前对神经根型颈椎病根性痛的确切机制尚不完全清楚,但机械压迫因素和生物化学因素在发病机制中的作用得到越来越多的认同。椎间盘退变引起的骨赘增生压迫神经根,引起无神经外膜保护的神经根产生以神经内水肿和缺血为主的病理改变。大量文献表明,退变的椎间盘及损伤的神经根存在多种炎性递质,如细胞因子、白三烯(LT)、肿瘤坏死因子(TNF)、免疫球蛋白和白介素(IL)等,机械性压迫与炎性刺激的协同作用造成神经根更严重的损伤。因此,保护和修复受损的神经根成为防治神经根型颈椎病根性痛的有效方法。目前对于神经根型颈椎病根性痛尚无有效的治疗药物,多采用牵引、针灸、手法推拿按摩、理疗等综合治疗,但只能暂时缓解疼痛,病人依从性差。对于一些病情严重者可选择外科手术治疗,但受到适应症的限制,风险大,费用昂贵,还存在复发的可能。
如何发挥中医药治疗神经根型颈椎病就成了摆在我们面前的课题。本课题组根据神经根型颈椎病“多风、多寒、多湿、多瘀、多虚”的特点,自拟“颈椎舒”方临床运用多年取得满意的疗效。动物实验和临床研究提示,“颈椎舒”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。葛根为方中君药之一,葛根素为葛根的主要有效成分,其化学名8-β-D-葡萄吡喃糖-4',7-二羟基异黄酮。葛根素治疗神经根型颈椎病已有报道,研究证明,葛根素具有调节骨代谢、改善血液循环、降低心肌耗氧量、降低血糖、防治高血压及动脉硬化、保护神经细胞、抗肝脏毒性、抗炎、祛痰、解热、提高机体免疫力、抗菌、抗病毒等多种药理作用。
为了研究葛根素治疗神经根型颈椎病根性痛的可能机制,探讨葛根素对受损的颈神经根的修复作用作用,本课题组从神经行为学、神经形态学和神经电生理学角度进行研究,既为了探讨葛根对神经根型颈椎病根性痛及其受损神经根修复的作用,也为明确中药复方“颈椎舒”的疗效机制提供实验依据。
二、方法与内容
1.葛根药材的鉴定
薄层色谱鉴定照薄层色谱法(附录VIB)试验,吸取上述两种溶液各10μl,分别点于同—以羧甲基纤维素钠为黏合剂的硅胶H薄层板上,使成条状,以三氯甲烷-甲醇-水(7:2.5:0.25)为展开剂,展开,取出,晾干,置紫外光灯(365nm)下检视。供试品色谱中,在与对照品色谱图相应的位置上,显示相同颜色的荧光斑点。
葛根中葛根素含量的测定根据2005版《中国药典(一部)》中规定,按照高效液相色谱法测定,葛根干燥品中葛根素含量不低于2.4%。
2.葛根素最佳提取工艺的确定及质量控制标准的建立
2.1最佳提取工艺的确定
以葛根素为指标,以料液比(14:1、16:1、18:1)g、提取时间(1、1.5、2)h、提取次数(1、2、3)次为考察因素,按L_9(3~4)正交表进行正交试验,比较水提取法和乙醇提取法,确定葛根提取的最佳工艺及质量控制标准
2.2质量控制标准的建立
按照最佳提取工艺做三批验证试验求得RSD值,计算平均浓度。
3.模型的建立及干预
所有大鼠在造模术前行左侧SLSEP检测,获取参考值。仿照文献施行椎管插线造模手术,略作改进:以0.8cm长、直径0.55 mm尼龙渔线制备成插线,C7椎板开窗后,分别沿脊髓纵轴插入至C7-T1,及C5-C7神经根处,部分重叠,牢固嵌入骨性椎管内。术中及术后死亡7只。余53只大鼠在术后第5天接受评价,筛选符合以下条件:①造模前后术侧E'-C6 Lat的增长为0.2-0.4ms;②步态评分为2-3分,无脊髓压迫症状,共47只大鼠入组。随机编号,使用excel均衡生成5个处理组,每组8只:生理盐水组、葛根提取物低、中、高剂量组、颈复康组。颈复康组用颈复康颗粒配制成密度为1:1溶液,所有处理组给药均定容到3ml灌胃。治疗期间对大鼠进行行为学观察记录自发疼痛行为和步态异常评分,治疗10天后行正中神经短潜伏期体感诱发电位(SLSEP)测定和压线段脊髓及神经根病理切片的制作与观察。
4.葛根提取物对受损神经根修复作用的评价
行为学观察实验开始及结束时分别测量大鼠体重;每日在9:00-10:30间将大鼠放置在观察箱中30min后观察:有无自噬、舔咬、嘶叫等;记录5min(300s)内患肢离地的累积时间,取三次的平均值;结合Kawakami与Dubuisson法对其由疼痛挛缩造成的步态障碍进行评估:1分=正常步态、足无畸形;2分=受损伤足轻触玻璃台面,不持重或轻微持重,走时跛行;足内收蜷缩畸形;3分=受损足抬起,走动时不着台面;伴明显足内收蜷缩畸形。
正中神经短潜伏期体感诱发电位(SLSEP)测定方法如文献,刺激和记录均使用2402号针灸针,参考电极置于附近1cm处皮下,刺激正中神经。记录并计算术侧Erb's点与C6椎板上点潜伏期的差值(E'-C6 Lat)、C6椎板上点波幅(C6Amp),Erb's点与C6椎板测量点距离,计算体感诱发电位传导速度(MCV)。
病理切片的制作与观察大鼠各项指标检测完成后,大剂量麻醉致死,迅速取出压线段脊髓及神经根,固定、石蜡包埋,进行HE染色,光镜下观察轴索、髓鞘和神经元的形态及神经内膜及间质内血管的变化、是否有异常物质的沉积和炎症细胞的浸润等来明确病变性质;分别统计各组:神经细胞空泡样变、坏死灶、轴突扭曲髓鞘脱失呈鱼骨状等阳性病变的病例数。
5.数据处理
提取工艺的优化使用正交设计(Orthogonal design);使用SPSS11.5行单因素重复测量数据的方差分析(Repeated Measures),Mauchly球形检验结果,若P＞0.05,满足球形假设,无需校正;若P≤0.05,则不满足球形假设,用ε校正系数为Greenhouse-Geisser校正的结果。计量数据用均数±标准差((?)±s)描述,治疗前后用配对t检验,各组间比较用单向方差分析(One-Way ANOVA)。
三、结果
实验大鼠均无明显的体重减轻,伤口愈合良好,无自噬及舔咬,无脓性分泌物。
1.葛根提取物对大鼠颈神经根性疼痛行为的影响
在术后5天时,分别评价各组自发疼痛行为和步态异常评分差异无显著性意义(P＞0.05)。实验结束时,生理盐水组的大鼠上肢仍弯曲紧贴胸口,其前肢避免触地,触地部位为肘部,其5分钟内前肢离地时间值无明显变化,步态蹒跚,躯体倾斜;EP高、中、低剂量治疗组的大鼠疼痛相关行为明显减轻,四肢趋向于平等支持体重,腕部或爪背部触地,呈稍不平衡步态。
2.葛根提取物对大鼠受损神经根修复的影响
生理盐水组大鼠的组织病理切片形态学观察见神经元坏死,假单极神经细胞结构消失,形成卫星细胞环绕的空洞;后根神经轴突及膜细胞结构不清,雪旺细胞核堆积,轴突有断裂,周围髓鞘消失,形成空洞;脊髓白质的后索、外侧索呈空泡样变性,脊髓灰质、白质处均有胶质细胞增生。葛根提取物高、中、低剂量组和颈复康组神经损伤程度较轻,出现节细胞空泡变性,坏死及髓鞘鱼骨变的例数较少,提示葛根提取物对大鼠受损神经有修复和保护作用。
3.葛根提取物对大鼠受损神经根电生理的影响
3.1对C6椎板上点波幅(C6 Amp)的影响不同组之间评分结果无显著性差异(F=6.878,P＜0.001),治疗前后评分结果有显著差异(F=1394.719,P＜0.001);在不同分组之间,除生理盐水组(t=0.890,P=0.403)无显著性差异,其余各组经配对t检验,结果均有显著差异,t值分别为28.714、17.540、12.529和21.616,均为P＜0.001。从不同组来看,治疗前差异没有统计学意义(F=0.822,P=0.520),治疗后评分结果差异有显著差异(F=14.432,P＜0.001),其中以颈复康组疗效最显著。各组与治疗前后之间存在交互效应(F=93.059,P＜0.001),由表可知,治疗后波幅的最大值在颈复康组,最小值在治疗后生理盐水组;
3.2对潜伏期的差值(E'-C6Lat)的影响不同组之间评分结果无显著性差异(F=20.025,P＜0.001),治疗前后评分结果有显著差异(F=384.062,P＜0.001);在不同分组之间,除生理盐水组(t=0.314,P=0.763)无显著性差异,其余各组经配对t检验,结果均有显著差异,t值分别为11.081、9.178、16.024和10.299,均为P＜0.001。从不同组来看,治疗前差异没有统计学意义(F=0.368,P=0.830),治疗后评分结果差异有显著差异(F=39.430,P＜0.001),其中以颈复康组疗效最显著。各组与治疗前后之间存在交互效应(F=27.133,P＜0.001),由表可知,治疗后潜伏期差值的最大值在生理盐水组,最小值在治疗后颈复康组;
3.3对体感诱发电位传导速度(MCV)的影响不同组之间评分结果有显著性差异(F=3.183,P=0.025),治疗前后评分结果有显著差异(F=187.437,P＜0.001);在不同分组之间,生理盐水组(t=0.708,P=0.502)无显著性差异,高剂量组(t=9.642,P＜0.001)、中剂量组(t=5.170,P=0.001)和低剂量组(t=14.803,P＜0.001)结果均有显著性差异。从不同组来看,治疗前差异没有统计学意义(F=0.394,P=0.812),治疗后评分结果差异有显著差异(F=9.327,P＜0.001),其中以高剂量组疗效最显著。各组与治疗前后之间存在交互效应(F=16.372,P＜0.001),由表可知,治疗后传导速度最大值在颈复康组,最小值在治疗后生理盐水组;
四、结论
1.葛根提取物对神经根型颈椎病神经根性疼痛大鼠有较好的止痛作用,当葛根提取物中含葛根素剂量为266.1、133.0、66.5mg·kg~(-1)·d~(-1)时可以显著减轻大鼠疼痛行为,并且存在一定的量效关系;
2.葛根提取物各剂量组可以改善根性痛大鼠受损神经根形态结构,减少出现节细胞空泡变性、坏死及髓鞘鱼骨变的例数,存在一定的量效关系;
3.对神经电生理的影响研究中发现,葛根提取物各剂量组能升高波幅、缩短潜伏期及加快体感诱发电位传导速度,并且提示存在一定的量效关系。
Objective and significance
Cervical disc disease is the neck,bone,cartilage,ligaments became degeneration, the secondary stimulation or around the organizational structure of oppression such as the spinal cord and nerve roots,blood vessels,soft tissue,sympathetic and so on, caused a group of sick syndrome eventually.Epidemiological investigations revealed that the adults in our country in the rate of cervical syndrome approximately 3.8%to 17.6%.
Conservative projections based on the current population,there are at least 50 to 100 million people are afflicted by cervical syndrome.Cervical spondylotic radiculopathy is the highest incidence of the cervical syndrome,about the total incidence of 60%to 70%.CSR is the nerve root injury by various factors which led invasion and its dominant regional sensory,motor and reflex obstacles for the pain caused to the main clinical manifestations of a disease.In the medical field on cervical syndrome understanding of the concept of the earliest,most from cervical spondylotic radiculopathy.CSR's main symptoms of nerve root pain.
At present,the type of cervical nerve root pain of the illness the exact mechanism is not yet entirely clear,but more and more people think that oppression mechanical and biochemical factors in the pathogenesis of factors play an important role.Disc degeneration caused Osteophyte proliferation,proliferation of osteophyte nerve root compression caused nerve membrane without the protection of the nerve root to produce nerve edema and ischemia-based pathological changes.
Lots of documents indicate that the disc degeneration of the nerve root injury and multiple inflammatory neurotransmitters,such as cytokines,leukotrienes,and tumor necrosis factor,immunoglobulin and interleukin and etc.However,machinery of oppression and stimulate synergies inflammatory nerve root caused more serious damage.Therefore,the prevention and treatment of cervical nerve root pain of the illness is the most effective way to protect and repair damaged nerve root.At present, there is no effective drug treatment of cervical nerve root pain of the illness;doctors use the combination therapy treatments such as traction,acupuncture,massage manipulation,physical therapy,etc.The severity of the patients may choose surgical treatment,but indications are the restrictions,and surgery risky,costly,and there still exist the possibility of recurrence.
How to play in medical treatment of cervical nerve root disease on before us has become the subject.According to the nerve root of the characteristics of cervical disease,we developed a "JingZhuiShu" clinical use for many years to achieve a satisfactory effect.Animal experiments and clinical studies suggest that "JingZhuiShu " to improve microcirculation and reduce platelet aggregation,lower blood viscosity, increased flexibility vessel wall,expanding blood vessels,can significantly improve the nerve root in patients with cervical pain,numbness,stiffness,cold,The inability of the symptoms of muscular dystrophy.Puerarin the main contain isoflavones, triterpene saponins and alkaloids,etc.Of these components,puerarin(Pur) as its main active ingredient,the chemical name is 8 -β-D-glucopyranosyl-4',7 - dihydroxy Isoflavones.Studies have shown that puerarin regulate bone metabolism and improve the body blood circulation,reducing myocardial oxygen consumption,lower blood sugar,prevent hypertension and arteriosclerosis,protect nerve cells,anti-liver drug, anti-inflammatory,expectorant,antipyretic and improve body immunity,antibacterial, antiviral and other pharmacological effect.
In order to study the puerarin treatment of cervical nerve root pain Binggen of the possible mechanisms of puerarin on the cervical nerve root damage to repair,we learn from the behavior of nerve morphological,physiological and neurological perspective.Our aim is not only to explore both the puerarin cervical nerve root of the root of pain and damage repair of nerve root,but also to clear Chinese herbal compound,"JingZhuiShu" of the effect of mechanisms to provide experimental basis.
Method and content
1.Puerarin Lobata(Willd.) Ohwi Identification.
TLC Identification According to TLC(AppendixⅥB) demands of a test solution of the lessons of these two 10μl,respectively,at the same point in sodium carboxymethyl cellulose for the silicone adhesive H plate,into strips,chloroform -methanol - water(7:2.5:0.25) for the launch of start,removed,dried,home UV light (365 nm) under review.In the chromatogram of the corresponding position for the test and reference substance two chromatograms of the same color have shown that the fluorescence spots.According to the 2005 edition of "Chinese Pharmacopoeia (1)",Puerarin in dry Puerarin Lobata(Willd.) Ohwi content of not less than 2.4%.
2.Puerarin optimum extraction of the establishment and the establishment of standards for quality control.
Puerarin to target for inspection,the proportion of raw materials solution (14:1,16:1,18:1),the extraction time(1,1.5,2) h,extraction times(1,2,3) for the inspection,by L9(34) orthogonal table orthogonal test,compare water extraction and ethanol extraction method to determine the best Puerarin extraction technology and quality control standards.Extraction Process in accordance with the best verification test done three batches obtained RSD value,calculating the average concentration.
3.Model and intervention.
All rats in the left-SLSEP preoperative detection,detection of the normal access. According to the literature Oxfam spinal surgery modeling interpolation line,slightly improved:to 0.8 cm in length,has a diameter of 0.55 mm nylon fishing line interpolation of a line,C7 laminectomy fenestration,were inserted along the longitudinal axis to the spinal cord C7-T1 and C5-C7 nerve roots,some overlapping, firmly embedded in the bone of the spinal canal.A total of seven rats in surgery or died after surgery.More than 53 rats in the first five days after receiving evaluation, selection meet the following conditions:①model around the side of E'-C6 Lat for the growth of 0.2-0.4 ms;②gait score of 2-3 points,no symptoms of spinal cord compression,a total of 47 rats into the group.Random numbers,use excel balanced generate five treatment groups,each with eight:saline group,puerarin extract of low, medium and high-dose group,control group.Control group with "Jingfukang" of particle density of 1:1 made solution,all treatment groups will be concentrated liquid capacity to 1 ml,by gavages' method of administration.
4.The evaluation of the extraction of Puerarin lobata(Willd.) Ohwi to restoration of the injured nerve behavior observed in rats.
Measurement of body weight in the experimental rats at the beginning and end; 9-10.30 everyday observed in rats placed in the box observation after 30 min:See whether there are rats autophagy,licking bite,scream and recorded within 5 min of the affected limbs from the cumulative time,the calculation of the average of three times;Use the method of Dubuisson and Kawakami and evaluate pain from the contraction of gait disturbance:1 = normal gait,no foot deformities;= 2 damage feet touch the glass surface,walking limp;foot or huddling malformation;3 = damaged foot lift,walking cannot touch the table,foot deformity or crouch obviously.Median nerve short-latency somatosensory evoked potentials(SLSEP) Determination.
Use acupuncture needles stimulate and record the reference electrode placed 1 cm subcutaneous rats and stimulating the median nerve.Recorded and calculated operated side Erb's point C6 laminectomy,and the latency point margin(E'-C6 Lat), the C6 laminectomy point amplitude(C6 Amp),and C6 Erb's point distance measurement points laminectomy,somatosensory evoked potentials calculated conduction velocity(MCV).
Detection of indicators after the completion of large doses of narcotic death, pressure line quickly removed spinal cord and nerve roots,fixed,paraffin-embedded, HE staining,was observed under light microscope axonal,and the myelin sheath of the neurons and neural patterns and endometrial stromal vascular changes,whether there are abnormal substances deposition and the infiltration of inflammatory cells, such as to the nature of specific diseases;each group respectively statistics:nerve cells vacuolar changes,necrotic foci showed loss of myelin axons distorted fish bone-shaped lesions,such as the number of cases positive.
5.The statistical analysis of experimental data.
Extraction of optimizing the use of orthogonal design,SPSS 11.5 single factor to the use of repeated measurement data analysis of variance(Repeated Measures), Mauchly spherical test results if P＞0.05 meets spherical assumption,without correction;if P≤0.05,spherical not meet the assumptions used s correction coefficient of Greenhouse-Geisser correction results.Measurement data with mean±standard deviation((?)±s) described before and after treatment using paired t test, each group compared with one-way analysis of variance(One-Way ANOVA).
Result
The rats had no significant weight loss,wound healing well,no self-licking macrophages and bite,no Nongxing secretions.
1.The EP on the treatment of pain in rats.
After 5 days,the evaluations of each group were spontaneous acts of pain and abnormal gait score difference was not significant(P＞0.05).The end of the experiment,the rats saline group still bending close upper chest,to avoid conflict with its foreleg,the touchdown site for the elbow,five minutes from the time percentile forelimb no significant changes in gait stumble,body tilt;EP high,medium and low dose treatment group pain-related behavior in rats reduced significantly limbs tend to support equal weight,or claw back contravention of the wrist,a slightly uneven gait;
2.The EP on rat nerve root injury treatment.
Saline group of rats observed histologically visible morphological neuronal death,false unipolar nerve cell structure disappeared,a satellite cells surrounded by empty;axons after nerve cell membrane structure and unclear,Schwann cell accumulation shaft a sudden rupture,the surrounding myelin disappeared,a hollow; spinal cord white matter after the cable,a cable lateral vacuolar degeneration of spinal cord gray matter,white matter gliosis were.Puerarin extract high,medium and low dose group and neck nerve injury rehabilitation lesser extent,there ganglion cell degeneration and necrosis and myelin fish bone changes were fewer,suggesting that the extract of Pueraria damage in rats nerve restoration and protection.
3.The EP on the nerve root injury Electroneurophysiological therapeutic effect.
On the group on rat(C6 Amp) impact.Score results among different groups had no significant difference(F = 6.878,P＜0.001),and score the results before and after treatment were significantly different(F = 1394.719,P＜0.001) between different groups,in addition to the normal saline group(t = 0.890,P = 0.403) with no significant difference in the rest of the group,paired t test,the results were significantly different,t = 28.714,17.540,12.529 and 21.616,respectively,P＜0.001. From different groups,the treatment did not statistically significant difference(F = 0.822,P = 0.520),post-treatment score difference in the results were significantly different(F = 14.432,P＜0.001),in which the carotid rehabilitation of the most significant group.Groups with the interaction between before and after treatment effect(F = 93.059,P＜0.001),by the table,we can see that after treatment the maximum amplitude in the neck rehabilitation group,the minimum in the saline group after treatment;
On the group on rat(E'-C6Lat) impact.Score results among different groups had no significant difference(F = 20.025,P＜0.001),and score the results before and after treatment were significantly different(F = 384.062,P＜0.001) between different groups,in addition to the normal saline group(t = 0.314,P = 0.763) with no significant difference in the rest of the group,paired t test,the results were significantly different,t = 11.081,9.178,16.024 and 10.299,respectively,P＜0.001. From different groups,the treatment did not statistically significant difference(F = 0.368,P = 0.830),post-treatment score difference in the results were significantly different(F = 39.430,P＜0.001),in which the carotid rehabilitation of the most significant group.Groups with the interaction between before and after treatment effect(F = 27.133,P＜0.001),by the table,we can see that the maximum incubation period after treatment in the saline group,the minimum rehabilitation in the treatment of persistent group;
On the group on rat(MCV) impact.The results among different groups score There was a significant difference(F = 3.183,P = 0.025),the score results before and after treatment were significantly different(F = 187.437,P＜0.001) between different groups,and normal saline group(t = 0.708,P = 0.502) with no significant difference between the high dose group(t = 9.642,P＜0.001) in the dose group(t = 5.170,P = 0.001) and low dose group(t = 14.803,P＜0.001) results there was significant difference.From different groups,the treatment did not statistically significant difference(F=0.394,P = 0.812),post-treatment score difference in the results were significantly different(F = 9.327,P＜0.001),with the high dose group of the most notable.Groups with the interaction between before and after treatment effect(F = 16.372,P＜0.001),by the table,we can see that the maximum rate after treatment in the neck rehabilitation group,the minimum in the saline group after treatment;
Conclusions
1.Extraction of Puerarin lobata(Willd.) Ohwi have better analgesic effect on nerve root type of cervical nerve root pain.When the Puerarin of Puerarin lobata(Willd.) Ohwi in the concentration of 266.1,133.0,66.5 mg ~ kg"l ~ dl,can significantly reduce pain in rats,and there are certain dose-effect relationship;
2.EP each dose group can significantly improve the structure of nerve damage, reduced ganglion cell degeneration and necrosis,and the myelin sheath of fish bone changes;
3.In the impact of electrophysiology study,EP-dose groups can increase the volatility,reduce the incubation period and accelerate somatosensory evoked potential conduction velocity,and prompts a certain dose-effect relationship.
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