Mechanism Study of the Promoting Action of Elongated Needle Acupuncture on Functional Repair in Rats with Acute Spinal Cord Injury

目的 观察芒针对急性脊髓损伤炎症反应和神经细胞凋亡的影响,研究PI3K/Akt和MAPK/ERK信号转导途径是否参与芒针的神经保护作用,探讨芒针促脊髓损伤修复的具体作用机制.方法 将150只成年雄性SD大鼠随机分为A组、B组、C组、D组和E组,每组30只.采用改良Allen＇s法制作大鼠脊髓中度损伤模型,A组为假手术组,不予以损伤脊髓及芒针治疗;B组造模后不予以芒针治疗;C组造模后采用芒针治疗;D组造模前0.5 h鞘内注射LY294002,造模后采用芒针治疗;E组造模前0.5 h鞘内注射PD98059,造模后采用芒针治疗.分别采用BBB评分检测大鼠的自发活动;ELISA检测炎性因子TNF-α 、IL-6、IL-1β、NF-κB的含量;TUNEL检测细胞凋亡的程度;免疫组化检测Bcl-2和Bax阳性细胞水平;Western印迹分析p-Akt和p-ERK在脊髓组织的表达,RT-PCR分析Cyt-C和Caspase-3在体内的表达.相对的下行Akt和ERK信号通路通过LY294002和PD98059特异性抑制剂处理分析在体内的抑制作用.结果 炎症反应和PI3K/Akt和MAPK/ERK信号通路抑制的神经元凋亡参与了脊髓损伤大鼠模型的损害,芒针介导的神经保护作用与Bax蛋白阳性神经元数目的减少及Bcl-2蛋白阳性神经元数量的增加有关.芒针治疗能改善大鼠的运动功能,PI3K/Akt和MAPK/ERK信号通路中p-Akt和p-ERK的激活,通过下调Bax蛋白和Bcl-2表达上调,抑制了线粒体凋亡途径关键因子Cyt-C的表达.TUNEL法检测并抑制激活神经元凋亡的Caspase-3的级联.PI3K/Akt和MAPK/ERK信号通路特异性抑制剂LY294002和PD98059的应用抑制了p-Akt和p-ERK的表达.结论 芒针促脊髓损伤修复的神经保护作用可能与炎症反应和PI3K/Akt和MAPK/ERK信号通路的激活、通过下调Bax蛋白和Bcl-2表达上调以及抑制线粒体途径诱导的凋亡有关.Objective To observe the effect of elongated needle acupuncture on inflammatory reactions and apoptosis in acute spinal cord injury, investigate if PI3K/Akt and MAPK/ERK signal transduction pathways are involved in the neuroprotective effect of elongated needle acupuncture and explore the promoting action of elongated needle acupuncture on spinal cord injury repair.Method One hundred and fifty adult male SD rats were randomized into groups A, B, C, D and E, 30 each. A model of moderate spinal cord injury was made by modified Allen＇s method. Group A received a sham operation without spinal cord injury and no elongated needle acupuncture. Group B did not receive elongated needle acupuncture after model making. Group C received elongated needle acupuncture after model making. Group D received an intrathecal injection of LY294002 at 0.5 hour before model making and elongated needle acupuncture after model making. Group E received an intrathecal injection of PD98059 at 0.5 hour before model making and elongated needle acupuncture after model making. Rat spontaneous activity was examined using the BBB rating. Inflammatory cytokines TNF-α, IL-6, IL-1β and NF-κB contents were measured by ELISA. The degree of apoptosis was determined by TUNEL. Bcl-2- and Bax-positive cell levels were measured by an immunohistochemical method. Spinal p-Akt and p-ERK expressions were determined by Western blot. In vivo expressions of Cyt-C and Caspase-3 were determined by RT-PCR. The in vivo inhibitory effect on downstream Akt and ERK signaling pathways was investigated using specific inhibitors LY294002 and PD98059.Result Inflammatory reactions and neuronal apoptosis due to the inhibition of PI3K/Akt and MAPK/ERK signal pathways were involved in the damage in a rat model of spinal cord injury. The neuroprotective effect of elongated needle acupuncture was related to a decrease in the number of Bax protein-positive neurons and an increase in the number of Bcl-2 protein-positive neurons. Elongated needle acupuncture treatment improved r浙江省中医药科研计划项目（2008CB067

Effects of electro-acupuncture on neuronal apoptosis and associative function in rats with spinal cord injury

目的 :探讨电针对大鼠急性脊髓损伤后膀胱功能改善的影响及作用机制。方法 :取健康成年雄性SPf级Sd大鼠60只,体重220~250 g,适应性饲养1周后,将大鼠按照随机数字表法随机分为假手术组、模型组、电针组和电针对照组,各15只。假手术组不予任何刺激,模型组、电针组和电针对照组大鼠采用改良AllEnS法制作脊髓损伤中度损伤Sd大鼠模型,模型组不予治疗,电针组给予秩边与水道穴电针治疗,电针对照组给予秩边与水道穴旁开0.5寸电针治疗,频率2/100 Hz,电流1 M A,刺激15 MIn,电针左右隔次交替,每日1次,共7次;分别于术后1、7 d观察大鼠残余尿量、排尿量的变化;术后7 d处死大鼠取伤段脊髓观察各组大鼠凋亡情况,检测bCl-2、bAX、bAd含量的变化。结果:造模后3组大鼠均出现不同程度的膀胱功能障碍。术后7 d,电针组、电针对照组残余尿量较术后1 d明显降低(P<0.001),且电针组与电针对照组比较差异有统计学意义(P<0.01);电针组、电针对照组较模型组在术后7 d排尿量增加,且电针组与电针对照组比较差异有统计学意义(P<0.001);TunEl发现电针可以抑制脊髓神经细胞的凋亡,电针组、电针对照组与模型组相比在术后7 d脊髓神经细胞凋亡率显著增加(P<0.01,P<0.05),且电针组与电针对照组比较差异有统计学意义(P<0.05);与模型组相比,电针组、电针对照组bAX、bAd的阳性表达率降低(P<0.01,P<0.05),而bCl-2的阳性表达率升高(P<0.01);且电针组与电针对照组比较差异有统计学意义(P<0.05)。结论 :电针能明显促进急性脊髓损伤的修复,其机制可能为通过增加bCl-2、抑制bAX、bAd的表达,从而抑制脊髓神经元细胞的凋亡发生作用的。Objective:To explore the effect of electro acupuncture to improve the bladder function after acute spinal cord injury in rats and its possible mechanism.Methods:Sixty healthy adult male SD rats of SPF grade,with body weight of 220 to250 g,one week after feeding adaptation,were randomly divided into sham operation group,model group,electro acupuncture group,electro acupuncture control group with 15 rats in each group.Sham operation group underwent no stimulation,and the moderate damage model of spinal cord injury were made in other three groups according to modified Allens method.The model group were not treated,electro acupuncture group were treated with electro acupuncture on Zhibianxue and Shuidaoxue,and electro acupuncture control group were treated with electro acupuncture on 0.5 inch next to Zhibianxue and Shuidaoxue.The frequency of 2 / 100 Hz,current of 1 m A,stimulation time of 15 min,once a day,left and right alternately stimulate every time,for a total of 7 times.The changes of residual urine volume and urine output in rats at the 1st and the 7th days after operation were observed.And 7 d later,the rats were sacrificed and the injured spinal cord were taken out to observe the apoptosis,and to detect the changes of Bcl-2,Bax,Bad content.Results:After modeling,the rats of three groups showed different bladder dysfunction.In electro acupuncture group and electro acupuncture control group,the residual urine volume of the 7th day after operation was significant lower than the 1st day after operation(P<0.001),and there was statistically significant difference on the 7th day after operation between two groups(P<0.001).Compared with model group,the urine output of electro acupuncture group and electro acupuncture control group was significantly increased on the 7th day after operation,and there was significant difference between electro acupuncture group and electro acupuncture control group(P<0.001).Electro acupuncture can inhibit apoptosis of spinal cord neurons by TUNEL detection.Postoperative at 7 d,the rate of nerve cell apoptosis in electro acupuncture group and electro acupuncture control group was significant increased than model group(P<0.01,P<0.05),and there was significant difference between electro acupuncture group and electro acupuncture control group(P<0.005).Compared with model group,the positive expression rate of Bax,Bad decreased(P<0.01,P<0.05),and Bcl-2 increased(P<0.01)in electro acupuncture group and electro acupuncture control group,there was significant difference between electro acupuncture group and electro acupuncture control group(P<0.01).Conclusion:Electro acupuncture can obviously promote the repair of acute spinal cord injury,its mechanism may be through increasing Bcl-2,inhibiting the expression of Bax,Bad,which inhibits the apoptosis of spinal cord neurons.浙江省中医药科研计划项目(编号:2008CB067)~

猪圆环病毒2型抗体免疫金标检测试纸的研制

应用酶联免疫原理和胶体金层析技术,采用特殊的生产工艺,在玻璃纤维膜包被胶体金标记PCV-2抗原,在硝酸纤维素膜上检测线和对照线处分别包被PCV-2抗原和兔抗PCV-2抗体,制成猪圆环病毒2型抗体免疫金标检测试纸。当待检样品阳性时,在检测线处形成抗原抗体的免疫复合物而凝聚显色;当待检样品阴性时,检测线处不形成抗原抗体免疫复合物不显色。整个试验过程只需15min。试纸与ELISA试剂比较,两者都具有微量、特异、准确的优点,且金标试纸独具操作方便、快速和结果直观、容易判定的优点