针刺联合鼠神经生长因子对脑瘫幼鼠神经行为和脑组织生长代谢相关蛋白表达的影响

Study of acupuncture combined with rat nerve growth factor on neurobehavioral ability of cerebral palsy infant rats and its brain tissue growth and metabolism associated proteins

目的:探讨针刺联合鼠神经生长因子(NGF)对脑瘫幼鼠的疗效及对其脑组织神经凋亡、修复生长及能量代谢相关蛋白表达的影响。方法:选取70只SD幼鼠,其中50只采用结扎单侧颈动脉使脑组织缺血+密闭缺氧法建立脑瘫幼鼠模型,20只设立空白对照组和假手术组,每组10只。30只造模成功的幼鼠随机分为模型组、NGF组和联合治疗组。空白对照组不予处理;假手术组:切开颈部皮肤分离出左侧颈动脉而后缝合并消毒创口;NGF组:腹腔注射NGF 2000 U·kg^(^(-1))·d^(-1);联合治疗组腹腔注射给药NGF 2000 U·kg^(-1)·d^(-1)的基础上进行针刺治疗,穴取"百会"、左侧"颞Ⅰ针""大椎""脊中""曲池""涌泉""合谷""肘节""膝前穴",每天1次;模型组腹腔注射同体积的0.9%NaCl溶液,均连续干预14d。治疗完成后进行神经行为能力评分,而后取各组幼鼠脑组织TUNEL法检测细胞凋亡率、PCR检测凋亡相关基因Bax、Bcl-2及Casp3的表达;Western blot检测各组幼鼠脑组织中神经生长相关蛋白(GAP-43)和单羧酸转运蛋白1(MCT1)的表达量。结果:干预后,空白对照组和假手术组幼鼠各项神经行为能力均无异常,而模型组、NGF组及联合治疗组均有不同程度的神经行为异常情况,其中联合治疗组和NGF组神经行为能力评分低于模型组(均P<0.05),且联合治疗组低于NGF组(均P<0.05)。联合治疗组及NGF组左侧脑细胞凋亡率、Bax及Casp3表达水平均低于模型组(均P<0.05),且两组Bcl-2均高于模型组(均P<0.05),联合治疗组较NGF组Bax及Casp3降低更明显(均P<0.05);联合治疗组和NGF组GAP-43及MCT 1蛋白表达量高于模型组(均P<0.05),且联合治疗组高于NGF组(均P<0.05)。结论:针刺联合NGF治疗脑瘫幼鼠可有效改善神经行为能力,抑制神经细胞凋亡,并通过上调GAP-43和MCT 1表达促进脑组织损伤修复和能量代谢。

Objective To study the effects of acupuncture combined with rat nerve growth factor（NGF） on the cerebral palsy infant rats and the proteins which associated with growth, apoptosis and metabolism. Methods Seventy infant rats were selected, Fifty infant rats of which were made the cerebral palsy infant model by the ligation of unilateral carotid artery for cerebral ischemia and oxygen-deficient environment, then the 30 model rats were randomly divided into a model group, a NGF group and a combined group, 10 rats in each group. Twenty infant rats were used in the sham-operated group and the blank control group, 10 rats in each group. The treatment was not given in the blank control group. The rats in the sham-operated group were cut the neck skin and separated the left carotid artery, and then sutured and disinfected the wound. The intraperitoneal injection of NGF（2000 U·kg^-1·d^-1） was used in the NGF group. Based on the injection in the NGF group, acupuncture was used in the combined group, once a day, and the acupoints were ＂Baihui＂（GV 20）, left ＂nieⅠ＂（extra）, ＂Dazhui＂（GV 14）, ＂Jizhong＂（extra）, ＂Quchi＂（LI 11）, ＂Yongquan＂（KI 1）, ＂Hegu＂（LI 4）, ＂Zhoujie＂（extra） and ＂Xiqianxue＂（extra）. The same volume of saline was intraperitoneally injected in the model group for continuous 14 days. Neurobehavioral ability score was evaluated after treatment. TUNEL were conducted to detect the brain cell apoptosis rate and the expressions of apoptosis associated gene Bax, Bcl-2 and Casp3 were detected by PCR. The level of nerve growth associated protein（GAP-43） and energy metabolism-related protein monocarboxylate transporter protien 1（MCT 1） were detected by Western blot. Results After intervention, the neurobehavioral ability of baby rats in the blank control and sham-operated group was normal, but there was various degrees of abnormity in the model group, NGF group and combined group.