“腰三针”干预腰多裂肌损伤大鼠多种氧化应激因子的表达

Effect of“Lumbar Three Needles”on expression of oxidative stress factors in rats with multifidus muscles injury

背景:腰椎后路手术是目前临床进行诸多手术如椎间盘切除、椎管减压等的主要手术入路,但有10%-40%的腰椎后路手术患者在术后不久可再次出现腰背部疼痛及相关功能障碍,这与手术过程中对椎旁肌如多裂肌的过度牵拉或钝性损伤有关。目的:观察腰三针对大鼠腰多裂肌损伤后氧化应激因子丙二醛、活性氧、超氧化物歧化酶、谷胱甘肽过氧化物酶及磷酸化蛋白激酶B表达的影响。方法:将24只雄性SD大鼠随机分为3组,每组8只。模型组、腰三针组分别采用0.5%布比卡因盐酸盐肌内注射复制大鼠腰多裂肌损伤模型;对照组予生理盐水肌内注射。对照组与模型组造模后不进行针刺干预,腰三针组造模后给予针刺靳三针之"腰三针",即大肠俞、肾俞、委中,针刺后连接电针,波形选用疏密波,电针频率采用2 Hz/10 Hz,电流强度选择1 mA,持续干预20 min,每天干预1次,共干预7 d。电针干预7 d后,通过苏木精-伊红染色观察损伤部位多裂肌形态学变化,采用试剂盒检测各组大鼠多裂肌中丙二醛、活性氧、超氧化物歧化酶、谷胱甘肽过氧化物酶的表达;采用Western-blot法检测多裂肌中磷酸化蛋白激酶B蛋白的表达。实验经广东省第二中医院实验动物伦理委员会批准,伦理批准号:048617。结果与结论:①苏木精-伊红染色显示,干预7d后,模型组可见肌纤维损伤明显,但已出现部分修复,但炎性细胞数量仍较多;腰三针组可出现较多新生的肌纤维,炎性细胞明显减少;②腰三针组肌纤维横截面积明显优于模型组(P<0.01);③干预后,模型组丙二醛、活性氧表达量明显高于对照组(P<0.01);腰三针组丙二醛、活性氧表达明显低于模型组(P<0.01),超氧化物歧化酶、谷胱甘肽过氧化物酶表达高于模型组(P<0.01或P<0.05);腰三针组磷酸化蛋白激酶B蛋白表达明显高于模型组(P<0.01);④结果说明,腰三针干预可显著降低多裂肌损伤后氧化应激水平,可能与提高磷酸化蛋白激酶B的表达有关。

BACKGROUND:Posterior lumbar spine surgery is currently the main surgical approach for many surgical operations such as discectomy and spinal canal decompression.However,10%-40%of patients with posterior lumbar spine surgery can have low back pain and related dysfunctions soon after surgery,which is related to excessive stretching or blunt injury to the paraspinal muscles such as the multifidus muscle during the operation.OBJECTIVE:To explore the effect of"Lumbar Three Needles"on the expression of oxidative stress factors,including malondialdehyde,reactive oxygen species,superoxide dismutase,glutathione peroxidase and phosphorylated protein kinase B,in rats with multifidus muscles injury.METHODS:Twenty-four male Sprague-Dawley rats were randomly divided into control group,model group and"Lumbar Three Needles"group,with 8 rats in each group.An animal model of multifidus muscles injury was made by intramuscular injection of 0.5%bupivacaine hydrochloride in the model and treatment groups in control and"Lumbar Three Needles"group,while normal saline was injected in the control group.No acupuncture intervention was given in the control group and model group,while Dachangshu,Shenshu and Weizhong acupoints were electroacupunctured in the"Lumbar Three Needles"group.Needles were then stimulated electrically using a Han’s Acupoint Nerve Stimulator with density wave,frequency of 2 Hz/10 Hz and continuous current of 1 mA for 20?minutes,once daily for 7 days in total.The multifidus muscle was stained with hematoxylin-eosin staining to observe morphological changes,and kits were used to observe the expression of malondialdehyde,reactive oxygen species,superoxide dismutase,and glutathione peroxidase,while western blot was applied to observe the expression of phosphorylated protein kinase B.The study protocol was approved by the Animal Ethic Committee of Guangdong Second Traditional Chinese Medicine Hospital with an approval No.048617.RESULTS AND CONCLUSION:Hematoxylin-eosin staining results indicated that the muscle fibers were partially fixed in the model group and there were still a lot of macrophages after 7 days of intervention.Compared with the model group,more newborn muscle fibers and fewer macrophages could be seen in the"Lumbar Three Needles"group.After intervention,the cross-sectional area of the multifidus muscles in the"Lumbar Three Needles"group was significantly bigger than that in the model group(P<0.01).The expression levels of malondialdehyde and reactive oxygen species in the model group were significantly increased compared with the control group(P<0.01)and"Lumbar Three Needles"group(P<0.01).The expression of superoxide dismutase,glutathione peroxidase and phosphorylated protein kinase B in the"Lumbar Three Needles"group was significantly higher than that in the model group(P<0.01 or P<0.05).Therefore,"Lumbar Three Needles"can significantly decrease the oxidative stress level after multifidus muscle injury,which may be related to the improvement of phosphorylated protein kinase B.