魏氏核归丸对腰椎间盘突出大鼠髓核MMP2、MMP9、TIMP1表达的影响
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摘要
目的:探究魏氏核归丸对腰椎间盘突出症(Lumbar disc herniation,LDH)大鼠髓核基质金属蛋白酶2 (Matrix metalloproteinase 2,MMP2)、基质金属蛋白酶9 (Matrix metalloproteinase 9,MMP9)、基质金属蛋白酶抑制剂1 (Tissue inhibitor of metalloproteinase 1,TIMP1)表达的影响。方法:SPF级SD健康大鼠60只,随机分为假手术组、模型组以及魏氏核归丸低、中、高剂量组。通过自体椎间盘移植方法制备LDH大鼠模型,假手术组仅暴露神经根后缝合伤口。造模后5天后连续灌胃给药14天,低、中、高剂量组大鼠分别给予81、162、324 mg/mL魏氏核归丸;假手术组和模型组给予等量生理盐水。造模后观察大鼠的生物学行为,并在相应时间点测量大鼠左后肢机械刺激缩爪阈值,HE染色观察腰椎髓核形态变化;ELISA方法测量大鼠腰椎髓核MMP2、MMP9、TIMP1蛋白表达量变化。结果:造模后大鼠左后肢跛行、易激怒,无其它异常行为,魏氏核归丸干预后情况好转。造模5天,与假手术组比较,模型组大鼠左后肢缩爪阈值显著降低(P <0.05);灌胃7天,与模型组比较,魏氏核归丸高、中剂量组左后肢缩爪阈值均显著升高(P <0.05),高剂量组阈值上升最快,低剂量组差异无统计学意义(P> 0.05);灌胃14天,高剂量组阈值接近假手术组(P> 0.05)。HE染色显示,模型组髓核结构基本消失,形态遭到破坏,随着处理剂量的升高,髓核组织形态逐渐恢复正常。ELISA结果显示,灌胃后7天和14天,与假手术组比较,模型组、魏氏核归丸低、中、高剂量组大鼠髓核MMP2、MMP9、TIMP1蛋白表达水平均显著升高(P <0.05);与模型组比较,魏氏核归丸低、中、高剂量组大鼠MMP2、MMP9表达水平均显著降低(P <0.05),TIMP1表达水平均显著升高(P <0.05),且随着魏氏核归丸剂量的升高,MMP2、MMP9表达水平越来越低,TIMP1表达水平越来越高;与灌胃后7天比较,魏氏核归丸低、中、高剂量组MMP2、MMP9、TIMP1蛋白在灌胃后14天的表达水平均显著降低(P <0.05)。结论:魏氏核归丸能明显减轻LDH大鼠的病情,抑制大鼠髓核MMP2、MMP9蛋白表达,促进TIMP1表达。
Objective: To investigate the effect of Wei's Hegui pills on the expression of matrix metalloproteinase-2(MMP2),matrix metalloproteinase-9(MMP9) and matrix metalloproteinase inhibitor 1(TIMP1) of nucleus pulposus in rats with nucleus of lumbar disc herniation(LDH). Methods:Divided 60 healthy SD rats in SPF grade into the sham operation group,the model group, the group of low-dose Wei's Hegui pills, the group of middle-dose Wei's Hegui pills, and the group of high-dose Wei's Hegui pills randomly. Made LDH rat models by autologous intervertebral disc transplantation method. The sham operation group only sutured the wound after exposing the nerve root. Five days after modeling, applied drugs by gavage continuously for 14 days. The groups of low-dose, middle-dose and high-dose Wei's Hegui pills were given81,162,and 324 mg/m L of Wei's Hegui pills respectively,while the sham operation group and the model group were given the same amount of normal saline. Observed the biological behavior of the rats after modeling,and measured the threshold of mechanical stimulation of the left hind limbs of rats at the corresponding time points. Observed the morphological changes of the lumbar nucleus by HE staining. Measured the amount of expression changes of MMP2,MMP9 and TIMP1 proteins in the lumbar nucleus of rats. Results:After modeling,the rats were easily irritated with their left hind limbs paralyzed and without other abnormal behaviors. This situation improved after applying the intervention of Wei's Hegui pills. After 5 days of modeling,compared with the sham operation group,the threshold of paw withdrawal in the left hind limb of the rats in the model group was significantly decreased(P < 0.05). After 7 days of gavage,compared with the model group,the group of middle-dose Wei's Hegui pills and the group of high-dose Wei's Hegui pills were obviously increased(P < 0.05);the increase of threshold in the group of high-dose Wei's Hegui pills was the fastest, and there was no difference being found in the group of low-dose Wei's Hegui pills(P > 0.05). After 14 days of gavage,the threshold in the group of high-dose Wei's Hegui pills was close to that in the sham operation group(P > 0.05). HE staining showed that the nucleus pulposus structure in the model group disappeared and its morphology was destroyed,but it gradually returned to normal as the the dose increased in the treatment. The results of ELISA showed that the expression levels of MMP2,MMP9 and TIMP1 in the nucleus pulposus of rats in the model group,the groups of low-dose,middle-dose and high-dose Wei's Hegui pills were significantly higher than those in the sham operation group at the 7 th and 14 th day after gavage(P < 0.05);compared with the model group,the expression levels of MMP2 and MMP9 in the groups of low-dose,middle-dose and high-dose Wei's Hegui pills were clearly decreased(P < 0.05),while the expression level of TIMP1 was evidently increased(P < 0.05). With the increase of the dose of Wei's Hegui pills, the expression levels of MMP2 and MMP9 were getting lower and lower, and the expression level of TIMP1 was getting higher and higher. Compared with those after 7 days of gavage,the expression levels of MMP2,MMP9 and TIMP1 in the groups of low-dose,middle-dose and high-dose Wei's Hegui pills were decreased significantly after 14 days of gavage(P < 0.05). Conclusion: Wei's Hegui pills can significantly alleviate the condition of LDH rats, inhibit the expressions of MMP2 and MMP9 proteins in nucleus pulposus of rats,and promote the expression of TIMP1.
Objective: To investigate the effect of Wei's Hegui pills on the expression of matrix metalloproteinase-2(MMP2),matrix metalloproteinase-9(MMP9) and matrix metalloproteinase inhibitor 1(TIMP1) of nucleus pulposus in rats with nucleus of lumbar disc herniation(LDH). Methods:Divided 60 healthy SD rats in SPF grade into the sham operation group,the model group, the group of low-dose Wei's Hegui pills, the group of middle-dose Wei's Hegui pills, and the group of high-dose Wei's Hegui pills randomly. Made LDH rat models by autologous intervertebral disc transplantation method. The sham operation group only sutured the wound after exposing the nerve root. Five days after modeling, applied drugs by gavage continuously for 14 days. The groups of low-dose, middle-dose and high-dose Wei's Hegui pills were given81,162,and 324 mg/m L of Wei's Hegui pills respectively,while the sham operation group and the model group were given the same amount of normal saline. Observed the biological behavior of the rats after modeling,and measured the threshold of mechanical stimulation of the left hind limbs of rats at the corresponding time points. Observed the morphological changes of the lumbar nucleus by HE staining. Measured the amount of expression changes of MMP2,MMP9 and TIMP1 proteins in the lumbar nucleus of rats. Results:After modeling,the rats were easily irritated with their left hind limbs paralyzed and without other abnormal behaviors. This situation improved after applying the intervention of Wei's Hegui pills. After 5 days of modeling,compared with the sham operation group,the threshold of paw withdrawal in the left hind limb of the rats in the model group was significantly decreased(P < 0.05). After 7 days of gavage,compared with the model group,the group of middle-dose Wei's Hegui pills and the group of high-dose Wei's Hegui pills were obviously increased(P < 0.05);the increase of threshold in the group of high-dose Wei's Hegui pills was the fastest, and there was no difference being found in the group of low-dose Wei's Hegui pills(P > 0.05). After 14 days of gavage,the threshold in the group of high-dose Wei's Hegui pills was close to that in the sham operation group(P > 0.05). HE staining showed that the nucleus pulposus structure in the model group disappeared and its morphology was destroyed,but it gradually returned to normal as the the dose increased in the treatment. The results of ELISA showed that the expression levels of MMP2,MMP9 and TIMP1 in the nucleus pulposus of rats in the model group,the groups of low-dose,middle-dose and high-dose Wei's Hegui pills were significantly higher than those in the sham operation group at the 7 th and 14 th day after gavage(P < 0.05);compared with the model group,the expression levels of MMP2 and MMP9 in the groups of low-dose,middle-dose and high-dose Wei's Hegui pills were clearly decreased(P < 0.05),while the expression level of TIMP1 was evidently increased(P < 0.05). With the increase of the dose of Wei's Hegui pills, the expression levels of MMP2 and MMP9 were getting lower and lower, and the expression level of TIMP1 was getting higher and higher. Compared with those after 7 days of gavage,the expression levels of MMP2,MMP9 and TIMP1 in the groups of low-dose,middle-dose and high-dose Wei's Hegui pills were decreased significantly after 14 days of gavage(P < 0.05). Conclusion: Wei's Hegui pills can significantly alleviate the condition of LDH rats, inhibit the expressions of MMP2 and MMP9 proteins in nucleus pulposus of rats,and promote the expression of TIMP1.
引文
[1]陈少华,傅秀珍,张广清,等.腰椎间盘突出症中医护理模式研究[J].中国医药导报,2015,12(10):147-150.
[2]许电,许时良,陈慧珍.中西医结合治疗腰椎间盘突出症临床研究[J].中医学报,2017,32(5):879-883.
[3]Li Y,Fredrickson V,Resnick DK.How Should We Grade Lumbar Disc Herniation and Nerve Root Compression ASystematic Review[J].Clin Orthop Relat Res,2015,473(6):1896-1902.
[4]Baillet A,Grange L,TrocméC,et al.Differences in MMPs and TIMP-1 expression between intervertebral disc and disc herniation[J].Joint Bone Spine,2013,80(3):341-342.
[5]王涛,康汇,李红川.腰椎间盘突出患者组织MMP及TIMP指标的变化研究[J].湖南师范大学学报:医学版,2015,12(6):129-131.
[6]Ozkanli S,Kaner T,Efendioglu M,et al.The relation of matrix metalloproteinase 1,2,3 expressions with clinical and radiological findings in primary and recurrent lumbar disc herniations[J].Turkish Neurosurgery,2015,25(1):111-116.
[7]陆志东,金群华,陈志荣.大鼠非压迫性髓核突出模型的建立[J].中国矫形外科杂志,2003,11(2):1361-1363.
[8]郑国泉.中医针灸联合中医推拿综合治疗腰间盘突出症效果分析[J].中外医疗,2016,35(36):177-178,181.
[9]杨庆红.腰椎间盘突出症的中医护理体会[J].光明中医,2017,32(5):749-751.
[10]赵明宇,黄桂成.从腹论治腰椎间盘突出症的研究进展[J].中医学报,2012,27(2):217-219.
[11]李中锋,邓强,张彦军,等.中药熏蒸联合推拿治疗腰椎间盘突出症的临床效果[J].现代生物医学进展,2015,15(36):7067-7069,7110.
[12]王妍,李晗辉,罗敏,等.腰椎间盘突出症患者椎间盘镜技术术前心理状态与术后临床疗效的相关性分析[J].中国临床药理学杂志,2015,31(19):1931-1933.
[13]赖志刚,涂履超.针刀松解核归丸内服治疗腰椎间盘突出症[J].中医正骨,2001,13(3):44.
[14]甘维红.针刀治疗腰椎间盘突出症急性期的护理与康复指导[J].中国民族民间医药,2010,19(6):170.
[15]周臣安.蜜麻丸加腰背肌锻炼治疗腰椎间盘退行性病变[J].中国骨伤,2000,13(5):285.
[16]Xu H,Mei Q,He J,et al.Correlation of Matrix Metalloproteinases-1 and Tissue Inhibitor of Metalloproteinases-1 with Patient Age and Grade of Lumbar Disk Herniation[J].Cell Biochemistry&Biophysics,2014,69(3):439-444.
[17]Gupta RS,Wu XT,Hong X,et al.Technique of Percutaneous Transforaminal Endoscopic Discectomy for the Treatment of Lumbar Disc Herniation[J].Open Journal of Orthopedics,2015,5(7):208-216.
[18]Grzela K,Litwiniuk M,Zagorska W,et al.Airway Remodeling in Chronic Obstructive Pulmonary Disease and Asthma:the Role of Matrix Metalloproteinase-9[J].Archivum Immunologiae Et Therapiae Experimentalis,2016,64(1):1-9.
[19]Ming C.Cha,Peter P.Purslow.Expressions of matrix metalloproteinases and their inhibitor are modified by beta-adrenergic agonist ractopamine in skeletal fibroblasts and myoblasts[J].Canadian Journal of Animal Science,2017,92(2):159-166.
[20]Aras AB,Guven M,Balak N,et al.Evaluation of the Association Between Matrix Metalloproteinase 11 and Intervertebral Disc Disease[J].Turkish Neurosurgery,2015,26(2):274-279.
[2]许电,许时良,陈慧珍.中西医结合治疗腰椎间盘突出症临床研究[J].中医学报,2017,32(5):879-883.
[3]Li Y,Fredrickson V,Resnick DK.How Should We Grade Lumbar Disc Herniation and Nerve Root Compression ASystematic Review[J].Clin Orthop Relat Res,2015,473(6):1896-1902.
[4]Baillet A,Grange L,TrocméC,et al.Differences in MMPs and TIMP-1 expression between intervertebral disc and disc herniation[J].Joint Bone Spine,2013,80(3):341-342.
[5]王涛,康汇,李红川.腰椎间盘突出患者组织MMP及TIMP指标的变化研究[J].湖南师范大学学报:医学版,2015,12(6):129-131.
[6]Ozkanli S,Kaner T,Efendioglu M,et al.The relation of matrix metalloproteinase 1,2,3 expressions with clinical and radiological findings in primary and recurrent lumbar disc herniations[J].Turkish Neurosurgery,2015,25(1):111-116.
[7]陆志东,金群华,陈志荣.大鼠非压迫性髓核突出模型的建立[J].中国矫形外科杂志,2003,11(2):1361-1363.
[8]郑国泉.中医针灸联合中医推拿综合治疗腰间盘突出症效果分析[J].中外医疗,2016,35(36):177-178,181.
[9]杨庆红.腰椎间盘突出症的中医护理体会[J].光明中医,2017,32(5):749-751.
[10]赵明宇,黄桂成.从腹论治腰椎间盘突出症的研究进展[J].中医学报,2012,27(2):217-219.
[11]李中锋,邓强,张彦军,等.中药熏蒸联合推拿治疗腰椎间盘突出症的临床效果[J].现代生物医学进展,2015,15(36):7067-7069,7110.
[12]王妍,李晗辉,罗敏,等.腰椎间盘突出症患者椎间盘镜技术术前心理状态与术后临床疗效的相关性分析[J].中国临床药理学杂志,2015,31(19):1931-1933.
[13]赖志刚,涂履超.针刀松解核归丸内服治疗腰椎间盘突出症[J].中医正骨,2001,13(3):44.
[14]甘维红.针刀治疗腰椎间盘突出症急性期的护理与康复指导[J].中国民族民间医药,2010,19(6):170.
[15]周臣安.蜜麻丸加腰背肌锻炼治疗腰椎间盘退行性病变[J].中国骨伤,2000,13(5):285.
[16]Xu H,Mei Q,He J,et al.Correlation of Matrix Metalloproteinases-1 and Tissue Inhibitor of Metalloproteinases-1 with Patient Age and Grade of Lumbar Disk Herniation[J].Cell Biochemistry&Biophysics,2014,69(3):439-444.
[17]Gupta RS,Wu XT,Hong X,et al.Technique of Percutaneous Transforaminal Endoscopic Discectomy for the Treatment of Lumbar Disc Herniation[J].Open Journal of Orthopedics,2015,5(7):208-216.
[18]Grzela K,Litwiniuk M,Zagorska W,et al.Airway Remodeling in Chronic Obstructive Pulmonary Disease and Asthma:the Role of Matrix Metalloproteinase-9[J].Archivum Immunologiae Et Therapiae Experimentalis,2016,64(1):1-9.
[19]Ming C.Cha,Peter P.Purslow.Expressions of matrix metalloproteinases and their inhibitor are modified by beta-adrenergic agonist ractopamine in skeletal fibroblasts and myoblasts[J].Canadian Journal of Animal Science,2017,92(2):159-166.
[20]Aras AB,Guven M,Balak N,et al.Evaluation of the Association Between Matrix Metalloproteinase 11 and Intervertebral Disc Disease[J].Turkish Neurosurgery,2015,26(2):274-279.