[1]李记天,马言,罗漫丽.淫羊藿苷干预大鼠椎间盘源性腰痛的实验研究[J].中医正骨,2020,32(03):7-13.
　LI Jitian,MA Yan,LUO Manli.An experimental study of icariin for intervention of discogenic low back pain in rats[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2020,32(03):7-13.
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淫羊藿苷干预大鼠椎间盘源性腰痛的实验研究()
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《中医正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第32卷
期数:
2020年03期
页码:
7-13
栏目:
基础研究
出版日期:
2020-03-20

文章信息/Info

Title:
An experimental study of icariin for intervention of discogenic low back pain in rats
作者:
李记天; 马言; 罗漫丽
(河南省洛阳正骨医院/河南省骨科医院,河南 郑州 450016)
Author(s):
LI Jitian; MA Yan; LUO Manli
Luoyang Orthopedic-Traumatological Hospital,Zhengzhou 450016,Henan,China
关键词:
腰痛;  椎间盘退行性变;  淫羊藿甙;  大鼠; Sprague-Dawley;  动物实验
Keywords:
low back pain;  intervertebral disc degeneration;  icariin;  rats; Sprague-Dawley;  animal experimentation
摘要:
目的:探讨淫羊藿苷(icariin,ICA)干预大鼠椎间盘源性腰痛的效果及可能的作用机制。方法:将45只8周龄SPF级雄性SD大鼠随机分为5组,假手术组、生理盐水组、ICA 50 mg·kg-1组、ICA 100 mg·kg-1组各10只,塞来昔布组5只。生理盐水组、ICA 50 mg·kg-1组、ICA 100 mg·kg-1及塞来昔布组大鼠通过L4～5和L5～6椎间盘穿刺建立大鼠腰痛模型; 假手术组仅显露椎间盘,不进行穿刺。自造模后第7天开始进行药物干预,至造模后第21天结束。ICA 50 mg·kg-1组和ICA 100 mg·kg-1组以ICA溶液进行灌胃(ICA溶于去离子水中),每天剂量分别为50 mg·kg-1和100 mg·kg-1; 塞来昔布组以塞来昔布胶囊进行灌胃(塞来昔布胶囊粉剂溶于去离子水中),每天100 mg·kg-1; 生理盐水组以生理盐水灌胃,每天100 mg·kg-1; 假手术组常规饲养,不进行干预。从各组随机选取5只大鼠,分别于造模前、造模后第7天、第14天、第21天、第28天、第35天进行足底机械性疼痛阈值检测。造模后第35天,足底疼痛阈值测定结束后处死假手术组、生理盐水组、ICA 50 mg·kg-1组和ICA 100 mg·kg-1组的5只大鼠,以qRT-PCR法测定椎间盘组织中P物质(substance P,SP)mRNA和降钙素基因相关肽(calcitonin gene related peptide,CGRP)mRNA的含量。造模后第21天时,处死假手术组、生理盐水组、ICA 50 mg·kg-1组和ICA 100 mg·kg-1组其余的5只大鼠,以ELISA法测定大鼠椎间盘组织中细胞因子诱导的中性粒细胞趋化剂-1(cytokine-induced neutrophil chemoattractant-1,CINC-1)含量。结果:①足底机械性疼痛阈值。时间因素和分组因素存在交互效应(F=17.971,P=0.001)。5组大鼠的足底机械性疼痛阈值总体比较,差异有统计学意义,即存在分组效应(F=146.660,P=0.000)。造模前后不同时点间足底机械性疼痛阈值的差异有统计学意义,即存在时间效应(F=118.057,P=0.000)。假手术组足底机械性疼痛阈值随时间未见明显变化,一直处于较高水平[(14.60±0.89)g,(14.79±0.47)g,(15.00±0.00)g,(15.00±0.00)g,(15.00±0.00)g,(15.00±0.00)g,F=0.836,P=0.537]。生理盐水组足底机械性疼痛阈值造模后明显降低,后期一直维持在较低水平[(14.67±0.74)g,(3.44±2.22)g,(3.19±1.37)g,(2.84±0.96)g,(3.92±1.36)g,(3.02±0.98)g,F=58.882,P=0.000]。ICA 50 mg·kg-1组、ICA 100 mg·kg-1组和塞来昔布组造模前后的足底机械性疼痛阈值变化趋势基本一致,造模后均先降低,药物干预开始后逐渐回升,药物干预结束后又逐渐降低[(15.00±0.00)g,(2.78±0.81)g,(4.64±1.67)g,(5.59±1.25)g,(8.52±1.63)g,(6.11±1.49)g,F=56.088,P=0.000;(14.66±0.76)g,(2.53±1.37)g,(10.65±2.69)g,(9.67±2.41)g,(10.67±2.04)g,(8.59±2.95)g,F=16.684,P=0.000;(15.00±0.00)g,(2.28±1.03)g,(12.09±1.28)g,(12.37±1.34)g,(6.71±2.89)g,(5.18±1.88)g,F=44.668,P=0.000]。造模后第7天时,ICA 50 mg·kg-1组、ICA 100 mg·kg-1组和塞来昔布组的足底机械性疼痛阈值两两比较,组间差异均无统计学意义。造模后第14天时,ICA 100 mg·kg-1组和塞来昔布组的足底机械性疼痛阈值均高于ICA 50 mg·kg-1组(P=0.001; P=0.000); ICA 100 mg·kg-1组和塞来昔布组的足底机械性疼痛阈值比较,差异无统计学意义。造模后第21天时,ICA 100 mg·kg-1组和塞来昔布组的足底机械性疼痛阈值均高于ICA 50 mg·kg-1组(P=0.009; P=0.000); ICA 100 mg·kg-1组和塞来昔布组的足底机械性疼痛阈值比较,差异无统计学意义。造模后第28天时,ICA 100 mg·kg-1组的足底机械性疼痛阈值高于塞来昔布组(P=0.049); ICA 50 mg·kg-1组与ICA 100 mg·kg-1组、塞来昔布组比较,组间差异均无统计学意义。造模后第35天时,ICA 50 mg·kg-1组、ICA 100 mg·kg-1组和塞来昔布组的足底机械性疼痛阈值两两比较,组间差异均无统计学意义。②椎间盘组织CINC-1含量。造模后第21天,假手术组、生理盐水组、ICA 50 mg·kg-1组及ICA 100 mg·kg-1组大鼠椎间盘组织中CINC-1含量比较,差异有统计学意义[(534.2±142.6)pg·mL-1,(28 376.0±976.7)pg·mL-1,(21 866.0±1 536.0)pg·mL-1,(9 956.0±1 010.0)pg·mL-1,F=423.000,P=0.000]。生理盐水组的CINC-1含量高于假手术组、ICA 50 mg·kg-1组和ICA 100 mg·kg-1组(P=0.000; P=0.003; P=0.000); ICA 50 mg·kg-1组的CINC-1含量高于ICA 100 mg·kg-1组(P=0.000)。③椎间盘组织SP mRNA和CGRP mRNA含量。造模后第35天,假手术组、生理盐水组、ICA 50 mg·kg-1组及ICA 100 mg·kg-1组大鼠椎间盘组织中SP mRNA和CGRP mRNA含量比较,组间差异均有统计学意义(1.04±0.49,183.50±118.60,55.50±10.26,19.53±8.05,F=9.499,P=0.000; 0.74±0.21,6.29±2.06,1.55±0.69,1.19±0.37,F=27.180,P=0.000)。生理盐水组的SP mRNA含量高于假手术组、ICA 50 mg·kg-1组和ICA 100 mg·kg-1组(P=0.008; P=0.042; P=0.015); ICA 50 mg·kg-1组的SP mRNA含量高于ICA 100 mg·kg-1组(P=0.000)。生理盐水组的CGRP mRNA含量高于假手术组、ICA 50 mg·kg-1组和ICA 100 mg·kg-1组(P=0.000; P=0.001; P=0.000); ICA 50 mg·kg-1组和ICA 100 mg·kg-1组的CGRP mRNA含量比较,差异无统计学意义。结论:ICA可有效缓解大鼠椎间盘源性腰痛,其镇痛效果与剂量有关,与同剂量的塞来昔布相比其镇痛效果持续时间更长,降低大鼠椎间盘组织中CINC-1的水平可能是其镇痛的作用机制之一。
Abstract:
Objective:To explore the effects of icariin(ICA)on discogenic low back pain(DLBP)in rats and its possible mechanism of action.Methods:Forty-five 8-week-old SPF-grade male SD rats were selected and were randomly divided into sham-operated group(10),normal saline group(10),ICA 50 mg/kg group(10),ICA 100 mg/kg group(10)and celecoxib group(5).The lumbago models were built in rats of normal saline group,ICA 50 mg/kg group,ICA 100 mg/kg group and celecoxib group by puncturing L4/L5 and L5/L6 intervertebral disc,while the surgeries were performed on rats in sham-operated group to expose their intervertebral discs and no intervertebral disc puncture was performed.The drug intervention were performed on rats from day 7 to day 21 after modeling.The rats in ICA 50 mg/kg group were intragastric administrated with ICA solution(ICA was dissolved into deionized water)in daily dosages of 50 mg/kg.The rats in ICA 100 mg/kg group,celecoxib group and normal saline group were intragastric administrated with ICA solution,celecoxib solution(celecoxib capsule powders were dissolved into deionized water)and normal saline respectively in daily dosage of 100 mg/kg,while the rats in sham-operated group were bred normally and were not given any drug interventions.Five rats were randomly selected out from each group,and plantar mechanical pain threshold values were measured before modeling and at the 7th,14th,21st,28th and 35th day after the modeling respectively.At the 35th day after the modeling,the five rats in sham-operated group,normal saline group,ICA 50 mg/kg group and ICA 100 mg/kg group were executed after the last measurement of pelma pain threshold value and the contents of substance P(SP)mRNA and calcitonin gene related peptide(CGRP)mRNA in intervertebral disc tissues were measured by using qRT-PCR assays.At the 21st day after the modeling,the other 5 rats in sham-operated group,normal saline group,ICA 50 mg/kg group and ICA 100 mg/kg group were executed,and the content of cytokine-induced neutrophil chemoattractant-1(CINC-1)in intervertebral disc tissues of rats were measured by using ELISA.Results:There was interaction between time factor and group factor in plantar mechanical pain threshold values(F=17.971,P=0.001).There was statistical difference in plantar mechanical pain threshold values between the 5 groups in general,in other words,there was group effect(F=146.660,P=0.000).There was statistical difference in plantar mechanical pain threshold values between different timepoints before and after the modeling,in other words,there was time effect(F=118.057,P=0.000).No significant time-dependent change of plantar mechanical pain threshold values were found in sham-operated group,and the pain threshold values remained at a high level(14.60+/-0.89,14.79+/-0.47,15.00+/-0.00,15.00+/-0.00,15.00+/-0.00,15.00+/-0.00 g,F=0.836,P=0.537).The plantar mechanical pain threshold values decreased significantly in normal saline group after modeling,and then remained at a low level(14.67+/-0.74,3.44+/-2.22,3.19+/-1.37,2.84+/-0.96,3.92+/-1.36,3.02+/-0.98 g,F=58.882,P=0.000).The plantar mechanical pain threshold values were basically consistent with each other in the variation tendency in ICA 50 mg/kg group,ICA 100 mg/kg group and celecoxib group before and after the modeling,and they all decreased firstly after the modeling and increased gradually after the beginning of drug intervention and then decreased gradually after the end of drug intervention(15.00+/-0.00,2.78+/-0.81,4.64+/-1.67,5.59+/-1.25,8.52+/-1.63,6.11+/-1.49 g,F=56.088,P=0.000; 14.66+/-0.76,2.53+/-1.37,10.65+/-2.69,9.67+/-2.41,10.67+/-2.04,8.59+/-2.95 g,F=16.684,P=0.000; 15.00+/-0.00,2.28+/-1.03,12.09+/-1.28,12.37+/-1.34,6.71+/-2.89,5.18+/-1.88 g,F=44.668,P=0.000).At the 7th day after the modeling,further pairwise comparison showed that there was no statistical difference in the plantar mechanical pain threshold values between ICA 50 mg/kg group,ICA 100 mg/kg group and celecoxib group.At the 14th day after the modeling,the plantar mechanical pain threshold values were higher in ICA 100 mg/kg group and celecoxib group compared to ICA 50 mg/kg group(P=0.001; P=0.000),and there was no statistical difference between ICA 100 mg/kg group and celecoxib group.At the 21st day after the modeling,the plantar mechanical pain threshold values were higher in ICA 100 mg/kg group and celecoxib group compared to ICA 50 mg/kg group(P=0.009; P=0.000),and there was no statistical difference between ICA 100 mg/kg group and celecoxib group.At the 28th day after the modeling,the plantar mechanical pain threshold values were higher in ICA 100 mg/kg group compared to celecoxib group(P=0.049),and there was no statistical difference between ICA 50 mg/kg group and ICA 100 mg/kg group and between ICA 50 mg/kg group and celecoxib group.At the 35th day after the modeling,further pairwise comparison showed that there was no statistical difference in the plantar mechanical pain threshold values between ICA 50 mg/kg group,ICA 100 mg/kg group and celecoxib group.At the 21st day after the modeling,there was statistical difference in the content of CINC-1 in intervertebral disc tissues of rats between sham-operated group,normal saline group,ICA 50 mg/kg group and ICA 100 mg/kg group(534.2+/-142.6,28 376.0+/-976.7,21 866.0+/-1 536.0,9 956.0+/-1 010.0 pg/mL,F=423.000,P=0.000).The content of CINC-1 was higher in normal saline group compared to sham-operated group,ICA 50 mg/kg group and ICA 100 mg/kg group(P=0.000; P=0.003; P=0.000),and was higher in ICA 50 mg/kg group compared to ICA 100 mg/kg group(P=0.000).At the 35th day after the modeling,there was statistical difference in the contents of SP mRNA and CGRP mRNA in intervertebral disc tissues of rats between sham-operated group,normal saline group,ICA 50 mg/kg group and ICA 100 mg/kg group(1.04+/-0.49,183.50+/-118.60,55.50+/-10.26,19.53+/-8.05,F=9.499,P=0.000; 0.74+/-0.21,6.29+/-2.06,1.55+/-0.69,1.19+/-0.37,F=27.180,P=0.000).The content of SP mRNA in intervertebral disc tissues was higher in normal saline group compared to sham-operated group,ICA 50 mg/kg group and ICA 100 mg/kg group(P=0.008; P=0.042; P=0.015),and was higher in ICA 50 mg/kg group compared to ICA 100 mg/kg group(P=0.000).The content of CGRP mRNA in intervertebral disc tissues was higher in normal saline group compared to sham-operated group,ICA 50 mg/kg group and ICA 100 mg/kg group(P=0.000; P=0.001; P=0.000),and there was no statistical difference between ICA 50 mg/kg group and ICA 100 mg/kg group.Conclusion:ICA can effectively relieve DLBP in rats,and its analgesic effect is associated with the dose and lasted longer than that of same dose of celecoxib.It can decrease the level of CINC-1 in intervertebral disc tissues,which may be one of its analgesic mechanisms.

参考文献/References:

[1] HOY D,BROOKS P,BLYTH F,et al.The epidemiology of low back pain[J].Best Pract Res Clin Rheumatol,2010,24(6):769-781.
[2] Global Burden of Disease Study 2013 Collaborators.Global,regional,and national incidence,prevalence,and years lived with disability for 301 acute and chronic diseases and injuries in 188 countries,1990-2013:a systematic analysis for the global burden of disease study 2013[J].Lancet,2015,386(9995):743-800.
[3] 陈栋,陈春慧,胡志超,等.中国成人腰痛流行病学的系统评价[J].中国循证医学杂志,2019,19(6):651-655.
[4] MAHER C,UNDERWOOD M,BUCHBINDER R.Non-specific low back pain[J].Lancet,2017,389(170):736-747.
[5] WENGER H C,CIFU A S.Treatment of low back pain[J].JAMA,2017,318(8):743-744.
[6] MATHIESON S,KASCH R,CHRISTOPHER G M,et al.Combination drug therapy for the management of low back pain and sciatica:systematic review and meta-analysis[J].J Pain,2019,20(1):1-15.
[7] 刘海文,董宝强,李光明,等.从“肝肾-经筋”理论探讨非特异性腰痛[J].长春中医药大学学报,2019,35(6):1021-1023.
[8] 邓再冲,关宏刚,廖嘉明,等.补肾调三焦法治疗盘源性腰痛急性期的临床疗效观察[J].广州中医药大学学报,2020,37(2):256-261.
[9] 李建国,谢兴文,李鼎鹏,等.淫羊藿提取物淫羊藿苷在细胞水平防治骨质疏松症的研究概况[J].中国骨质疏松杂志,2019,25(1):132-135.
[10] 古建立,李东升,郭建刚.驻春胶囊治疗膝骨性关节炎68例疗效观察[J].国医论坛,2003,18(4):24-25.
[11] 杨公博,朱立国,何佩珊,等.药对淫羊藿-白芍治疗老年腰椎间盘突出症的临床研究[J].中华中医药杂志,2018,33(6):2710-2712.
[12] SHENG C,DENG X Y,MA K G,et al.Icariin improves the viability and function of cryopreserved human nucleus pulposus-derived mesenchymal stem cells[J].Oxid Med Cell Longev,2018[2020-01-01].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6040248/.
[13] HUA W B,ZHANG Y K,WU X H,et al.Icariin attenuates interleukin-1β-Induced inflammatory response in human nucleus pulposus cells[J].Curr Pharm Des,2018,23(39):6071-6078.
[14] JIAO Y C,YE Y,LIN Y Z,et al.Propionibacterium acnes induces discogenic low back pain via stimulating nucleus pulposus cells to secrete pro-algesic factor of IL-8/CINC-1 through TLR2-NF-κB p65 pathway[J].J Mol Med,2019,97(1):25-35.
[15] VAN HEESWIJK V M,THAMBYAH A,ROBERTSON P A,et al.Does an annular puncture influence the herniation path?:an in vitro mechanical and structural investigation[J].Spine(Phila Pa 1976),2018,43(7):467-476.
[16] LI Z,LIU H,YANG H,et al.Both expression of cytokines and posterior annulus fibrosus rupture are essential for pain behavior changes induced by degenerative intervertebral disc:an experimental study in rats[J].J Orthop Res,2014,32(2):262-272.
[17] QIAN J,GE J,YAN Q,et al.Selection of the optimal puncture needle for induction of a rat intervertebral disc degeneration model[J].Pain Physician,2019,22(4):353-360.
[18] MURALIDHARAN A,PARK T,MACKIE J T,et al.Establishment and characterization of a novel rat model of mechanical low back pain using behavioral,pharmacologic and histologic methods[J].Front Pharmacol,2017,8:493.
[19] AHMED A,BERG S,ALKASS K,et al.NF-κB-Associated pain-related neuropeptide expression in patients with degenerative disc disease[J].Int J Mol Sci,2019,20(3):658.
[20] ABBIE L B,ASHLEY A C,BREAKWELL L M,et al.Expression and regulation of neurotrophic and angiogenic factors during human intervertebral disc degeneration[J].Arthritis Res Ther,2014,16(4):416.
[21] PEDERSEN L M,SCHISTAD E,JACOBSEN L M,et al.Serum levels of the pro-inflammatory interleukins 6(IL-6)and -8(IL-8)in patients with lumbar radicular pain due to disc herniation:a 12-month prospective study[J].Brain Behav Immun,2015,46:132-136.
[22] BURKE J G,WATSON R W,MCCORMACK D,et al.Intervertebral discs which cause low back pain secrete high levels of proinflammatory mediators[J].J Bone Joint Surg Br,2002,84(2):196-201.
[23] DE SOUZA GRAVA A L,FERRARI L F,DEFINO H L.Cytokine inhibition and time-related influence of inflammatory stimuli on the hyperalgesia induced by the nucleus pulposus[J].Eur Spine J,2012,21(3):537-545.
[24] KROCK E,MILLECAMPS M,ANDERSON K M,et al. Interleukin-8 as a therapeutic target for chronic low back pain:upregulation in human cerebrospinal fluid and pre-clinical validation with chronic reparixin in the SPARC-null mouse model[J]. EBioMedicine,2019,43:487-500.

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[8]吴婷婷,杨京辉,汪亚群,等.针刺联合太极拳训练治疗慢性非特异性腰痛的临床研究[J].中医正骨,2017,29(06):32.
　WU Tingting,YANG Jinghui,WANG Yaqun,et al.Clinical study on acupuncture therapy combined with Taijiquan(太极拳)exercise for treatment of chronic nonspecific low back pain[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2017,29(03):32.
[9]熊国星,王鑫,程元辉,等.动推疗法与单纯推拿疗法治疗慢性非特异性腰痛的近期疗效对比研究[J].中医正骨,2017,29(11):33.
　XIONG Guoxing,WANG Xin,CHENG Yuanhui,et al.A comparative study of short-term clinical curative effect of massage during exercise versus monotherapy of TUINA for treatment of chronic nonspecific low back pain[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2017,29(03):33.
[10]李展新,刘建航,李锦威,等.韦氏脊柱整治手法结合核心肌群训练治疗非职业高尔夫运动者非特异性腰痛[J].中医正骨,2018,30(04):13.
　LI Zhanxin,LIU Jianhang,LI Jinwei,et al.Wei's spinal remediation manipulation combined with core muscle training for treatment of nonspecific low back pain in non-professional golfers[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2018,30(03):13.
[11]叶晓宇,徐卫星,王润民.非手术与微创手术治疗椎间盘源性腰痛的研究进展[J].中医正骨,2018,30(05):40.

备注/Memo

备注/Memo:
(收稿日期:2020-02-06 本文编辑:李晓乐) 基金项目:河南省中医药科学研究专项课题(2019ZYZD02) 通讯作者:李记天 E-mail:jitianlee@hotmail.com
更新日期/Last Update: 2020-03-10