MMPs抑制剂和手法治疗对大鼠腰椎间盘组织退变影响的实验研究
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摘要
椎间盘的退行性改变(intervertebral disc degeneration,IDD)是引起颈肩、腰腿疼痛的一个重要因素,患者因各种诱因,如急性扭伤、慢性劳损及受凉等作用下,导致纤维环破裂,液体状的髓核从破裂处溢出,沿椎间盘与神经根之间的通道扩散,髓核的糖蛋白与β蛋白对神经根有强烈的化学刺激性,同时大量的组织胺释放出来,产生化学性神经根炎。在炎症情况下,多种化学媒质使血管对蛋白质的通透性升高,在神经内、外膜及神经根、椎窦神经中渗出大量炎性蛋白,引起局部缺血与电解质紊乱,刺激神经根及椎窦神经,引起神经支配区的疼痛~([1])。疼痛使局部肌肉收缩,呈保护性体位、出现腰椎侧弯,使正常人体的生理性生物力学状态改变,加重疼痛、肌肉痉挛的恶性循环过程,肌肉挛缩可进一步出现椎体小关节功能紊乱、椎管狭窄等伴随症状。IDD的病理生理学机制至今尚未明了。随着生物力学、分子生物学、组织工程学的发展,生长因子疗法、基因疗法、细胞疗法以及组织工程等,为IDD的治疗提供了新的手段。
以往大多数人认为椎间盘的退行性改变是一种生物力学作用的结果,但新近的研究表明生物力学的作用也不是绝对的,发现生物化学机制也发挥着至关重要的作用。文献上不断报道MMPs对椎间盘的退行性改变所起的作用,从而对这一病理过程有了新的认识。椎间盘的基质成分与关节软骨相似,研究表明正常软骨内MMPs与TIMP的表达水平相接近,但在骨关节炎和类风湿关节炎时,MMPs的生物活性明显升高,且mRNA的表达也增高,MMPs与TIMP的平衡破坏引起软骨破坏。四环素类抗生素及其衍生物是很有效的MMPs抑制剂,能通过多种途径抑制MMP-1、MMP-3、MMP-13、MMP-2和MMP-9的活性。研究显示,四环素及结构相似的化合物能抑制胶原酶和其他MMPs的效应,抑制MMPs的基质降解活性~([2])从而延缓椎间盘退变程度,并且大量实验研究发现COL-2a,Col-1a,Aggreean,CILP,TGF-β1,VDR,MMP-3,MMP-13,Timp-3,Timp-1基因的表达情况与椎间盘的退行性改变密切相关。
祖国医学认为本病的发生有内因、外因两方面。内因由于随着年龄的增长,肝肾渐虚,气血不足,邪气留连,病久入深,着于筋脉、肌骨,致瘀血内阻,气血运行失调,荣卫凝涩不通;腰部筋脉骨髓失养,使腰椎间盘逐渐变性,致使腰椎间盘的软骨板变薄,纤维环弹性减弱,髓核含水量逐渐减少,而失去了弹性;继之使椎间隙变窄,周围韧带松弛,或产生裂隙。外因由于外伤劳损及风寒湿邪入侵,引起局部气血瘀滞,阻滞经络,致机体阴阳失调,不通则痛,而引发本病。正如《诸病源候论·腰腿疼痛候》曰:“肾气不足,受风邪之所为也。劳伤则肾虚,虚者受于风冷,风冷与正气之争,故腰腿痛”。现代医学认为,髓核的膨出、压迫,引起周围组织的炎症、充血、水肿,从而压迫或刺激脊神经根,而致腰腿疼痛麻木。《素问·痹论》曰:“痹在骨则重,在于脉则血凝而不流,在于筋则屈不伸,在于内则不仁”。《素问·逆调论》又曰:“营气虚则不仁,卫气虚则不用,营卫俱虚,则不仁而不用”。本病属中医学腰腿痛范畴。采用中医手法治疗可降低神经应激功能,增加腰部局部组织痛阈,改善腰肌高张力状态,具有解痉止痛的作用,但对其作用机理不明。
本课题通过MMPs抑制剂和中医手法对造模大鼠的干预,从影像学和病理学角度来进行椎间盘组织退变机理的大体形态学研究,并在基因水平来探讨MMPs抑制剂和中医手法与相关椎间盘退变基因表达的关系,从而揭示预防和延缓椎间盘退变的机理,为临床进一步量化手法治疗效果和规范手法治疗方法提供理论依据。
第一部分:外源性MMPs抑制剂-四环素、手法对造模大鼠腰椎间盘组织退变的形态学影响(摘要)
目的:探讨外源性MMPs抑制剂-四环素、手法对造模大鼠腰椎间盘组织退变的形态学影响,试图从影像学和形态学角度阐明它们对椎间盘早期退变抑制的作用机理。
方法:将36只雄性SD大鼠随机平均地分为四组:正常组、对照组、四环素组、手法组。将它们分别单笼饲养,并且使对照组、四环素组、手法组造模。动物模型建立4周后,正常组和对照组皮下注射生理盐水,四环素组每日皮下注射四环素25 mg,持续2周。手法组的大鼠腰椎进行手法治疗约10分钟。分别于于造模前、造模后第2、8周每组随机选取3只动物麻醉后CR拍片检查。动物模型建立4周后,在进行第一次注射药物和手法干预前,每组随机选取3只动物,给予10%水合氯醛腹腔注射麻醉,拍摄腰椎X线正侧位片。造模达8周,以过量10%水合氯醛腹腔注射麻醉将其处死,迅速从腰椎后正中线切开皮肤,逐层分离腰椎两旁的肌肉韧带,直达椎体。取下整个腰椎段脊柱迅速放在冰面上,用眼科剪刀、镊剥离椎间小关节和椎体前方的软组织,分别从L1椎体下缘和L2椎体上缘分离上、下软骨终板,取出L1/2椎间盘组织。用同样的方法取出L2/3、L3/4、L4/5、L5/6椎间盘组织,用于其它实验。将取下的L1/2椎间盘组织放入4%多聚甲醛中固定,5%硝酸中脱钙,常规处理后,石蜡包埋,正中冠状切片,5um厚,HE染色。在日本OLYMPUS公司倒置显微镜IX71及显微成像系统DP71-IPP6.0下观察。
结果:两个月后,观察大鼠腰椎X线结果,四环素组干预后较另两组椎间隙变窄,脊柱腰段变直,部分椎体前下角骨皮质增生硬化,部分椎间孔变小;手法组干预后较另两组生理弯曲圆滑,椎间隙无明显改变,部分上段腰椎椎间孔有狭窄;对照组干预后生理弯曲存在,部分椎体前下角骨质硬化。光镜下,与正常组相比,四环素组、手法组无明显变化,髓核位于中心,无固缩,外周被纤维环包绕;对照组髓核稍有固缩。
结论:造模两个月后,X线影像学表现与造模前比较有明显改变,其组织结构有向退变方向发展趋势,说明大鼠腰椎间盘开始发生轻度退变,并处于退变的早期阶段。从X线影像学和组织形态学来看,外源性MMPs抑制剂-四环素、手法具有维护腰椎结构和改善早期退变阶段椎间盘组织结构的作用。
第二部分:MMPs抑制剂和手法治疗对大鼠腰椎间盘组织退变影响的基因表达研究(摘要)
目的:探讨外源性MMPs抑制剂-四环素、手法对造模大鼠腰椎间盘组织退变基因表达的影响,试图从基因水平阐明它们对椎间盘早期退变抑制的作用机理,为临床进一步量化手法治疗效果和规范手法治疗方法提供理论依据。
方法:将36只雄性SD大鼠随机平均地分为四组:正常组、对照组、四环素组、手法组。将它们分别单笼饲养,对照组、四环素组、手法组实验动物造模。动物模型建立4周后,正常组和对照组皮下注射生理盐水,四环素组每日皮下注射四环素25 mg,持续2周。手法组的大鼠腰椎进行手法治疗约10分钟。分别于造模前、造模后第2、8周每组随机选取3只动物麻醉后CR拍片检查。动物模型建立4周后,在进行第一次注射药物和手法干预前,每组随机选取3只动物,给予10%水合氯醛腹腔注射麻醉,拍摄腰椎X线正侧位片。造模达8周,以过量10%水合氯醛腹腔注射麻醉将其处死,迅速从腰椎后正中线切开皮肤,逐层分离腰椎两旁的肌肉韧带,直达椎体。取下整个腰椎段脊柱迅速放在冰面上,用眼科剪刀、镊剥离椎间小关节和椎体前方的软组织,分别从L1椎体下缘和L2椎体上缘分离上、下软骨终板,取出L1/2椎间盘组织。用同样的方法取出L2/3、L3/4、L4/5、L5/6椎间盘组织。将取出的大鼠L2/3-L5/6椎间盘组织,迅速放入事先预冷并编好号的取材盒中,置于液氮中保存。采用荧光定量PCR法检测MMPs抑制剂和手法治疗后相关基因COL-2a1, COL-1a1,Aggrecan,CILP,TGF-β1,VDR,MMP-3,MMP-13,Timp-3,Tirap-1在大鼠腰椎间盘组织退变中的表达量,运用DPS9.5版统计软件包进行方差分析和大鼠腰椎X光正位片侧偏角度和相关基因的关联度系数分析。
结果: 1.运用DPS9.5版统计软件包选择完全随机设计的单因数方差分析的多重比较LSD法进行统计分析得到以下结果。
(1)COL-1a的表达量正常组最低,与手法组和四环素组间比较,P>0.05,故与该两组间差异无意义;对照组COL-1a的表达量较其它各组最高,P<0.01,故与其它各组差异有显著意义。
(2)COL-2a的表达量正常组较其它组高,P<0.01,故与其它各组差异有显著意义;手法组与四环素组问比较, P>0.05,故两组间差异无意义,即COL-2a在手法组的表达量与四环素组接近;对照组与手法组和四环素组间比较表达量低,P<0.01,故与该两组差异有显著意义。
(3)Aggrecan的表达量正常组最高,P<0.01,故与其它各组差异有显著意义;对照组的表达量最低,P<0.01,与手法组和四环素组间比较差异有显著意义;Aggrecan在手法组的表达量高于四环素组,P<0.01,故两组间差异有显著意义。
(4)CILP的表达量正常组最低,P<0.01,故与其它各组差异有显著意义;其它各组间的表达量相近,P>0.05,故各组间差异无意义。
(5)TGF-β1的表达量正常组最高,与四环素组和对照组比较,P<0.01,故与该两组间差异有显著意义;手法组TGF-β1的表达量与四环素组和对照组比较, P<0.01,故与该两组间差异有显著意义;正常组比手法组TGF-β1的表达量稍高,P<0.05,故两组间差异有意义;四环素组和对照组比较,P>0.05,故两组间差异无意义。
(6)VDR的表达量正常组最高,P<0.01,故与其它各组差异有显著意义;四环素组VDR的表达量较手法组和对照组低,P<0.01,故与该两组间差异有显著意义;手法组和对照组间比较,P>0.05,故两组间差异无意义。
(7)MMP-3的表达量正常组最低,P<0.01,与其它各组差异有显著意义;对照组MMP-3的表达量较手法组与四环素组高,P<0.01,故与该两组间差异有显著意义;手法组MMP-3的表达量比四环素组高,P<0.05,故两组间差异有意义。
(8)MMP-13的表达量正常组最低,P<0.05,与其它各组差异有意义;对照组MMP-13的表达量最高,P<0.01,故与其它各组差异有显著意义;四环素组较手法组MMP-13的表达量高,P<0.05,故两组间差异有意义。
(9)TIMP-1的表达量正常组最低,P<0.01,故与其它各组差异有显著意义;对照组TIMP-1的表达量高,与手法组和四环素组比较,P<0.01,故与该两组差异有显著意义;四环素组TIMP-1的表达量高于手法组,P<0.01,故两组间差异有显著意义。
(10)TIMP-3的表达量正常组与手法组相近,P>0.05,故两组间差异无意义;正常组和手法组TIMP-3的表达量与对照组和四环素组相比,P<0.01,故正常组和手法组TIMP-3的表达量与对照组和四环素组相比差异有显著意义;对照组TIMP-3的表达量最高,四环素组TIMP-3的表达量最低,该两组相比,P<0.01,故两组间差异有显著意义。
2.测量得到大鼠腰椎的侧偏角均值分别为:正常大鼠0°;对照组14°;四环素组12°;手法组10°,运用DPS9.5 0版统计软件包选择其它的灰色系统方法下的关联度分析得到以下结果见表5。
表5大鼠腰椎X光正位片侧偏角度和相关基因的关联度系数注:CILP的表高达量对大鼠腰椎侧弯的影响最大,而TGF-β1的高表达量对大鼠腰椎侧弯的影响最小。
结论:
(1)中医手法和四环素相似的抑制COL-1a基因表达可知两种治疗方法都可延缓大鼠腰椎间盘的退变,具有相同作用,而对照组COL-1a的高表达更能说明这一点,并且与文献关于椎间盘出现退变时Ⅰ型胶原增加相符。
(2)中医手法和四环素能使COL-2a基因表达量增加,而其增加可延缓大鼠腰椎间盘的退变,这与对照组COL-2a型胶原基因的低表达应证。
(3)中医手法增加Aggrecan的表达量比四环素更明显,可见中医手法对延缓大鼠腰椎间盘的退变的治疗效果比四环素更明显。
(4)CILP的表达量正常组最低,其它各组间的表达量相近,说明此动物模型造模相当成功(椎间盘退变时CILP的含量高于正常水平),从中医手法可使TGF-β1的表达量增加可间接推知中医手法可抑制CILP的表达,进而延缓椎间盘退变。四环素也有类似效果。
(5)中医手法可使TGF-β1的表达量增加,而其在椎间盘内增多能促进细胞增殖和细胞外基质合成,维护椎间盘内环境稳定可延缓大鼠腰椎间盘的退变,四环素无此作用。
(6)中医手法使VDR的表达量较四环素高,而VDR的表达可以直接参与软骨细胞的分化、增殖和成熟。而椎间盘中富含软骨细胞,细胞间质中富含Aggrecan,因此中医手法升高VDR的表达量而通过软骨细胞增殖来延缓大鼠腰椎间盘的退变,减少了椎骨骨刺和椎间隙狭窄的可能性,这与影象学结果相一致。
(7)从MMP-3与TIMP-1的表达量可知,四环素可使MMP-3的表达量减少而使TIMP-1的表达量增高,这种平衡可以延缓椎间盘的退变;中医手法也可减少MMP-3的表达量,并且在1%极显著水平表达量与四环素相同说明中医手法和四环素治疗效果相同,都可以抑制MMP-3的表达而使TIMP-1的表达量增高,从而减少细胞外基质的降解延缓椎间盘的退变。
(8)四环素因其负反馈作用可减少TIMP-3的表达量,则MMP-13的表达量升高;而中医手法可使TIMP-3的表达量升高并与正常组接近,则MMP-13的表达量降低,这样中医手法就可减少MMPs的产生,从而减少细胞外基质的降解,也就可以延缓椎间盘的退变。
(9)从大鼠腰椎X光正位片侧偏角度和相关基因的关联度系数可知:CILP的高表达量与侧偏角度最相关,而TGF-β1的高表达量与侧偏角度最不相关,以上恰恰说明CILP与TGF-β1的相互抑制作用。CILP的表达量增高,而TGF-β1的表达量降低时,基质合成减少而破坏软骨细胞进而引起椎间关节退变而发生侧弯。
综上所述,以上各组基因的表达均参与基质的合成与降解,由此可见椎间盘的退变与细胞外基质非常相关,MMPs抑制剂可以调节相关基因的表达延缓椎间盘的退变,中医手法也有类似效果,有时中医手法的疗效更好。
Intervertebral disc degeneration (IDD) are an important cause of neck, shoulder, back, leg pain. Annulus fibrosus broken is caused by a variety of incentives such as acute sprains, chronic fatigue and cold etc.So liquid-like nucleus pulposus spills, diffuses along the channel between the disc and nerve root. And chemical radicular neuritis is induced by glycoprotein andβprotein within the nucleus pulposus which are strong chemically irritant and the following released histamine nucleus pulposus. Because of inflammation, a lot of inflammatory protein which exudes from high permeable vascular, accumulates in the intima and outima of nerve root and vertebral sinus nerve. Protein exudation causes local ischemia and electrolyte disorders and results in stimulation of nerve root and vertebral sinus nerves, causing corresponding innervation zone pain. The pain causes local muscle contraction and protective lumbar scoliosis , and the physiological biomechanical changes takes place and aggravate pain, so resulting in a vicious cycle. Furthermore, muscle spasm can aggravate vertebral facet joint dysfunction, spinal canal stenosis and so on. IDD pathophysiological mechanism is still under investigation. With development of biomechanics and molecular "biology and tissue engineering , growth factor therapy, gene therapy, cell therapy and tissue engineering etc. provide some choice for the treatment of IDD.
In the past, it is well accepted that IDD is a result of the biomechanical change, but recent studies have shown that biochemical factors also play an important role. MMPs are often reported in the development of IDD and provide a new sight in the pathological process. Intervertebral disc has a similar matrix components comparing with articular cartilage. It is reported that expression of MMPs and TIMP is similar in the normal cartilage , but the balance of MMPs and TIMP is interrupted in osteoarthritis and rheumatoid arthritis with a significantly higher MMPs activity and mRNA expression, and this imbalance results in cartilage damage . Tetracyclines and their derivatives are very effective inhibitors of MMPs, can inhibit MMP-1, MMP-3, MMP-13, MMP-2 and MMP-9 activity through different mechanisms. Studies have shown that tetracycline and their derivatives can inhibit collagenase and other MMPs effects and slow down the disc degeneration. And a large number of experimental study found that intervertebral disc degeneration are closely related with COL-2a , Col-1a , Aggrecan, CILP, TGF-β1, VDR, MMP-3, MMP-13, Timp-3, Timp-1 gene expression.
The present investigation focused on the effects of intervention using the MMPs inhibitors and traditional Chinese medicine massage on rat IDD model. The general morphology was investigated by imaging and pathology, and real time PCR was used to study gene expression of degenerated intervertebral disc before and after the MMPs inhibitors and massage, respectively. This research aimed to reveal the mechanism of intervention and provide theoretical basis to quantify and standardize the treatment approach.
PartⅠ: morphological impact of exogenous MMP inhibitors tetracycline and massage on lumbar IDD in rat model
Abstract Objective: To investigate the morphological impact of exogenous MMPs inhibitor tetracycline and massage on lumbar IDD in rat model, to elucidate mechanism of their inhibition in terms of imaging and morphology. Methods: 36 male SD rats were randomly divided into four groups: normal group, control group, tetracycline group and massage group. All the 36 rats were single-cage feed and the control, tetracycline,model group were modeled.4 weeks after modeling, subcutaneous injection of normal saline was administrated to control group daily, subcutaneous injection of tetracycline 25 mg to tetracycline group, and 10 minutes massage to the massage group daily, Respectively. All sustained two weeks. 3 rats were randomly selected in each group for CR-Xray before and 2weeks, 8 weeks after modeling.4 weeks, 3 rats were randomly selected in each group for lumbar CR-Xray before first injection of massage, anesthetized by 10% chloral hydrate through intraperitoneal injection. 8 weeks after modeling, rats were executed by overdose 10% chloral hydrate intraperitoneal injection. vertebral column were excided through dorsal meso-incision. the removed vertebral column were put on ice as soon as possible andL1/2, L2/3, L3/4, L4/5, L5/6 intervertebral disc tissue were procured. Half of L1/2 intervertebral disc tissue was fixed in 4% paraformaldehyde, decalcified in 5% nitric acid, embedded in paraffin, 5um thick section was obtained, and went on HE staining. OLYMPUSBH optical microscope was used for observation. The remaining intervertebral disc tissue were used for other experiments. Results: Two months after modeling, lumbar spine X-ray shown: tetracycline group had narrower intervertebral space, smaller foraminal, worse spine curvature and more obvious bone sclerosis than the other two groups after the intervention; massage group had better spine curvature, but narrower intervertebral space, smaller foraminal in some upper segment than the other two groups after the intervention; the control group showed no changes in physiological curvature, bone sclerosis in some anterior ventro-rvertebrae. No significant difference was found among the normal group, the tetracycline group and the massage group under optical microscope: nucleus pulposus is at the center, no shrinkage, enveloped by the peripheral annulus f ibrosus; the control group showed a slight shrinkage of nucleus pulposus. Conclusion: two months after modeling, X-ray imaging showed significantly changes and mild degeneration and indicated a trend of degeneration. From the X-ray and morphological study, the exogenous MMPs inhibitor-tetracycline, massage can inhabit the early stages of disc degeneration.
PartⅡ: gene expression research of the impact of MMP inhibitors - tetracycline and massage on lumbar intervertebral disc degeneration in rats
Abstract Objective : To investigate gene expression of degenerated intervertebral disc treated the exogenous MMPs inhibitors and massage, to reveal mechanism of intervention from the genetic level and provide theoretical basis to further quantify and standardize the treatment approach. Methods: 36 male SD rats were randomly divided into four groups: normal group, control group, tetracycline group and massage group. All the 36 rats were single-cage feed and the control , tetracycline, model group were modeled. 4 weeks after modeling, subcutaneous injection of normal "saline was administrated to control group daily, subcutaneous injection of tetracycline 25 mg to tetracycline group, and 10 minutes massage to the massage group daily, Respectively.All sustained two weeks. 3 rats were randomly selected in each group for CR-Xray before and 2weeks, 8 weeks after modeling. 4 weeks, 3 rats were randomly selected in each group for lumbar CR-Xray before first injection of massage , anesthetized by 10% chloral hydrate through intraperitoneal injection. 8 weeks after modeling, rats were executed by overdose 10% chloral hydrate intraperitoneal injection. Vertebral column were excided through dorsal meso-incision. the removed vertebral column were put on ice as soon as possible and L1/2, L2/3, L3/4, L4/5, L5/6 intervertebral disc tissue were procured. The procured L2/3, L3/4, L4/5, L5/6 intervertebral disc tissue were stored in liquid nitrogen. Gene expression level of COL-2a, COL-1a, Aggrecan, CILP, IGF-(β1, VDR, MMP-3, MMP-13, Timp-3, Timp-1 in intervertebral disc were tested using real time PCR assay after treatment of MMPs inhibitors-tetracycline and massage.DPS9.5 statistical software package were used to do variance analysis and correlation coefficient analysis between scoliosis angle in posterior-anterior X-ray and associated gene expression. Results: 1. Statistical analysis was performed with statistical package DPS9. 50. Completely random designed one-way ANOVA ( LSD method ) was used for comparison between groups. (1) The expression of COL-1a showed no difference in normal group, chirismus group and tetracycline group (P > 0. 05) and significantly higher in control compared with other groups (P < 0. 01). (2) The expression of COL-2a was significantly higher in normal group compared with other groups (P < 0. 01). There was no difference between chirismus group and tetracycline group (P> 0. 05). The expression of COL-2a was significantly lower in control compared with chirismus group and tetracycline group (both P < 0.01). (3) The expression of aggrecan was significantly higher in normal group compared with other groups (P < 0.01) , significantly lower in control than those in chirismus group and tetracycline group (both P < 0.01), and significantly higher in chirismus group compared with tetracycline group (P<0. 01). (4) The expression of CILP was significantly lower in normal group compared with other groups (P< 0. 01) and showed no difference in control, chirismus group and tetracycline group (P>0. 05). (5) The expression of TGF-β1 was significantly higher in normal group compared with tetracycline group and control (both P<0. 01), significantly higher in chirismus group than those in tetracycline group and control (both P<0.01), and significantly higher in normal group compared with chirismus group (P<0.05). The expression of TGF-β1 showed no difference between tetracycline group and control (P>0.05). (6) The expression of VDR was significantly higher in normal group compared with other groups (P<0.01), and significantly lower in tetracycline group than those in chirismus group and control (both P<0.01). There was no difference between chirismus group and control (P>0.05). (7) The expression of MMP-3 was significantly lower in normal group compared with other groups (P<0.01) , significantly higher in control than those in chirismus group and tetracycline group (both P<0.01), and significantly higher in chirismus group compared with tetracycline group (P< 0.05). (8) The expression of MMP-13was significantly lower in normal group compared with other groups (P<0.05), significantly higher in control than those in other groups (P<0. 01), and significantly higher in tetracycline group compared with chirismus group (P < 0.05). (9) The expression of TIMP-1 was significantly lower in normal group compared with other groups (P＜0.01) , significantly higher in control than those in chirismus group and tetracycline group (both P<0.01), and significantly higher in tetracycline group compared with chirismus group (P <0.01). (10) The expression of TIMP-3 showed no difference between normal group and chirismus group (P>0.05).The expression of TIMP-3 was significantly higher in control compared with other groups (P<0.01) , and significantly lower in tetracycline group compared with other groups (P <0.01). 2. The means of Cobb angle of rat lumbar spine are 0°in normal group, 14°in control, 12°in tetracycline group, and 10°in chirismus group, respectively. Correlation analysis was performed under other grey system methods with statistical package DPS9. 50 (Table 5). Table 5 Correlation of Cobb angle of rat lumbar spine in anteroposterior radiograph with related genes
Conclusions: (1)Chirismus and tetracycline have a similar therapeutic effect to relieve the lumbar disc degeneration of rats via inhibiting the expression of COL-1a. The higher expression of COL-1a in control approves this point and is consistent to the literatures which report the increase of collagen I after IDD. (2)Chirismus and tetracycline can up-regulate the expression of C0L-2a and relieve the lumbar disc degeneration of rats , which is contrary to the low expression of COL-2a in control. (3)Chirismus has a stronger effect to induce the expression of aggrecan than tetracycline, which indicates chirismus has a better therapeutic effect on the lumbar disc degeneration of rats comparing with tetracycline. (4) The expression of CILP is significantly lower in normal group than other groups in which show no statistical difference. That means the animal model is valid because the expression of CILP is up-regulated after IDD. The up-regulation of TGF-β1 by chirismus indirectly shows that chirismus could inhibit the expression of CILP and relieve IDD. Tetracycline has a similar effect. (5)Chirismus could induce the expression of TGF-β1. The increase of TGF-β1 in intervertebral disc could promote cell proliferation and extracellular matrix synthesis as well as maintain the homeostasis of intervertebral disc for relieving the lumbar disc degeneration of rats. Tetracycline has no such effect. (6) The expression of VDR was significantly higher in chirismus group than tetracycline group. VDR could directly participate the differentiation, proliferation, and maturity of chondrocytes. There are abundant chondrocytes in the intervertebral disc and plenty aggrecan in the intercellular substance. Chirismus could up-regulate the expression of VDR and relieve the lumbar disc degeneration of rats via chondrocytes as well as decrease the possibility of vertebrae spur and disc stenosis, which is consistent to the imageological conclusions. (7)Tetracycline could down-regulate MMP-3 meanwhile up-regulate TIMP-1 to relieve IDD. Chirismus has a similar effect on regulation of these genes comparing with tetracycline and relieves IDD via inhibiting the degradation of extracellular matrix. (8)Tetracycline could down-regulate the expression of TIMP-3 via negative feedback and MMP-13 would be up-regulated. Chirismus could up-regulate TIMP-3 to approach normal group and MMP-13 would be down-regulated. Then chirismus inhibits the degradation of extracellular matrix and relieves IDD via down-regulating MMPs. (9) The expression of CILP is correlated highly witn Cobb angle meanwhile TGF-β1 has a low correlation with Cobb angle (Table 1). High expression of CILP and low expression of TGF-β1 inhibit matrix synthesis as well as destroy chondrocytes, which induce intervertebral joint degeneration that produce scoliosis. Here we conclude that the expressions of above genes all participate the synthesis and degradation of matrix. IDD has a close relation with extracellular matrix. MMPs inhibitors could relieve IDD via regulating related genes. Chirismus has a similar even sometimes better therapeutic effects.
以往大多数人认为椎间盘的退行性改变是一种生物力学作用的结果,但新近的研究表明生物力学的作用也不是绝对的,发现生物化学机制也发挥着至关重要的作用。文献上不断报道MMPs对椎间盘的退行性改变所起的作用,从而对这一病理过程有了新的认识。椎间盘的基质成分与关节软骨相似,研究表明正常软骨内MMPs与TIMP的表达水平相接近,但在骨关节炎和类风湿关节炎时,MMPs的生物活性明显升高,且mRNA的表达也增高,MMPs与TIMP的平衡破坏引起软骨破坏。四环素类抗生素及其衍生物是很有效的MMPs抑制剂,能通过多种途径抑制MMP-1、MMP-3、MMP-13、MMP-2和MMP-9的活性。研究显示,四环素及结构相似的化合物能抑制胶原酶和其他MMPs的效应,抑制MMPs的基质降解活性~([2])从而延缓椎间盘退变程度,并且大量实验研究发现COL-2a,Col-1a,Aggreean,CILP,TGF-β1,VDR,MMP-3,MMP-13,Timp-3,Timp-1基因的表达情况与椎间盘的退行性改变密切相关。
祖国医学认为本病的发生有内因、外因两方面。内因由于随着年龄的增长,肝肾渐虚,气血不足,邪气留连,病久入深,着于筋脉、肌骨,致瘀血内阻,气血运行失调,荣卫凝涩不通;腰部筋脉骨髓失养,使腰椎间盘逐渐变性,致使腰椎间盘的软骨板变薄,纤维环弹性减弱,髓核含水量逐渐减少,而失去了弹性;继之使椎间隙变窄,周围韧带松弛,或产生裂隙。外因由于外伤劳损及风寒湿邪入侵,引起局部气血瘀滞,阻滞经络,致机体阴阳失调,不通则痛,而引发本病。正如《诸病源候论·腰腿疼痛候》曰:“肾气不足,受风邪之所为也。劳伤则肾虚,虚者受于风冷,风冷与正气之争,故腰腿痛”。现代医学认为,髓核的膨出、压迫,引起周围组织的炎症、充血、水肿,从而压迫或刺激脊神经根,而致腰腿疼痛麻木。《素问·痹论》曰:“痹在骨则重,在于脉则血凝而不流,在于筋则屈不伸,在于内则不仁”。《素问·逆调论》又曰:“营气虚则不仁,卫气虚则不用,营卫俱虚,则不仁而不用”。本病属中医学腰腿痛范畴。采用中医手法治疗可降低神经应激功能,增加腰部局部组织痛阈,改善腰肌高张力状态,具有解痉止痛的作用,但对其作用机理不明。
本课题通过MMPs抑制剂和中医手法对造模大鼠的干预,从影像学和病理学角度来进行椎间盘组织退变机理的大体形态学研究,并在基因水平来探讨MMPs抑制剂和中医手法与相关椎间盘退变基因表达的关系,从而揭示预防和延缓椎间盘退变的机理,为临床进一步量化手法治疗效果和规范手法治疗方法提供理论依据。
第一部分:外源性MMPs抑制剂-四环素、手法对造模大鼠腰椎间盘组织退变的形态学影响(摘要)
目的:探讨外源性MMPs抑制剂-四环素、手法对造模大鼠腰椎间盘组织退变的形态学影响,试图从影像学和形态学角度阐明它们对椎间盘早期退变抑制的作用机理。
方法:将36只雄性SD大鼠随机平均地分为四组:正常组、对照组、四环素组、手法组。将它们分别单笼饲养,并且使对照组、四环素组、手法组造模。动物模型建立4周后,正常组和对照组皮下注射生理盐水,四环素组每日皮下注射四环素25 mg,持续2周。手法组的大鼠腰椎进行手法治疗约10分钟。分别于于造模前、造模后第2、8周每组随机选取3只动物麻醉后CR拍片检查。动物模型建立4周后,在进行第一次注射药物和手法干预前,每组随机选取3只动物,给予10%水合氯醛腹腔注射麻醉,拍摄腰椎X线正侧位片。造模达8周,以过量10%水合氯醛腹腔注射麻醉将其处死,迅速从腰椎后正中线切开皮肤,逐层分离腰椎两旁的肌肉韧带,直达椎体。取下整个腰椎段脊柱迅速放在冰面上,用眼科剪刀、镊剥离椎间小关节和椎体前方的软组织,分别从L1椎体下缘和L2椎体上缘分离上、下软骨终板,取出L1/2椎间盘组织。用同样的方法取出L2/3、L3/4、L4/5、L5/6椎间盘组织,用于其它实验。将取下的L1/2椎间盘组织放入4%多聚甲醛中固定,5%硝酸中脱钙,常规处理后,石蜡包埋,正中冠状切片,5um厚,HE染色。在日本OLYMPUS公司倒置显微镜IX71及显微成像系统DP71-IPP6.0下观察。
结果:两个月后,观察大鼠腰椎X线结果,四环素组干预后较另两组椎间隙变窄,脊柱腰段变直,部分椎体前下角骨皮质增生硬化,部分椎间孔变小;手法组干预后较另两组生理弯曲圆滑,椎间隙无明显改变,部分上段腰椎椎间孔有狭窄;对照组干预后生理弯曲存在,部分椎体前下角骨质硬化。光镜下,与正常组相比,四环素组、手法组无明显变化,髓核位于中心,无固缩,外周被纤维环包绕;对照组髓核稍有固缩。
结论:造模两个月后,X线影像学表现与造模前比较有明显改变,其组织结构有向退变方向发展趋势,说明大鼠腰椎间盘开始发生轻度退变,并处于退变的早期阶段。从X线影像学和组织形态学来看,外源性MMPs抑制剂-四环素、手法具有维护腰椎结构和改善早期退变阶段椎间盘组织结构的作用。
第二部分:MMPs抑制剂和手法治疗对大鼠腰椎间盘组织退变影响的基因表达研究(摘要)
目的:探讨外源性MMPs抑制剂-四环素、手法对造模大鼠腰椎间盘组织退变基因表达的影响,试图从基因水平阐明它们对椎间盘早期退变抑制的作用机理,为临床进一步量化手法治疗效果和规范手法治疗方法提供理论依据。
方法:将36只雄性SD大鼠随机平均地分为四组:正常组、对照组、四环素组、手法组。将它们分别单笼饲养,对照组、四环素组、手法组实验动物造模。动物模型建立4周后,正常组和对照组皮下注射生理盐水,四环素组每日皮下注射四环素25 mg,持续2周。手法组的大鼠腰椎进行手法治疗约10分钟。分别于造模前、造模后第2、8周每组随机选取3只动物麻醉后CR拍片检查。动物模型建立4周后,在进行第一次注射药物和手法干预前,每组随机选取3只动物,给予10%水合氯醛腹腔注射麻醉,拍摄腰椎X线正侧位片。造模达8周,以过量10%水合氯醛腹腔注射麻醉将其处死,迅速从腰椎后正中线切开皮肤,逐层分离腰椎两旁的肌肉韧带,直达椎体。取下整个腰椎段脊柱迅速放在冰面上,用眼科剪刀、镊剥离椎间小关节和椎体前方的软组织,分别从L1椎体下缘和L2椎体上缘分离上、下软骨终板,取出L1/2椎间盘组织。用同样的方法取出L2/3、L3/4、L4/5、L5/6椎间盘组织。将取出的大鼠L2/3-L5/6椎间盘组织,迅速放入事先预冷并编好号的取材盒中,置于液氮中保存。采用荧光定量PCR法检测MMPs抑制剂和手法治疗后相关基因COL-2a1, COL-1a1,Aggrecan,CILP,TGF-β1,VDR,MMP-3,MMP-13,Timp-3,Tirap-1在大鼠腰椎间盘组织退变中的表达量,运用DPS9.5版统计软件包进行方差分析和大鼠腰椎X光正位片侧偏角度和相关基因的关联度系数分析。
结果: 1.运用DPS9.5版统计软件包选择完全随机设计的单因数方差分析的多重比较LSD法进行统计分析得到以下结果。
(1)COL-1a的表达量正常组最低,与手法组和四环素组间比较,P>0.05,故与该两组间差异无意义;对照组COL-1a的表达量较其它各组最高,P<0.01,故与其它各组差异有显著意义。
(2)COL-2a的表达量正常组较其它组高,P<0.01,故与其它各组差异有显著意义;手法组与四环素组问比较, P>0.05,故两组间差异无意义,即COL-2a在手法组的表达量与四环素组接近;对照组与手法组和四环素组间比较表达量低,P<0.01,故与该两组差异有显著意义。
(3)Aggrecan的表达量正常组最高,P<0.01,故与其它各组差异有显著意义;对照组的表达量最低,P<0.01,与手法组和四环素组间比较差异有显著意义;Aggrecan在手法组的表达量高于四环素组,P<0.01,故两组间差异有显著意义。
(4)CILP的表达量正常组最低,P<0.01,故与其它各组差异有显著意义;其它各组间的表达量相近,P>0.05,故各组间差异无意义。
(5)TGF-β1的表达量正常组最高,与四环素组和对照组比较,P<0.01,故与该两组间差异有显著意义;手法组TGF-β1的表达量与四环素组和对照组比较, P<0.01,故与该两组间差异有显著意义;正常组比手法组TGF-β1的表达量稍高,P<0.05,故两组间差异有意义;四环素组和对照组比较,P>0.05,故两组间差异无意义。
(6)VDR的表达量正常组最高,P<0.01,故与其它各组差异有显著意义;四环素组VDR的表达量较手法组和对照组低,P<0.01,故与该两组间差异有显著意义;手法组和对照组间比较,P>0.05,故两组间差异无意义。
(7)MMP-3的表达量正常组最低,P<0.01,与其它各组差异有显著意义;对照组MMP-3的表达量较手法组与四环素组高,P<0.01,故与该两组间差异有显著意义;手法组MMP-3的表达量比四环素组高,P<0.05,故两组间差异有意义。
(8)MMP-13的表达量正常组最低,P<0.05,与其它各组差异有意义;对照组MMP-13的表达量最高,P<0.01,故与其它各组差异有显著意义;四环素组较手法组MMP-13的表达量高,P<0.05,故两组间差异有意义。
(9)TIMP-1的表达量正常组最低,P<0.01,故与其它各组差异有显著意义;对照组TIMP-1的表达量高,与手法组和四环素组比较,P<0.01,故与该两组差异有显著意义;四环素组TIMP-1的表达量高于手法组,P<0.01,故两组间差异有显著意义。
(10)TIMP-3的表达量正常组与手法组相近,P>0.05,故两组间差异无意义;正常组和手法组TIMP-3的表达量与对照组和四环素组相比,P<0.01,故正常组和手法组TIMP-3的表达量与对照组和四环素组相比差异有显著意义;对照组TIMP-3的表达量最高,四环素组TIMP-3的表达量最低,该两组相比,P<0.01,故两组间差异有显著意义。
2.测量得到大鼠腰椎的侧偏角均值分别为:正常大鼠0°;对照组14°;四环素组12°;手法组10°,运用DPS9.5 0版统计软件包选择其它的灰色系统方法下的关联度分析得到以下结果见表5。
表5大鼠腰椎X光正位片侧偏角度和相关基因的关联度系数注:CILP的表高达量对大鼠腰椎侧弯的影响最大,而TGF-β1的高表达量对大鼠腰椎侧弯的影响最小。
结论:
(1)中医手法和四环素相似的抑制COL-1a基因表达可知两种治疗方法都可延缓大鼠腰椎间盘的退变,具有相同作用,而对照组COL-1a的高表达更能说明这一点,并且与文献关于椎间盘出现退变时Ⅰ型胶原增加相符。
(2)中医手法和四环素能使COL-2a基因表达量增加,而其增加可延缓大鼠腰椎间盘的退变,这与对照组COL-2a型胶原基因的低表达应证。
(3)中医手法增加Aggrecan的表达量比四环素更明显,可见中医手法对延缓大鼠腰椎间盘的退变的治疗效果比四环素更明显。
(4)CILP的表达量正常组最低,其它各组间的表达量相近,说明此动物模型造模相当成功(椎间盘退变时CILP的含量高于正常水平),从中医手法可使TGF-β1的表达量增加可间接推知中医手法可抑制CILP的表达,进而延缓椎间盘退变。四环素也有类似效果。
(5)中医手法可使TGF-β1的表达量增加,而其在椎间盘内增多能促进细胞增殖和细胞外基质合成,维护椎间盘内环境稳定可延缓大鼠腰椎间盘的退变,四环素无此作用。
(6)中医手法使VDR的表达量较四环素高,而VDR的表达可以直接参与软骨细胞的分化、增殖和成熟。而椎间盘中富含软骨细胞,细胞间质中富含Aggrecan,因此中医手法升高VDR的表达量而通过软骨细胞增殖来延缓大鼠腰椎间盘的退变,减少了椎骨骨刺和椎间隙狭窄的可能性,这与影象学结果相一致。
(7)从MMP-3与TIMP-1的表达量可知,四环素可使MMP-3的表达量减少而使TIMP-1的表达量增高,这种平衡可以延缓椎间盘的退变;中医手法也可减少MMP-3的表达量,并且在1%极显著水平表达量与四环素相同说明中医手法和四环素治疗效果相同,都可以抑制MMP-3的表达而使TIMP-1的表达量增高,从而减少细胞外基质的降解延缓椎间盘的退变。
(8)四环素因其负反馈作用可减少TIMP-3的表达量,则MMP-13的表达量升高;而中医手法可使TIMP-3的表达量升高并与正常组接近,则MMP-13的表达量降低,这样中医手法就可减少MMPs的产生,从而减少细胞外基质的降解,也就可以延缓椎间盘的退变。
(9)从大鼠腰椎X光正位片侧偏角度和相关基因的关联度系数可知:CILP的高表达量与侧偏角度最相关,而TGF-β1的高表达量与侧偏角度最不相关,以上恰恰说明CILP与TGF-β1的相互抑制作用。CILP的表达量增高,而TGF-β1的表达量降低时,基质合成减少而破坏软骨细胞进而引起椎间关节退变而发生侧弯。
综上所述,以上各组基因的表达均参与基质的合成与降解,由此可见椎间盘的退变与细胞外基质非常相关,MMPs抑制剂可以调节相关基因的表达延缓椎间盘的退变,中医手法也有类似效果,有时中医手法的疗效更好。
Intervertebral disc degeneration (IDD) are an important cause of neck, shoulder, back, leg pain. Annulus fibrosus broken is caused by a variety of incentives such as acute sprains, chronic fatigue and cold etc.So liquid-like nucleus pulposus spills, diffuses along the channel between the disc and nerve root. And chemical radicular neuritis is induced by glycoprotein andβprotein within the nucleus pulposus which are strong chemically irritant and the following released histamine nucleus pulposus. Because of inflammation, a lot of inflammatory protein which exudes from high permeable vascular, accumulates in the intima and outima of nerve root and vertebral sinus nerve. Protein exudation causes local ischemia and electrolyte disorders and results in stimulation of nerve root and vertebral sinus nerves, causing corresponding innervation zone pain. The pain causes local muscle contraction and protective lumbar scoliosis , and the physiological biomechanical changes takes place and aggravate pain, so resulting in a vicious cycle. Furthermore, muscle spasm can aggravate vertebral facet joint dysfunction, spinal canal stenosis and so on. IDD pathophysiological mechanism is still under investigation. With development of biomechanics and molecular "biology and tissue engineering , growth factor therapy, gene therapy, cell therapy and tissue engineering etc. provide some choice for the treatment of IDD.
In the past, it is well accepted that IDD is a result of the biomechanical change, but recent studies have shown that biochemical factors also play an important role. MMPs are often reported in the development of IDD and provide a new sight in the pathological process. Intervertebral disc has a similar matrix components comparing with articular cartilage. It is reported that expression of MMPs and TIMP is similar in the normal cartilage , but the balance of MMPs and TIMP is interrupted in osteoarthritis and rheumatoid arthritis with a significantly higher MMPs activity and mRNA expression, and this imbalance results in cartilage damage . Tetracyclines and their derivatives are very effective inhibitors of MMPs, can inhibit MMP-1, MMP-3, MMP-13, MMP-2 and MMP-9 activity through different mechanisms. Studies have shown that tetracycline and their derivatives can inhibit collagenase and other MMPs effects and slow down the disc degeneration. And a large number of experimental study found that intervertebral disc degeneration are closely related with COL-2a , Col-1a , Aggrecan, CILP, TGF-β1, VDR, MMP-3, MMP-13, Timp-3, Timp-1 gene expression.
The present investigation focused on the effects of intervention using the MMPs inhibitors and traditional Chinese medicine massage on rat IDD model. The general morphology was investigated by imaging and pathology, and real time PCR was used to study gene expression of degenerated intervertebral disc before and after the MMPs inhibitors and massage, respectively. This research aimed to reveal the mechanism of intervention and provide theoretical basis to quantify and standardize the treatment approach.
PartⅠ: morphological impact of exogenous MMP inhibitors tetracycline and massage on lumbar IDD in rat model
Abstract Objective: To investigate the morphological impact of exogenous MMPs inhibitor tetracycline and massage on lumbar IDD in rat model, to elucidate mechanism of their inhibition in terms of imaging and morphology. Methods: 36 male SD rats were randomly divided into four groups: normal group, control group, tetracycline group and massage group. All the 36 rats were single-cage feed and the control, tetracycline,model group were modeled.4 weeks after modeling, subcutaneous injection of normal saline was administrated to control group daily, subcutaneous injection of tetracycline 25 mg to tetracycline group, and 10 minutes massage to the massage group daily, Respectively. All sustained two weeks. 3 rats were randomly selected in each group for CR-Xray before and 2weeks, 8 weeks after modeling.4 weeks, 3 rats were randomly selected in each group for lumbar CR-Xray before first injection of massage, anesthetized by 10% chloral hydrate through intraperitoneal injection. 8 weeks after modeling, rats were executed by overdose 10% chloral hydrate intraperitoneal injection. vertebral column were excided through dorsal meso-incision. the removed vertebral column were put on ice as soon as possible andL1/2, L2/3, L3/4, L4/5, L5/6 intervertebral disc tissue were procured. Half of L1/2 intervertebral disc tissue was fixed in 4% paraformaldehyde, decalcified in 5% nitric acid, embedded in paraffin, 5um thick section was obtained, and went on HE staining. OLYMPUSBH optical microscope was used for observation. The remaining intervertebral disc tissue were used for other experiments. Results: Two months after modeling, lumbar spine X-ray shown: tetracycline group had narrower intervertebral space, smaller foraminal, worse spine curvature and more obvious bone sclerosis than the other two groups after the intervention; massage group had better spine curvature, but narrower intervertebral space, smaller foraminal in some upper segment than the other two groups after the intervention; the control group showed no changes in physiological curvature, bone sclerosis in some anterior ventro-rvertebrae. No significant difference was found among the normal group, the tetracycline group and the massage group under optical microscope: nucleus pulposus is at the center, no shrinkage, enveloped by the peripheral annulus f ibrosus; the control group showed a slight shrinkage of nucleus pulposus. Conclusion: two months after modeling, X-ray imaging showed significantly changes and mild degeneration and indicated a trend of degeneration. From the X-ray and morphological study, the exogenous MMPs inhibitor-tetracycline, massage can inhabit the early stages of disc degeneration.
PartⅡ: gene expression research of the impact of MMP inhibitors - tetracycline and massage on lumbar intervertebral disc degeneration in rats
Abstract Objective : To investigate gene expression of degenerated intervertebral disc treated the exogenous MMPs inhibitors and massage, to reveal mechanism of intervention from the genetic level and provide theoretical basis to further quantify and standardize the treatment approach. Methods: 36 male SD rats were randomly divided into four groups: normal group, control group, tetracycline group and massage group. All the 36 rats were single-cage feed and the control , tetracycline, model group were modeled. 4 weeks after modeling, subcutaneous injection of normal "saline was administrated to control group daily, subcutaneous injection of tetracycline 25 mg to tetracycline group, and 10 minutes massage to the massage group daily, Respectively.All sustained two weeks. 3 rats were randomly selected in each group for CR-Xray before and 2weeks, 8 weeks after modeling. 4 weeks, 3 rats were randomly selected in each group for lumbar CR-Xray before first injection of massage , anesthetized by 10% chloral hydrate through intraperitoneal injection. 8 weeks after modeling, rats were executed by overdose 10% chloral hydrate intraperitoneal injection. Vertebral column were excided through dorsal meso-incision. the removed vertebral column were put on ice as soon as possible and L1/2, L2/3, L3/4, L4/5, L5/6 intervertebral disc tissue were procured. The procured L2/3, L3/4, L4/5, L5/6 intervertebral disc tissue were stored in liquid nitrogen. Gene expression level of COL-2a, COL-1a, Aggrecan, CILP, IGF-(β1, VDR, MMP-3, MMP-13, Timp-3, Timp-1 in intervertebral disc were tested using real time PCR assay after treatment of MMPs inhibitors-tetracycline and massage.DPS9.5 statistical software package were used to do variance analysis and correlation coefficient analysis between scoliosis angle in posterior-anterior X-ray and associated gene expression. Results: 1. Statistical analysis was performed with statistical package DPS9. 50. Completely random designed one-way ANOVA ( LSD method ) was used for comparison between groups. (1) The expression of COL-1a showed no difference in normal group, chirismus group and tetracycline group (P > 0. 05) and significantly higher in control compared with other groups (P < 0. 01). (2) The expression of COL-2a was significantly higher in normal group compared with other groups (P < 0. 01). There was no difference between chirismus group and tetracycline group (P> 0. 05). The expression of COL-2a was significantly lower in control compared with chirismus group and tetracycline group (both P < 0.01). (3) The expression of aggrecan was significantly higher in normal group compared with other groups (P < 0.01) , significantly lower in control than those in chirismus group and tetracycline group (both P < 0.01), and significantly higher in chirismus group compared with tetracycline group (P<0. 01). (4) The expression of CILP was significantly lower in normal group compared with other groups (P< 0. 01) and showed no difference in control, chirismus group and tetracycline group (P>0. 05). (5) The expression of TGF-β1 was significantly higher in normal group compared with tetracycline group and control (both P<0. 01), significantly higher in chirismus group than those in tetracycline group and control (both P<0.01), and significantly higher in normal group compared with chirismus group (P<0.05). The expression of TGF-β1 showed no difference between tetracycline group and control (P>0.05). (6) The expression of VDR was significantly higher in normal group compared with other groups (P<0.01), and significantly lower in tetracycline group than those in chirismus group and control (both P<0.01). There was no difference between chirismus group and control (P>0.05). (7) The expression of MMP-3 was significantly lower in normal group compared with other groups (P<0.01) , significantly higher in control than those in chirismus group and tetracycline group (both P<0.01), and significantly higher in chirismus group compared with tetracycline group (P< 0.05). (8) The expression of MMP-13was significantly lower in normal group compared with other groups (P<0.05), significantly higher in control than those in other groups (P<0. 01), and significantly higher in tetracycline group compared with chirismus group (P < 0.05). (9) The expression of TIMP-1 was significantly lower in normal group compared with other groups (P＜0.01) , significantly higher in control than those in chirismus group and tetracycline group (both P<0.01), and significantly higher in tetracycline group compared with chirismus group (P <0.01). (10) The expression of TIMP-3 showed no difference between normal group and chirismus group (P>0.05).The expression of TIMP-3 was significantly higher in control compared with other groups (P<0.01) , and significantly lower in tetracycline group compared with other groups (P <0.01). 2. The means of Cobb angle of rat lumbar spine are 0°in normal group, 14°in control, 12°in tetracycline group, and 10°in chirismus group, respectively. Correlation analysis was performed under other grey system methods with statistical package DPS9. 50 (Table 5). Table 5 Correlation of Cobb angle of rat lumbar spine in anteroposterior radiograph with related genes
Conclusions: (1)Chirismus and tetracycline have a similar therapeutic effect to relieve the lumbar disc degeneration of rats via inhibiting the expression of COL-1a. The higher expression of COL-1a in control approves this point and is consistent to the literatures which report the increase of collagen I after IDD. (2)Chirismus and tetracycline can up-regulate the expression of C0L-2a and relieve the lumbar disc degeneration of rats , which is contrary to the low expression of COL-2a in control. (3)Chirismus has a stronger effect to induce the expression of aggrecan than tetracycline, which indicates chirismus has a better therapeutic effect on the lumbar disc degeneration of rats comparing with tetracycline. (4) The expression of CILP is significantly lower in normal group than other groups in which show no statistical difference. That means the animal model is valid because the expression of CILP is up-regulated after IDD. The up-regulation of TGF-β1 by chirismus indirectly shows that chirismus could inhibit the expression of CILP and relieve IDD. Tetracycline has a similar effect. (5)Chirismus could induce the expression of TGF-β1. The increase of TGF-β1 in intervertebral disc could promote cell proliferation and extracellular matrix synthesis as well as maintain the homeostasis of intervertebral disc for relieving the lumbar disc degeneration of rats. Tetracycline has no such effect. (6) The expression of VDR was significantly higher in chirismus group than tetracycline group. VDR could directly participate the differentiation, proliferation, and maturity of chondrocytes. There are abundant chondrocytes in the intervertebral disc and plenty aggrecan in the intercellular substance. Chirismus could up-regulate the expression of VDR and relieve the lumbar disc degeneration of rats via chondrocytes as well as decrease the possibility of vertebrae spur and disc stenosis, which is consistent to the imageological conclusions. (7)Tetracycline could down-regulate MMP-3 meanwhile up-regulate TIMP-1 to relieve IDD. Chirismus has a similar effect on regulation of these genes comparing with tetracycline and relieves IDD via inhibiting the degradation of extracellular matrix. (8)Tetracycline could down-regulate the expression of TIMP-3 via negative feedback and MMP-13 would be up-regulated. Chirismus could up-regulate TIMP-3 to approach normal group and MMP-13 would be down-regulated. Then chirismus inhibits the degradation of extracellular matrix and relieves IDD via down-regulating MMPs. (9) The expression of CILP is correlated highly witn Cobb angle meanwhile TGF-β1 has a low correlation with Cobb angle (Table 1). High expression of CILP and low expression of TGF-β1 inhibit matrix synthesis as well as destroy chondrocytes, which induce intervertebral joint degeneration that produce scoliosis. Here we conclude that the expressions of above genes all participate the synthesis and degradation of matrix. IDD has a close relation with extracellular matrix. MMPs inhibitors could relieve IDD via regulating related genes. Chirismus has a similar even sometimes better therapeutic effects.
引文
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