摘要
目的 :构建大鼠自体尾椎破裂型椎间盘突出冲吸收动物模型,探讨P38丝裂原活化蛋白激酶(P38 mitogen-activated protein kinases,P38MAPK)信号通路在破裂型椎间盘突出重吸收中的作用。方法 :将40只3月龄SD大鼠随机分为对照组和实验组,每组20只。采用刺破大鼠自体尾椎椎间盘,包埋至L4-5背部肌肉的方法制作破裂型椎间盘突出重吸收模型。实验组从造模至取材之日予腹腔注射P38MAPK特异性阻断剂SB203580;对照组予腹腔注射生理盐水,分别在造模1周和4周后处死并取出包埋的椎间盘,电子天平称重。计算1周末和4周末椎间盘减少质量,镜下观察椎间盘形态退变情况。Real Time PCR法检测P38MAPK、肿瘤坏死因子α(tumour necrosis factor-α,TNF-α)、白细胞介素1β(interleukin-1β,IL-1β)、基质金属蛋白酶3(matrix metalloproteinase-3,MMP-3)、基质金属蛋白酶9(matrix metalloproteinase-9,MMP-9)的m RNA表达。Western Blot法检测TNF-α、IL-1β、MMP-3、MMP-9及磷酸化P38丝裂原活化蛋白激酶(phosphorylated p38mitogen-activated protein kinase,p-p38MAPK)的蛋白表达。结果 :实验组1周时椎间盘质量减小0.47±2.90mg,4周时减少1.11±3.05mg,两组之间无统计学差异(P>0.05),对照组1周时椎间盘质量减小4.15±1.84mg,4周时减少10.56±3.29mg,两组之间存在统计学差异(P<0.05)。镜下可见,实验组4周时与1周时相比,组织形态无明显退变,对照组4周时与1周时相比,组织形态出现明显退变。Real Time PCR检测,1周时,实验组椎间盘髓核组织中TNF-α、p38MAPK、MMP-3、MMP-9的m RNA表达低于对照组(P<0.05);4周时,实验组TNF-α、IL-1β、p38MAPK、MMP-3的m RNA表达低于对照组(P<0.05)。Western Blot法检测,4周时,实验组椎间盘组织中TNF-α、P38MAPK、P-p38MAPK、MMP-3的蛋白表达低于对照组(P<0.05);对照组4周时椎间盘组织中TNF-α、IL-1β、P-p38MAPK的蛋白表达均高于1周时(P<0.05)。对照组髓核组织中P-p38MAPK与MMP-3、IL-1β、TNF-α的蛋白表达呈正相关(0<r<1,P<0.05)。结论 :P38阻断剂可能通过抑制髓核组织中P38MAPK的磷酸化,降低TNF-α、IL-1β、MMP-3、MMP-9的m RNA和蛋白体表达,从而抑制突出椎髓核组织重吸收。
Objectives： To investigate the role of P38 mitogen-activated protein kinases（P38MAPK） signal phosphorylation in the lumbar intervertebral disc herniation spontaneous resorption. Methods： 40 Sprague Dawley（SD） rats were divided into experimental group（P38 MAPK inhibition） and control group（non-contained）. The model of ruptured lumbar intervertebral disc herniation resorption was established on SD rat by putting two coccygeal intervertebral disc（IVDs） which were removed and injured prior into the subcutaneous space of the back where the L4-5 spinous and all vertebral plates were chewed and the dural sac was ruptured. Furthermore, experimental group was injected intraperitoneally by SB203580, and control group was injected by saline. At 1 and weeks 4 post-surgery, the rats were sacrificed, and the relocated discs were taken out for further testing. Morphological changes were examined by HE staining, and the mRNA and protein expressions of P38 mitogen-activated protein kinase（P38MAPK）, phosphorylated p38 mitogen-activated protein kinase（P-p38MAPK）, tumour necrosis factor-α（TNF-α）, interleukin-1β（IL-1β）, matrix metalloproteinase-3（MMP-3）, matrix metalloproteinase-9（MMP-9） in intervertebral disc tissue were statistically determined by Real Time-PCR and Western-Blot. Results： In control group, the disc had a mass reduction of 4.15±1.84mg over the weekend, and 10.56±3.29mg decreased by 4 weeks（P〈0.05）. In experimental group, the reduction value of disc mass at 4 weeks and 1 week was respectively 1.11±3.05mg and 0.47±2.90mg, the difference was not statistically significant（P〉0.05）. Under microscope, the tissue morphology of transplanted disc in control group showed obvious degeneration. The morphological degeneration of intervertebral disc tissue was not obvious in experimental group. Real Time PCR test results： over the week, the mRNA expression of TNF-α, p38MAPK, MMP-3 and MMP-9 of intervertebral discs in experimental group was significantly lower than that in control group（P〈0.05）. At 4 weeks, the mRNA expression of TNF-α, IL-1β, p38MAPK and MMP-3 in experimental group was significantly lower than that in control group（P〈0.05）. Western Blot test results： the protein expression of TNF-α, P38MAPK, P-p38MAPK and MMP-3 of intervertebral discs in experimental group was lower than that in control group at 4 weeks（P〈0.05）. In control group, the protein expression of TNF-α, IL-1β and P-p38MAPK of the intervertebral disc nucleus at 4 weeks was significantly higher than that at 1 weekend（P〈0.05）. The protein expressions of MMP-3, IL-1β, TNF-α were positively correlated with P38MAPK time-dependently in control group（0〈r〈1, P〈0.05）. Conclusions： The phosphorylation of P38MAPK signaling pathway can regulate inflammatory factor agglomeration, improve the expression of matrix metalloproteinase, and promote the resorption of nucleus pulposus. The P38MAPK-specific blocker can reduce the phosphorylation of P38MAPK and then reduce tthe expression levels of mRNA and protein of TNF-α, IL-1β, MMP-3 and MMP-9. The resorption effect was inhibited.
出处
《中国脊柱脊髓杂志》
CAS
CSCD
北大核心
2017年第10期938-945,共8页
Chinese Journal of Spine and Spinal Cord
基金
国家自然科学基金资助项目(编号:81473691)
