大强度力竭运动和PDTC干预对大鼠关节软骨损伤的影响及NF—κB信号途径的探讨

黄伟

摘 要：观察一次大强度力竭运动和补充NF-κB抑制剂——吡咯烷二巯基氨甲酸（PDTC）对大鼠膝关节软骨诱导型一氧化氮合酶（iNOS）、一氧化氮（NO）含量，基质金属蛋白酶-13酶原（pro-MMP-13）和活性MMP-13（active-MMP-13）的影响，探讨过度运动诱导iNOS表达上调并造成软骨损伤的可能机制。将40只SD大鼠随机分为4组：对照组（C组）、给药组（P组）、运动组（E组）、运动+给药组（EP组）。C组给予1 mL生理食盐水腹腔注射，P组给予200 mg/kg PDTC腹腔注射，E组腹腔注射1 mL生理盐水后进行1次大强度力竭运动，EP组腹腔注射PDTC后进行1次力竭运动。实验后2 h，提取关节液，硝酸还原酶法测定NO含量；HE染色观察软骨形态学变化并进行Mankins评分；实时荧光定量PCR检测iNOS和MMP-13 mRNA水平；western blot法测定iNOS、pro-MMP-13和active-MMP-13蛋白表达量；凝胶电泳迁移率分析（EMSA）测定NF-κB与DNA结合活性。结果显示与C组比较，E组和EP组Mankins评分、关节液NO含量、iNOS和MMP-13 mRNA水平、iNOS和active-MMP-13蛋白水平以及NF-κB与DNA结合活性均非常显著性升高（P<0.01）；与E组比较，EP组上述指标均非常显著性降低（P<0.01）。pro-MMP-13蛋白水平在各组均无显著性差异（P>0.05）。结果说明一次大强度力竭运动通过NF-κB信号途径介导iNOS表达上调，促进NO大量释放和MMP-13激活，从而参与了软骨损伤的过程。

关 键 词：运动医学；力竭运动；核因子-κB；诱导型一氧化氮合酶；软骨；创伤性骨关节炎；大鼠

中图分类号：G804.5 文献标志码：A 文章编号：1006-7116（2014）01-0138-07

Effects of high intensity exhaustive exercise and PDTC intervention on rats

articular cartilage injury as well as NF-κB signal pathway exploration

HUANG Wei

（School of Physical Education，Zhengzhou University，Zhengzhou 450044，China）

Abstract： In order to observe the effects of one-time high intensity exhaustive exercise and pyrrolidine dithiocarbamate （PDTC， an NF-κB inhibitor） supplementation on rats knee articular cartilage inducible nitric oxide synthase （iNOS）， nitric oxide （NO） content， matrix metalloproteinase-13 proenzyme （pro-MMP-13） and active-MMP-13， so as to probe into a possible mechanism of over-exercise inducing iNOS expression increase and causing cartilage injury， the author divided 40 SD rats randomly into 4 groups， namely， a control group （group C）， a PDTC fed group （group P）， an exercise group （group E）， and an exercise + PDTC fed group （group EP）， injected 1 mL of normal saline into the abdominal cavity of the rats in group C， injected 200 mg/kg of PDTC into the abdominal cavity of the rats in group P， injected 1ml of normal saline into the abdominal cavity of the rats in group E and then let them do an one-time high intensity exhaustive exercise， injected PDTC into the abdominal cavity of the rats in group EP and then let them do an one-time exhaustive exercise， 2 h after the experiment， extracted synovia， measured NO content by using the nitrate reductase method， observed morphological cartilage changes by means of HE dyeing and determined Mankins scores， measured iNOS and MMP-13 mRNA levels by means of real-time fluorescent quantification PCR， measured iNOS， pro-MMP-13 and active-MMP-13 protein expression levels by using the western blot method， measured the activity of combination of NF-κB and DNA by means of electrophoretic mobility shift assay （EMSA）， and revealed the following findings： comparing the rats in groups E and EP with the rats in group C， Mankins scores， synovial NO content， iNOS and MMP-13 mRNA levels， iNOS and active-MMP-13 protein levels， and the activity of combination of NF–κB and DNA increased very significantly （P<0.01）； comparing the rats in group EP with the rats in group E， all the said indexes decreased very significantly （P<0.01）； there was no significant difference in protein pro-MMP-13 level between the rats in various groups. The said findings indicated that one-time high intensity exhaustive exercise mediated iNOS expression increase via NF-κB signal pathway， boosted the mass discharge of NO and the activation of MMP-13， thus participated the process of cartilage injury.endprint