【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Mankin’s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Mankin’s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。更多还原

【Abstract】 In order to observe the effects of one-time high intensity exhaustive exercise and pyrrolidine dithiocarbamate(PDTC, an NF-κB inhibitor) supplementation on rat’s 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 de- termined Mankin’s 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, Mankin’s 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.termined Mankin’s 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, Mankin’s 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.更多还原