大強度力竭運動和PDTC干預對大鼠關節軟骨損傷的影響及NF—κB信號途徑的探討

黃偉

摘 要：觀察一次大強度力竭運動和補充NF-κB抑制劑——吡咯烷二巰基氨甲酸（PDTC）對大鼠膝關節軟骨誘導型一氧化氮合酶（iNOS）、一氧化氮（NO）含量，基質金屬蛋白酶-13酶原（pro-MMP-13）和活性MMP-13（active-MMP-13）的影響，探討過度運動誘導iNOS表達上調并造成軟骨損傷的可能機制。將40只SD大鼠隨機分為4組：對照組（C組）、給藥組（P組）、運動組（E組）、運動+給藥組（EP組）。C組給予1 mL生理食鹽水腹腔注射，P組給予200 mg/kg PDTC腹腔注射，E組腹腔注射1 mL生理鹽水后進行1次大強度力竭運動，EP組腹腔注射PDTC后進行1次力竭運動。實驗后2 h，提取關節液，硝酸還原酶法測定NO含量；HE染色觀察軟骨形態學變化并進行Mankins評分；實時熒光定量PCR檢測iNOS和MMP-13 mRNA水平；western blot法測定iNOS、pro-MMP-13和active-MMP-13蛋白表達量；凝膠電泳遷移率分析（EMSA）測定NF-κB與DNA結合活性。結果顯示與C組比較，E組和EP組Mankins評分、關節液NO含量、iNOS和MMP-13 mRNA水平、iNOS和active-MMP-13蛋白水平以及NF-κB與DNA結合活性均非常顯著性升高（P<0.01）；與E組比較，EP組上述指標均非常顯著性降低（P<0.01）。pro-MMP-13蛋白水平在各組均無顯著性差異（P>0.05）。結果說明一次大強度力竭運動通過NF-κB信號途徑介導iNOS表達上調，促進NO大量釋放和MMP-13激活，從而參與了軟骨損傷的過程。

關 鍵 詞：運動醫學；力竭運動；核因子-κB；誘導型一氧化氮合酶；軟骨；創傷性骨關節炎；大鼠

中圖分類號：G804.5 文獻標志碼：A 文章編號：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