Expermental Study Of The Role Of TNF-α,MMP-9/TIMP-1 In IP Relieving I/R Injruy Of Skeletal Muscle Of Rabbits

Objective:To investigate the mechanism of ischemic preconditioning protecting against ischemia-reperfusion injury of skeletal muscle by studying the expression changes of TNF-α,MMP-9/TIMP-1 in skeletal muscle of rabbits'hind limbs which were treated with ischemic preconditioning(or not) before carrying out ischemia reperfusion, so as to lay a base of clinical application of ischemia preconditioning and provide a new thinking and theoretical evidence for other preconditioning methods.Methods:Twenty-four healthy male rabbits were divided into three groups randomly. Every group had eight rabbits. The models of ischemia preconditioning and ischemia reperfusion injury were established by separating the arteria femoralis and vena femoralis by operation and blocking them by artery clamps. It was only to separate arteria femoralis and vena femoralis but not block them by artery clamps in Sham group. The serumal level of CK, LDH, AST in every group were detected before and after ischemia reperfusion. All skeletal muscle samples were made into pathological sections and were stained by Hematoxylin and eosin staining. To observe the pathological changes of every pathological section( mainly about integrity of skeletal muscle fibers, edema extent of skeletal muscle cells and interstitial substance and inflammatory reaction) and to observe the three samples which were selected from every group randomly and respectively by electron microscope. The expression levels of TNF-α, MMP-9 and TIMP-1 in all skeletal muscle samples were detected by immunohistochemistry staining and were denoted by optical density value(OD value).Results : 1. 24 hours After reperfusion, Sham group: CK 365.2±68.0(U/L), LDH 129.5±13.3(U/L),AST 88.4±7.9(U/L) ; IP+I/R group : CK 1278.6±84.7(U/L), LDH 491.1±50.5(U/L), AST 169.7±18.6(U/L) ; I/R group : CK 2666.1±125.3(U/L), LDH 1184.4±97.8(U/L), AST 273.9±25.2(U/L). Compared with Sham group, the serumal level of CK, LDH, AST in IP+I/R group and I/R group were heightened at different degree(P<0.05), especially in I/R group. The serumal level of CK, LDH, AST in IP+I/R group were lower than I/R group(P<0.05). Pathological sections of skeletal muscle samples showed: Morphous and structure of skeletal muscle in Sham group were normal. Arrangement disorder of skeletal muscle fibers, disintegration of some skeletal muscle fibers, obstruct of lumen of blood vessel, effusion of a large quantity of neutrophils, edema of skeletal muscle cells, significant engorgement of intercellular substance were saw in I/R group. Compared with I/R group, the degree of engorgement of intercellular substance and edema of skeletal muscle cells in IP+I/R group were relieved. Small focal necrosis of skeletal muscle fibers were saw casually and effusion of neutrophils relieved significantly in IP+I/R group. Examination of transmission electron microscope showed: Myofilament lysis, severe edema of mitochondria, dilataltion of sarcoplasmic reticulum, endothelial cells'notable swelling and basement membrane rupture of microvessel, interstitial edema and leukocytic infiltrate were saw in I/R group. Compared with I/R group, the foregoing injuries in IP+I/R group were relieved. 2. OD value of Sham group: TNF-α0.133±0.014,MMP-9 0.162±0.021, TIMP-1 0.148±0.017; OD value of I/R group: TNF-α0.435±0.047,MMP-9 0.488±0.055,TIMP-1 0.165±0.020;OD value of IP+I/R group TNF-α0.249±0.036, MMP-9 0.266±0.027, TIMP-1 0.159±0.121. Compared with Sham group, the expression of TNF-αand MMP-9 in IP+I/R group and I/R group were heightened at different degree and had significant difference by computer image analysis (P<0.05), especially in I/R group. The expression of TNF-αand MMP-9 in IP+I/R group were lower than I/R group(P<0.05). The expression of TIMP-1 in I/R group and IP+I/R group increased slightly, but their OD values among every group had no significant difference by analysis of variance(F=1.546,P=0.236>0.05).Conclusions:1.The method of separating the arteria femoralis and vena femoralis by operation and blocking them by artery clamps is a very successful way to establish the models of ischemia preconditioning and ischemia reperfusion injury. It can be used to carry out in the empirical studies of ischemia preconditioning and ischemia reperfusion injury. 2. Ischemia preconditioning could relieve the ischemia reperfusion injury of skeletal muscle significantly. It is an effective clinical way to prevent and relieve the ischemia reperfusion injury of tissues and organs. 3. Ischemia preconditioning could decrease the expression of TNF-αso as to inhibit the expression and activity of MMP-9 and keep the balance of MMP-9/TIMP-1, so the injury of tissues and organs resulted by the increase of MMP-9 was relieved. It could be one of the effective mechanisms that ischemia preconditioning relieves the ischemia reperfusion injury of skeletal muscle.