Protection Induced By Ischemic Postconditioning Against Ischemia-reperfusion Injury In Rat Skeletal Muscle And Preliminary Study On Risk Pathway

BACKGROUND:The mechanisms underlying ischemic postconditioning protection against ischemia-reperfusion injury in rat skeletal muscle have not been intensively investigated,so we made a preliminary study on RISK signaling pathway mechanism and further tested the protective effect of an optimal protocol of ischemic postconditioning.OBJECTIVE:To further investigate the protective effective of an optimized protocol of ischemic postconditioning on ischemia-reperfusion injury in rat skeletal muscle and make a preliminary study on RISK pathway.METHODS:Healthy adult male Sprague-Dawley rats were randomly divided into 3 Groups（n=6）:Ischemia Reperfusion Group（IR Group）,Ischemic Postconditioning Group（IPostC Group）,Normal Group（N Group）.Rats in each Group were given occlusion/disocclusion on the right femoral artery for ischemia-reperfusion of right lower limbs.The rats in IR Group were subjected to 4 hours of ischemia,followed by 24 hours of reperfusion;The rats in IPostC Group were subjected to 4 hours of ischemia,and immediate 4 cycles of 30 seconds reperfusion/30 seconds ischemia,followed by 24 hours of reperfusion;The rats in N Group were undergone sham surgery.Morphological changes in soleus in each group were evaluated by HE staining;infarct size of gastrocnemius was measured with method of TTC staining;expression of key proteins in RISK pathway from tibialis anterior were detected with Western Blot;and muscular Ca²⁺-induced mPTP opening were evaluated by spectrophotometer.RESULTS:The HE staining showed that,compared with N Group,muscle fiber arrangement in IR Group was much more disordered and fiber interval more widened,some of the muscle fibers ruptured,a large number of inflammatory focuses and edema could be seen under light microscope;The above pathophysiological changes in IPostC Group were much improved,and edema was much more slight compared with IR Group.Infarct size with TTC staining in IPostC Group was much decreased compared to IR Group[（22.27±11.69）%vs（43.32±11.44）%（P<0.05）],there was no difference between the IPostC Group and the N Group[（22.27±11.69）%vs（15.26±7.13）%（P>0.05）].Western blot analysis showed the expression levels of T-Akt and T-eNOS in both IPostC and IR Group were higher than that in N Group,but there was no significant difference between that in IPostC and IR groups（P>0.05）;the expression levels of P-Akt,P-eNOS-S1177 in IPostC Group were all increased compared to that in IR Group（P<0.05）.Interestingly,the expression level of P-eNOS-Thr495 in IR Group was much increased,when compared with that in both N Group and IPostC Group（P<0.01）.The mPTP measurement results showed that the change in absorbance at 520nm over 2min,4min,6min in IPostC Group displayed smaller degree of reduction compared with that in N Group and IR Group（P<0.05）.CONCLUSION:The results indicated that ischemia-reperfusion injury in rat skeletal muscle can be reduced by applying optimal protocol of ischemic postconditioning.The mechanism underlying ischemic postconditioning may be ssociated with the activation of RISK signaling pathway（PI3K-Akt-eNOS）,inhibition or delay of mPTP opening,resulting in enhanced tolerance of ischemia-reperfusion injury in rat skeletal muscle.