| Objectives:We used in vivo I/R model of SD rats to observe the cardioprotective effects of sevoflurane and explore the mechanism of SpostC; Autophagy may be the mechanism responsible for SpostC. To study the effects of SpostC on autophagy, we detect the number of autophagic vacuoles, the autophagic protein expression and lysosomal function. To further confirm that SpostC accelerates autophagic flux, we used CQ to abolish the effects of SpostC. We want to reveal that SpostC can accelerate autophagic flux by improving the lysosomal function and reduce the opening of mPTP, then less apoptosis of myocytes。 We focus on the autophagic effects of SpostC in order to provide new molecular targets for exploring intervention against I/R injury.Methods:1. Experimental protocol:Rats were randomly assigned into seven groups. To determine the effects of SpostC on autophagy, rats were divided into the Sham, Sham+SpostC, I/R group, and I/R+SpostC group. To determine whether the efficacy of SpostC was maintained with CQ treatment, additional rats were administered intra-peritoneal injections of CQ (10mg/kg)1h before surgical operation. Therefore, additional three groups (CQ+Sham group, CQ+I/R group,CQ+I/R+SpostC group) were used.2. Rats I/R in vivo model with SpostC treatment:Rats underwent30min of LAD ligation and then either30or120min of reperfusion and received2.4%(v/v) sevoflurane during the first5min of reperfusion.3. ECG and hemodynamics:using a rat limb-lead monitor heart rate and rhythm continuously, monitoring blood pressure by the right femoral artery catheterization.4. Heart function:At the end of120min reperfusion,±dp/dtmax was evaluated by an direct catheterization in left ventricluar.5. cTnl:At the end of120min reperfusion, blood was collected for ELISA test of cTnI;6. Evans blue staining:At the end of120min reperfusion, LAD was reoccluded, and administered1mLEvans blue through the right carotid venous, the normal perfusion area was stained blue, while the myocardium in area at risk remained pale.7. TTC staining:At the end of120min reperfusion, heart was removed for TTC staining, necrosis cardiac tissue is pale, while vital cardiac tissue was stained red. 8. Quantitative morphometric analysis of autophagic vacuoles by a transmission electron microscope:At the end of30min reperfusion, heart was removed for ultrastructure examination.9. Western blotting:At the end of30min reperfusion, heart was removed for examination the expression level of autophagic protein and apoptotic protein.10. Chloroquine was used to block autophaic flux:rats were administered intra-peritoneal injections of CQ (10mg/kg)1h before surgical operation.Quantitative morphometric analysis of autophagic vacuoles by a transmission electron microscope to conform that autophagic flux was influenced on the cardioprotective effects of SpostC.11. Tunel test:At the end of120min reperfusion, heart was removed for Tunel test, fixed in4%paraformaldehyde solution and preparation of paraffin-embedded sections.12. mPTP opening:At the end of30min reperfusion, heart was removed, embedded in OCT, and sliced into frozen sections. MPTP opening was determined with calcein-AM in the presence of cobalt chloride, green fluenrence was observed under a microscope at a wave lenth of505nm.13. Cathepsin B activity:At the end of30min reperfusion, heart was removed,the tissue samples were completely homogenized by a Dounce homogenizer. The supernatant was used for enzymatic assay by kit,and the relative fluorescence units (RFUs) were measured by spectrofluorometric analyses by using a UV-fluorescent spectrophotometer with excitation and emission settings of400and505nm, respectively.14. Statistical analysisThe data are presented as the mean±SD. GraphPad Prism5.0(GraphPad, San Diego, CA, USA) was used to analyze the data. The significant differences among multiple groups were analyzed by ANOVA followed by the Newman-Keuls multiple comparison test. Differences in hemodynamic parameters over time in each group were analyzed by two-way RM ANOVA matching by columns followed by Bonferroni post-tests. P<0.05was considered to be statistically significant.Result:1. After LAD occlusion-release, the color change of epicardium, hemodynamics and ECG changed after cardiac ischemia and reperfusion.2. Mean artery blood pressure was improved in I/R+SpostC group (P<0.05); At the90min of reperfusion, heart rate was lower in I/R+SpostC compared with I/R group (P <0.05),however, at the120min of reperfusion,heart rate had no difference between I/R+SpostC and I/R group (P>0.05) 3. At the end of120min reperfusion,±dp/dtmax was improved in I/R+SpostC group compared with I/R group (4.316±0.422X103VS.3.863±0.299X103mmHg/s,3.430±0.410×103VS.2.954±0.247×103mmHg/s, P<0.05)4. At the end of120min reperfusion, infarct size in I/R+SpostC group decreased compared with I/R group.(38.7±1.1%vs.17.1±3.8%, P<0.05)5. At the end of30min reperfusion, the number of autophagic vacuoles increased in I/R group compared with Sham group (20.0±3.4vs.1.0±1.7/100μm2, P<0.05), the number of autophagic vacuoles decreased in I/R+SpostC group compared with I/R group (11.8±1.8vs.20.0±3.4/100μm2, P<0.05)6. At the end of30min reperfusion, the expression level of LC3B-II and Beclin-1increased in I/R group compared with Sham group (P<0.05), the expression level of LC3B-II and Beclin-1decreased in I/R+SpostC group compared with I/R group (P<0.05)7. At the end of30min reperfusion, the expression level of p62increased in I/R group compared with Sham group (P<0.05), the expression level of p62decreased in I/R+SpostC group compared with I/R group (P<0.05)8. At the end of30min reperfusion, the expression level of cathepsinB increased in I/R group compared with Sham group (P<0.05), the expression level of cathepsinB increased in I/R+SpostC group compared with I/R group (P<0.05)9. chloroquine(CQ)inhibit autophagic flux,the number of autophagic vacuoles in CQ+Sham group increased compared with Sham group(p<0.05).The number of autophagic vacuoles had no difference between CQ+I/R and I/R.The number of autophagic vacuoles increased in CQ+I/R+SpostC compared with I/R+SpostC group(p<0.05).10. the expression level of LC3B-II increased in CQ+Sham group compared with Sham group (P<0.05), the expression level of LC3B-II had no difference between I/R group and CQ+I/R group (P>0.05); SpostC decreased the accumulation of LC3B-II in CQ+I/R+SpostC compared with CQ+I/R(p<0.05).11. Beclin-1was elevated in I/R group compared with Sham group(p<0.05), Beclin-1decreased in I/R+SpostC group compared with I/R group(p<0.05); However, there was no difference between CQ+Sham and Sham group, CQ+I/R and I/R group CQ+I/R+SpostC and I/R+SpostC group(p>0.05)12. p62was elevated in CQ+Sham group compared with Sham group(p<0.05).there was no difference between CQ+I/R and I/R group (p>0.05). p62increased in CQ+I/R+SpostC group compared with I/R+SpostC group(p>0.05). p62decreased in CQ+I/R+SpostC group compared with CQ+I/R group (p>0.05)13. cathepsinB was elevated in I/R group compared with Sham group(p<0.05).cathepsinB was reduced in CQ+Sham group compared with Sham group(p<0.05).cathepsinB was elevated in I/R+SpostC group compared with I/R group(p<0.05).there was no difference between CQ+I/R and I/R group,and CQ+I/R+SpostC and I/R+SpostC group(p>0.05).14. cTnI:Sham group (11.95±3.11ng/L), CQ+Sham group (15.15±2.59),I/R group (28.73±4.12),CQ+I/R group (28.03±2.79), I/R+SpostC group (19.34±2.64ng/L), CQ+I/R+SpostC group (23.42±2.26ng/L), there was no difference between Sham and CQ+Sham, I/R and CQ+I/R group; cTnI increased in CQ+I/R+SpostC compared with I/R+SpostC group (P<0.05)15. Area at risk:The percentage of AAR/LV in Sham, CQ+Sham,I/R,CQ+I/R, I/R+SpostC, CQ+I/R+SpostC group were52.9±8.0%,56.2±4.9%,54.5±3.0%,54.9±3.8%,53.6±5.4%,54.0±9.4%.There were no difference among those groups (P>0.05)16. Infarct size:The percentage of infarct size/AAR had no difference between Sham and CQ+Sham, I/R (44.2±3.1%) and CQ+I/R group (45.3±4.0%)(P>0.05); Infarct size increased in CQ+I/R+SpostC (39.5±4.0%) compared with I/R+SpostC (21.1±4.7%)(P<0.05)17. Compared with the I/R group, the myocardial fluorescence intensity of rats in the I/R+SpostC group was significantly increased (P<0.05). After CQ treatment, the myocardial fluorescence intensity of rats in the CQ+I/R+SpostC group was weak compared with the I/R+SpostC group (P<0.05). CQ had no effect on the myocardial fluorescence intensity of sham-operated rats.18. The percentage of Tunel positive nuclues in Sham, CQ+Sham,I/R,CQ+I/R, I/R+SpostC, CQ+I/R+SpostC group were:0%,4.7±2.1%,25.8±3.8%,28.0±2.7%,13.6±4.1%,23.4±3.0%; SpostC decreased the number of Tunel positive nuclues in I/R+SpostC compared with I/R group (P<0.05); The number of Tunel positive nuclues in CQ+Sham group was more than Sham group (P<0.05); There was no difference in the number of Tunel positive nuclues between I/R and CQ+I/R group (P>0.05); The number of Tunel positive nuclues in CQ+I/R+SpostC group increased compared with I/R+SpostC group (P<0.05)Conclusion:1. SpostC reduced infarct size after ischemia reperfusion injury. 2. SpostC promoted autophagic flux and less autophagic vacuoles accumulated.3. SpostC enhanced the lysosomal cathepsinB activity, improved lysosomal function and increased the fusion between autophagosome and lysosome.4. SpostC decreased the apoptosis of myocytes through less accumulation of autophagosomes; SpostC attenuated the opening of the mPTP, which may contributes to the mechanism of SpostC.In summary, our results showed that SpostC promoted autophagic flux after ischemia reperfusion in rat hearts, reduced the accumulation of autophagosomes. SpostC improved the lysosomal function to enhance autophagic activity, maintain mitochondrion homeostasis, reduced I/R injury, and decreased the myocytes apoptosis. Moreover, chloroquine was used to inhibit the clearance of autophagosomes, which reduced the cardioprotection of SpostC, and further supported the conclusion that increased clearance of autophagosomes is required by SpostC. The present result showed that autophagy may be a new therapeutic targets for cardioprotection. |
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