| Background:Acute myocardial infarction (AMI) has become one of the high morbidity and mortality of cardiovascular disease. At present, the most common treatment for AMI is recanalization of occluded vessels to of save the risk region of heart. However, the reperfusion therapy can cause the death of cardiomyocytes which is called ischemia reperfusion injury (IRI). Therefore, how to prevent and treat the reperfusion injury has become one of important tasks in the treatment of patients with acute myocardial infarction myocardial ischemia.High mobility group protein 1 (HMGB1), interleukin (IL)-17A and autophagy are involved in the process of myocardial ischemia reperfusion injury. The study found that there is a closely relationship between HMGB1, IL-17A and autophagy. The increasing expression of HMGB1 and IL-17A in myocardial ischemia reperfusion may cause the excessive activation of autophagy, resulting in the aggravation of myocardial cell injury. Therefore, the research mainly focuses on the autophagy caused by HMGB1-IL-17A axis and its role in myocardial ischemia reperfusion injury in rats and its possible mechanism, this would be helpful to clarify the mechanism of myocardial ischemia reperfusion injury, and will give a new sight to prevent the myocardial ischemia reperfusion injury.Methods:In vitro:Neonatal rat cardiomyocytes were incubated in DMEM F12 for 1 h anoxia followed by 4 h reoxygenation, then were treated with exogenous HMGB1, IL-17A factor and its monoclonal antibody, myocardial damage indexes of LDH, CK and oxidative stress parameters SOD, MDA were measured, apoptosis rate of cardiomyocytes was assessed by flow cytometry. The expression of HMGB1, IL-17A, autophagy associated protein LC3 and Beclin-1 were detected by Western Blot Construction of shuttle plasmid carrying HMGB1 and IL-17A gene, homologous recombination in HEK293 cells after adenovirus packaging the formation of Ad-HMGB1 and Ad-IL-17A, amplified, purified virus titer reached liquid in vitro experimental requirements. In vivo:90 SD rats were randomly divided into 6 groups receiving the following treatments:Group 1:Sham operation (SO) group (n=15), rats were subjected to surgical manipulation without the induction of myocardial ischemia; Group 2:Ischemia and reperfusion (I/R) group (n=15), rats were subjected to the left anterior descending coronary artery occlusion for 30 min followed by reperfusion for 4 h; Group 3:I/R+Anti-HMGB1 group (n=15), rats were injected by neutralizing antibody of HMGB1 (200?L) 30min before subjected to I/R model; Group 4: I/R+Anti-IL-17A group (n=15), rats were injected by neutralizing antibody of IL-17A (200?L) 30min before subjected to I/R model; Group 5:I/R+Ad-HMGB1 group (n=15), rats were injected by Ad-HMGB1 (100?L,1×1011 PFU/mL) 3 days before subjected to I/R model; Group 6:I/R+Ad-HMGB1 group (n=15), rats were injected by Ad-IL-17A (100?L,1×1011 PFU/mL) 3 days before subjected to I/R model. Transfection efficiency was evaluated by fluorecent microscope 3 days after adenovirus injection, and 4 h after reperfusion, myocardial infarct size were defined by TTC and Evans' Blue; apoptosis in heart was detected by TUNEL staining; the concentration of serum enzyme LDH, CK content in myocardial injury were measured; the SOD activity, MDA concentration, inflammatory factor of TNF-alpha, IL-6 contained in heart tissue were measured; The expression of HMGB1, LC3, Beclin-1 and apoptosis related protein of Bax, Bcl-2 were detected by Western Blot, the expression of IL-17A was detected by Western Blot and Real-Time PCR.Results:1. In vitro experiments:Compared with the control group, the concentration of LDH, CK and MDA were significantly increased and the SOD activity was decreased in H/R group, Western Blot analysis revealed that the expression of HMGB1, IL-17A, Beclin-1 and LC3-?/LC3-? rate were significantly increased (all P<0.05); when compared with H/R group, the concentration of LDH, CK and MDA were decreased and the SOD activity was increased in H/R+Anti-HMGB1 and H/R+Anti-IL-17A group and the expression of IL-17A, Beclin-1, LC3-?/LC3-? rate were decreased (all P<0.05); On the contrary, the concentration of LDK, CK and MDA were increased and the SOD activity was decreased in H/R+HMGB1 and H/R+IL-17A group and the expression of IL-17A, Beclin-1, LC3-?/LC3-? rate were increased (all P<0.05).2. Recombiant Ad-HMGB1 and Ad-IL-17A were constructed successfully with the titer of 1×1011 pfu/ml.3. In vitro experiments:Compared with the SO group, the apoptisis rate was higher, the serum concentration of LDH and CK were significantly increased in I/R group, meanwhile, the SOD activity was decreased and the concentration of MDA, TNF-alpha, IL-6 were increased in heart tissue, and the expression of HMGB1, IL-17A, Beclin-1, LC3-?/LC3-? and Bax/Bcl-2 rate were were significantly increased (all P<0.05). After comparison of I/R group and Anti-HMGB1 group and Anti-IL-17A group, we found that the infarct size and apoptisis rate were decreased, the serum concentration of LDH and CK were decreased, meanwhile, the SOD activity was increased and the concentration of MDA, TNF-alpha, IL-6 were decreased in heart tissue, and the expression of HMGB1, IL-17A, Beclin-1, LC3-?/LC3-? and Bax/Bcl-2 rate were also decreased (all P<0.05). There was a opposite result when after comparison of I/R group and Ad-HMGB1 group and Ad-IL-17A group, the infarct size and apoptisis rate were increased, the serum concentration of LDH and CK were increased, meanwhile, the SOD activity was decreased and the concentration of MDA, TNF-alpha, IL-6 were increased in heart tissue, and the expression of HMGB1, IL-17A, Beclin-1, LC3-II /LC3-1 and Bax/Bcl-2 rate were also increased (all P<0.05).Conclusions:1. In vitro experiments we confirmed that HMGB1 and IL-17A can induce autophagy in myocardial hypoxia reoxygenation. HMGB1 could up regulate the expression level of IL-17A, then exacerbate of H/R on myocardial cells injury2. Construction of shuttle plasmid carrying HMGB1 and IL-17A gene, homologous recombination in HEK293 cells after adenovirus packaging the formation of Ad-HMGBl and Ad-IL-17A, amplified, purified virus titer up to 1×1011 PFU/mL.3. In vitro experiments we further confirmed that HMGB1 could upregulate the expression level of IL-17A in transcription and post transcription, thereby promoting myocardial autophagy and aggravate myocardial ischemia reperfusion injury, and this may be related to increased oxidative stress, inflammatory reaction and promote cell apoptosis. |
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