Study On Inhibition Of HMGB1 Regulate Autophagy Protect Against Myocardial Ischemia/reperfusion Injury In Diabetic Mice

Significance and Objective: People with diabetes have heavy intravascular plaques load and poor myocardial tolerance,which seriously effect coronary blood flow and myocardial resuscitation ability after ischemia/reperfusion(I/R),resulting in a high incidence of postoperative restenosis and complications.Autophagic dysfunction in diabetes status directly expedite the death process of injuried cardiac myocytes.Therefore,Regulation of autophagy to profitable transformation is the key to alleviate cardiac I/R injury in diabetes.High mobility group box 1(HMGB1)can regulate autophagy and participate in cardiac I/R injury.Inhibition of HMGB1-mediated inflammatory response can mitigate diabetic cardiac I/R damage in a short-term.However,no relevant studies have been reported on whether the potential association between inhibition of HMGB1 and autophagy in diabetic myocardial I/R injury.Therefore,to probe into the protection approach in diabetic cardiac I/R injury,seeking efficacious interference target,and for the sake of a preliminary theoretical basis for solving the trouble of cardiac I/R injury in glycuresis.This study establish cardiac I/R model in diabetic mice(DM),the 24-hour is used as the reperfusion endpoint to preliminarily evaluate the protective effect of inhibiting HMGB1,the expression of autophagic related molecules are analyzed.Methods: Male C57BL/Ks J mice and db/db mice(6-8 weeks old)were selected and compartmentalized into control(CON)group and I/R group,separately.I/R model was pre-builted by ischemia for 30 minutes/reperfusion for 24 hours.The changes of cardiac tissue modality were viewed by hematoxylin and eosin(H&E)dying;the degree of cardiac infarction was analysed by Evans blue/TTC double dying and the indexes of cardiac enzymology;the m RNA expression levels of HMGB1,Beclin1,microtubule-associated proteins 1 light chain 3(LC3),and p62 were detected by RT-q PCR;the protein expression levels of HMGB1,Beclin1,p62,and lysosomal-associated membrane protein 2(LAMP2)and the ratio of LC3II/I were determined by Western blot,so as to explore the vulnerability of reperfusion myocardium in diabetic status and its latent molecular mechanism.db/db mice were further compartmentalized into anti-HMGB1antibody(H-Ig)group and anti-Ig G antibody(Ig G)group.Anti-HMGB1 antibody or anti-Ig G antibody were injected through caudal vein after cardiac I/R for 15 minutes,and the above experimental techniques were implemented at the end of reperfusion.The protein of cardiac HMGB1 in the DM subgroups were located by immunohistochemistical analysis,and the proteins of cardiac LC3 and p62 in the DM subgroups were located by immunofluorescence assay,so as to verify the protective action of inhibiting HMGB1 on diabetic cardiac I/R injury and its effect on cardiac autophagy.Results: 1.In comparison to the I/R group,the cardiac lesion in the I/R(DM)group was notably worse.2.In comparison to the I/R group,the m RNA expression levels of HMGB1,LC3,and p62 in the I/R(DM)group were considerably up-regulated.The protein expression levels of HMGB1,Beclin1,LAMP2,and the ratio of LC3II/I in the I/R(DM)group were remarkably higher than those in the I/R group,while the protein expression level of p62 was substantially lessened.3.Immunohistochemical analysis results showed that a large number of HMGB1 proteins existed in the myocardium of Ig G(DM)group compared with the CON(DM)group,and mainly in cytoplasm and extracellular;in contrast to the Ig G(DM)group,HMGB1 proteins was notably diminished in the myocardium of H-Ig(DM)group.4.In comparison to the Ig G(DM)group,the cardiac lesion was substantially ameliorated in the H-Ig group.5.The m RNA expression levels of Beclin1,LC3,and p62 in the H-Ig(DM)group were remarkably down-regulated in comparison to those in the Ig G(DM)group;the protein expression levels of Beclin1,LAMP2,and the ratio of LC3II/I in the H-Ig(DM)group were considerably lower than those in the Ig G(DM)group,while the protein expression level of p62 was notably increased.Immunofluorescence dying results showed that in comparison to the CON(DM)group,LC3 expression was enhanced and p62 expression was diminished in the Ig G(DM)group;in contrast to the Ig G(DM)group,the expression of LC3 and p62 was inversed in the H-Ig(DM)group.Conclusion: 1.After cardiac I/R in diabetes status,the expression of HMGB1 was enhanced,autophagy was abnormally sensitized,and cardiac reperfusion injury was substantially aggravated;2.Inhibition of HMGB1 activity may usefully protect the cardiac damaged via supressing the over-activation of autophagy after cardiac I/R in diabetes status.