Role Of Targeted Microbubbles Carrying IL-6 And IL-8 Antibodies In Alleviating Myocardial Ischemia/Reperfusion Injury In Rabbits

The First PartExpression of IL-6 and IL-8 in myocardialischemia/reperfusion injury in rabbitsObjective:To explore a simple and effective method for preparing rabbit model of myocardial ischemia/reperfusion injury(MIRI),establish MIRI model successfully,and detect the levels of IL-6 and IL-8 in rabbit MIRI.Methods:168 Japanese rabbits were randomly divided into 3 groups:112 in the myocardial ischemia/reperfusion group(I/R group),28 in the sham operation group(S group)and 28 in the normal group(N group).The I/R group was further divided into four subgroups according to the reperfusion time,which were reperfusion 30 min,reperfusion 60 min,reperfusion 120 min,and reperfusion 180 min.In I/R group,the left anterior descending coronary artery was blocked for 30 min and then the occlusion was relieved and reperfused.The same part of the S group was only blocked by threading.Electrocardiogram and echocardiography were performed on each group before and after surgery.ELISA was used to detect the levels of IL-6 and IL-8 in serum.Four rabbits were sacrificed from each group.Myocardial tissue was taken for HE staining and scanning electron microscopy.Results:1.ElectrocardiogramThe ST segment was elevated upwards at 30 min of ischemia.There was no significant decrease in ST segment within 30 min after reperfusion.ST segment decreased slightly after 45 minutes of reperfusion.ST segment decreased significantly to equipotential line at 60 min after reperfusion.The ST segment was reduced to the equipotential line at 120 min reperfusion and 180 min of reperfusion,and the electrocardiogram was almost normal.2.EchocardiographyAbnormal wall motion was observed in the anterior wall of left ventricle,and the rate of wall thickening was significantly reduced at 30 min after ischemia and 30 min of reperfusion.After 60 min of reperfusion,the wall motion and wall thickening rate in the ischemic area returned to normal.3.ELISAThe levels of IL-6 and IL-8 in serum of I/R groups were significantly higher than those in S group and N group,and the difference was statistically significant(P<0.05).With the prolongation of reperfusion time,the levels of IL-6 and IL-8 in serum of I/R groups increased gradually.The level of IL-6 peaked at 180 min of reperfusion,and the difference was statistically significant(all P=0.000).With the prolongation of reperfusion time,the expression level of IL-8 in serum increased gradually,reaching the peak at 120 min of reperfusion and continuing to 180 min of reperfusion.The difference was statistically significant(P=0.000).The level of IL-8 peaked at 120 min of reperfusion and 180 min of reperfusion,with statistical significance(P=0.000).There was no significant difference between 120 min reperfusion and 180 min reperfusion(P=0.336).There were no significant difference between S group and the N group(all P>0.05).4.Histopathological examinationHE staining and SEM showed myocardial injury in I/R group.With the prolongation of reperfusion time,the degree of injury increased gradually.The most severe injury occurred at 120 min and 180 min of reperfusion,and the degree was similar.Conclusions:The MIRI model was successfully established blocking the left anterior descending coronary artery for 30 minutes and releasing the blockade.Ischemia-reperfusion can lead to myocardial damage and elevated serum levels of IL-6 and IL-8 in rabbits.This study lays a foundation for the next step of MIRI treatment research.The second partEvaluation of myocardial ischemia/reperfusion injury in rabbits by targeted microbubbles with IL-6 and IL-8 antibodiesObjective:To prepare targeted microbubbles carrying IL-6 and IL-8 monoclonal antibodies and double antibodies and evaluate myocardial ischemia/reperfusion injury of rabbits by targeted myocardial contrast echocardiography(MCE).Methods:Following the remaining 144 animal models in the first part,the animal models were randomly divided into four subgroups:the bare microbubble group(MBc),the microbubble group with IL-6 monoclonal antibody(MBIL-6),the microbubble group with IL-8 monoclonal antibody(MBIL-8)and the double antibody microbubble group with IL-6 and IL-8(MBd),36 in each group.Flow cytometry was used to detect the binding rate of different doses of antibodies to microbubbles,and the optimal compatibility ratio was obtained.According to this compatibility ratio,biotin-avidin bridging method was used to conjugate biotinylated IL-6 and IL-8 monoclonal antibodies to Targestar-SA microbubbles respectively and jointly,to construct targeted microbubbles with monoclonal antibodies and double antibodies.MCE was performed by intravenous injection of the above-mentioned ultrasound contrast agent into the ear margin of rabbits.Qlab software was used to analyze the video intensity(VI)of anterior myocardium.The results were correlated with the serum levels of IL-6 and IL-8 detected by ELISA in the first part.Results:1.Detection of binding rate by flow cytometryThe optimal binding efficiency of antibody to Targestar-SA microbubbles was over 80%of three groups of targeted ultrasound contrast agent.2.Preparation of targeted ultrasound contrast agentTargeted microbubbles with monoclonal antibodies and double antibodies were successfully constructed by biotin-avidin bridging method.The size,morphology and properties of the four groups of microbubbles were basically similar.The microbubbles were uniform in size and uniform in distribution.3.Targeting MCEThere were no significant differences in ? of axterior wall between I/R groups and S group and N group of MBc microbubbles(P>0.05).The ? of anterior wall in I/R groups was significantly higher than that in S group and N group of three targeted microbubbles.With the prolongation of reperfusion time,? increased gradually.MBIL-6 microbubbles and MBd microbubbles reached the peak at 180 min of reperfusion,and MBIL-8 mierobubbles reached the peak at 120 min and 180 min of reperfusion.At the same reperfusion time,the ? of the four microbubbles ranged from high to low:MBd>MBIL-8>MBIL-6>MBc.4.Correlation analysisThere were strong correlations between IL-6 and IL-8 levels detected by ELISA and ? in anterior wall of three targeted microbubbles in I/R groups(all r>0.7 and P<0.05).Conclusions:Targeted microbubbles carrying IL-6 and IL-8 monoclonal antibody and double IL-6 and IL-8 monoclonal antibodies can be successfully prepared by using biotin-avidin bridging method.Targeted microbubbles are more sensitive and targeted to MIRI imaging.The dual-antibody targeted microbubbles has better imaging characteristics than the single-antibody targeted microbubbles.Targeted MCE can indirectly reflect the levels of IL-6 and IL-8 in MIRI by quantitative analysis the ? of anterior wall,achieving non-invasive and quantitative evaluation of inflammatory response in MIRI.The third partRole of targeted microbubbles carrying IL-6 and IL-8 antibodies in alleviating myocardial ischemia/reperfusion injury in rabbitsObjective:To investigate the role of ultrasound targeted microbubble destruction mediated by monoclonal antibodies and double antibodies carrying IL-6 and IL-8 in alleviating myocardial ischemia/reperfusion injury in rabbits.Methods:Following the 144 animal models examined by MCE in the second part.Rabbits in each group were injected with naked microbubbles(MBc),monoclonal antibody microbubbles carrying IL-6(MBIL-6),monoclonal antibody microbubbles earrying IL-8(MBIL-8),and double-antibody microbubbles carrying IL-6 and IL-8(MBd)via ear vein respectively.The myocardial imaging were observed and stored images.After stable attachment of microbubbles,the low-power focused ultrasound experimental device was used to perform ultrasoruc irradiation on the chest wall of rabbits.After irradiation,blood was collected through the middle ear artery of rabbits.The levels of IL-6 and IL-8 in serum were detected by ELISA.The animals were sacrificed 3 days later and myocardial tissue was taken for histopathological examination.Results:1.After irradiation with MBc microbubbles,there were no significant change in? and the contents of IL-6 and IL-8 in serum of all groups,and the difference were not statistically significant(all P>0.05).2.After irradiation with MBIL-6 microbubbles,? and the levels of IL-6 in serum of I/R groups were significantly lower than those before irradiation(all P<0.05).There were no significant changes in the levels of IL-8,and the difference were not statistically significant(all P>0.05).3.After irradiation with MBIL-8 microbubbles,? and the levels of IL-8 in serum of I/R groups were significantly lower than those before irradiation(all P<0.05).There were no significant changes in the levels of IL-6,and the difference were not statistically significant(all P>0.05).4.After irradiation with MBd microbubbles,? and the levels of IL-6 and IL-8 in serum of I/R groups were significantly lower than those before irradiation(all P<0.05),and the differences were statistically significant(all P<0.05).5.Compared the difference of VI before and after irradiation of four microbubbles,MBd>MBIL-6 and MBIL-8>MBc,the differences were statistically significant(all P<0.05).There were no significant difference between MBIL-6 microbubbles and MBIL-8 microbubbles in each group(all P>0.05).6.The change rate of video intensity and the neutralization rate of IL-6 and IL-8 of three targeted ultrasound microbubbles of I/R groups before and after irradiation were the highest at 30 min of reperfusion.With the prolongation of reperfusion time,the change rate of video intensity and the neutralization rate of inflammatory factors decreased gradually.Conclusions:By using UTMD technology to mediate targeted microbubbles carrying IL-6 and IL-8 monoclonal antibodies and double antibodies,IL-6 and IL-8 monoclonal antibodies can be released in the ischemia-reperfusion injury area to neutralize inflammatory factors in the target area.By antigen-antibody neutralization,MIRI inflammation can be effectively alleviated.The intervention effect of targeted microbubbles with dual-antibody is better than that with single-antibody.The earlier the irradiation intervention,the better the effect of reducing inflammation.These results provide a more powerful experimental basis for clinical search for safe and efficient methods to reduce MIRI,and is expected to become a new method for clinical treatment of MIRI.