Preliminary Study Of Bortezomib In Mitigatin Of Myocardial Ischemia Reperfusion Injury

Part Ⅰ The effect of Bortezomib pretreatment on MIRI Background: Myocardial ischemia reperfusion injury(MIRI)is the major risk events of clinically AMI patients who has been treated with reperfusion related procedures.Finding target for reperfusion theray has become a significant research hotspot for improvement of long term prognosis and survival rates.Studies have reported that various proteasome inhibitors(PIs)can improve MIRI.Bortezomib(Bor)is a novel and highly selective PI,but its specific role in MIRI and its molecular mechanism are not yet clear.Combined with previous studies reporting differential effects of PIs on cells at different doses,this part of the study aims to explore the effects of Bor pretreatment on MIRI.Methods: The mouse atrial cardiomyocyte cell line HL-1 was cultured in vitro and a cellular hypoxia-reoxygenation(H/R)model was established to simulate cardiomyocyte MIRI injury.Three concentrations of Bor at 10,50 and 100 n M were added to the cell culture medium and pretreated for 24 h before giving H/R treatment for modeling.The level of lactate dehydrogenase(LDH)release in the culture supernatant of each H/R cell model was measured using the LDH cytotoxicity assay kit,and the level of LDH release was used to assess the degree of cell damage.Meanwhile,a mouse MIRI model was established by transient ischemia of the left anterior descending coronary artery(LAD)of C57BL/6 mice for 30 min,and the effects of Bor on MIRI were explored at two time points of administration,0 h before ischemia and 2 h before ischemia,and at two concentrations of 0.1 mg/kg and 0.5mg/kg,respectively.Mice were tested for cardiac function using echocardiography24 h after reperfusion,and the changes in cardiac function in different treatment groups were assessed by the changes in left ventricular ejection fraction(LVEF)and left ventricular fractional shortening(LVFS).Hematoxylin and eosin staining(HE staining)and Myeloperoxidase(MPO)staining of heart sections were used to assess the degree of histopathological damage and inflammatory response in mice in different treatment groups after 24 h of reperfusion.The changes in infarct size and cardiac injury indexes were detected and evaluated after 24 h of reperfusion using Evans Blue/TTC staining and serum troponin T assay kits.The changes of 20 S in cardiac lysates of each treatment group after Bor administration were detected using the Proteasome 20 S assay kit.Results: The results of cell experiments showed that when Bor pretreatment concentration was 10 n M and 50 n M,the cell mortality rate of both hypoxic 6h/reoxygenated 18 h(H6R18)and hypoxic 6 h/reoxygenated 24 h(H6R24)groups was significantly decreased compared with the PBS group.When Bor pretreatment concentration was 100 n M,Bor significantly reduced cell mortality in the H6R18 group,but had no significant effect on cell mortality in the H6R24 group.The results of animal experiments showed that Bor,when administered 2 h before ischemia at a concentration of 0.5 mg/kg,significantly increased mice LVEF and LVFS,improved LV systolic function,and reduced the extent of myocardial cell injury and inflammatory response after MIRI compared with the control group.Interestingly,Bor,when administered 0 h before ischemia and at a concentration of 0.5 mg/kg,significantly decreased LVEF and LVFS,damaged LV systolic function,and aggravated the degree of myocardial cell injury and inflammatory response after MIRI compared with the control group.Further,mice pretreated with Bor administered at a concentration of 0.5 mg/kg 2 h before MIRI surgery showed significant decrease in myocardial infarct size and cardiac injury marker levels and significant inhibition of cardiac 20 S proteasome activity compared to the control group.When administered at a concentration of 0.1 mg/kg,there was no significant differences in left ventricular systolic function compared with the control in Bor 2 h pretreatment and Bor 0 h pretreatment group mice.Conclusions: Bor has differential effects on MIRI at different concentrations and pretreatment time.Bor significantly alleviated MIRI when administered 2 h before MIRI and at a concentration of 0.5 mg/kg,but Bor significantly aggravated MIRI when administered 0 h before MIRI at a concentration of 0.5 mg/kg.Part Ⅱ Preliminary exploration of the mechanism of Bortezomib in alleviation of MIRIBackground: During reperfusion period after acute myocardial infarction(AMI),oxidative stress can severely damage the heart.Intervention of reperfusion-related oxidative stress injury has become an important research direction for myocardial protection after MIRI.Nuclear factor erythroid 2-related factor 2(Nrf2),an important component of the endogenous antioxidant pathway,has recently been reported to be possibly involved in the protective effects of PIs against oxidative stress-related diseases.In the previous part of the study,we found that Bor administered at a concentration of 0.5 mg/kg 2 h before MIRI surgery exerted a protective effect against MIRI,but the specific mechanism of its protective effect has not been reported yet.Combined with the previous study,this section aims to explore the molecular mechanism of Bor’s protective effect in MIRI.Methods: A mouse MIRI model was established by transient ischemia in C57BL/6mice with LAD for 30 min.0.5 mg/kg of Bor was injected intraperitoneally 2 h before MIRI modeling,and the cardiac ROS release level was detected by Dihydroethidium(DHE)probe,cardiac Glutathione(GSH)and cardiac Malonaldehyde(MDA)expression levels were detected by biochemical kits,and myocardial Superoxide dismutase [Cu-Zn](SOD1),Catalase(CAT),Nrf2 and Heme Oxygenase 1(HO-1)protein expression changes were detected by Western Blot to evaluate the degree of oxidative stress injury in different treatment groups after 3 h of reperfusion;ML-385,an inhibitor of Nrf2,was administered before MIRI modeling,and the expression changes of each index were detected after intraperitoneal injection of Bor.Results: Compared with control group,the mice in the Bor group had significantly lower ROS levels,significantly higher GSH,significantly lower MDA,and significantly higher SOD1 and CAT in the heart tissue after MIRI modeling.Nuclear Nrf2 and HO-1 protein level was significantly increased compared with the control group in Bor pretreated mice;After the administration of Nrf2 inhibitor ML-385 to Bor group mice,their nuclear Nrf2 protein level was significantly decreased,HO-1protein level was significantly decreased,GSH level was significantly decreased and MDA level was significantly increased compared with Bor administration alone group.Conclusions: Bor significantly alleviated the oxidative stress response after MIRI when administered 2 h before MIRI modeling and at a concentration of 0.5 mg/kg,and the protective effect of Bor on MIRI was achieved,at least in part,by activating the Nrf2/HO-1 antioxidant pathway.