A Preliminary Mechanism Study Of PPolyHb On Myocardial Cell Ischemia-reperfusion Injury

Acute ischemic cardiovascular disease is caused by the formation of thrombus or embolism within blood vessels,which can cause acute organ circulation disorder and damage of tissues.Symptoms of clinical manifestations are acute heart failure,varying degrees of angina and myocardial infarction and other symptoms.Major clinic characters include high incidence rate and death rate.Only a small part of acute patients can be diagnosed and receive revascularization within three hours of onset.More than 95%of patients have lost the best timing of recanalization due to their disease/condition.In addition,sudden increase of blood and oxygen can also cause hypoxia tissue reperfusion injury.Therefore,supplying oxygen to obstructed tissues and organs is the core of the treatment of acute ischemic cardiovascular and cerebrovascular diseases.It can protect the hypoxic tissues and organs,and prolong the optimal treatment time as well.Meanwhile,the supply of oxygen is controlled to avoid reperfusion injury caused by oxygen,inflammatory factors and free radicals.Hemoglobin-based oxygen carriers(HBOCs)is a type of macro molecules that was crosslinked or intermolecularly polymerized with a cross-linking agent or a polymeric agent on the basis of free hemoglobin molecules.It has a series of advantages such as easy transportation,stable storage,insusceptible virus microbial infection and good prospect in development and application.pPolyHb is a kind of hemoglobin oxygen carrier,which uses porcine hemoglobin as raw material and cross-linked with glutaraldehyde.Its diameter is only about 20nm,much smaller than red blood cells(7-8?m).In theory,it can pass through endovascular narrow gap and supply oxygen for the ischemic areas and tissues in time.pPolyHb contains heme groups,heme has been reported to induce the synthesis of hemoglobin oxygenase HO-1.HO-1 has antioxidant and anti-inflammatory ability.In addition,ischemia/reperfusion could produce a large number of ROS lead to mitochondrial dysfunction,and further promote apoptosis.Therefore,the cells must remove the damaged mitochondria to prevent the accumulation of ROS.Mitochondrial autophagy can effectively remove damaged mitochondria and excess ROS to ensure the stability of intracellular mitochondria.In this study,the cell hypoxia-reoxygenation model was established.The Cell viability were detected by MTT assay.LDH and CK activity in supernatant fluid were detected.Two groups of pPolyHb(10?M and 20?M)were chosen according to these results.The effects of pPolyHb on ischemia-reperfusion injury in H9C2 cells were investigated.The levels of TNF-?,EL-1?,anti-inflammatory cytokines IL-10 were detected by immunohistochemistry.The expression of Bax,Bcl-2 and CytC and the induction of pPolyHb on HO-1 were detected by western blot.In order to reveal the mechanism of pPolyHb in mitochondrial autophagy induced by hypoxia/reoxygenation injury in H9C2 cells.The expression of mitochondrial autonomic protein Parkin and Pinkl together with the apoptosis of mitochondrial autophagy were detected.The results showed that the pPolyHb can effectively improve the viability of the cells.The activity of LDH and CK in cell supernatant of pPolyHb cells were significantly lower than that in the ischemia-reperfusion injury group,and the apoptotic rate was also decreased.The apoptosis-related proteins Bax and Bcl-2 and the expression level of CytC indicated that pPolyHb could increase the expression of Bcl-2 and inhibit the the expression of CytC and Bax to reduce the apoptosis of cardiomyocytes.Meanwhile,pPolyHb can reduce the inflammatory factors TNF-?,IL-1?,and increase the expression of IL-10 playing an anti-inflammatory effect,and pPolyHb can induce HO-1 expression in the regulation of ischemia-reperfusion.In addition,the results showed that pPolyHb could effectively induce the expression of mitochondrial autophagy-related protein Pinkl and Parkin,and the protein expression was decreased gradually with the time of reoxygenation.When the mitochondrial inhibitor was used to inhibit mitochondrial autophagy,the content of anti-apoptotic protein Bcl-2 was decreased and the expression of pro-apoptotic protein Bax and CytC increased.It was confirmed that pPolyHb reoxygenation can modulate apoptosis by activating mitochondrial autophagy,which plays a protective effect on ischemia-reperfusion injury.