An Exploratory Study On The Regulation Of Oxygen Level On Cardiac Fibroblasts Involved In The Repair Of Fibrous Scars After Myocardial Infarction And The Possible Protective Effect Of Danshen Injection

Objective:Coronary heart disease is the leading cause of death worldwide.Myocardial infarction（MI）is casued by acute or chronic blockage of coronary artery due to a variety of pathophysiological insults,which results in myocardial injury and is followed by fibrous scar repair.The reparation process of MI is closely related to the biological activities of cardiac fibroblasts（CFs）.Previous studies have shown that inflammatory cells and cytokines,cell debris,and mechanical stretch pose significant regulatory effects on CFs.However,it remains unclear whether local tissue oxygen supply in the infarcted area impacts on the CFs bioactivities or not.Therefore,in order to clarify the effect of oxygen level changes on the biological activities of fibroblasts after myocardial infarction,we explored the spatial and temporal relationship between local oxygen supply and the biological activities of CFs in MI mouse model,and further verified the direct effect of oxygen level on the biological behaviors of CFs in vitro.Meanwhile,existing studies have shown that Danshen Injection（DSI）and its main effective components exhibited beneficial effect of MI by improving ventricular remodeling,microcirculation,and anti-arrhythmia,etc.To further clarify whether Salvia miltiorrhiza has a protective effect on acute myocardial infarction,we explored the possible protective role of DSI and its main effective components on acute infacted heart and their direct effect on CFs.Methods:1.In Vivo experiments:8-week-old C57BL/6J mice were randomly divided into sham plus saline group（sham+saline）and MI plus saline group（MI+saline）.The morphology and function of the hearts were evaluated at 1,3,7 and 28 days postoperatively,and immunohistochemistry and immunofluorescent staining techniques were used to evaluate the cellular and molecular changes in the firbrous scar.2.In Vitro experiments:CFs were isolated from neonatal C57BL/6J mice and passage 2 cells were exposed to normoxia（21%O₂）,hypoxia（1%O₂）,or hypoxia reoxygenation（1%→21%O₂）conditions.Cell proliferation was assessed by cell counting kit 8（CCK-8）,migration activity of CFs was observed by scratch test,and the phenotypic transformation of CFs was detected by gel contraction and immunofluorescence assay.Western blotting were used to further detect the changes of proteins related and intracellular signals.In addition,drug blocking,adenovirus overexpression,or antibody neutralization techiniques were also applied to explore the key regulatory molecules involved in hypoxia regulated CFs bioactivities switch.3.Intervention experiments of Danshen injection and its main effetive components:Mice were randomly divided in to sham+DSI group and MI+DSI group.The morphology and function of the heart were evaluated by echocardiogram,and immunohistochemistry and immunofluorescent staining techniques were used to further evaluate cellular effect of DSI and its main effective components.In vitro CFs proliferation activity was further explored.Results:1.In vivo results:Compared with sham group,histological observation in MI group showed time-dependent increment of myocardial collagen deposition in the infarct area,and matured fibrous scar was formed 28 days after surgery.Meanwhile,infarcted area showed obvious hypoxia at the early stage of MI（within 7 days）,together with elevated expression of vimentin（CFs marker）.The expression ofα-smooth muscle actin（α-SMA）,as marker of activated CFs that is the myofibroblasts,increased along with alleviation of hypoxia due to massive angiogenesis（7 days later）in the infarct area.2.In vitro experiment results:Hypoxia significantly promoted proliferation and migration of CFs,while blunted secretion and transdifferentiation of CFs.Inhibition of HIF1αby 2-Me OE2,overexpression of HIF1αby adenovirus,or transformation growth factorβ（TGFβ）antibody neutralization test demonstrated that hypoxia promoted proliferation and migration of CFs in a HIF1αdependent manner,but not HIF2αor TGF-βdependent way.Reoxygenated CFs was proved to be transdifferentiated to myofibroblasts via the activation of TGF-β/Smads signaling,which exhibited enhanced capability of collagen production.3.Intervention experiments of Danshen injection and its main effetive components:Both MI+saline and MI+DSI group showed impaired cardiac function at 7 days after operation,but there were no difference between this two groups.Meanwhile,DSI and its effective components had no toxic effect on CFs,but high dosages of these drugs showed inhibition of CFs proliferation under normoxia,but promoted CFs proliferation under hypoxia.Conclusion:1.Dynamic change of oxygen levels in infarcted area was found to be a significant modulator in CFs biological activity.On the one hand,insufficient oxygen was proved to promote proliferation and migration of CFs via HIF1α,which provided a cellular preparation for fibrous scar formation in infarcted area.On the other hand,as the local oxygen recovered due to angiogenesis after MI,TGF-β/Smads signal promoted CFs transdifferentiation to myofibroblasts,which orchestrated the fibrous scar formation afterword.Therefore,changes of oxygen level in the infarcted area is the one of the key factors for in modulating CFs bioactivities during MI scar reparation.2.Danshen injection showed no significant protective effect on cardiac function in the model of acute myocardial infarction,although high dosage of DSI and its effective components were revealed to inhibit CFs proliferation under normoxia,and promote CFs proliferation under hypoxia.