Effect And Mechanism Of Rosamultin By Hypoxia Myocardial Injury

Object:The purpose of this experiment is to use hypoxia Rats and H9c2 myocardial hypoxia model respectively,from the level of the whole animal and in vitro cellular level to further explore the protective effect of Rosamultin on hypoxia induced myocardial cell injury and its mechanism.Provide theoretical and experimental basis for the development of new anti-anoxia medicines.Methods:The experiment is divided into two parts.Part ?:Protective effects of Rosamultin on acute hypoxia-induced myocardial injury in rats and its mechanism.1 Grouping:50 clean male rats,weighing(200± 20)g,were randomly divided into normal control group(C),hypobaric hypoxia model group(M),Rosamultin's high concentration group(H),Rosamultin's low concentrations(L),Dexamethasone positive control group(N).2 Establish hypobaric hypoxia injury model:Adjust parameter settings hypobaric chamber to simulate high altitude 7000 m environment and maintain the hypobaric hypoxia environment 18 h,in order to establish hypobaric hypoxia injury model.3 To use the chemical and biological detection kit for cardiac tissue homogenates of superoxide dismutase(SOD),malondialdehyde(MDA),glutathione(GSH),lactate dehydrogenase(LDH)content were detected.4 Observation of pathological changes of lesion myocardial tissue stained Nagar-Olsen staining.5 To use of Western-blot technique to detect cardiac tissue associated with apoptotic protein expression levels of Cyt C.Part II:Protective effects of Rosamultin on cardiac cells H9c2 hypoxic injury and its mechanism.1 Experimental groups:Experimental groups including normal control group(C),hypoxia model group(M),Rosamultin group(R),Tormentic acid Group(T),Verapamil Group(W).To determine the optimal concentration of Rosamultin,Tormentic and Verapamil by MTT assay.2 Establish hypoxia model:To use the three gas incubator established model of myocardial hypoxia,and determine the best anoxia time by MTT assay.3 To use the chemical and biological detection kits detect cell supernatant lactate dehydrogenase leakage amount(LDH),intracellular superoxide dismutase(SOD)content,malondialdehyde(MDA)content,intracellular glutathione(GSH)content.4 Structural changes in cell morphology was observed by inverted microscope.Trypan blue staining and Hoechst-PI staining for the apoptosis inhibition of Rosamultin and Tormentic acid.5 To use the flow cytometry to detect cell apoptosis.6 Western-Blot method to detect changes in the mitochondrial apoptosis pathway associated protein Bcl-2,Bax,Cyt C,Caspase 9,cleaved-Caspase 3 protein levels.After adding ERK blocker PD98059,check p-ERK 1/2 expression and apoptosis-related proteins.Results:The results is divided into two parts.Part1:Protective effects of Rosamultin on acute hypoxia-induced myocardial injury in rats and its mechanism.1 Hypobaric hypoxia model building process.Hypobaric chamber simulated altitude of 7000 m height hypoxia 18 h,after the end of hypoxia mice in each group had no deaths occurred.Hypoxia group compared with the normal activity of rat model rats slowed respiratory rate.Hypoxia model group than the normal group activity slowed,respiratory rate,the state administration of Rosamultin group has improved compared with rats in the model group.2 Biochemical Indexes Results.Hypoxia induced plasma SOD activity and GSH content decreased significantly increased MDA content and LDH leakage.Rosamultin hypoxia group compared with the model group,significantly increased the activity of SOD and GSH content and decreased MDA content and LDH leakage.3 Staining:1)HE staining:Rosamultin and Dexamethasone interventions to improve vasodilation caused by hypoxia,tissue edema,local inflammation sexual assault Run;deletion of part of the heart structure,hierarchy is unknown;myocardial fibers sloppy,disordered arrangement,cell swelling deformation.2)Nagar-0lsen staining:Myocardial hypoxia red dye area were significantly reduced by Rosamultin and Dexamethasone.Filaments aligned were more closely in Rosamultin group compared to the model group.Part ?:Protective effects of Rosamultin on cardiac cells H9c2 hypoxic injury and its mechanism.1 Establish hypoxia model.Hypoxia 1 h,2 h and 4 h after,MTT showed that the survival rate of myocardial cells hypoxia at any time increase in sustained reductions.Anoxia time was 2 h,nearly half suppress cell survival rate,there is selected as the best 2 h hypoxia.2 Determination of drug concentration.In determining hypoxia time,were given 1×10-8mol/L?1×10-12mol/L Rosamultin for drug intervention,by the MTT results showed that when given the concentration of Rosamultin 1×10mol?1×10-10mol the gradual increase in cell survival,cell viability at the highest 1×10-10 mol/L,and when to give 1×10-10 mol/L?1×10-12 mol/L concentration,cell viability decreased.In summary,so I chose 1×10-10mol/L 1 × 10-11 mol/L 1×10-12 mol/L as the optimal drug concentration of Rosamultin.3 Biochemical Indexes Results.When the cells were subjected to hypoxia,the cell supernatant lactate dehydrogenase leakage was significantly increased.Intracellular superoxide dismutase,catalase and glutathione content decreased,MDA content significantly increased.When given Rosamultin and Tormentic acid intervention,cell supernatant lactate dehydrogenase leakage was significantly decreased.Intracellular superoxide dismutase,catalase and glutathione content increased significantly,the content of MDA significantly decreased.4 Morphological observation.1)Morphological observation indicates that under an inverted microscope can be observed when myocardial damage from lack of oxygen,the cell shrinkage and round,the space between cells was significantly increased,nuclear condensation.Portion of the cell suspension off,lost the basic form normal myocardial cells.After giving Rosamultin and Tormentic acid intervention significantly improves the above-mentioned cell damage morphology.2)Trypan blue staining showed that Rosamultin and Dexamethasone can significantly reduce the hypoxic necrosis of blue cells,and cell morphology can be improved.3)Hoechst-PI staining showed that Rosamultin and Tormentic acid can significantly reduce the hypoxic necrosis of the red cells,and can improve the nuclei were segmented,fragmented and nuclear condensation and other morphological changes.5 Flow cytometry showed Rosamultin and Tormentic acid can significantly reduce myocardial cell apoptosis,increased cell survival;6 Western-Blot results showed that when cells by hypoxia,compared with normal control group,Bcl-2 protein was down-regulated,Bax,Cyt C,Caspase 9,cleaved-Caspase 3 and ERK1/2 expression regulation.After giving Rosamultin and Tormentic acid intervention reversed the above results.After addition of ERK1/2 blocker PD98059,Rosamultin and Tormentic acid of regulation over several proteins is suppressed.Conclusions:1 Rosamultin can play a protective role on myocardial hypoxia injury by improve myocardial free radical scavenging ability.2 Rosamultin can effectively protect myocardial ultrastructure in rats,reduce hypoxia-induced myocardial injury.3 Rosamultin and Tormentic acid play a protective effect on myocardial hypoxia injury by increasing myocardial antioxidant enzymes and reduce the content of lipid peroxidation.4 Rosamultin and Tormentic acid can effectively reduce the apoptosis and morphological changes of myocardial cell structure induced by hypoxia.5 Rosamultin and Tormentic acid use ERK1/2 MAPK signaling pathway regulates mitochondrial apoptosis pathway,altered expression of apoptosis-related protein,thereby inhibiting myocardial apoptosis,myocardial play a protective role.