| ObjectiveOxidative stress is a state of imbalance between oxidative and anti-oxidative system in the body.The over-production of reactive oxygen species(ROS)might attack some important macromolecules such lipids,proteins and DNA,leading to abnormal structure and function of cells and even death,which is called oxidative stress injury.Oxidative stress injury is closely related with pathophysiology of many cardiovascular diseases such as cardiac ischemia/reperfusion(I/R)injury.7,8-Dihydroxyflavone(DHF),as a natural flavonoid,selectively activates the tyrosine kinase B receptor(Trk B)to exert powerful neuroprotective and nutritional effects.Some studies also have showed that DHF might regulate cardiovascular functions.For example,DHF was found to relax the aorta of rats and have anti-hypertensive effect,DHF was also found to protect endothelial cells against cytotoxicity of hydrogen peroxide(H2O2).In this study,H9c2 cardiac cells were treated with a high concentration of H2O2 to establish oxidative stress-induced cell injury,while a rat model of myocardial I/R injury was established by ligating left anterior descending coronary artery(LAD)for 30 min followed by reperfusion for 3 h.In both cell and animal models,we explored the protective effects of DHF on oxidative stress-induced cardiac cell injury and the underlying mechanism,especially effect of anti-endoplasmic reticulum stress(ERS)of DHF.Methods and Contents1.H9c2 cardiomyocyte experiment:The cells were pretreated with DHF for 1 h and then incubated with 700 μmol/L H2O2 for 24 h.The protective effect of DHF was observed by the MTT method and TUNEL staining.Furthermore,to clarify the protective mechanism of DHF,the changes of Nrf2/ heme oxygenase-1(HO-1)pathway,Trk B/Akt pathway and ERS-related proteins were determined by Western blot analysis.2.Animal experimentsA total of 21 rats were randomly divided into three groups: Sham group,I/R group and I/R+DHF group,with 6-7 rats in each group.In the sham group,the rat’s LAD was only threaded and not ligated.In the I/R group and I/R+DHF group,LAD was ligated for 30 min followed by reperfusion for 3 h.The drug treatment was initiated 10 min before reperfusion by intraperitoneal(i.p.)injection of DHF(10 mg/kg)or solvent solution(PBS with 5% DMSO).After sacrificed,the blood was taken from the heart and the myocardial tissue below the ligature was taken for various tests,These tests were as follows:(1)TTC staining was used to confirm the I/R injury,(2)Serum myocardial enzyme assays: the serum lactate dehydrogenase(LDH)was determined by the rate method,and the serum creatine kinase isoenzyme(CK-MB)was determined by immunosuppression method,(3)the changes of Trk B/Akt and ERS-related proteins were dertermined by Western blot analysis.Results1.H2O2 treatment(100-800 μmol/L)for 24 h induced obvious oxidative stress injury in H9c2 cardiac cells,of which 700 μmol/L H2O2 decreased the cell survival rate to(0.57± 0.01,P<0.01).Pretreatment with DHF(5-20 μmol/L,1 h)showed protective effect in H2O2(700 μmol/L)-treated cells in a dose-dependent manner,among which 10 μmol/L DHF was found to have the maximal protective effect,as evidenced by the highest survival rate to(0.72±0.02,P<0.01).In the follow-up experiments,the concentrations of DHF and H2O2 were 10 μmol/L and 700 μmol/L.2.The results of TUNEL fluorescence staining of H9c2 cardiac cells showed that the number of TUNEL positive cells increased significantly after H2O2 treatment(55.7%±9.4% v.s 2.1%±0.4%,P<0.01),compared with the control group.DHF pretreatment significantly reduced the apoptotic cells(23.9%±5.4% v.s 55.7%±9.4%,P<0.05),and the apoptotic cells in DHF group was similar to that in the control group.3.The results of detecting the Nrf2/HO-1 signaling pathway showed that the levels of Nrf2 and HO-1 protein both in H2O2 group and DHF-pretreated groups were not significantly changed,compared with the control group.Nrf2/HO-1 signaling pathway is an important intracellular antioxidant stress pathway.These results suggest that Nrf2/HO-1 signaling pathway might not be involved in the protective effect of DHF against H2O2 induced H9c2 cell injury.4.The results of Trk B/Akt signaling pathway in H9c2 cardiac cells showed that compared with the control group,H2O2 significantly reduced the expression of p-Trk B protein(0.77±0.02 v.s 1.06±0.06,P<0.05)and p-Akt(0.47±0.04 v.s 0.89±0.06,P<0.05),while DHF pretreatment markedly increased p-Trk B(1.10±0.08 v.s 0.77±0.02,P<0.05)and p-Akt protein expression level(0.81±0.06 v.s 0.47±0.04,P<0.05).5.Detection of ERS-related proteins in H9c2 cardiac cells revealed that,compared with the control group,H2O2 significantly increased the protein expression of GRP78(0.98±0.17 v.s 0.42±0.07,P<0.01)and CHOP(1.20±0.12 v.s 0.79±0.07,P<0.05).Meanwhile,the expression of inactive Caspase12 protein was decreased(0.73±0.04 v.s 1.23±0.16,P<0.05).DHF pretreatment increased the expression of GRP78(0.52±0.03v.s 0.98±0.17,P<0.05)and CHOP(0.83±0.05 v.s 1.20±0.12,P<0.05).but the protein level of Caspase12 was increased(1.25±0.14 v.s 0.73±0.04,P<0.05).These results suggest that H2O2 induces ERS and ERS-related apoptosis in H9c2 cells,these changes might be counteracted by DHF.6.TTC staining of rat hearts was as follows: After ligation of the LAD for 30 min followed by reperfusion for 3 h,the heart slices were cut.The red area was normal myocardial tissue,and the white area was ischemic tissue.It suggests that the myocardial I/R injury model of rats was successfully established.7.The contents of LDH and CK-MB in rat serum were determined,and the results were as follows: compared with Sham group,LDH and CK-MB in I/R group were increased by 55%(2466±75.66 v.s 1596±145.5,P<0.01)and 97.9%(1215±154.8 v.s 614±104.1,P<0.05)respectively.Compared with the I/R group,the LDH content(1868±103.9 v.s 2466±75.66,P<0.05)and CK-MB content(1726.4±102.6 v.s 1215±154.8,P<0.05)were significantly decreased in DHF treatment group.These results suggested that DHF had a protective effect in rats with I/R injury.8.Trk B/Akt signaling pathway was detected in myocardial tissue of rats.Compared with Sham group,p-Trk B expression in the I/R group was significantly decreased(1.44±0.06 v.s 1.94±0.07,P<0.05),and p-Akt expression was also significantly decreased(0.49±0.09 v.s 1.16±0.03,P<0.05).After intraperitoneal administration of DHF(10mg/kg)before reperfusion,p-Trk B level in I/R+DHF group was significantly increased(1.93±0.21 v.s 0.85±0.09,P<0.05),and p-Akt expression was also increased(1.02±0.18 v.s 0.49±0.09,P<0.05).The results indicated that the protective effect of DHF might be related to the activation of Trk B/Akt signaling pathway.9.Detection of ERS-related proteins in cardiac tissue revealed that compared with the Sham group,I/R injury resulted in significantly increased levels of GRP78(1.14±0.24 v.s 0.33±0.05,P<0.05)and CHOP(1.59±0.31 v.s 0.57±0.09,P<0.05).The activation of e IF2α was also significantly increased(1.57±0.22 v.s 0.75±0.08,P<0.05).DHF pretreatment significantly decreased the expression levels of GRP78(0.49±0.04 v.s 1.14±0.24,P<0.05)and CHOP(0.73±0.15 v.s 1.59±0.31,P<0.05).In addition,the level of Caspase12 in myocardium of the I/R group was decreased(1.14±0.06 v.s 1.57±0.09,P<0.05),while the level of Caspase12 in DHF-treated group was increased to that of the Sham group(1.49±0.11 v.s 1.14±0.06,P<0.05).These results suggest that the protective effect of DHF against myocardial I/R injury might be related to its anti-ERS effect.ConclusionsIn conclusion,7,8-Dihydroxyflavone(DHF)exerts protective effects both in H2O2-treated H9c2 cells and in rats with cardiac I/R injury.These effects might be closelyrelated with its activating Trk B/Akt signaling pathway and its anti-ERS effect. |
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