| Objectives To observe the relationship between the protection of Sulforaphane(SFN)on myocardial cells,H9C2 cold hypoxia/reoxygenation injury and dose and treatment time also the related mechanism.Methods 1 Establishment and grouping of experimental models: First part is the discussion on SFN's dose: The experiment is divided into Sham group,H9C2 myocardial cell's routine culture and cold H/R group.After routine cell culture medium is replaced with D-hanks solution,the H9C2 myocardials are placed in the anoxic box.Then add 95%N2 and 5% CO2 for thirty minutes.After ten hours' culture in 4? environment,change the D-hanks solution into a conventional cell culture medium and the culture in the cell culture box for four hours.SFN5?M pretreatment group,SFN10?M pretreatment group,SFN25?M pretreatment group and SFN100 u M pretreatment group.Twelve hours before they hypoxia,put the cells into the SFN medium with a final concentration of 5?M,10?M,25?M and 100?M.And then take the same steps of cold H/R group.Second part is the discussion on the pretreatment time of SFN.The experiment is divided into cold H/R group,SFN is pretreated for twenty-four hours group,twelve hours group,eight hours group,four hours group and half hour group.Twenty-four hours,twelve hours,eight hours,four hours and half hour before they hypoxia,put the cells into the SFN medium with a final concentration of 10?M.And then take the same steps of cold H/R group.2 Detect the index: The changes of H9C2 myocardial cells' morphology and the degree od apoptosis in inverted high power microscope is observed.The viability of H9C2 cells in each group through the CCK8 method is detected,and the expression of NQO1,HO-1,Bcl-2 and Bax protein by the means of western blot is detected.Results The survival of cells are increased in different dose of SFN pretreatment group(5?M,10?M,25?M,100?M group)compared with the cold H/R group.The cell survival rate is improved in different degree(70.49±2.77,76.16±1.38,67.70±3.34,60.08±2.24 vs54.95±1.54,P<0.05).The survival of cells is increased in different time of pretreatment group(24h,12 h,8h,4h,0.5h group)compared with the cold H/R group.The cell survival rate is improved in different degree(70.12±3.24,75.78±3.23,68.75±4.15,64.45±3.26,60.21±2.24 vs 56.04±2.23,P<0.01).Expression of HO-1 and NQO1 protein of myocardial cells in each group.Sham group: HO-1 and NQO1 protein expressions are weak.Compared with Sham group,the levels of HO-1 and NQO1 protein are significantly increased in cold H/R group(P<0.05).Compared with the cold H/R group,the HO-1 and NQO1 protein levels in the SFN pretreatment group are significantly increased at different time in the pretreatment group with different doses of SFN(P<0.05).Among them,the most obvious is in SFN10?M group and SFN12 h group.Expressions of Bcl-2and Bax protein in myocardial cells of each group: Sham group: Bcl-2 and Bax protein expressions are weak.Compared with Sham group,the levels of Bcl-2 and Bax protein are significantly increased in cold H/R group(P<0.05).Compared with the cold H/R group,there is no difference in the level of Bcl-2 protein in the SFN100?M group(P<0.05),but in other groups it is significantly increased(P<0.05)and the Bax protein level decreased significantly(P<0.05).Among them,the most obvious is also in this case.Conclusions 1 SFN can increase the expression of enzyme II HO-1 and NQO1 to protect the H9C2 myocardial cells' cold H/R injury.2 SFN can protect the H9C2 myocardial cells' H/R injury by the increase of Bcl-2 protein,decrease of Bax protein and increase of Bcl-2/Bax ratio.3.The optimal dose of SFN for protection is 10?M.The best pretreatment time of SFN is 12 hours. |
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