| Objective:To explore how Ang-(1-7)play a role of protection on the H/R injurying myocardial cell and whether the role regulate the endoplasmic reticulum stress by means of Mas receptor in Cellular level.Methods:1.H9C2 cell culture,subculture and cryopreservation;Establish H/R model;2.H9C2 were treated medium with Ang-(1-7)or/and A-779.Experient was divided into five groups:(1)the Control group: no intervention;(2)H/R group: cells were exposed to hypoxia 3 hours after reoxygenation 6 hours;(3)Ang-(1-7)+H/R group: cells were exposed to 1000nmol/L Ang-(1-7)30 minutes before hypoxia 3 hours after reoxygenation 6 hours;(4)Ang-(1-7)+A-779+H/R group: cells were exposed to1000nmol/L A-779 1 hour before ahypoxia 3 hours,then cells were exposed to1000nmol/L Ang-(1-7)30 minutes before hypoxia 3 hours after reoxygenation 6 hours;(5)A-779+H/R group: cells were exposed to 1000nmol/L A-779 1 hour before ahypoxia3 hours.3.Detecting myocardial cell apoptosis,myocardial enzymes cell on supernatant and myocardial cells endoplasmic reticulum stress related indicators,multi-level detections research protective effect of Ang-(1-7)on myocardial cell ischemia reperfusion injury and its mechanism of action.Results:1.CK-MB:The groups CK-MB: 429.11±260.55,1145.87±58.50,601.94±153.38,1081.88±184.39,1085.65±134.28.Compared with the control group,the H/R group increases(P<0.01),Ang-(1-7)+H/R group increased(P<0.05),Ang-(1-7)+A-779+H/R group increases(P<0.01),A-779+H/R group increased(P<0.01);Compared to the H/R group,Ang-(1-7)+H/R group decreased(P<0.05),Ang-(1-7)+A-779+H/R group was no statistically significant difference(P>0.05),A-779+H/R was no statistically significant difference(P>0.05).2.The caspase 3 spectrophotometric method to detect apoptosis:Relatively OD of 5 groups : 2.07±0.18,2.66±0.14,2.13±0.09,2.59±0.29,2.59±0.15.Compared with the control group,the H/R group increased(P<0.01),Ang-(1-7)+H/R group increased(P<0.05),Ang-(1-7)+A-779+H/R increases(P<0.01),A-779+H/R group increases(P<0.01);Compared to H/R group,Ang-(1-7)+H/R group decreased(P<0.05),Ang-(1-7)+A-779+H/R group,there was no statistically significant difference(P>0.05),A-779+H/R group,there was no statistically significant difference(P>0.05).3.The Western blot detecting apoptosis related proteins:3.1 Bcl-2 protein levels of groups(×10-2): 6.16±0.74,2.21±0.60,3.59±0.38,2.38±0.72,2.41±0.70.Compared with the control group,the H/R group decreases(P<0.01),Ang-(1-7)+H/R group decreased(P<0.01),Ang-(1-7)+A-779+H/R group decreases(P<0.01),A-779+H/R group decreases(P<0.01);Compared with H/R group,Ang-(1-7)+H/R group increased(P<0.05),Ang-(1-7)+A-779+H/R group had difference of no statistical significance(P>0.05),the A-779+H/R group difference had no statistical significance(P>0.05).3.2 Bim protein levels of groups(×10-2): 7.47±0.54,13.02±0.85,10.28±0.74,13.85±2.4,12.58±1.96.Compared with the control group,the expression of H/R group increases(P<0.01),Ang-(1-7)+H/R group increased(P<0.05),Ang-(1-7)+A-779+ H/R group increased(P< 0.01),the expression of A-779+H/R group increases(P<0.01);Compared with H/R group,Ang-(1-7)+H/R group reduced(P<0.05),the Ang-(1-7)+A-779+H/R group expressed difference of no statistical significance(P>0.05),the A-779+H/R group differences had no statistical significance(P> 0.05).4.The Western blot detecting ERS related proteins:4.1 GRP78 protein levels of groups(×10-2): 197.57±15.27,258.96±30.29,194.13±18.75,268.89±24.92,268.13±53.00.Compared with the control group,the expression of H/R group increased(P<0.01),Ang-(1-7)+H/R group quantity differences had no statistical significance(P>0.05),Ang-(1-7)+A-779+H/R group increased(P<0.01),the expression of A-779+H/R group increased(P<0.01);Compared with H/R group,Ang-(1-7)+H/R group reduced(P<0.05),Ang-(1-7)+A-779+H/R group expressed difference of no statistical significance(P>0.05),the expression of A-779+H/R group quantity differences had no statistical significance(P>0.05).4.2 CHOP protein levels of groups(×10-2): 23.52±5.68,60.32±11.34,42.57±7.62,63.05±6.65,65.46±4.41.Compared with the control group,the H/R group increased(P<0.01),Ang-(1-7)+H/R group increased(P<0.05),Ang-(1-7)+A-779+H/R group increased(P<0.01),the A-779 H/R group increased(P< 0.01);Compared with H/R group,Ang-(1-7)+H/R group reduced(P<0.05),Ang-(1-7)+A-779+H/R group expressed difference of no statistical significance(P>0.05),the expression of A-779+H/R group difference had no statistical significance(P>0.05).4.3 Caspase 12 protein levels of groups(×10-2): 46.32±10.12,95.87±13.94,68.71±10.89,84.19±12.24,100.28±14.58.Compared with the control group,the expression of H/R group increased(P<0.01),Ang-(1-7)+H/R group expression increased(P<0.05),Ang-(1-7)+A-779+H/R group increased(P<0.01),the A-779+H/R group increased(P<0.01);Compared with H/R group,Ang-(1-7)+H/R group reduced(P< 0.05),Ang-(1-7)+A-779+H/R group expression difference of no statistical significance(P>0.05),the A-779+H/R group difference had no statistical significance(P>0.05).4.4 JNK protein levels of groups(×10-2): 28.02±6.74,72.07±9.36,48.31±10.05,74.03±18.30,68.74±9.26.Compared with the control group,the H/R group increased(P<0.01),Ang-(1-7)+H/R group increased(P<0.05),Ang-(1-7)+A-779+H/R group increased(P<0.01),the A-779+H/R group increased(P<0.01);Compared with H/R group,Ang-(1-7)+H/R group reduced(P<0.05),Ang-(1-7)+A-779+H/R group expressiondifference of no statistical significance(P>0.05),the A-779+H/R group difference of no statistical significance(P>0.05).Conclusion:1.The MIRI is protected by Ang-(1-7)though inhibiting the endoplasmic reticulum stress-apoptotic pathways.2.Ang-(1-7)play a role of the above by its specific receptors--Mas receptors. |
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