The Protective Effects Of Ang-(1-7) On Myocardial H/R Injury Through "PI3K/Akt-SERVA-Endoplasmic Reticulum Stress-Apoptosis" Pathway

Objective:To explore whether the protective effect of Ang-（1-7）on hypoxia/reoxygenation injury of H9C2 cardiomyocytes is through inhibition of endoplasmic reticulum stress pathway and further explore whether it acts through Akt signaling pathway.Methods:1.H9C2 cardiomyocyte culture,subculture and seeded 6-well plates;establish of hypoxia/reoxygenation（H/R）model;2.When the cell density in the 6-well plate is more than 80%,the Ang-（1-7）is treated according to the experimental protocol:（1）control:without any intervention;（2）H/R group:The cells were hypoxic for 3 hours and then reoxygenated for 6 hours;（3）Ang-（1-7）+H/R group:Ang-（1-7）was given for 30 minutes before hypoxia and reoxygenation,followed by hypoxia for 3 hours and reoxygenation for 6 hours.The apoptosis of each group was detected:caspase3 activity was detected by Spectrophotometry,and the expression levels of apoptotic proteins（BIM,p-Bcl-2）were detected by Western blotting.3.When the cell density in the 6-well plate is more than 80%,the Ang-（1-7）and/or Akt-siRNA are treated according to the experimental protocol:（1）control:without any intervention;（2）H/R group:The cells were hypoxic for 3 hours and then reoxygenated for 6 hours;（3）Ang-（1-7）+H/R group:Ang-（1-7）was given for 30 minutes before hypoxia and reoxygenation,followed by hypoxia for 3 hours and reoxygenation for 6 hours;（4）Ang-（1-7）+Akt-siRNA+H/R group:Akt-siRNA was administered 1 hour before hypoxia-reoxygenation,andAng-（1-7）wasgiven30minutesbefore hypoxia-reoxygenation,followed by hypoxia for 3 hours and reoxygenation for 6 hours;（5）Akt-siRNA+H/R group:Akt-siRNA was incubated 30 min before hypoxia and reoxygenation,followed by hypoxia for 3 hours and reoxygenation for 6 hours.Firstly,detecting the expression of Akt and P-Akt protein,and verifying the Akt–siRNA interference effect.Secondly,Multi-level detection of cardiomyocyte apoptosis;Detection of CK-MB content in supernatant of each group by Elisa.Thirdly,detecting the expression of endoplasmic reticulum stress-related proteins.Finally,SERCA activity and the intracellular calcium concentration in each group.Results:1.Detection of the effect of Ang-（1-7）on apoptosis:1.1 The Caspase3 activity of each group was examined,and the results showed:Compared with control group,the H/R group increased（P<0.01）.Compared with the H/R group,the Ang-（1-7）+H/R group decreased（P<0.01）.1.2 Western blotting was used to detect the relative expression of apoptotic proteins in each group.The results showed that:Compared with control group,the H/R group increased BIM（P<0.01）and p-Bcl-2（P<0.01）.Compared with the H/R group,the Ang-（1-7）+H/R group decreased BIM（P<0.01）and p-Bcl-2（P<0.01）.2.Detection of the influence of Ang-（1-7）on four key points of path:2.1 Evaluation of interference effect,detecting the expression of Akt and p-Akt in each group by western blotting.The results showed that:There were no significant difference in the relative expression of Akt between the groups（P>0.05）.Compared with Ang-（1-7）+H/R group,the relative expression of p-Akt in Ang-（1-7）+Akt-siRNA+H/R group was significantly reduced（P<0.01）.Compared with H/R group,the p-Akt in the Akt-siRNA+H/R group decreased（P<0.01）.2.2 Multi-level detection of cardiomyocyte apoptosis（1）Determination of Caspase3 activity by spectrophotometry.The results showed that:Compared with control group,the H/R group increased the relative OD values（P<0.01）.Compared with the H/R group,the Ang-（1-7）+H/R group decreased the relative OD values（P<0.01）and the Akt-siRNA+H/R group was no statistically significant difference（P>0.05）.ComparedwithAng-（1-7）+H/Rgroup,the Ang-（1-7）+Akt-siRNA+H/R group increasde the relative OD values（P<0.01）.（2）The western blotting detecting apoptosis related proteins.The results showed that:Compared with control group,the H/R group decreased Bcl-2（P<0.01）and increased BIM（P<0.01）and p-Bcl-2（P<0.01）.Compared with the H/R group,the Ang-（1-7）+H/R group increased Bcl-2（P<0.01）and decreased BIM（P<0.01）and p-Bcl-2（P<0.01）.Compared with Ang-（1-7）+H/R group,the Ang-（1-7）+Akt-siRNA+H/R group decreased Bcl-2（P<0.01）and increased BIM（P<0.01）and p-Bcl-2（P<0.01）.2.3 Determination of CK-MB content in cell supernate of each group by ELISA.The results showed that:Compared with the H/R group,the CK-MB content in Ang-（1-7）+H/R group decreased（P<0.01）and there was no statistically significant difference in Akt-siRNA+H/R group（P>0.05）.Compared with Ang-（1-7）+H/R group,the CK-MB content in Ang-（1-7）+Akt-siRNA+H/R group increasde（P<0.01）.2.4 Detection of endoplasmic reticulum stress-related proteins by Western blotting.The results showed that:Compared with control group,the H/R group increased CHOP（P<0.01）,Caspase-12（P<0.01）and p-JNK（P<0.01）.Compared with the H/R group,the Ang-（1-7）+H/R group decreased CHOP（P<0.01）,Caspase-12（P<0.01）and p-JNK（P<0.01）but the expression of caspase12 was no statistically significant difference in Akt-siRNA+H/R group（P>0.05）.Compared with Ang-（1-7）+H/R group,the Ang-（1-7）+Akt-siRNA+H/R group increasde CHOP（P<0.01）,Caspase-12（P<0.01）and p-JNK（P<0.01）.2.5 SERCA activity of cells in each group.Compared with control group,the SERCA activity in H/R group reduced（P<0.01）.Compared with the H/R group,the SERCA activity in Ang-（1-7）+H/R group increased（P<0.01）but there was no statistically significant difference in Akt-siRNA+H/R group（P>0.05）.Compared with Ang-（1-7）+H/R group,the SERCA activity in Ang-（1-7）+Akt-siRNA+H/R group reduced（P<0.01）.6.Fluorescent probe Fluo-3/AM for intracellular calcium concentrationFrom picture,Compared with control group,the H/R group increased.Compared with the H/R group,the Ang-（1-7）+H/R group decreased.Compared with Ang-（1-7）+H/R group,the Ang-（1-7）+Akt-siRNA+H/R group increasde.Conclusing:1.Ang-（1-7）can reduce apoptosis during H/R injury.2.When cardiomyocytes are injury under hypoxic-reoxygenated,Ang-（1-7）can activate Akt signaling pathway,improves SERCA activity,maintain intracellular Ca²⁺,inhibits endoplasmic reticulum stress,and decrease ERS-induced apoptosis,then reduce H/R injury.