The Research On The Protective Effect And Mechanism Of Pcsk9 Inhibitor On Hypoxic Cardiomyocytes

Objective: This study mainly explores the protective effect of PCSK9 inhibitors on hypoxic cardiomyocytes and possible mechanisms of action,so as to provide a theoretical basis for the development of more effective therapeutic drugs for the protection of reperfusion-induced myocardium.Methods: 1.The mouse myocardial ischemia-reperfusion injury model was established by ligating the left anterior descending(LAD)and opening the left anterior descending coronary artery.2.Animal grouping and administration: male healthy C57/BL6 mice,6-8 weeks old,body weight(20±2)g,randomly divided into 7 groups,each with 8 mice.(1)Control group(sham operation group,namely Sham group);(2)Surgery+ normal saline group(I/R+NS group);(3)Surgery + low-dose PCSK9 inhibitor group(I/R+LPCSK9i group));(4)Surgery + high-dose PCSK9 inhibitor group(I/R+HPCSK9i group);(5)Surgery + LY294002(PI3K inhibitor)group(I/R+LY group);(6)Surgery + LY294002 Group +PCSK9 inhibitor group(I/R+LY+PCSK9i group);(7)Surgery + statin group(I/R+Ator group).3.Observe the myocardial injury through myocardial TTC and Evans blue staining,color Doppler ultrasound detection of heart function,serum myocardial enzymes,BNP,and TNI.4.The occurrence of apoptosis was detected by TUNEL staining of paraffin sections of myocardial tissue;the protein expression levels of Caspase 3,m TOR,AMPK,AKT and apoptosis-related proteins(Bcl-2,Bax)were detected by western blot.4.Perform statistical analysis.Normally distributed measurement data are expressed as (?)±s.Each group of experiments needs to be repeated three times.The comparison between the two groups uses t-test.The comparison between multiple groups uses single-factor analysis of variance,and multiple comparisons afterwards The Dunnett method was used for analysis.Using a two-sided test,P<0.05 indicates that the difference is statistically significant.Results: 1.The results of Evans blue/TTC staining showed that compared with the sham operation group,the myocardial infarction area and ischemic risk area of the mice in the I/R+NS group,I/R+LY group and I/R+LY+PCSK9i group The area was significantly increased(P<0.05);compared with the mice in the I/R+NS group,the IS and AAR of the mice in the I/R+LPCSK9i group,I/R+HPCSK9i group and I/R+Ator group were significantly reduced(P<0.05),but there was no significant difference in myocardial infarction area and AAR between the three groups of mice(P>0.05;Figure 1).The LVEF value of the mice in each group was detected by the small animal ultrasonic diagnostic instrument.Compared with the sham operation group,I/R significantly reduced the cardiac ejection fraction of the mice(P<0.05);compared with the I/R group,After the application of PCSK9 inhibitor,the cardiac ejection fraction of mice was significantly improved(P<0.05).2.ELISA method to detect the levels of myocardial injury markers in the plasma of each group of mice showed: compared with the Sham group,the LDH in the I/R+NS group,I/R+LY group and I/R+LY+PCSK9i group The content of TNI,CK-MB and BNP increased significantly(P<0.05);compared with the I/R+NS group,the I/R+LPCSK9i group,the I/R+HPCSK9i group and the I/R+Ator group were smaller The contents of LDH,CK-MB,BNP,and TNI of mice were significantly decreased(P<0.05);compared with I/R+LY+LPCSK9i group,I/R+LPCSK9i group,I/R+HPCSK9i group and I The contents of LDH,CK-MB,BNP and TNI of the three groups of mice in the I/R+Ator group were significantly decreased(P<0.05).There was no significant difference between the I/R+HPCSK9i group and the I/R+Ator group and the I/R+LPCSK9i group(P>0.05).3.PCSK9 inhibitors reduced the apoptotic rate of cardiomyocytes in mice with ischemia-reperfusion.The TUNEL labeling method detected the apoptosis rate of mouse cardiomyocytes.The results showed that the apoptosis rate in the Sham group was(5.10±2.05)%.Compared with the Sham group,the apoptosis rate in the I/R+NS group was(40.2±3.21)% significantly increased.(P<0.05),compared with I/R+NS group,I/R+LPCSK9i group(25.1±3.27)%,I/R+HPCSK9i group(23.5±2.53)% and I/R+Ator group(26.5± 2.41)% apoptosis rate was significantly reduced(P<0.05);compared with I/R+LY+PCSK9i group(38.6±2.89)%,I/R+LPCSK9i group(25.1±3.27)%,I/R+HPCSK9i The apoptosis rate of(23.5±2.53)% in the group was also significantly reduced(P<0.05).4.Western blot test results: Compared with the Sham group,the expression of AMPK,Bax and Caspase 3 in the I/R+NS group increased significantly(P<0.05),while m TOR,AKT and Bcl-2 decreased significantly(P<0.05);Compared with the I/R+NS group,the expressions of AMPK,Bax and Caspase 3 in the I/R+LPCSK9i group,I/R+HPCSK9i group and I/R+Ator group were reduced significantly(P<0.05),while m TOR,AKT and Bcl-2 significantly increased(P<0.05);compared with the I/R+LY+PCSK9i group,the PCSK9 inhibitor treatment group(I/R+LPCSK9i group and I/R+HPCSK9i group)had AMPK,Bax and Caspase 3 Expression was significantly reduced(P<0.05),m TOR,AKT and Bcl-2 were significantly increased(P<0.05);compared with I/R+NS group,I/R+LY group and I/R+LY+PCSK9i group There was no significant difference in the expression of m TOR,AMPK,AKT,Bcl-2,Bax and Caspase 3(P>0.05).Conclusion: 1.PCSK9 inhibitor reduces the area of myocardial infarction in ischemia-reperfusion(I/R)mice,improves heart function,reduces myocardial damage and inhibits myocardial apoptosis caused by I/R in mice.2.PCSK9 inhibitors can inhibit cell apoptosis induced by myocardial endoplasmic reticulum stress caused by ischemia-reperfusion injury by activating the PI3K/AKT/m TOR signaling pathway.