Effects Of Salvianolic Acid B Combined With Hydroxysafflor Yellow A On Myocardial Ischemia Reperfusion Injury

Objective To study the effect and mechanism of salvianolic acid B(Sal B),one of the main active components of Salvia miltiorrhiza,and hydroxysafflower yellow a(HSYA),one of the effective components of safflower.To establish a model of myocardial cell injury induced by oxygen glucose deprivation/reperfusion(OGD/R),and to explore the protective effect of the combination of the two compotent in the model,and to reveal its mechanism,in order to explore the basic theory of Sal B combined with HSYA.OGD/R model can simulate the pathophysiological process of myocardial ischemia-reperfusion injury(MIRI)to a certain extent,so this study can provide experimental basis for clinical treatment of MIRI and other heart diseases.Methods(1)The OD value was measured by CCK-8 cytotoxicity test.According to the OD value,the safe concentration range of Sal B,HSYA and Danhong Injection(DHI)was calculated,and then the optimal action concentration was determined within the safe concentration range for subsequent experimental study.(2)Establishment of OGD/R injury model of H9c2 cardiomyocytes.H9c2 cardiomyocytes can be divided into Normal group,Model group,Danhong injection group(2%),Sal B + HSYA group(Sal B 20μmol/L,HSYA 80μmol/L)and Sal B+HSYA+LY294002 group(Sal B 20μmol/L,HSYA 80μmol/L,LY294002 20μmol/L),OGD 12h/R 6h,during reoxygenation,the corresponding reagents or drugs were added to the cells in each group to observe the morphological changes of H9c2 cardiomyocytes in each group.In DHI group add 2% DHI,Sal B+HSYA group add Sal B 20 μmol/L and HSYA 80 μmol/L,Sal B+HSYA+LY294002 group add Sal B 20μmol/L,HSYA 80 μmol/L and and LY294002 20μmol/L.In model group,add DMEM without any drugs.At the end of culture,the morphological changes of H9c2 cardiomyocytes in each group were observed and recorded under inverted microscope,and the expression of inflammation related factors(such as TNF-α,IL-1,IL-6)and the changes of oxidative stress related markers(MDA and SOD)were detected by ELISA..(3)Establishment of OGD/R injury model of H9c2 cardiomyocytes.H9c2 cardiomyocytes can be divided into Normal group,Model group,Danhong injection group(2%),Sal B + HSYA group(Sal B 20μmol/L,HSYA 80μmol/L)and Sal B+HSYA+LY294002 group(Sal B 20μmol/L,HSYA 80μmol/L,LY294002 20μmol/L),OGD 12h/R 6h,during reoxygenation,the corresponding reagents or drugs were added to the cells in each groups.In DHI group add 2% DHI,Sal B+HSYA group add Sal B 20 μmol/L and HSYA 80 μmol/L,Sal B+HSYA+LY294002 group add Sal B 20μmol/L,HSYA 80 μmol/L and and LY294002 20μmol/L.In model group,add DMEM without any drugs.Detection of LDH relative activity and CK activity in cell supernatant.The cells were stained with Hoechst 33342 and the morphological changes were observed and recorded under fluorescence microscope.Results(1)The results of CCK-8 showed that the non-cytotoxic concentration ranges of Sal B,HSYA and DHI were 0-40 μmol/L,0-160 μmol/L and 0-2%,respectively.(2)The results of anti-inflammatory and anti-oxidation effects of Sal B combined with HSYA on the model of OGD/R injury of H9c2 cardiomyocytes:the cell morphology showed that Sal B+HSYA group and DHI group were significantly improved,but there was no significant difference between the two groups.At the same time,TNF-α,IL-1,IL-6,MDA levels were significantly reduced in Sal B+HSYA group and DHI group,but the SOD levels were increascd,there was no significant difference between the two groups.The contents of TNF-α,IL-1 and IL-6 and the contents of MDA in Sal B+HSYA+LY group were significantly higher than those in Sal B + HSYA + LY294002 group,but the level of SOD in Sal B+HSYA+LY294002group was significantly lower than those in Sal B+HSYA group.(3)The results showed that LDH relative activity and CK activity in supernatant of H9c2 cardiomyocytes in OGD/R model were significantly higher than those in normal group,while LDH and CK content in Sal B+HSYA and DHI group were significantly lower than those in model group,and LDH and CK content in Sal B+HSYA+LY294002 group were higher than that in Sal B+HSYA+LY294002 group.Hoechst33342 staining showed that the morphology of nucleus and chromatin in Sal B+HSYA group and DHI group were significantly improved compared with the model group,LY294002 could inhibit the effect of Sal B+HSYA on improving the morphology of nucleus and chromatin.Conclusion(1)The optimal concentration of Sal B+HSYA is Sal B20μmol/L+HSYA 80 μmol/L.(2)Sal B+HSYA can protect H9c2 cardiomyocytes from injury caused by OGD/R through anti-inflammatory,anti-oxidation and anti-apoptosis effects.(3)The activation of PI3K/Akt signaling pathway interferes with the injury of H9c2 cardiomyocytes induced by OGD/R by Sal B+HSYA to a certain extent.