Effect Of Qishen Granule On Different Types Of Epoxygenase Subtype COXs Mediated Cardiomyocyte Inflammation Injury

Objective:This study using hypoxia and reoxygen (H/R) inflammatory injury model with H9C2 cells in vitro on the basis of previous research. It was evaluated after used QSKL and Quercetin in H9C2 cells the anti-inflammatory efficacy and revealed the inflammatory reaction of the two drugs in PLA2/COXs/PGS pathway on the protein of SPLA2, COX1, COX2 and inflammatory factor of NF-κB、TNF-α、IL-6、iNOS. It fully reveals the effect mechanism of QSKL on inhibit inflammatory damage of cardiac muscle cells by regulating the different subtypes of COXs pathway from the effect of protein and active ingredients.Method:1. The survival rate of H9C2 cells by the MTT method in hypoxia and reoxygen damage model of H9C2 myocardial cells using different concentrations of QSKLand Quercetin. The H9C2 cells were randomly divided into Control group, Model group, QSKL group and Quercetin group. The relative protein expression of TNF-a in H9C2 cells was detected by ELISA kit.2. RT-qPCR was detected the gene expression of SPLA2, COX1, COX2 in Control group, Model group, QSKL group and Quercetin group. Western Blot was detected the protein expression levels of sPLA2,COX1, COX2 of H9C2 cells in Control group, Model group, QSKL group and Quercetin group. ELISA was detected the protein expression levels of PGE2 of H9C2 cells in Control group, Model group, QSKL group and Quercetin group.3. RT-qPCR was detected the gene expression of NF-κB、TNF-α、IL-6 and iNOS of H9C2 cells in Control group, Model group, QSKL group and Quercetin group.Result:1. To establish the myocardial hypoxia and reoxygen damage model in H9C2 cells, the survival rate of H9C2 cells was detected by MTT. H9C2 cell viability was decreased linearly compared with the Control group for 4h,6h,8h (P<0.01).The survival rate of the H9C2 cells was 46.89%. Hypoxia and reoxygen myocardial damage model of H9C2 cells mainly through the biological changes in the inflammatory pathway. So, we choice the 8h,12h for the time of the subsequent pharmacological study of H9C2 cells inflammatory damage model.2. Experimental study on efficacy evaluation of QSKL in H9C2 myocardial hypoxia and reoxygenation model:(1) The cell viability gradually increased after using different concentrations of QSKL in H9C2 cells (except for the 1000μg·mL-1), which the concentrations of QSKL with 800μg·mL-1 and 600μg·mL-1, the cell viability become the highest and between the two concentrations have not statistical difference (P< 0.01), so we choice 600μg·mL-1 as the effective dose in H9C2 cells inflammatory injury model. (2) ELISA method was used to detect the relative TNF-α protein expression in H9C2 cells in each group. The expression of TNF-α protein in H9C2 cells was significantly lower than the Model group (P＜0.01). Among them, TNF-α protein content was significantly lower than Quercetin group (P<0.05).3. Experimental study on the influence of arachidonic acid (AA) pathway for QSKL in H9C2 myocardial hypoxia and reoxygenation model:RT-qPCR and Western Blot were detected the gene expression of sPLA2, COX1 and COX2. The results were the protein expression of sPLA2, COX1 and COX2 in QSKL group and Quercetin group were generally lower relative content compared with the Model group (P<0.05). At the same time, the gene expression with the level of the gene of COX1 in QSKL group was significantly lower than that of Quercetin group (P< 0.05). However, the protein expression with the level of the gene of COX2 in QSKL group was significantly lower than Quercetin group (P< 0.05). The protein expression detected by ELISA kit of PGE2 in H9c2 cells with QSKL group and Quercetin group was significantly lower than Model group (P＜0.01). Among them, The PGE2 of QSKL group was obviously lower than Quercetin group (P＜0.05).4. Experimental study on anti-inflammatory mechanism of QSKL in H9C2 myocardial hypoxia and reoxygenation model:RT-qPCR was detected the gene expression of NF-κB、 TNF-α、IL-6 and iNOS in H9C2 cells. The results was the gene expression of NF-κB、TNF-α、 IL-6 and iNOS in H9C2 cells with QSKL group and Quercetin group was significantly lower than Model group (P＜0.01). Among them, the gene expression of TNF-α and IL-6 were significantly lower than Quercetin group (P＜0.05); however, the expression of iNOS was significantly higher than QSKL group (P<0.01).Conclusion:1. To established H9C2 cells inflammatory injury model induced by the hypoxia and reoxygen injury, the subsequent pharmacological study of hypoxia and reoxygen injury for H9C2 cells inflammatory damage model was 8h and 12h.600μg·mL-1 for QSKL and 10μmol·L-1 for Quercetin as the effective dose in H9C2 cells inflammatory injury model. It was found that the administration group could effectively inhibit the expression of TNF-a in H9C2 cells. At the same time, the content of TNF-a in the QSKL group was lower than the Quercetin group, which showed that the QSKLcompound has good effect on the inhibition of inflammatory reaction.2. At the level of gene and protein, the SPLA2, COX1 and COX2 expression levels in H9C2 cells could be effectively reduced by QSKL and Quercetin. At the same time, the expression of PGE2 in the drug group was significantly lower than the Model group. The experiment showed that the drug group may be mediated by the sPLA2/COXs/PGs pathway, which could be effectively protected the inflammatory damage of H9C2 cells. The expressions of PGE2 and COXs in the QSKL compound were significantly lower than Quercetin.3. The drug group could reduced genes expression of NF-κB、TNF-α、IL-6、iNOS in H9C2 cells and inhibited the inflammatory reaction process. NF-κB、TNF-α、IL-6 and other proinflammatory factor involved in inflammation, oxidative stress, platelet aggregation, thrombosis, disorder of lipid metabolism and other pathological response. The results showed that QSKL compound and Quercetin could effectively improve4he occurrence, development and prognosis of myocardial ischemic coronary heart disease. The QSKL compound was better in reducing the expression of TNF-a and IL-6. However, Quercetin was more significantly than QSKLcompound in the regulation of iNOS gene expression.