| Objective:Studies found that disorders of lipid metabolism and inflammatory are important in the pathogenesis of coronary heart disease. Danqi Pill (DQP) is made of Danshen and Sanqi and has been routinely prescribed in the treatment of coronary heart disease. DQP has lipid-lowering and anti-inflammatory effects. However, the pharmacological mechanisms are not yet fully understood. In this study, we induced an animal model of myocardial ischemia to study the effects of DQP. Factors in inflammatory signaling pathways and lipid metabolism pathways were assessed by western blot (WB) and real-time real-time Polymerase chain reaction (real-time PCR). In particular, expressions of different subtypes of PPARs and molecules in phosphate idylinositol 3-kinase (PI3K)-protein kinase B (AKT1/2)-extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway were quantified. The objective of this study is to reveal the pharmacological mechanism of DQP in the treatment of coronary heart disease.Methods:1.120 SPF male Sprague-Dawley (SD) rats, weighing 220±10g, were randomly divided into sham operation group and operation group after being fedon a regular diet for a week. Animals in operation group underwent left coronary artery ligation. After ligation surgery, animals in operation group were further randomly divided into model group, positive group (aspirin group and captopril group) and DQP group. Drugs were administered to aminals consecutively for 28 days since the second day after operation. The dose of DQP was 1.5 gkg-1, the dose of positive drug captopril was 1.2 mg·kg-1 and the dose of positive drug aspirin was 53.3 mg·kg-1. All drugs were dissolvedin waterand the volume given to each rat was 10 ml·kg-1 body. Animals in sham operation group and model group were given the same volume of saline.2. Echocardiography was applied to test the changes of ejection fraction (EF), fractional shortening (FS), left ventricular end-diastolic diameter (LVEDd) and left ventricular end-stolic diameter (LVEDs). Plasma lipid levels were evaluated by blood biochemical test.3. Change of proteins in inflammatory signaling pathway, such as phospholipase A2 (PLA2), cyclooxygenase 2 (COX2), nuclear factor-KB (NF-κB) and signal transducer and activator of transcription 3 (STAT3), were assessed by WB.4. Indicators in lipid metabolic pathways, such as fattty acid tranclocase (FAT/CD36), apolipoproteinC-III (ApoC-III) and ATP-binding cassette protein 1 (ABCA1), were detected by real-time PCR. Expressions of the factors of lipid absorption-transport-modified proteins, such asapolipoprotein A-1 (ApoA-1), fatty acid-binding protein (FABP), carnitine palmitoyl transferase 1A (CPT-1 A),3-hydroxy-3methyl-glutaryl-CoA reductase (HMGCR) and cytochrome P450 (CYP450) were assessed by WB.5. Expressions of of different subtypes of PPARs (PPARa and PPARβ/δ) were detected by real-time PCR. Expressions of proteins in PI3K-AKT1/2-ERK1/2 signaling pathway were assessed by WB method. Expressions of transcriptional factors such as PPARs (PPARa and PPARy), retinoid X receptor A (RXRA) and peroxisome proliferator-activated receptor co-stimulating factor 1-a (PGC-la) were also detected by WB.Results:1. Echocardiography showed that LVEDd value increased by 53.53%(P<0.01) and LVEDs value increased by 156.73%(P<0.01) in model groups compared with that in sham group, EF value decreased by 49.96%(P<0.05) and FS value decreased by 58.29%(P<0.01) in model group compared with those in sham group. LVEDd value decreased by 32.55% (P>0.05) in DQP group compared with that in model group, EF value increased by 43.00% (P<0.05) and FS value increased by 54.06%(P<0.05) in DQP group compared with those in model group. These results suggested that DQP could improve cardiac function.2. Blood biochemical tests showed that dyslipidemia in rats with myocardial infarction was regulated by DQP. Compared with sham group, the content of total cholesterol (TC) was increased by 19.95%(P>0.05), the content of triglycerides (TG) was increased by 136.88% (P<0.01), the content of low-density lipoprotein cholesterol (LDL-C) was increased by 127.82% (P<0.05), and the content of very low-density lipoprotein cholesterol (VLDL-C) was increased by140.00%(P<0.01) in model group. The content of high-density lipoprotein cholesterol (HDL-C) was decreased by 43.46%(P<0.05) in model group compared with that in sham group. Compared with model group, the content of TC was decreased by 3.37% (P>0.05), the content of TG was decreased by 58.23%(P<0.01), the content of LDL-C was decreased by 52.09%(P<0.01), and the content of VLDL-C was decreased by 75.00% (P<0.01) in DQP group. The content of HDL-C was increased by 62.50%(P<0.05) in DQP group compared with that in model group.These results demonstrated that DQP could regulate lipid metabolism.3. WB tests showed that, compared with sham group, the content of PLA2, COX2 and STAT3 in model group were increased by 47.5%(P<0.01),60.13%(P<0.05) and 113.9% (P<0.01), respectively. WB results showed that expressions of proteins in inflammatory pathways (PLA2, COX2, NF-κB) were down-regulated significantly in DQP group compared with those in model group. In DQP group, expressions of PLA2 and COX2 were down-regulated by 25.11%(P<0.05) and 59.74%(P<0.01), respectively, compared with those in the model group. The inflammatory protein NF-κB was down-regulated by 56.2%(P<0.01), compared with the model group. Level of STAT3 was increased by 49.6%(P<0.01). The anti-inflammatory effects of DQP may not be regulated by STAT3, but may be regulated by PLA2-COX2 signaling pathway mediated by NF-κB. Positive drug showed similar effects on NF-κB.4.Real time-PCR test showed that DQP could regulate expressions of proteins in lipid metabolic pathways (CD36, ApoC-Ⅲ, ABCA1).WB result indicated that DQP could regulate proteins in lipid absorption-transport-modified pathways(ApoA-I, FABP, CPT-1 A, HMGCR, P450). In DQP group, lipid metabolism was regulated towards normal level. Compared with sham group,the content of ApoA-1, FABP and CPT-1 A were decreased by 12.63%(P<0.05),10.00%(P>0.05) and 28.44%(P<0.05), respectively. Compared with model group, the content of ApoA-1, FABP and CPT-1 A were increased by 59.53% (P<0.01),28.97%(P<0.05) and 54.93%(P<0.01), respectively. Compared with sham group, the content of HMGCR and P450 were increased by 45.11%(P<0.05) and 79.79%(P<0.01), respectively. Compared with model group, the content of HMGCR and P450 in DQP group were decreased by73.25%(P<0.01) and 48.86%(P<0.01), respectively. DQP also showed effects on factors such as CD36, ApoC-Ⅲ and ABCA1, but the effects were not significant (P>0.05).5. Expressions of proteins were evaluated by WB. Compared with sham group, the levels of PPARa and PPARy were decreased by 24.25%(P>0.05) and 64.8%(P<0.01) in model group. Compared with sham group, the level of RXRA and PGC-la were decreased by 42.0! (P<0.05) and 61.75%(P<0.05) in model group. The results showed that the content of PPARa was increased by 141.31%(P<0.01), the content of PPARy was increased by 352.5’ (P<0.01), the content of RXRA was increased by 123.71%(P<0.01), and the content of PGC-la was increased by 103.41%(P>0.05) in DQP group compared with those in model group. Expressions of different subtypes of PPARs (PPARa and PPARβ/δ) were assessed by real-time PCR method. The results showd that, compared with model group, the content of PPARa was increased by 507.77%(P<0.05) and the content of PPARβ/δ was increased by 667.23%(P<0.05) in DQP group. It was consistent with the results of WB tests. These results demonstrated that DQP could exert cardioprotective effect by regulating expressions of PPARs.6. Proteins in PI3K-AKT1/2-ERK1/2 signaling pathway were detected by WB. The result: showed that the contents of PI3K, AKT1/2 and ERK1/2 were decreased by 63.41%(P<0.05), 60.23%(P<0.01) and 52.12%(P<0.01), respectively. The content of PI3K was increased by 89.62%(P>0.05), the content of ERK1/2 was increased by 138.67%(P<0.01) and the content of AKT1/2 was increased by 96.39%(P<0.01) in DQP group compared with those in model group.DQP was shown to have effect on PI3K-AKT1/2-ERK1/2 pathway.Conclusion:Disorders of lipid metabolism and inflammatory reactions occurred in rats in myocardial infarction model group. The content of TC, TG, LDL-C and VLDL-C were up-regulated and the content of HDL-C was down-regulated in the model group. After intervention with DQP, we found that DQP could regulate lipid metabolism and inhibit inflammatory reactions. DQP can improve heart function in rats with ischemic coronary heart disease. DQP can modulate PLA2-COX2 pathways and alleviate inflammation by acting on NF-κB. DQP regulates lipid metabolism by regulating proteins (CD36, ApoC-III, ABCA1,ApoA-1, FABP, CPT-1A, HMGCR, P450) in lipid uptake-transport-metabolism pathway. Moreover, different subtypes of PPARs can be up-regulated by DQP. DQP could also regulate PI3K-AKT1/2-ERK1/2 pathway. This study also provides insight into the mechanisms of DQP in the treatment of coronary heart diseases by PPARs. Our study reveals the anti-inflammatory and lipids-lowering mechanisms of DQP and provides the experimental basic for refinement of DQP. |
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