OBJECTIVE: To investigate the effects of matrine combined with LY294002 on proliferation, apoptosis and cell cycle of human myeloid leukemia K562 cells and explore the underlying mechanism. METHODS: The effects of different concentrations of matrine alone and in combination with LY294002 on the proliferation of K562 cells were examined with CCK-8 assay. The changes in morphology of K562 cells were observed following treatment for 48 h with 0.4 g/L matrine and 10 μmol/L Y294002, either alone or in combination, and cell apoptosis was detected using flow cytometry with annexin V-FITC/PI double labeling; the changes in cell cycle was detected with PI labeling. Western blotting was performed to examine the effect of matrine combined with LY294002 on expressions of p-mTOR, p-PI3K, Akt, p-Akt, cyclinD1, Bcl-2 and caspase-9 in the cells. RESULTS: Treatment with different concentrations of matrine, both alone and in combination with LY294002, inhibited the proliferation of K562 cells in a time- and concentration-dependent manner. Compared with matrine treatment alone, the combined treatment caused more obvious morphological changes of the cells, significantly increased cell apoptosis (P < 0.01), and induced cell cycle arrest in G₀/G₁ (P < 0.01). Western blotting showed that the protein expression levels of p-mTOR, cyclinD1, p-PI3K, p-Akt and Bcl-2 in K562 cells increased while the expression level of caspase-9 decreased significantly after the combined treatment (P < 0.01). CONCLUSION Matrine combined with LY294002 produces a synergistic inhibitory effect on K562 cells possibly by down-regulating the p-Akt expression in PI3K/Akt signaling pathway, reducing the expressions of p-mTOR, cyclinD1 and Bcl-2, and increasing the expression of caspase-9.
Humans ; K562 Cells ; Caspase 9 ; Matrines ; Phosphatidylinositol 3-Kinases ; Cell Cycle ; Cell Division ; Leukemia, Myeloid ; Apoptosis ; Proto-Oncogene Proteins c-bcl-2

Qishen Yiqi Dripping Pills(QSYQ) are used clinically to treat various myocardial ischemic diseases, such as angina pectoris, myocardial infarction, and heart failure; however, the molecular mechanism of QSYQ remains unclear, and the scientific connotation of traditional Chinese medicine(TCM) compatibility has not been systematically explained. The present study attempted to screen the critical pathway of QSYQ in the treatment of myocardial ischemia by network pharmacology and verify the therapeutic efficacy with the oxygen-glucose deprivation(OGD) model, in order to reveal the molecular mechanism of QSYQ based on the critical pathway. The key targets of QSYQ were determined by active ingredient identification and target prediction, and underwent pathway enrichment analysis and functional annotation with David database to reveal the biological role and the critical pathway of QSYQ. Cell counting Kit-8(CCK-8), lactate dehydrogenase(LDH), and Western blot tests were launched on high-content active ingredients with OGD cell model to reveal the molecular mechanism of QSYQ based on the critical pathway. The results of network pharmacology indicated that QSYQ, containing 18 active ingredients and 82 key targets, could protect cardiomyocytes by regulating biological functions, such as nitric oxide biosynthesis, apoptosis, inflammation, and angiogenesis, through TNF signaling pathway, HIF-1 signaling pathway, PI3 K-Akt signaling pathway, etc. HIF-1 signaling pathway was the critical pathway. As revealed by CCK-8 and LDH tests, astragaloside Ⅳ, salvianic acid A, and ginsenoside Rg_1 in QSYQ could enhance cell viability and reduce LDH in the cell supernatant in a concentration-dependent manner(P<0.05). As demonstrated by the Western blot test, astragaloside Ⅳ significantly down-regulated the protein expression of serine/threonine-protein kinase(Akt1) and hypoxia-inducible factor 1α(HIF-1α) in the HIF-1 signaling pathway, and up-regulated the protein expression of vascular endothelial growth factor A(VEGFA). Salvianic acid A significantly down-regulated the protein expression of upstream phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha(PIK3 CA) and downstream HIF-1α of Akt1. Ginsenoside Rg_1 significantly down-regulated the expression of HIF-1α protein and up-regulated the expression of VEGFA. The therapeutic efficacy of QSYQ on myocardial ischemia was achieved by multiple targets and multiple pathways, with the HIF-1 signaling pathway serving as the critical one. The active ingredients of QSYQ could protect cardiomyocytes synergistically by regulating the targets in the HIF-1 signaling pathway to inhibit its expression.
Drugs, Chinese Herbal/pharmacology* ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics* ; Myocardial Ischemia/genetics* ; Signal Transduction ; Vascular Endothelial Growth Factor A

Objective::The effects of three different doses of borneol on acute myocardial infarction (AMI) model rats and the effects on oxidative stress factors were compared to provide reference for elucidation of the dose-effect relationship and mechanism of anti-myocardial infarction. Method::Healthy adult male SPF SD rats were randomly divided into sham operation group, model group, solvation model group, nitroglycerin group, Borneolum high, medium and low dose(0.6, 0.3, 0.15 g·kg^-1) group, l-Borneolum and Borneolum syntheticum high, medium, low dose(0.2, 0.1, 0.05 g·kg^-1) group, a total of 13 groups, 20 in each group. Gavage was performed at 20 mL·kg^-1 once a day for 3 days of continuous preventive administration. The sham operation group and the model group were given the same volume of distilled water, and the solvation model group was given the same volume of 5% polysorbate 80.On the third day of the pre-administration, 30 minutes after the last dose, the left anterior descending coronary artery was ligated to make a model, and the successful rats were treated for 3 days. BL-420N biological system analyzer was used to record the ST-segment amplitude and hemodynamic changes. Rat body weight and cardiac weight were weighed to calculate cardiac viscera coefficients, 2, 3, 5-triphenyl tetrazolium chloride (TTC) staining was used to calculate the myocardial infarction rate. Hematoxylin-eosin (HE) staining was used to evaluate the degree of myocardial pathological damage. According to the kit requirements, serum levels of lactate dehydrogenase (LDH), aspartate amino-transaminase (AST), creatine kinase isoenzyme (CK-MB) and oxidative stress factors superoxide dismutase (SOD), malondialdehyde (MDA) were detected. Result::Compared with the sham operation group, the ST segment amplitude of the model group significantly increased after 5 minutes, the left ventricular diastolic blood pressure (LVDP) value increased significantly, and the measured maximum shortening velocity (Vpm) value of the left ventricular myocardial contraction component significantly decreased. The organ coefficient and myocardial infarction rate were extremely significantly increased, and the myocardial pathological tissue was severely damaged. The serum CK-MB, AST, LDH, and MDA contents were significantly increased (P<0.05, P<0.01). Compared with the solvation model group, the Borneolum and l-Borneolum in the middle and low, and the Borneolum syntheticum high dose groups could significantly inhibited the abnormal elevation of ST segments at different time points. The Borneolum and l-Borneolum high, medium, low, and Borneolum syntheticum high dose groups significantly increased the left ventricular systolic blood pressure (LVSP) value and decrease the LVDP value (P<0.01). The Borneolum medium, low, and l-Borneolum high, medium, Borneolum syntheticum high dose groups significantly increased the maximum rate of left ventricular pressure rise (dp/dt max) and Vpm value (P<0.05, P<0.01). The Borneolum and l-Borneolum medium, low dose groups significantly reduced rat cardiac organ coefficients. The Borneolum high, medium, low and l-Borneolum, Borneolum syntheticum medium, low dose groups significantly improved myocardial infarction in rats (P< 0.05, P<0.01). The Borneolum low, l-Borneolum high, medium, and Borneolum syntheticum high groups also significantly improved the degree of pathological damage (P<0.01). High dose of l-Borneolum significantly reduced CK-MB content, medium and low dose of l-Borneolum significantly reduced AST activity, medium and low dose of l-Borneolum, high, medium and low dose of Borneolum syntheticum significantly reduced LDH activity (P<0.05, P<0.01). Serum SOD activity of rats in l-Borneolum high, medium, and Borneolum syntheticum high dose groups increased significantly (P<0.05, P<0.01). Serum MDA levels in Borneolum high, medium, low, and l-Borneolum high, middle dose groups significantly decreased (P<0.01). Conclusion::Three kinds of borneol in different dose groups can play different degrees of myocardial protection. Under the experimental conditions, there was a trend of l-Borneolum>Borneolum>Borneolum syntheticum in improving the efficacy of myocardial infarction, the dose-effect of Borneolum was negatively correlated, Borneolum syntheticum was positively correlated, and no significant dose-effect relationship between l-Borneolum.