Effect of miltirone on proliferation and apoptosis of leukemia THP-1 cells / 中国中药杂志

To investigate the toxicity and related mechanism of miltirone to human acute myeloid leukemia THP-1 cells. To be specific, the active components and targets of miltirone were retrieved from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP), and the target proteins were converted into standard gene names with UniProt. Acute leukemia-rela-ted target genes were screened from GeneCards and DisGeNET. Venn diagram was constructed with Venny 2.1 to yield the common targets of the disease and the drug. The protein-protein interaction(PPI) network was constructed by STRING and Cytoscape 3.8.2. THP-1 cells in the logarithmic growth phase were treated with dimethyl sulfoxide(DMSO), and 2.5, 5, 10, 15, and 20 μmol·L~(-1) miltirone for 24 h, respectively. The proliferation rate of cells was analyzed by carboxyfluorescein diacetate succinimidyl ester(CFSE), apoptosis rate by flow cytometry with Annexin V-PE/7 AAD staining, and cell morphology by acridine orange staining. Real-time quantitative PCR(qPCR) was employed to detect the mRNA levels of nuclear receptor coactivator 2(NCOA2), poly(ADP-ribose) polymerase-1(PARP1), B-cell lymphoma-2(Bcl-2)-associated X protein(Bax), Bcl-2, and cysteine aspartyl protease-3(caspase-3). The effect of miltirone on apoptosis was detected in presence of caspase inhibitor Z-VAD-FMK. A total of 26 targets of miltirone, 1 046 genes related to acute leukemia, and 6 common targets of the two were screened out. Flow cytometry result showed miltirone at 10 μmol·L~(-1) can inhibit proliferation and promote apoptosis of THP-1 cells. The typical manifestations of apoptosis, such as cell shrinkage, nuclear rupture, and chromatin agglomerate were displayed by acridine orange staining. The decreased mRNA levels of NCOA2 and PARP1 and increased Bax/Bcl-2 ratio and the activity of pro-apoptotic protein caspase-3 were observed. Z-VAD-FMK can attenuate the apoptosis-inducing effect of miltirone. This study indicates that miltirone can inhibit the proliferation and promote the apoptosis of THP-1 cells, by down-regulating NCOA2 and PARP1, raising Bax/Bcl-2 ratio, and activating caspase-3.

Pharmacological actions of miltirone in the modulation of platelet function / 中国药理学与毒理学杂志

OBJECTIVE Salvia miltiorrhiza bunge contains various active constituents, some of which have been developed as commercially available medicine. Moreover, some other ingredients in Salvia miltiorrhiza play great roles in anti-platelet activity. The aim of the present study was to investi-gate the effects and the underlying mechanism of miltirone,a lipophilic compound of Salvia miltiorrhiza Bunge. METHODS The ability of miltirone to modulate platelet function was investigated by a variety of in vitro and in vivo experiments.Platelet aggregation and dense granule secretion induced by various agonists were measured with platelet aggregometer.Clot retraction and spreading were imaged by digital camera and fl uorescence microscope. Ferric chloride-induced carotid injury model and pulmonary thromboembolism model were used to check miltirone effect in vivo. To elucidate the mechanisms of anti-platelet activity of miltirone,flow cytometry and Western blotting were performed. RESULTS Miltirone (2,4,8 μmol·L-1)was shown to suppress platelet aggregation,dense granule and α granule secretion in a dose-dependent manner. Meanwhile, miltirone inhibited the clot retraction and spreading of washed platelets.It reduced the phosphorylation of PLCγ2,PKC,Akt,GSK3β and ERK1/2 in the down-stream signal pathway of collagen receptor.It also reduced the phosphorylation of Src and FAK in the integrin αⅡbβ3 mediated"outside-in"signaling,while it did not suppress the phosphorylation of β3.In addition, miltirone prolonged the occlusion time and reduced collagen/epinephrine induced pulmonary thrombi. CONCLUSION Miltirone suppresses platelet "inside-out" and "outside-in" signaling by affecting PLCγ2/PKC/ERK1/2, PI3K/Akt and Src/FAK signaling. Therefore, miltirone might represent a potential anti-platelet candidate for the prevention of thrombotic disorders.