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Objective: The study was designed to investigate the antitumor effect of Pulsatilla chinensis saponins components and the synergistic effect of its effect components on tumor glycolysis. Methods: NCI-H460 cell lines were cultured in vitro, MTT assay was used to detect the inhibitory rate of various components of Pulsatilla chinensis saponins. The compatibility and synergistic antitumor effect of different effect components were studied by Calcusyn 3.0 software. Biochemical assay and Elisa assay were used to detect the glycolysis related metabolites (pyruvate, lactic acid, and glucose) and enzymes (GLUT1, HK, PKM2, and LDHA). Energy metabolism related gene members from KEGG pathway database and key protein (ERK1/2, Ras, GLUT1, MCT4) were detected by qRT-PCR and Western blotting. Results: The antitumor results in vitro showed that PSD, R13 and PSA in Pulsatilla chinensis saponins had the strongest antitumor activity for human lung cancer NCI-H460 cells and the IC50 were 5.2, 4.6, and 7.9 μg/mL, respectively. Using Calcusyn 3.0 software to confirm the distribution ratio of three effective monomers in Pulsatilla chinensis saponins and determine the synergistic antitumor effect after compatibility. The results of biochemical and Elisa assay showed that the content of pyruvate, lacate, glucose, HK, PKM2 and LDHA were significantly decreased and the GLUT1 content was significantly increased compared with the blank group (P < 0.05),; Compared with each monomer group, the content of pyruvic acid, lactic acid, glucose, HK, PKM2 and LDHA in the combined group were significantly decreased and the content of GLUT1 was significantly increased (P < 0.05). Real-time PCR results show that the combinations group has the most nodes in the network diagram, and its target was more than each monomer group. Western blotting results showed that compared with the blank group, the combinations group significantly decreased the expression of ERK1/2, Ras, GLUT1, and MCT4 protein (P < 0.05). Conclusion: The combination of effect components of Pulsatilla chinensis saponins has synergistic antitumor effect on NCI-H460 cells and the antitumor mechanism may be associated with the regulation of glycolysis.
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Objective: To screen the differentially expressed proteins of saponins in Pulsatillae Radix inhibiting the proliferation and induce apoptosis on NCI-H460 tumor cells based on proteome technology using nano LC-LTQ-Orbitrap-MS/MS, and preliminarily speculate the potential mechanism. Method: NCI-H460, SK-OV-3 and SGC-7901 tumor cells were cultured in vitro. Methylthiazoletetrazolium (MTT) assay was used to detect the inhibitory rate of saponins in Pulsatillae Radix on three tumor cell lines. Effect of saponins in Pulsatillae Radix on apoptosis was analyzed by Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining flow cytometry and 4',6-diamidino-2-phenylindole (DAPI) staining. Apoptosis was analyzed using flow cytometry and DAPI stain. Nano LC-LTQ-Orbitrap-MS/MS was used to investigate the changes in the protein profiles on NCI-H460 cells treated with saponins in Pulsatillae Radix. Proteins exhibiting differential expression were analyzed by DAVID Bioinformatics Resources 6.8 and Kyoto encyclopedia of genes and genomes (KEGG) database. The differentially expressed proteins were verified by Western blot. Result: Saponins in Pulsatillae Radix could inhibit the proliferation of NCI-H460, SK-OV-3 and SGC-7901 tumor cells and induce apoptosis of NCI-H460 tumor cells. Effect of Saponins in Pulsatillae Radix on the proliferation and apoptosis of NCI-H460 tumor cells was mainly related to the regulation of biological function of ribosome, glycolysis/gluconeogenesis and other biological processes. It was possible to induce apoptosis of NCI-H460 tumor cells by interfering mitogen-activated protein kinase (MAPK) signaling pathway and regulating the Caspase pathway. Conclusion: Saponins in Pulsatillae Radix can inhibit the proliferation and induce the apoptosis of NCI-H460 tumor cells, the mechanism may be related to the intervention of MAPK signaling pathway and the regulation of Caspase pathway. These findings are helpful to elucidate the molecular mechanism of the anti-tumor effect of saponins in Pulsatillae Radix.
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Objective: The study was designed to investigate the molecular mechanism of quercitrin on osteogenic differentiation and adipogenic differentiation of rBMSCs. Methods: rBMSCs were harvested from SD rats, and determination of alkaline phosphatase (ALP) activity, quantification of mineralization by Alizarin Red S staining, and the mRNA expression of osteogenic differentiation markers (Runx2, BMP-2, and OSX) by RT-PCR after rBMSCs stimulated by osteogenic induction with (0.1–10) µg/mL of quercitrin, quantification of Lipid droplet by Oil Red O staining and the mRNA expression of adipogenic differentiation marker (PPARγ C/EBPα and aP2) by RT-PCR after rBMSCs stimulated by adipogenic induction with (0.1-10) µg/mL of quercitrin. Results: Quercitrin can up-regulate the mRNA expression of osteogenic differentiation markers (Runx2, BMP-2, and OSX) and increase ALP activity and mineralization after osteogenic induction, on the other hand quercitrin can suppress the mRNA expression of adipogenic differentiation markers (PPARγ C/EBPα and aP2) and decrease lipid droplet after adipogenic induction. Conclusion: This study suggested that quercitrin not only stimulated osteogenic differentiation but also inhibited adipogenic differentiation of rBMSCs, which was associated with the up-regulation of Runx2, BMP-2, and OSX mRNA expression and the down-regulation of PPARγ C/EBPα and aP2 mRNA expression.
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The present study was designed to evaluate the cardioprotective effect of latifolin on pituitrin(Pit) or isoproterenol(ISO)-induced myocardial injury in rats, and further investigate its underlying mechanisms. Rats were administrated sublingually with pituitrin or subcutaneously with isoproterenol to induce acute myocardial ischemia in rats, and lead II electrocardiograph was recorded. In rats with isoproterenol, ELISA assay or colorimetric method was used to detect the content or activity of myocardial injury markers in serum, and the SOD activity and MDA content in myocardium were detected by colorimetric assay; histopathological examination was conducted by HE staining; the frozen section of myocardial tissues was used for DCFH-DA fluorescent staining to detect the content of ROS in myocardium; Western blot was used to detect the protein expression levels of Nrf2, Keap1, HO-1 and NQO1 in myocardium. Results showed that latifolin significantly inhibited ST-segment changes induced by pituitrin or isoproterenol, and increased heart rate. Further mechanism study showed that latifolin reduced cardiac troponin I(cTnI) level, aspartate transaminase(AST) and lactate dehydrogenase(LDH) activities in serum, increased myocardial superoxide dismutase(SOD) activity and reduced myocardial malondialdehyde(MDA) level, and protected myocardium with less necrosis, infiltration of inflammatory cells and fracture of myocardial fibers. Furthermore, latifolin obviously reduced ROS level in myocardium, inhibited the expression of Kelch-like ECH-associated protein-1(Keap1), increased the nuclear translocation of nuclear factor erythroid 2 related factor 2(Nrf2), and promoted the expression of Heme oxygenase-1(HO-1) and NAD(P)H quinone oxidoreductase-1 (NQO1) in myocardial tissues. Our data suggest that latifolin has a potent protective effect against pituitrin or isoproterenol-induced myocardial injury, which may be related to inhibition of oxidative stress by activating Nrf2 signaling pathway.