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Aim To investigate the hepatotoxicity of rutecarpine(RUT)by using high-content screening technology.Methods HepG2 cells were exposed to different concentrations of RUT for different time, then cell viability was detected by MTT method.Cell count, nucleus injury, mitochondrial membrane potential(MMP), reactive oxygen species(ROS), internal flow of calcium, cell membrane integrity(DIR)were measured by high-content screening technology.The activation of MAPK, NF-κB and JAKs-STATs was assayed by high-content screening technology.The apoptosis was detected by flow cytometry.Results The viability was significantly reduced by 100 μmol·L-1 RUT(P<0.01)after HepG2 cell exposure to RUT for 24 h, the nuclear area decreased and the nuclear morphology was uneven, and after 48 h, the cell count was significantly reduced(P<0.01), the early apoptosis was detected(P<0.01).After HepG2 cell exposure to RUT for 6 h, the levels of ROS and internal flow of calcium significantly increased(P<0.01), and the cell membrane integrity was obviously damaged(P<0.01).After exposure to 100 μmol·L-1 RUT for 24 h, the phosphorylation of ERK, JNK, STAT3 and p38 significantly increased(P<0.01, P<0.05), but there was no significant change in total protein level.The expression of c-Jun and c-Fos was up-regulated at 3 h(P<0.01), and at 3h time point, the phosphorylation of NF-κB p65 significantly increased(P<0.01), but nuclear translocation was not significant.Conclusions Under certain conditions, RUT shows cytotoxicity on HepG2 cells, and its toxic mechanism is mainly related to injury caused by oxidative stress and inflammatory response.
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Aim To investigate the protective effect of baicalin on inflammatory response of human microvascular endothelial cells ( HMECs) induced by lipopo- lysaccharides ( LPS) and its mechanism. Methods LPS was applied to establish inflammatory model on HMECs in this work. HMECs were pretreated with different concentrations of baicalin ( 1. 0 x 10 "6 , 1. 0 x 10 ~7 and 1. 0 x 10 "8 mol • L"1) , and then exposed to LPS. The supernatant was removed and assayed for expression of the adhesion molecule ICAM-1, the inflam- atory mediator IL-6 and MCP-1 by using ELISA reagent kits. The inward flow of calcium ion was observed by fluorescence microscope, and the intracellular calcium ion level was measured by flow cytometry. The fluorescence intensity of nucleus NF-kB p65 was detected by immunoflourescent technique. The nucleus transcriptional activity of NF-kB was measured by the dual lu- ciferase reporter assay system. Moreover, the expression of protein NF-kB p65, phospo-NF-kb p65 ( p p65 ) and Toll like receptor 4 (TLR4 ) were detected by Western blot. Results The internal flow of calcium, the phosphorylation of NF-kB p65 and the transcription activity significantly increased, and the expression of ICAM-1, IL-6 and MCP-1 up-regulated after HMECs were exposed to LPS. Baicalin inhibited the inward flow of calcium ion, the nuclear translocation of NF-kB p65 and the up-regulation of transcriptional activity, decreased the expression of ICAM-1, IL-6 and MCP-1, and down-regulated the expression of NF-kB p65, p- p65 and TLR4 in a dose-dependent manner. Conclusions Baicalin possesses a protective effect on inflammatory response of endothelial cells induced by LPS, and its mechanism may be highly related to the inhibition of the internal flow of calcium and the activation of NF-kB signaling pathway, and thus reduce the level of inflammatory response.
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AIM@#To study the bufadienolides in the Chinese traditional drug "Ch'an Su" and their cytotoxic activity.@*METHOD@#Various chromatographic techniques were used to isolate the constituents, and their structures were elucidated through physical and spectroscopic data.@*RESULTS@#Twenty compounds were isolated, and eighteen were evaluated in vitro for their cytotoxic activity against A-549 and K-562 cells.@*CONCLUSION@#Compound 1 (bufalin 3β-acrylic ester) was a new bufadienolide and exhibited the most potent activity against the two tumor cell lines with IC50 values of 7.16 and 6.83 nmol · L(-1). The relationships between structure and activity are discussed.