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Aim To investigate the effect of phillygenin ( PHI) on lipopolysacchride ( LPS) and normal human plasma ( NHP) induced inflammatory injury on alveolar type II epithelial A549 cells and the related mechanism. Methods A549 cells were exposured to 1 mg • L
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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 effect of proliferation and activation of vascular smooth muscle cells (VSMCs) induced by activated the complement alternative pathway and intervention. Methods Normal human plasma was specifically activated the complement alternative pathway by incubated with cobra venom factor (CVF). Exposed VSMCs to the activated complement products, the change of cell morphology and the cell viability were assayed by inverted phase contrast microscope and MTT method, respectively. The supernatant was assayed for expression of E-selectin, ICAM-1 and VCAM-1 by using ELISA reagent kits. The protein expression levels of p-NF-kB p65, NF-kB p65 and IKK were assayed by Western blot. The nucleus transcriptional activity of NF-ΚB p65 was tested by the dual luciferase reporter assay system. Pyrrolidine dithiocarbamate (PDTC) was used to intervene the proliferation and activation of VSMCs. Results VSMCs were activated and induced to proliferation after exposed to the products of activated complement alternative pathway. The expressions of E-selectin, VCAM-1 and ICAM-1 were up-regulated. The contents of ICAM-1 and VCAM-1 reached the peak at 6 h and the E-selectin increased significantly at 12 h. Meanwhile, the phosphorylation level of NF-ΚB p65, nucleus transcriptional activity of NF-ΚB p65 and the protein expression of IKK and N F - Κ B p65 increased. The above mentioned changes were clearly inhibited by PDTC. Conclusions The activated complement alternative pathway can initiate proliferation and activation of VSMCs, and its mechanism goes hand in hand with activation of N F - Κ B signaling pathway. PDTC, a specific inhibitor of N F - K B, can effectively inhibit the proliferation and activation of VSMCs.