Protection of kinsenoside against AGEs-induced endothelial dysfunction in human umbilical vein endothelial cells.

AIMS: Kinsenoside is the major ingredient of Anoectochilus roxburghii which is a traditional Chinese herb using for the treatment of diabetes. The present study investigated the safety and vascular protection of kinsenoside related to advanced glycation end products (AGEs) in human umbilical vein endothelial cells (HUVECs) and the underlying mechanisms. MATERIALS AND METHODS: HUVECs were pre-incubated with AGEs (200µg/mL) for 1h, and then co-treated with different concentrations of kinsenoside (10-30µg/mL) for another 48h. After the supernatant was collected, the contents of nitric oxide (NO), the levels of reactive oxygen species (ROS) and inflammatory cytokines, and the expressions of AGEs receptor (RAGE) and nuclear factor kappa B (NF-κB) were measured. KEY FINDINGS: No significant changes in cell viability were found in kinsenoside-treated cells at the range of 10-70µg/mL. Pretreatment with kinsenoside induced a significant increase in NO production in AGEs-induced cells. In addition, kinsenoside not only inhibited the expression of RAGE but also decreased intracellular ROS generation induced by AGEs. Furthermore, kinsenoside suppressed the protein and gene expression of NF-κB, and reduced the release of intercellular adhesion molecule-1 (ICAM-1) and human monocyte chemoattractant protein-1 (MCP-1) in a dose-dependent manner remarkably. SIGNIFICANCE: These results indicated that kinsenoside might attenuate AGEs-induced endothelial dysfunction via AGEs-RAGE-NF-κB pathway. Considering the relatively low toxicity of kinsenoside, it might be a promising agent for treatment of vasculopathy in diabetic patients.

Involvement of mitochondria and caspase pathways in N-demethyl-clarithromycin-induced apoptosis in human cervical cancer HeLa cell.

AIM: To study the mechanisms by which N-demethyl-clarithromycin (NDC) induces human cervical cancer HeLa cell apoptosis in vitro. METHODS: The viability of N-demethyl-clarithromycin-induced HeLa cells was measured by MTT assay. Apoptotic cells with condensed nuclei were visualized by phase contrast microscopy. Nucleosomal DNA fragmentation was assayed by agarose gel electrophoresis. Measurement of mitochondrial transmembrane potential was analyzed by a FACScan flowcytometer. Caspase-3, poly-(ADP-ribose) polymerase (PARP), caspase-activated DNase (ICAD), Bcl-2, Bax, p53, and SIRT1 protein expression and the release of cytochrome c were detected by Western blot analysis. RESULTS: N-demethyl-clarithromycin, an anti-inflammatory substance, inhibited HeLa cell growth in a dose- and time-dependent manner. N-demethyl-clarithro-mycin induced HeLa cell death through the apoptotic pathways. The pan-caspase inhibitor (z-VAD-fmk), caspase-3 inhibitor (z-DEVD-fmk) and the caspase-9 inhibitor (z-LEHD-fmk) partially enhanced cell viability induced by N-demethyl-clarithromycin, but the caspase-8 inhibitor (z-IETD-fmk) had almost no effect. Caspase-3 was activated then followed by the degradation of caspase-3 substrates, the inhibitor of ICAD and PARP. Simultaneously, mitochondrial transmembrane potential was markedly reduced and the release of cytochrome c in the cytosol was increased. N-demethyl-clarithromycin upregulated the expression ratio of mitochondrial Bax/Bcl-2, and significantly increased the expression of the p53 protein. It also downregulated anti-apoptotic protein SIRT1 expression. CONCLUSION: N-demethyl-clarithromycin induced apoptosis in HeLa cells via the mitochondrial pathway.