Ketamine ameliorates hypoxia-induced endothelial injury in human umbilical vein endothelial cells

OBJECTIVES: Hypoxia leads to endothelial cell inflammation, apoptosis, and damage, which plays an important role in the complications associated with ischemic cardiovascular disease. As an oxidoreductase, p66Shc plays an important role in the regulation of reactive oxygen species (ROS) production and apoptosis. Ketamine is widely used in clinics. This study was designed to assess the potential protective effect of ketamine against hypoxia-induced injury in human umbilical vein endothelial cells (HUVECs). Moreover, we explored the potential mechanism by which ketamine protected against hypoxia-induced endothelial injury. METHODS: The protective effects of ketamine against hypoxia-induced injury was assessed using cell viability and adhesion assays, quantitative polymerase chain reaction, and western blotting. RESULTS: Our data showed that hypoxia reduced HUVEC viability, increased the adhesion between HUVECs and monocytes, and upregulated the expression of endothelial adhesion molecules at the protein and mRNA levels. Moreover, hypoxia increased ROS accumulation and upregulated p66Shc expression. Furthermore, hypoxia downregulated sirt1 expression in HUVECs. Alternatively, ketamine was shown to reverse the hypoxia-mediated reduction of cell viability and increase in the adhesion between HUVECs and monocytes, ameliorate hypoxia-induced ROS accumulation, and suppress p66Shc expression. Moreover, EX527, a sirt1 inhibitor, reversed the protective effects of ketamine against the hypoxia-mediated reduction of cell viability and increase in adhesion between HUVECs and monocytes. CONCLUSION: Ketamine reduces hypoxia-induced p66Shc expression and attenuates ROS accumulation via upregulating sirt1 in HUVECs, thus attenuating hypoxia-induced endothelial cell inflammation and apoptosis.

Roles of PKCβ/P66Shc oxidative stress signal pathway in mediating hyperoxia-induced ROS production in alveolar epithelial cells / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To explore the roles of PKCβ/P66Shc oxidative stress signal pathway in mediating hyperoxia-induced reactive oxgen species (ROS) production in alveolar epithelial cells (A549) and the protective effects of PKCβ inhibitor on hyperoxia-induced injuries of alveolar epithelial cells.</p><p><b>METHODS</b>A549 cells were cultured in vitro and randomly divided into three groups: control, hyperoxia and PKCβ inhibitor LY333531 treatment. The hyperoxia group was exposed to a mixture of O2 (950 mL/L) and CO2 (50 mL/L) for 10 minutes and then cultured in a closed environment. The LY333531 group was treated with PKCβ inhibitor LY333531 of 10 µmol/L for 24 hours before hyperoxia induction. Cells were collected 24 hours after culture and the levels of PKCβ, Pin1, P66Shc and P66Shc-Ser36 were detected by Western blot. The intracellular translocation of P66Shc, the production of ROS and cellular mitochondria membrane potential were measured using the confocal microscopy.</p><p><b>RESULTS</b>Compared with the control group, the levels of PKCβ, Pin1, P66Shc and P-P66Shc-Ser36 in A549 cells 24 hours after culture increased significantly in the hyperoxia group. These changes in the hyperoxia group were accompanied with an increased translocation rate of P66Shc from cytoplasm into mitochondria, an increased production of mitochondrial ROS, and a reduced mitochondrial membrane potential. Compared with the hyperoxia group, the levels of Pin1, P66Shc and P66Shc-Ser36 in A549 cells, the translocation rate of P66Shc from cytoplasm into mitochondria and the production of mitochondrial ROS decreased significantly, while the mitochondrial membrane potential increased significantly in the LY333531 treatment group. However, there were significant differences in the above mentioned measurements between the LY333531 treatment and control groups.</p><p><b>CONCLUSIONS</b>Hyperoxia can increase the expression of PKCβ in alveolar epithelial cells and production of mitochondrial ROS and decrease mitochondrial membrane potential. PKCβ inhibitor LY333531 can partially disrupt these changes and thus alleviate the hyperoxia-induced alveolar epithelial cell injury.</p>

Regulatory effects of Shc-related phosphotyrosine adaptor proteins on aging / 药学学报

Aging-related oxidative stress and free radical theory has become accepted increasingly as explaination, at least in part of the aging process. In murine models of aging, a genetic deficiency of the p66(Shc) (66-kilodalton isoform of Shc gene products) gene, which encodes a phosphotyrosine signal adapter protein, extends life span by 30%, and confers resistance to oxidative stress. Upon oxidative stress, p66(Shc) is phosphorylated at Ser36, contributing to inactivation of the forkhead-type transcription factors (FKHR/ FoxO1), which regulates the gene expression of cellular antioxidants. The p66(Shc) has a direct connection with the life span related signaling, which is conserved evolutionarily. Shc is basically not expressed in mature neurons of the adult brain and spinal cord. Instead, two Shc homologues, Sck/ShcB and N-Shc/ ShcC, which have been proved to be effective on oxidative stress and aging, are expressed in neural system. The expression of Shc-related genes is affected in the aging process, which may be relevant to cellular dysfunction, stress response and/or cognitive decline during aging.