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.

Protective Effect of Liraglutide on Hypoxia and High Glucose-induced Oxidative Stress Injury in Cardiomyocyte / 中国药师

Objective:To explore the effects and possible mechanism of liraglutide on hypoxia and high glucose-induced oxidative stress injury in cardiomyocytes. Methods:The neonatal rat cardiomyocytes were separated and cultured in vitro. The hypoxia and high glucose-induced injury model was established in neonatal rat cardiomyocytes. The cells were divided into six groups:the normal control group, liraglutide control group, hypoxia and high glucose model group, liraglutide treatment group, GLP-1R antagonist group and hyperosmotic control group. The metabolic ability of the cells was detected by MTT assay, the activities of LDH and CK-MB were detected by colorimetric method,SOD activity and MDA content were determined by xanthine oxidase method and thiobarbituric acid method,ROS level was measured by chemiluminescence method. The mRNA and protein expression of adaptor protein p66Shc was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. Results:Compared with those in the normal control group, the cells in hypoxia and high glucose model group had poorly metabolic ability,the content of LDH, CK-MB, MDA and ROS increased (P < 0.01), the activity of SOD decreased (P <0.01), and the expression of adaptor protein p66Shc greatly increased(P <0.01). After the treatment with liraglutide,the above mentioned parameters were all improved(P < 0.01). Exendin(9-39),an antagonist of GLP-1R,attenuated the protective effect of liraglutide. Conclusion:Liraglutide has a protective effect on cardiomyocytes by down-regulating adaptor protein p66Shc expression and reducing ROS formation.

Expression of p66Shc in heart failure patients and its relationship with oxidative stress / 中国综合临床

Objective To investigate the expression of cardiac muscle protein p66Shc in patients with congestive heart failure and its relationship with oxidative stress.Methods From October 2016 to March 2017, eighty patients with acute heart failure treated in the cardiology department of Shanghai Pudong Hospital were collected in this study,including 22 cases of grade II,26 cases of grade III,32 cases of grade IV,and 56 healthy subjects were selected in the control group.SOD activity,MDA content and p66Shc mRNA expression of the acute stage of heart failure,the recovery stage of heart failure and the control group were measured.The changes of SOD,MDA and p66Shc in patients with heart failure and the control group were analyzed.The expression of p66Shc,along with MDA and SOD were analyzed.Results There were significant differences in the SOD,MDA and p66Shc mRNA of patients in the acute stage of heart failure,the recovery stage and the control group were (SOD:(65.67±8.31)kU/L vs.(75.77±7.56)kU/L vs.(90.03±7.76)kU/L,MDA:(10.22±1.56)μmol/L vs.(8.34 ± 1.75)μmol/L vs.(5.21 ± 2.16)μmol/L,p66Shc mRNA:(5.79 ± 1.71)vs.(3.95 ± 1.24) vs.1.87±0.93))(F=2.903,2.762,2.696,P<0.05).With the aggravation of heart failure,the level of SOD decreased significantly(NYHA grade Ⅱ:(67.54 ± 7.29)kU/L;grade Ⅲ:(61.62 ± 6.96)kU/L;gradeⅣ:(57.94±6.75)kU/L,F=2.872,P<0.05)and the levels of MDA(NYHA grade Ⅱ:(9.32±1.18)μmol/L;grade Ⅲ:(10.95 ± 1.26)μmol/L;grade Ⅳ:(12.13 ± 1.07)μmol/L,F=2.703,P<0.05)and p66Shc (NYHA grade Ⅱ:(4.92 ± 0.96);grade Ⅲ:(5.66 ± 1.17);grade Ⅳ:(6.28 ± 1.15),F=2.681,P<0.05) increased significantly.The expression of p66Shc and mRNA was positively correlated with MDA in acute attack (r=0.722,P<0.05),and negatively correlated with SOD(r=-0.670,P<0.05).Conclusion P66shc is closely related to oxidative stress in heart failure,and p66Shc may be involved in the process of heart failure oxidative stress,which can provide a basis for the new treatment of heart failure.

Expression of p66Shc in heart failure patients and its relationship with oxidative stress / 中国综合临床

Objective To investigate the expression of cardiac muscle protein p66Shc in patients with congestive heart failure and its relationship with oxidative stress.Methods From October 2016 to March 2017, eighty patients with acute heart failure treated in the cardiology department of Shanghai Pudong Hospital were collected in this study,including 22 cases of grade II,26 cases of grade III,32 cases of grade IV,and 56 healthy subjects were selected in the control group.SOD activity,MDA content and p66Shc mRNA expression of the acute stage of heart failure,the recovery stage of heart failure and the control group were measured.The changes of SOD,MDA and p66Shc in patients with heart failure and the control group were analyzed.The expression of p66Shc,along with MDA and SOD were analyzed.Results There were significant differences in the SOD,MDA and p66Shc mRNA of patients in the acute stage of heart failure,the recovery stage and the control group were (SOD:(65.67±8.31)kU/L vs.(75.77±7.56)kU/L vs.(90.03±7.76)kU/L,MDA:(10.22±1.56)μmol/L vs.(8.34 ± 1.75)μmol/L vs.(5.21 ± 2.16)μmol/L,p66Shc mRNA:(5.79 ± 1.71)vs.(3.95 ± 1.24) vs.1.87±0.93))(F=2.903,2.762,2.696,P<0.05).With the aggravation of heart failure,the level of SOD decreased significantly(NYHA grade Ⅱ:(67.54 ± 7.29)kU/L;grade Ⅲ:(61.62 ± 6.96)kU/L;gradeⅣ:(57.94±6.75)kU/L,F=2.872,P<0.05)and the levels of MDA(NYHA grade Ⅱ:(9.32±1.18)μmol/L;grade Ⅲ:(10.95 ± 1.26)μmol/L;grade Ⅳ:(12.13 ± 1.07)μmol/L,F=2.703,P<0.05)and p66Shc (NYHA grade Ⅱ:(4.92 ± 0.96);grade Ⅲ:(5.66 ± 1.17);grade Ⅳ:(6.28 ± 1.15),F=2.681,P<0.05) increased significantly.The expression of p66Shc and mRNA was positively correlated with MDA in acute attack (r=0.722,P<0.05),and negatively correlated with SOD(r=-0.670,P<0.05).Conclusion P66shc is closely related to oxidative stress in heart failure,and p66Shc may be involved in the process of heart failure oxidative stress,which can provide a basis for the new treatment of heart failure.

Protein kinase C beta II upregulates intercellular adhesion molecule-1 via mitochondrial activation in cultured endothelial cells

Activation of protein kinase C (PKC) is closely linked with endothelial dysfunction. However, the effect of PKCβII on endothelial dysfunction has not been characterized in cultured endothelial cells. Here, using adenoviral PKCβII gene transfer and pharmacological inhibitors, the role of PKCβII on endothelial dysfucntion was investigated in cultured endothelial cells. Phorbol 12-myristate 13-acetate (PMA) increased reactive oxygen species (ROS), p66shc phosphorylation, intracellular adhesion molecule-1, and monocyte adhesion, which were inhibited by PKCβi (10 nM), a selective inhibitor of PKCβII. PMA increased the phosphorylation of CREB and manganese superoxide dismutase (MnSOD), which were also inhibited by PKCβi. Gene silencing of CREB inhibited PMA-induced MnSOD expression, suggesting that CREB plays a key role in MnSOD expression. Gene silencing of PKCβII inhibited PMA-induced mitochondrial ROS, MnSOD, and ICAM-1 expression. In contrast, overexpression of PKCβII using adenoviral PKCβII increased mitochondrial ROS, MnSOD, ICAM-1, and p66shc phosphorylation in cultured endothelial cells. Finally, PKCβII-induced ICAM-1 expression was inhibited by Mito-TEMPO, a mitochondrial ROS scavenger, suggesting the involvement of mitochondrial ROS in PKC-induced vascular inflammation. Taken together, the results suggest that PKCβII plays an important role in PMA-induced endothelial dysfunction, and that the inhibition of PKCβII-dependent p66shc signaling acts as a therapeutic target for vascular inflammatory diseases.

Influence of chronic fluorosis on protein kinase Cβ/p66shc signal pathway in the brain of rats / 中华地方病学杂志

Objective To investigate the influence of chronic fluorosis on protein kinase Cβ (PKCβ)/p66shc signal pathway in the brain of rats,and reveal the molecular mechanism of brain damage.Methods According to body weight by the random number table method thirty SD rats were divided into three groups of 10 each (half females and half males),the normal control group [less than 0.5 mg/L of fluorine (prepared with NaF) in drinking water],low fluoride exposure group (10.0 mg/L fluorine),and high fluoride exposure group (50.0 mg/L fluoride).The experiment period was 6 months.The protein level of PKCβ,p66shc,phospho-p66shc and preserved ammonia acyl isomerase (Pin1) in rat brain was detected by Western blotting.The level of neuron nuclear antigen (NeuN),p66shc and phospho-p66sh in brain of rats was detected by immunohistochemistry.Results By Western blotting,the levels of PKCβ,Pin1 and phospho-p66shc protein in brain tissue in high fluoride exposure group [(193.00 ± 57.53)％,(228.21 ± 71.14)％,(201.54 ±:50.86)％] were higher than those of the normal control groups [(100.00 ± 21.24)％,(100.00 ± 40.55)％,(100.00 ± 13.35)％,all P ＜ 0.05].By immunohistochemistry,the numbers of NeuN staining in brain tissue of the rats in both high and low fluoride exposure groups [(49.50 ± 12.57)％,(65.66 ±14.58)％] were lower than that of the control group [(100.00 ± 18.32)％,all P ＜ 0.01].The level of phospho-p66shc protein in brain tissue in high fluoride exposure group [(242.66 ± 93.01)％] was higher than those of the low fluoride exposure and the normal control groups [(152.53 ± 60.65)％,(100.00 ± 25.63)％,all P ＜ 0.01].Conclusion Chronic fluorosis has increased the expressions of PKCβ,Pin1 and phospho-p66shc at protein level in brain of rats,which may be related to the molecular mechanism of brain damage resulted from chronic fluorosis.

Study on lentiviral vector target inducing p66 shc gene silencing / 重庆医学

Objective To construct p66shc gene interfering lentivirus vectors recombination and transfect it to 293T cells ,RNA interfering was carried out to induce p66shc gene silence ,so as to provide basis for further study of the p66shc function .Methods Screening of three RNA targets which were named after p66shc‐shc1 ,p66shc‐shc2 ,p66shc‐shc3 ,cloned into the pLenR‐GPH vec‐tor ,which contained green fluorescent protein(GFP) and transformed into DH5αcells .The positive clone were picked out for right sequencing and transfected to 293T cells with pRsv‐REV ,pMDlg‐pRRE ,pMD2G .The expression of GFP in inverted fluorescence microscope confirmed the virus packaging success .Fluorescence quantitative PCR and Western blot technology were used to investi‐gate the expression of p66shc at the molecular and protein levels ,p66shc‐shc1 target of effective silencing p66shc gene was selected to prepare for subsequent tests .Results The shRNA lentivirus vector was constructed which could express p66shc and was trans‐fected into 293T cells successfully .Fluorescence quantitative PCR and Western blot technology were used to investigate p66shc gene silence by RNA interference .Conclusion The lentivirus RNAi vector of targeted expression p66shc could induce p66shc gene si‐lence at the molecular and protein levels after transfected into 293T cells by RNA interference .

Nafamostat Mesilate Inhibits TNF-alpha-Induced Vascular Endothelial Cell Dysfunction by Inhibiting Reactive Oxygen Species Production

Nafamostat mesilate (NM) is a serine protease inhibitor with anticoagulant and anti-inflammatory effects. NM has been used in Asia for anticoagulation during extracorporeal circulation in patients undergoing continuous renal replacement therapy and extra corporeal membrane oxygenation. Oxidative stress is an independent risk factor for atherosclerotic vascular disease and is associated with vascular endothelial function. We investigated whether NM could inhibit endothelial dysfunction induced by tumor necrosis factor-alpha (TNF-alpha). Human umbilical vein endothelial cells (HUVECs) were treated with TNF-alpha for 24 h. The effects of NM on monocyte adhesion, vascular cell adhesion molecule-1 (VCAM-1) and intracellular adhesion molecule-1 (ICAM-1) protein expression, p38 mitogen-activated protein kinase (MAPK) activation, and intracellular superoxide production were then examined. NM (0.01~100 microg/mL) did not affect HUVEC viability; however, it inhibited the increases in reactive oxygen species (ROS) production and p66shc expression elicited by TNF-alpha (3 ng/mL), and it dose dependently prevented the TNF-alpha-induced upregulation of endothelial VCAM-1 and ICAM-1. In addition, it mitigated TNF-alpha-induced p38 MAPK phosphorylation and the adhesion of U937 monocytes. These data suggest that NM mitigates TNF-alpha-induced monocyte adhesion and the expression of endothelial cell adhesion molecules, and that the anti-adhesive effect of NM is mediated through the inhibition of p66shc, ROS production, and p38 MAPK activation.

Trichostatin A Modulates Angiotensin II-induced Vasoconstriction and Blood Pressure Via Inhibition of p66shc Activation

Histone deacetylase (HDAC) has been recognized as a potentially useful therapeutic target for cardiovascular disorders. However, the effect of the HDAC inhibitor, trichostatin A (TSA), on vasoreactivity and hypertension remains unknown. We performed aortic coarctation at the inter-renal level in rats in order to create a hypertensive rat model. Hypertension induced by abdominal aortic coarctation was significantly suppressed by chronic treatment with TSA (0.5 mg/kg/day for 7 days). Nicotinamide adenine dinucleotide phosphate-driven reactive oxygen species production was also reduced in the aortas of TSA-treated aortic coarctation rats. The vasoconstriction induced by angiotensin II (Ang II, 100 nM) was inhibited by TSA in both endothelium-intact and endothelium-denuded rat aortas, suggesting that TSA has mainly acted in vascular smooth muscle cells (VSMCs). In cultured rat aortic VSMCs, Ang II increased p66shc phosphorylation, which was inhibited by the Ang II receptor type I (AT1R) inhibitor, valsartan (10 microM), but not by the AT2R inhibitor, PD123319. TSA (1~10 microM) inhibited Ang II-induced p66shc phosphorylation in VSMCs and in HEK293T cells expressing AT1R. Taken together, these results suggest that TSA treatment inhibited vasoconstriction and hypertension via inhibition of Ang II-induced phosphorylation of p66shc through AT1R.

Tat-Mediated p66shc Transduction Decreased Phosphorylation of Endothelial Nitric Oxide Synthase in Endothelial Cells

We evaluated the role of Tat-mediated p66shc transduction on the activation of endothelial nitric oxide synthase in cultured mouse endothelial cells. To construct the Tat-p66shc fusion protein, human full length p66shc cDNA was fused with the Tat-protein transduction domain. Transduction of TAT-p66shc showed a concentration- and time-dependent manner in endothelial cells. Tat-mediated p66shc transduction showed increased hydrogen peroxide and superoxide production, compared with Tat-p66shc (S/A), serine 36 residue mutant of p66shc. Tat-mediated p66shc transduction decreased endothelial nitric oxide synthase phosphorylation in endothelial cells. Furthermore, Tat-mediated p66shc transduction augmented TNF-alpha-induced p38 MAPK phosphorylation in endothelial cells. These results suggest that Tat-mediated p66shc transduction efficiently inhibited endothelial nitric oxide synthase phosphorylation in endothelial cells.

Oxidative stress in human skin fibroblasts induced by UVB irradiation / 中华皮肤科杂志

Objective To observe the aging,apoptosis,cell cycle arrest and oxidative stress in human skin fibroblast(HSF)induced by UVB,and to detect the expression profiles of p66Shc,a determinant of oxidative stress response and life span,in this process.Methods HSF cells were exposed to UVB at a subcytotoxic dosage twice a day for three days.The cells without exposure served as control.After another 24-hour culture,SA-β-Gal staining was performed to evaluate the senescence state of the cells,flow cytometry to observe cell apoptosis;cell cycle arrest was detected by serum starvation and flow cytometry:ELISA was applied to detect intracellular levels of superoxide dismutase(SOD)and malondialdehvde(MDA),and Western blotting to analyze the expression of p66Shc protein.Results The percentage of cells positive for SA-β-Gal staining increased from 0 to 98.3% after UVB radiation,which strongly suggested an aging state of HSF cells.The percentage of apoptotic cells increased from 0.96% to 37%.and 80.07% of the HSF cells were arrested in G0/G1 phase following the irradiation.Intracellular SOD activity decreased from(52.35±4.97)ng/g to(7.81±0.68)ng/g(P＜0.01).while intracellular MDA was found to increase from(3.52±0.34)ng/g to(33.91±3.20)ng/g(P＜0.05).The p66Shc protein was found to be weakly expressed in HSF in 24 hours following the exposure to UVB,and a stronger expression was noted 48 hours later.Conclusions HSF cells are induced into a state of senescence associated with oxidative stress after UVB irradiation,which may be applied as an in vitro model in aging research.The expression of p66Shc is increased in HSF during this process,and further studies are needed to explore the relation between p66Shc and oxidative stress as well as cellular aging.

p66shc Adaptor Protein Suppresses the Activation of Endothelial Nitric Oxide Synthase in Mouse Embryonic Fibroblasts

Among the Shc proteins, p66shc is known to be related to oxidative stress responses and regulation of the production of reactive oxygen species (ROS). The present study was undertaken to investigate the role of p66shc on endothelial nitric oxide synthase (eNOS) activity in the mouse embryonic fibroblasts (MEFs). When wild type (WT) or p66shc (-/-) MEFs were transfected with full length of eNOS cDNA, the expression and activity of eNOS protein were higher in the p66shc (-/-) MEFs. These phenomena were reversed by reconstitution of p66shc cDNA transfection in the p66shc (-/-) MEFs. The basal superoxide production in the p66shc (-/-) MEFs was not significantly different from that of WT of MEFs. However, superoxide production induced by NADPH in the p66shc (-/-) MEF was lesser than that in WT MEFs. When compared with WT MEFs, cell lysate of p66shc (-/-) MEFs showed significantly increased H-ras activity without change of endogenous H-ras expression. Our findings suggest the pivotal role of p66shc adaptor protein played in inhibition of endothelial nitric oxide production via modulation of the expression and/or activity of eNOS protein.

Regulatory Mechanism of p66 Shc Expression by TSH in FRTL-5 Cells / 대한내분비학회지

BACKGROUND: Thyroid goiters are very common, however, the mechanism of development is not fully understood. A TSH receptor has been known to activate two different signaling pathways the cAMP/protein kinase A(PKA) and phospholipase C(PLC)/protein kinase C(PKC) systems. However, both systems are limited in the degree to which they explain the discrepancy between a goiter and TSH receptor activation. It has recently been reported that the expression of p66 Shc was increased by TSH stimulation in thyrocytes, suggesting that the p66 Shc molecule may play a critical role in the transition of the TSH-induced growth signals. METHODS AND RESULTS: In this study, we examined the expression of p66 Shc by stimulation of TSH, and the regulatory mechanisms of the TSH-induced expression of the p66 Shc in FRTL-5 cells. In FRTL-5 cells, TSH could increase the expression of the p66 Shc, and the this expression was decreased to basal levels after the removal of TSH. The TSH-induced p66 Shc expression was competitively inhibited by TSH receptor blocking antibodies. The increments of the expression of the p66 Shc protein caused by TSH were both time and concentration dependent, and it was same in the mRNA levels. Cholera toxin increased the expression of the p66 Shc, while pertussis toxin did not. The activators of the cAMP/PKA pathway (8-bromo-cAMP and forskolin) also stimulated the expression of p66 Shc, and the PKA inhibitor H89 decreased the expression, while the inhibition of the PKC pathway by GF109203X, or PMA, affected the expression of p66 Shc very little. CONCLUSION: Our data suggests that p66 Shc may play an important role in regulating the growth of thyrocytes. The TSH receptor - Gs protein - adenylate cyclase - cAMP - PKA pathway mainly mediates the TSH effects on the expression of p66 Shc molecules.