Minmin Zhu

Propofol protects against high glucose-induced endothelial dysfunction in human umbilical vein endothelial cells.

BACKGROUND: Hyperglycemia, via peroxynitrite-mediated endothelial nitric oxide synthase (eNOS) enzymatic uncoupling, induced endothelial dysfunction. Propofol has been reported to improve high glucose–induced endothelial dysfunction. However, its mechanisms of action remain unclear. We hypothesized that propofol could improve hyperglycemia-induced endothelial dysfunction by decreasing the peroxynitrite level and thus restoring eNOS coupling. METHODS: At the end of 3 days of incubation in medium with 30 mM glucose, human umbilical vein endothelial cells were treated with different concentrations (0.2, 1, 5, and 25 &mgr;M) of propofol for different times (0.5, 1, 2, and 4 hours). In parallel experiments, cells were cultured in 5 mM glucose for 3 days as a control. Nitric oxide (NO) production was measured with a nitrate reductase assay. Superoxide anion (O2·−) accumulation was measured with the reduction of ferricytochrome c and dihydroethidine fluorescence assay. The treatment that had maximal effect on 30 mM glucose–induced NO production and O2·− accumulation was applied in the following studies to examine the underlying signaling pathways. eNOS total protein, eNOS dimer and monomer expression, eNOS phosphorylation at Ser1177, inducible NO synthase total protein, inducible NO synthase dimer and monomer expression, peroxynitrite, and guanosine triphosphate cyclohydrolase I expression were measured by Western blot. Tetrahydrobiopterin (BH4) level was measured with liquid chromatography–mass spectrometry. RESULTS: Compared with 5 mM glucose treatment, 30 mM glucose significantly decreased NO production by 60% (P < 0.001) and increased O2·− accumulation by 175% (P = 0.0026), which were both attenuated by propofol in a concentration- and time-dependent manner. Compared with 5 mM glucose treatment, total eNOS protein expression was increased by 30 mM glucose (P < 0.001), whereas the ratio of eNOS dimer/monomer (P = 0.0001) and eNOS phosphorylation (P < 0.001) were decreased by 30 mM glucose. Propofol did not affect 30 mM glucose–induced total eNOS protein expression, but restored the ratio of eNOS dimer/monomer (P = 0.0005) and increased eNOS phosphorylation (P < 0.001). 30 mM glucose–induced O2·− accumulation was inhibited by the eNOS inhibitor hydrochloride. Furthermore, compared with 5 mM glucose treatment, 30 mM glucose decreased the BH4 level (P = 0.0001) and guanosine triphosphate cyclohydrolase I expression (P < 0.001), whereas it increased peroxynitrite level (P = 0.0003), which could all be reversed by propofol (P = 0.0045, P < 0.001, P = 0.0001 vs 30 mM glucose treatment, respectively). CONCLUSIONS: Propofol has beneficial effects on 30 mM glucose–induced NO reduction and O2·− accumulation in human umbilical vein endothelial cells. This may be mediated through inhibiting peroxynitrite-mediated BH4 reduction, and restoring eNOS coupling.

Propofol attenuates angiotensin II-induced apoptosis in human coronary artery endothelial cells

BACKGROUND Angiotensin II (Ang II) induces oxidative stress and apoptosis in vascular endothelial cells. We hypothesized that propofol may attenuate Ang II-induced apoptosis in human coronary artery endothelial cells (HCAECs) and aimed to identify the underlying mechanisms. METHODS Endothelial cells were pre-treated with propofol and then stimulated with Ang II. Apoptosis was examined by TUNEL, DNA laddering, and caspase-3 activity assays. The effect of propofol on Ang II-modulated NADPH oxidase expression and activity, nitric oxide synthase III (NOSIII) expression and phosphorylation and activity, lipid peroxidation, superoxide anion generation, nitric oxide production, caspase activity, and protein expression of cytochrome c, Bcl-2, and C-IAP-1 were measured. RESULTS Ang II induced apoptosis, which was attenuated by 50 µM propofol (P<0.05). Propofol ameliorated Ang II-induced NADPH oxidase expression and activation (P<0.01), lipid peroxidation (P<0.05), and superoxide anion generation (P<0.05), whereas restoring NOSIII phosphorylation and activity (P<0.01) were down-regulated by Ang II. Propofol attenuated Ang II-modulated cytochrome c release, and the expression of Bcl-2 and C-IAP-1. In addition, propofol inhibited Ang II-induced caspase-9 (P<0.01) and caspase-3 activity (P<0.01). CONCLUSIONS Propofol protected HCAECs from Ang II-induced apoptosis by interfering with the generation of oxidative stress and redox-sensitive apoptotic pathways.

Propofol Protects Against High Glucose–induced Endothelial Apoptosis and Dysfunction in Human Umbilical Vein Endothelial Cells

BACKGROUND:Perioperative hyperglycemia is a common clinical metabolic disorder. Hyperglycemia could induce endothelial apoptosis and dysfunction. Propofol is a widely used IV anesthetic drug in clinical settings. In the present study, we examined whether and how propofol reduced high glucose–induced endothelial apoptosis and dysfunction in human umbilical vein endothelial cells (HUVECs). METHODS:HUVECs were cultured with different concentrations (5, 10, 15, and 25 mM) of glucose for different times (4, 8, 12, and 24 hours). To study the effect of propofol, cells were incubated with different concentrations (0.2, 1, 5, and 25 &mgr;M) of propofol for 2 hours. In parallel experiments, cells were incubated in 5 mM glucose as control. Nitric oxide (NO) production was measured with a nitrate reductase assay. Cell viability was determined with a Cell Counting Kit-8. Protein expression of active caspase 3, cytochrome c, endothelial NO synthase (eNOS), p-eNOS-Thr495, p66Shc, protein kinase C &bgr;II (PKC&bgr;II), and p-PKC&bgr;II-Ser660 was measured by Western blot analysis. Accumulation of superoxide anion (O2˙−) was measured with the reduction of ferricytochrome c. Cell apoptosis was determined with terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling staining. RESULTS:Compared with control, high glucose decreased NO production (P < 0.0001) and reduced cells viability (P < 0.0001) in HUVECs. Compared with high glucose treatment, pretreatment of cells with propofol (5 &mgr;M, 2 hours) reduced high glucose–induced inhibitory p-eNOS-Thr495 phosphorylation (P < 0.0001), increasing NO production (P = 0.0007), decreased high glucose–induced p66Shc expression (P < 0.0001) and p66Shc mitochondrial translocation (P < 0.0001), O2˙− accumulation (P < 0.0001), mitochondrial cytochrome c release (P < 0.0001), active caspase 3 expression (P < 0.0001), and enhancing endothelial viability (P < 0.0001). Furthermore, propofol inhibited high glucose–induced PKC&bgr;II expression (P = 0.0002) and p-PKC&bgr;II-Ser660 phosphorylation (P < 0.0001). Moreover, the observed protective effect of propofol was quite similar to that of PKC&bgr;II inhibitor. CONCLUSIONS:Propofol, by a mechanism of decreasing high glucose–induced PKC&bgr;II expression and p-PKC&bgr;II-Ser660 phosphorylation, inhibits high glucose–induced p66Shc mitochondrial translocation, therefore protecting HUVECs from high glucose–induced endothelial dysfunction and apoptosis.

MFHAS1 promotes colorectal cancer progress by regulating polarization of tumor-associated macrophages via STAT6 signaling pathway

Malignant fibrous histiocytoma amplified sequence 1 (MFHAS1) is a predicted oncoprotein that demonstrates tumorigenic activity in vivo; however, the mechanisms involved are unknown. Macrophages are divided into the pro-inflammatory M1 and anti-inflammatory/protumoral M2 subtypes. Tumor cells can induce M2 polarization of tumor-associated macrophages (TAMs) to promote metastasis; but the underlying pathways require to be elucidated. In this study, we detected a positive association between MFHAS1 expression in TAMs and human colorectal cancer (CRC) TNM stage. Supernatant of CT26 murine CRC cells induced MFHAS1 expression in RAW264.7 murine macrophages. Additionally, CT26 supernatant induced the M2 marker CD206 and activated the pro-M2 STAT6 and KLF4 signaling in control but not MFHAS1-silenced RAW264.7 macrophages. Moreover, supernatant of control, but not MFHAS1-silenced macrophages promoted CT26 cell proliferation, migration and epithelial-mesenchymal transition. Compared with control macrophages, MFHAS1-silenced macrophages showed significantly reduced protumoral effects in vivo. Together, these results suggested that CRC cells induce M2 polarization of TAMs through MFHAS1 induction and subsequent STAT6 and KLF4 activation to promote CRC progress. Finally, similar to CT26 supernatant stimulation, peroxisome proliferator-activated receptor-γ (PPARγ) activation by rosiglitazone induced M2 polarization of RAW264.7 macrophages through MFHAS1-dependent pathway. Our results highlight the role of MFHAS1 as a regulator of macrophages polarization and CRC progress.

Angiotensin II-induced mouse hippocampal neuronal HT22 cell apoptosis was inhibited by propofol: Role of neuronal nitric oxide synthase and metallothinonein-3.

BACKGROUND The activation of renin angiotensin system is involved in multiple pathological processes. The neuroprotective effect of propofol has been reported. We hypothesized that propofol may attenuate Angiotensin II (Ang II)-induced apoptosis in mouse hippocampal HT22 cells and aimed to identify the underlying mechanisms. METHODS Mouse hippocampal HT22 cells were pre-treated with propofol, and stimulated with Ang II. Apoptosis was examined by transferase dUTP nick end labeling (TUNEL) staining and caspase-3 activity assay. The effect of propofol on Ang II-modulated neuronal nitric oxide synthase (nNOS) expression, nitric oxide (NO) production, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase expression and activity, caspase activity and metallothinonein-3 (MT-3) expression were measured. RESULTS Compared with control, Ang II concentration- and time-dependently induced apoptosis, which was attenuated by propofol in a concentration-dependent manner. Ang II (1 μM, 3 h) induced the expression of nNOS and NADPH oxidase, caused NO and superoxide anion accumulation, thus leading to excessive oxidative stress. Ang II also induced cytochrome C release and the activation of caspase 9 as well as caspase 3. In addition, Ang II reduced the expression of MT-3. Importantly, these effects were alleviated by 50 μM propofol, nNOS inhibitor S-methyl-l-thiocitrulline (SMTC) and angiotensin type 1 receptor (AT1R) blocker losartan, but not AT2R blocker PD123319. CONCLUSIONS Ang II via AT1R induced oxidative stress and apoptosis in hippocampal HT22 cells, and the neuroprotective anti-apoptotic effect of propofol was mediated through inhibiting oxidative stress.

Propofol Protects Against Angiotensin II-Induced Mouse Hippocampal HT22 Cells Apoptosis Via Inhibition of p66Shc Mitochondrial Translocation

Abstract Hippocampal neuronal oxidative stress and apoptosis have been reported to be involved in cognitive impairment, and angiotensin II could induce hippocampal oxidative stress and apoptosis. Propofol is a widely used intravenous anesthetic agent in clinical practice, and it demonstrates significant neuroprotective activities. In this study, we investigated the mechanism how propofol protected mouse hippocampal HT22 cells against angiotensin II-induced oxidative stress and apoptosis. Cell viability was evaluated with CCK8 kit. Protein expressions of active caspase 3, cytochrome c, p66Shc, p-p66shc–Ser36, protein kinase C βII (PKCβII), Pin-1 and phosphatase A2 (PP2A) were measured by Western blot. Superoxide anion (O2.−) accumulation was measured with the reduction of ferricytochrome c. Compared with the control group, angiotensin II up-regulated expression of PKCβII, Pin-1 and PP2A, induced p66Shc–Ser36 phosphorylation, and facilitated p66Shc mitochondrial translocation, resulting in O2.− accumulation, mitochondrial cytochrome c release, caspase 3 activation, and the inhibition of cell viability. Importantly, we found propofol inhibited angiotensin II-induced PKCβII and PP2A expression and improved p66Shc mitochondrial translocation, O2.− accumulation, mitochondrial cytochrome c release, caspase 3 activation, inhibition of cell viability. On the other hand, propofol had no effects on angiotensin II-induced Pin-1 expression and p66Shc–Ser36 phosphorylation. Moreover, the protective effects of propofol on angiotensin II-induced HT22 apoptosis were similar with calyculin A, an inhibitor of PP2A and CGP53353, an inhibitor of PKCβII. However, the protective effect of propofol could be reversed by FTY720, an activator of PP2A, rather than PMA, an activator of PKCβII. Our data indicated that propofol down-regulated PP2A expression, inhibiting dephosphorylation of p66Shc–Ser36 and p66Shc mitochondrial translocation, decreasing O2.− accumulation, reducing mitochondrial cytochrome c release, inhibiting caspase 3 activation. By these mechanisms, it protects mouse hippocampal HT22 cells against angiotensin II-induced apoptosis.

Propofol attenuates pancreatic cancer malignant potential via inhibition of NMDA receptor.

ABSTRACT Propofol is a commonly used intravenous anesthetic, and could attenuate cancer cells malignant potential via inhibiting hypoxia‐inducible factor‐1&agr; (HIF‐1&agr;) expression. However, the mechanism is still inclusive. In the present study, we mainly focus on the mechanism by which propofol down‐regulated HIF‐1&agr; expression and malignant potential in pancreatic cancer cells. Human pancreatic cancer cells (Miapaca‐2 and Panc‐1) in vitro and murine pancreatic cancer cell (Panc02) in vivo were used to assess the effect of propofol on vascular endothelial growth factor (VEGF) expression and migration of pancreatic cancer cells. Propofol inhibited cells migration, expression of VEGF and HIF‐1&agr;, phosphorylation of extracellular regulated protein kinases (ERK), AKT, Ca2+/calmodulin dependent protein kinases II (CaMK II), and Ca2+ concentration in a concentration‐dependent manner (5, 25, 50, 100 &mgr;M). Furthermore, MK801, an inhibitor of NMDA receptor, and KN93, an inhibitor of CaMK II, could inhibit the expression of VEGF, HIF‐1a, p‐AKT, p‐ERK, p‐CaMK II in vitro, growth of tumor and VEGF expression in vivo, which were similar to the effect of propofol. In addition, the anti‐tumor effect of propofol could be counteracted by rapastinel, an activator of NMDA receptor. Our study indicated that propofol suppressed VEGF expression and migration ability of pancreatic cancer cells in vitro and in vivo, probably via inhibiting NMDA receptor.

The protective effects of propofol against CoCl 2 -induced HT22 cell hypoxia injury via PP2A/CAMKIIα/nNOS pathway

BackgroundPerioperative cerebral ischemia/hypoxia could induce hippocampal injury and has been reported to induce cognitive impairment. In this study, we used cobalt chloride (CoCl2) to build a hypoxia model in mouse hippocampal cell lines. Propofol, a widely used intravenous anesthetic agent, has been demonstrated to have neuroprotective effect. Here, we explored whether and how propofol attenuated CoCl2-induced mouse hippocampal HT22 cell injury.MethodsMouse hippocampal HT22 cells were pretreated with propofol, and then stimulated with CoCl2. Cell viability was measured by cell counting kit 8 (CCK8). The effect of propofol on CoCl2-modulated expressions of B-cell lymphoma 2 (Bcl-2), BAX, cleaved caspase 3, phosphatase A2 (PP2A), and the phosphorylation of Ca2+/Calmodulin (CaM)-dependent protein kinase II (pCAMKIIα), neuron nitric oxide synthase at Ser1412 (pnNOS-Ser1412), neuron nitric oxide synthase at Ser847 (pnNOS-Ser847) were detected by Western blot analysis.ResultsCompared with control, CoCl2 treatment could significantly decrease cell viability, which could be reversed by propofol. Further, we found CoCl2 treatment could up-regulate the expression of PP2A, BAX, cleaved caspase three and cause the phosphorylation of nNOS-Ser1412, but it down-regulated the expression of Bcl-2 and the phosphorylation of CAMKIIα and nNOS-Ser847. More importantly, these CoCl2-mediated effects were attentuated by propofol. In addition, we demonstrated that propofol could exert similar effect to that of the PP2A inhibitor (okadaic acid). Further, the PP2A activator (FTY720) and the CAMKIIα inhibitor (KN93) could reverse the neuroprotective effect of propofol.ConclusionOur data indicated that propofol could attenuate CoCl2-induced HT22 cells hypoxia injury via PP2A/CAMKIIα/nNOS pathway.

Propofol ameliorates endothelial inflammation induced by hypoxia/reoxygenation in human umbilical vein endothelial cells: Role of phosphatase A2

Hypoxia/reoxygenation (H/R) induces endothelial inflammation with augmentation of endothelial adhesion molecules over-expression. Propofol was reported to attenuate endothelial adhesion molecule expression in some situations. Here, we examined the molecular mechanism for how propofol restored H/R-mediated up-regulation of endothelial adhesion molecules in human umbilical vein endothelial cells (HUVECs). Compared with the control group, H/R up-regulated expression of Pin-1 and PP2A, increased p66(Shc)-Ser(36) phosphorylation, induced p66(Shc) mitochondrial translocation, O2(-) accumulation and NF-κB activation, and decreased eNOS-Ser(1177) phosphorylation and nitric oxide (NO) production, thus up-regulating expression of endothelial adhesion molecules and increasing mononuclear-endothelial interaction. More importantly, except that propofol had no effect on H/R-induced p66(Shc)-Ser(36) phosphorylation, most of H/R-mediated changes were alleviated by propofol, resulting in the reduction of endothelial adhesion molecules expression and mononuclear-endothelial adhesion. Moreover, we demonstrated the protective effect of propofol on H/R-induced endothelial inflammation was similar to that of calyculin A, an inhibitor of PP2A. In contrast, FTY720, an activator of PP2A, antagonized the effect of propofol. Our data indicated that propofol down-regulated PP2A expression, leading to reduced dephosphorylation of p66(Shc)-Ser(36) and eNOS-Ser(1177), which is associated with ROS accumulation and NO reduction, resulting in inhibition of endothelial adhesion molecule expression and mononuclear-endothelial interaction.

Propofol attenuates high glucose‐induced superoxide anion accumulation in human umbilical vein endothelial cells

Perioperative hyperglycemia is a common clinical metabolic disorder. Hyperglycemia could induce endothelial apoptosis, dysfunction, and inflammation, resulting in endothelial injury. Propofol is a widely used anesthetic drug in clinical settings. Our previous studies indicated that propofol attenuated high glucose‐induced endothelial apoptosis, dysfunction, and inflammation via inhibiting reactive oxygen species (ROS) accumulation. However, the mechanisms by which propofol reduces high glucose‐induced endothelial ROS accumulation are still obscure. In this study, we examined how propofol attenuates high glucose‐induced endothelial ROS accumulation. Compared with 5 mm glucose treatment, 15 mm glucose upregulated the expression of pin‐1, phosphatase A2 (PP2A), p66shc and mitochondrial p66shc expression, increased p66shc‐Ser36 phosphorylation, and O2·− accumulation. More importantly, although propofol had no effect on 15 mm glucose‐induced p66shc‐Ser36 phosphorylation and pin‐1 expression, propofol could downregulated PP2A expression and p66shc expression in whole‐cell and mitochondrion, resulting in the reduction of O2·− accumulation. Moreover, we demonstrated that the antioxidative effect of propofol was similar to that of calyculin A, an inhibitor of PP2A. In contrast, FTY720, an activator of PP2A, antagonized the effect of propofol. Our data indicated that the antioxidative effect of propofol was achieved by downregulating PP2A expression, resulting in the inhibition of p66shc‐Ser36 dephosphorylation and mitochondrial p66shc expression.