The Experimental Study Of HO-1Upregulation In Human Umbilical Vein Endothelial Cell By Propofol And DMSO

Propofol is characterized by a phenolic structure and presents antioxidant properties that have been demonstrated both in vitro and in vivo. The protective effects of propofol were previously attributed to its capability of scavenging hydrogen peroxide (H2O2), reducing the formation of lipid peroxides, decreasing the expression levels of nitric oxide synthase, and stabilizing the mitochondrial membrane. Recently, growing evidence suggests that another possible mechanism for the protective effects of propofol may be stimulation of heme oxygenase-1(HO-1), HO-1is a stress-induced protein also known as heat shock protein. Several studies have shown the antioxidant and anti-inflammatory effects of HO-1. However, whether propofol increases HO-1expression and activity at clinically relevant concentrations has not been well examined. Many of the substances that induce HO-1expression do so by the activation of mitogen-activated protein kinase (MAPKs) and NF-κB (Nuclear factor-KB) and Nrf2(Nuclear factor-E2related factor2). It has been reported that propofol differentially activates certain MAPKs, including extracellular signal-regulated kinases (ERKs),cJun-N-terminal kinases (JNKs), and p38-MAPK, depending on cell species and treatment conditions. Whether these MAPKs and NF-κB or Nrf2are involved in propofol-induced HO-1expression has not been clearly defined. In this context, we investigated the effect of propofol on HO-1expression and activity in human umbilical vein endothelial cells (HUVECs) under oxidative stress conditions or normal conditions. Furthermore, the signaling cascades for MAPKs/NF-κB/Nrf2were examined to understand the possible underlying mechanisms. We also examined whether increased HO-1is involved in propofol-mediated antiapoptotic effects in HUVECs.Dimethyl sulfoxide (DMSO) is the solvent of propofol in this studies. It has been used successfully in the treatment of pulmonary adenocarcinoma, rheumatologic diseases, chronicprostatitis, dermatologic diseases, and as a topical analgesic. Due to its anti-inflammatory actions and scavenging of reactive oxygen species, its use has been proposed in several gastrointestinal diseases. However, the mechanisms by which DMSO mediates its therapeutic effects are unclear. The accidental discovery of propofol study is DMSO may upregulates the HO-1expressiom when it at high concentration. Therefore, beside the study of propofol, we also investigated the capability of DMSO to up-regulate heme oxygenase-1expression, as well as the possible underlying mechanisms. We also designed the study to investigate whether DMSO-induced HO-1expression is associated with activation of MAPKs and NF-κB and Nrf2in human umbilical vein endothelial cells (HUVECs). Under mild oxidative stress conditions induced by H2O2, propofol significantly increased HO-1mRNA and protein expression in a dose-dependent manner. The upregulation effect of propofol was induced from2hours, and remained elevated for24hours. Given that HO-1protein levels do not represent HO-1activity, we further determined HO-1activity, a significant increase in HO-1activity was observed at10mmol/L of propofol under oxidative stress conditions, and HO-1activity was induced from2h. These data suggest that the increase in HO-1mRNA and protein is accompanied by enhanced HO-1activity. Under oxidative stressconditions, propofol-induced HO-1response was remarkably reduced by PD98059, whereas SB203580and SP600125had no such an effect. These data may suggest that ERKs are involved in propofol-induced HO-1upregulation. In the presence of H2O2, propofol activated ERKs in a dose-dependent manner, and this process was inhibited by PD98059. H2O2or propofol alone only slightly stimulated ERKs phosphorylation. These data further confirmed that propofol-induced HO-1expression is mediated through activation of ERKs pathways. Treatment with H2O2could significantly increase cell apoptosis, whereas the addition of propofol to H2O2-treated HUVECs resulted in a decrease in cell apoptosis. Pretreatment with ZnPPIX partly reversed the antiapoptotic role of propofol. These data suggested that the antiapoptotic protection of propofol may be partly mediated by HO-1. A significant increase in phosphorylation of cellular and nuclear NF-κBp65was measured in response to propofol, while propofol alone had no significant effect. The phosphorylation and nuclear translocation of NF-κBp65induced by propofol plus H2O2were inhibited by PD98059. H2O2induced relatively low levels of phosphorylation and nuclear translocation of NF-KBp65, and this process was not inhibited by PD98059.DMSO increased both HO-1mRNA and protein expression in a dose-and time-dependent manner. The up-regulation effect of DMSO wasinduced at1h and remained elevated for12h, A significant increase in HO-1activity was observed at0.2%of DMSO, and HO-1activity was increased at8h. These data suggest that the increase in HO-1mRNA and HO-1protein is accompanied by enhanced HO-1activity. The inhibition of the JNKs inhibitor significantly blocks DMSO induction of HO-1protein expression, while PD98059and SB203580had no detectable effects on this process. In the presence of0.8%DMSO, the phosphorylation of JNKs was increased in a time-dependent manner, while the phosphorylation of ERKs and p38-MAPK were not affected by DMSO. Together with the results of inhibitor assays, the data indicates that the JNKs pathway participates in the DMSO-mediated induction of HO-1in HUVECs. The nuclear accumulation of Nrf2was time-dependent and increased with DMSO. In the EMSA that was conducted using a binding sequence for Nrf2, no binding complex was detected in the untreated HUVECs, but the nuclear translocation and DNA binding of Nrf2were increased by DMSO treatment. The anti-oxidant response element-binding complex appeared after10min of DMSO treatment and was maintained for60min. To further verify the involvement of Nrf2in the DMSO induced up-regulation of HO-1expression, we knocked down Nrf2expression using its specific siRNA to examine HO-1expression. DMSO-induced HO-1expression was reduced by38%during transfection with Nrf2-siRNA. This finding confirms DMSO induced HO-1expression through Nrf2activation in human umbilical vein endothelial cells.To the best of our knowledge, this is the first study to provide evidence that propofol and DMSO increase HO-1expression and activity in vascular endothelial cells under oxidative stress conditions and this upregulation is partly mediated by the activation of ERKs pathway. Given the important role of vascular endothelium in maintaining system homeostasis (such as blood brain barrier), these results may shed light on the pharmacological basis for the clinical application of propofol in some oxidative stress conditions, such as in cerebral ischemic or reperfusion injury. Besides, we also found that DMSO up-regulates heme oxygenase-1expression in human umbilical vein endothelial cells and this process is related to the activation of JNKs and Nrf2signaling pathways. These results may shed new light on the pharmacological basis for the application of DMSO in clinical settings. In addition, the data from the present study emphasizes the necessity for appropriate solvent control groups to be developed in experimental models in which DMSO is being used to examine drug effects related to HO-1expression.