Ischemia reperfusion injury (IRI) is a common complication in the perioperative period and always causes perioperative patients to organ dysfunction and poor prognosis due to the lack of effective prevention and treatment. Vascular endothelial cells are the first-line defense cells in IRI. Previous studies have shown that the barrier function, paracrine, expression of adhesion molecules and angiogenesis of vascular endothelial cells play an important role in the regulation of IRI[13, 24]. Endothelial cell dysfunction is a complex pathophysiological event, in which oxidative stress is the key component. Some studies have shown that oxidative stress plays an important role in the development of IRI through ROS-mediated damages, such as endothelial dysfunction, inflammation, hypertrophy, apoptosis, cell migration, fibrosis and angiogenesis, and vascular remodeling caused by hypertension[12, 13].Therefore, the protective therapy based on vascular endothelial cells may become a promising direction for the prevention and treatment of perioperative IRI, and the search for new drugs and targets that can reduce oxidative stress injury of vascular endothelial cells has also become an important research hotspot.
In the process of exploring the prevention and treatment of IRI, the potential targets and preventive benefits of drugs based on narcotic drugs have become the subject of a large number of studies. So far, many narcotic drugs have been shown to have a protective effect,including Dex[21],propofol [25],sevoflurane [26] and sufentanil [27], etc. Among them, Dex has attracted the attention of many researchers. Dex is a highly selective α2-adrenoceptor agonist, which is widely used in the clinical. Currently, Dex has appeared in a variety of off-label application, such as examination of sedation (intranasal or oral administration)[28, 29] and as an analgesic adjuvant[30]. Furthermore, Dex is also widely used in medical research and has been applied in other clinical settings. It has been found that Dex can reduce the oxidative stress and apoptosis LPS-induced by improving the activity of superoxide dismutase, thereby attenuating the acute kidney injury in septicemic mice[21]. It can also alleviate the oxidative stress injury of cardiomyocytes by reducing the level of ROS[31]. Other studies have found that cardiac surgery patients who receive Dex infusion can achieve better short-term and long-term survival and reduce the incidence of postoperative cognitive impairment[32]. However, it has not been clearly whether Dex can alleviate the oxidative stress injury of vascular endothelial cells. Consequently, the current study sought to evaluate the effect of Dex on vascular endothelial cells under oxidative stress.
In this study, the H2O2- induced cell model of oxidative stress was established to mimic the IRI in vitro. Our study showed the cytotoxic effect of H2O2 on HUVECs in a dose-dependent manner. After treated with 550μM H2O2 for 12 h, the cultured HUVECs showed obvious oxidative stress injure, the cell activity significantly decreased and the LDH release increased a lot. Which is also consistent with previous work, but the concentration of H2O2 was slightly different. The reason may be due to differences in cell type, complexity (comparison of isolated cells / tissues with the entire organism), or time of exposure to H2O2. Previous studies showed human melanocytes induced with 400 μ M H2O2 for 24 hours will cause membrane blistering and cell contraction, resulting in cell death[33], while cardiomyocytes require only 12h with 500μM H2O2[31]. In addition, we also found that the intracellular ROS level, apoptosis rate and apoptosis-related protein caspase9 expression of HUVECs significantly increased after treatment with 550 μ M H2O2, but lead to a decrease in ERK1/2 phosphorylation. It is well known that oxidative stress is the result of the imbalance between ROS and antioxidant defense system. Existing studies have proved that, physiologically concentrations of H2O2 plays an important role in the signal transduction of oxidative stress. Higher concentration of H2O2 will lead to oxidative stress adaptation through NF- κ B pathway, while super-physiological concentration of H2O2 will cause a series of pathological processes such as lipid peroxidation, nucleic acid damage and inhibition of enzyme activity, which will change the molecular structure and function of cells, and eventually lead to growth arrest, apoptosis and necrosis. It may also cause aseptic inflammation and microvascular dysfunction[34, 35]. ERK1/2 is a survival-related kinase, which requires pre-ERK1/2 nuclear accumulation and ERK1/2-mediated gene transcription in the process of anti-apoptosis[36]. In recent studies, it has also been found that ERK1/2 signal transduction is involved in the regulation and expression of a variety of apoptotic proteins, which can control the promotion of apoptosis and anti-apoptosis[37]. Inhibiting the phosphorylation of ERK1/2 protein can effectively reduce cardiomyocyte apoptosis and treat myocardial hypertrophy and heart failure[38]. Therefore, this study and the above studies have proved that apoptosis is closely related to oxidative stress injury, and apoptosis is a certain outcome in the process of oxidative stress.
Next, we also observed that the phosphorylation of p38MAPK signal pathway in HUVECs was significantly enhanced after H2O2 treatment. Many studies have shown that oxidative stress is closely related to p38MAPK signaling pathway. High levels of ROS can activate p38MAPK phosphorylation,and mediate cell senescence or death. Conversely, activated p38MAPK can further increase ROS[39, 40].
When we pretreated HUVECs with 1 μ M and 10 μ M Dex, the results showed that the cell activity of HUVECs in Dex pretreatment group increased, the concentration of LDH in culture medium decreased, and the intracellular ROS level and apoptosis decreased significantly, and this effect was enhanced with the increase of Dex concentration. Western blot assay also showed that the phosphorylation of p38MAPK in HUVECs was inhibited and the phosphorylated of ERK1/2 was enhanced in Dex pretreatment group compared with H2O2 group. Our results are consistent with previous studies[41], which confirmed that Dex can reduce oxidative stress injury and apoptosis of HUVECs by inhibiting p38MAPK phosphorylation. In addition, we also compared Dex with p38MAPK signaling pathway inhibitor SB202190. After the application of SB202190, the activity of HUVECs increased significantly, while LDH, intracellular ROS and apoptosis decreased significantly. Western blot results showed that the expression of p-p38MAPK decreased significantly, while the expression of p-ERK1/2 increased significantly. In other studies, it has been found that Dex has a similar function to SB202190, which can alleviate hypoxia / reoxygenation injury of cardiomyocytes by inhibiting p38MAPK and downstream apoptosis signal pathway[42]. These results strongly suggest that Dex has the same effect as SB202190, and further confirm that Dex can protect HUVECs from oxidative stress by inhibiting p38MAPK pathway.