Abstract
BackgroundThis study was designed to test the hypothesis that κ-opioid receptor (κ-OR) stimulation reduces palmitate-induced HUVECs apoptosis and to investigate its mechanisms.MethodsHUVECs were subjected to sodium palmitate, apoptosis and cell viability were determined, HUVECs were treated with specific inhibitors to PI3K, Akt, eNOS and siRNAs targeting κ-OR and Akt. Groups were divided as follows: the control group, the sodium palmitate group, the sodium palmitate+U50,488H (a selective κ-OR agonist) group and the sodium palmitate+U50,488H + nor-BNI (a selective κ-OR antagonist) group.ResultsTreatment with sodium palmitate significantly reduced cell viability and increased apoptosis rate which were significantly alleviated by pretreatment with U50,488H, the effect of U50,488H was abolished by nor-BNI. Phosphorylation of Akt and eNOS, as well as NO production were attenuated and accompanied by an increased expression of caspase 3 when HUVECs were subjected to sodium palmitate, and all these changes were restored by pretreatment with U50,488H, the effects of U50,488H were abolished by nor-BNI, and specific inhibitors to PI3K, Akt, eNOS, respectively. SiRNAs targeting κ-OR or Akt abolished the effects of U50,488H on phosphorylation of Akt and eNOS as well as the expressions of caspase 3, Bax and Bcl-2. SiRNAs targeting Akt elicited no effect on the expression of κ-OR.ConclusionThis study provides the evidence for the first time that κ-OR stimulation possesses anti-palmitate-induced apoptosis effect, which is mediated by PI3K/Akt/eNOS signaling pathway.
Highlights
This study was designed to test the hypothesis that κ-opioid receptor (κ-OR) stimulation reduces palmitate-induced human umbilical vein endothelial cell lines (HUVECs) apoptosis and to investigate its mechanisms
U50,488H attenuated palmitate-induced apoptosis and increased HUVECs viability As shown in Fig. 1, after treatment of cells with sodium palmitate for 48 h, apoptosis was prominently increased as increased signal from Annexin V-fluorescein isothiocyanate (FITC) (Fig. 1a, b), cell survival rate was significantly decreased according to Cell counting kit 8 (CCK-8) assay (Fig. 1c)
The effects of U50,488H were blocked by nor-BNI, a selective κ-OR antagonist (Fig. 1b, c). These data indicated that κ-OR stimulation exerts an anti-apoptosis effect in HUVECs subjected to sodium palmitate
Summary
This study was designed to test the hypothesis that κ-opioid receptor (κ-OR) stimulation reduces palmitate-induced HUVECs apoptosis and to investigate its mechanisms. Endothelium dysfunction is associated with most forms of cardiovascular disease and is thought to play a vital role in the development of atherosclerosis, which remains a. An important risk factor in the pathogenesis of atherosclerosis is increased free fatty acids (FFAs) in serum and it is related to an increase in LDL, which has close relationship with the generation of reactive oxygen species (ROS) in endothelium [4]. ROS causes the suppression of Akt/eNOS signaling pathway, reduction in NO production, disturbance of the Bax/Bcl-2 family proteins and the following activation of caspase-3. It causes activation of the downstream apoptosis protease in the caspase cascade [5]. It has been reported that palmitate-induced endothelial apoptosis at least partly results from mitochondrial dysfunction [10]
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