The Role Of κ-opioid Receptors In Repressing High-fat Induced Vascular Endothelial Cell Apoptosis

The incidence of metabolic syndrome, which induced by hyperlipidemia, and performed as obesity, insulin resistance, hypertension, is increased with younger trend. The balance between production and release of vasoactive substances and cytokines by vascular endothelium is impaired in hyperlipidemia, which results in endothelial injuries. Endothelial injuries are considered as the major factor that promoting the initiation and development of macroangiopathy. Therefore, the improvement of endothelial function and inhibition of its injuries would effectively reduce the mortality rate of metabolic syndrome.k-opioid receptor(κ-OR) is widely expressed in the cardiovascular system. Endogenous k-opioid peptide is produced and released by heart, k-opioid peptide interacts with k-OR, thereby regulates the activities of the cardiovascular system. Our previous study showed that: ①the activation of κ-OR relaxed the abdominal aorta ring in a time, dose and an endothelium-dependent manner, which could significantly reduce the arterial blood pressure of hypertensive rats; ②the activation of k-OR upregulated the NO level in hypoxia condition, inhibited the expression of ET-1 and Ang II, and improved the pulmonary artery endothelial function; ③the activation of k-OR inhibited myocardial ischemia-induced inflammation and myocardial apoptosis. These effects are all associated with NO production, suggesting the activation of k-OR may have the function of endothelium-dependent vasodilation, regulate the balance of vasodilation/contraction factors,improve endothelial function and anti-inflammatory effects. However, the regulatory effects and molecular mechanism of k-OR in hyperlipidemic-induced endothelial injuries is still not clear. Therefore, on the basis of previous work, we constructed a high fat-induced endothelial cell model, focused on the study of the effect and molecular mechanism mediated by k-OR in repressing the high-fat- induced vascular endothelial cell apoptosis. Our study will expand the function of k-OR in improving hyperlipidemia-induced endothelial apoptosis, and reveal the molecular mechanisms of this process. It will also provide important theoretical significance for the treatment of endothelial injuries related diseases. [Objectives] 1. To illustrate the effects of k-OR activation on the biological behavior of endothelialcells in a high-fat-induced endothelial cell injury model; 2. To elucidate the molecular mechanisms underlying the effects of k-OR activation onthe biological behavior of endothelial cells. [Methods] 1. Human umbilical vein endothelial cell(HUVEC) was cultured and identified,sodiumpalmitate was selected for the high fat-induced injury of HUVEC. 2. Cells were treated with sodium palmitate for 48 hours and subsequent experimentswere performed. Groups were divided as follows: the control group, the high fat group,the high fat+U50,488H(a selective k-OR agonist) group and the highfat+U50,488H+nor-BNI(a selective k-OR antagonist)group. 3. To observe the cells growth situation of each group with optical microscope. 4. To analyze the lipid droplets production of each group with oil red staining. 5. To assess the cell viability of each group with CCK-8 assay. 6. To analyze the apoptosis rate of each group through flow cytometry. 7. To detect the expression of k-OR, p-Akt, total-Akt, p-e NOS, total-e NOS, and Caspase3 of each group with Western blot after specific inhibitors or si RNA treatment. 8. To examine the NO production in the culture supernatant of each group with NOdetection kit. [Results] 1. Effects of k-OR stimulation with U50,488 H on high fat-induced biological behaviorsof HUVEC.After treatment of sodium palmitate for 48 hours, the number of cells in the high fat group was decreased, cellular morphology was changed to typical apoptosis, and lipid droplets production was increased. Meanwhile, cell survival rate was significantly decreased and apoptosis was prominently upregulated. As the selective agonist of k-OR, U50,488 H significantly improved the high fat-induced cell growth situation, reduced the production of lipid droplets in cells, enhanced cell survival rate, and inhibited cell apoptosis. The effects mentioned above were blocked by nor-BNI, a selective k-OR antagonist, indicating that U50,488 H by means of activating k-OR, which mediated the activation of the downstream signaling pathway, led to a series of improvemnets of cell biological behavior. 2. Molecular mechanisms of the k-OR in reliefing high fat-induced endothelial apoptosis.According to the results in the different groups detected by Western blot and NO production assay, we found that k-OR, activated by U50,488 H, mediated the activation of the downstream PI3K-Akt-e NOS signaling pathway, promoted NO production, inhibited Caspase3 activation, and had the mitigative effect against high fat-induced cell damage and apoptosis thereby. Additionally, cells then were treated with si RNA and aimed to knockdown the expression of k-OR and Akt. We demonstrated that a significant increase of hyperlipidemia-induced apoptosis occurred in κ-OR si RNA transfected cells, and the anti-apoptotic effect of U50,488 H could be blocked by Akt knockdown. The data above indicating that the effect of anti-hyperlipidaemia-induced apoptosis which mediated by k-OR, is associated with the activation of PI3K-Akt-e NOS signaling pathway. [Conclusions]It is provided the evidence for the first time that k-OR stimulation with U50,488 H enhances the biological activity of e NOS, promotes NO production and inhibits cell apoptosis via the activation of PI3K-Akt-e NOS signaling pathway, and the endothelial apoptosis induced by high fat diet are therefore relieved. Our present study may provide theoretical basis for the prevention and treatment of endothelial injuries induced by high fat, and provide new ideas and strategies for the prevention of hyperlipidemia and metabolic syndrome developed to cardiovascular diseases.