Department of Anesthesiology, Shenzhen Shekou People's Hospital, Shenzhen, Guangdong 518067, China.
Chin Med J (Engl). 2010 Dec;123(24):3652-7.
Mu opioid receptor plays an important role in many physiological functions. Fentanyl is a widely used opioid receptor agonist for analgesia. This study was conducted to test the role of mu-opioid receptor on insulin release by determining whether fentanyl affected insulin release from freshly isolated rat pancreatic islets and if small interfering RNAs (siRNA) targeting mu-opioid receptor in the islets could knock down mu-opioid receptor expression.
Islets were isolated from ripe SD rats' pancreas by common bile duct intraductal collagenase V digestion and purified by discontinuous Ficoll density gradient centrifugation. The siRNA knock-down of mu-opioid receptor mRNA and protein in islet cells was analyzed by semi-quantitative real time-PCR and Western blotting. After siRNA-transfection for 48 hours, the islets were co-cultured with fentanyl as follows: 0 ng/ml, 3 ng/ml and 30 ng/ml for 48 hours. Then glucose-evoked insulin release was performed. As a control, the insulin release was also analyzed in islets without siRNA-trasfection after being co-cultured with fentanyl for 48 hours.
After 48 hours of transfections, specific siRNA targeting of mu-opioid receptors produced significant reduction of mu-opioid receptor mRNA and protein (P < 0.01). Fentanyl significantly inhibited glucose-evoked insulin release in islets in a concentration dependent manner (P < 0.01). But after siRNA-transfection for 48 hours, the inhibition on glucose-evoked insulin release was reversed (P < 0.01).
RNA interference specifically reduces mu-opioid receptor mRNA and protein expression, leading to reversal of the fentanyl-induced inhibition on glucose-evoked insulin release of rat islets. The activation of opioid receptor induced by fentanyl functions to inhibit insulin release. The use of RNAi presents a promising tool for future research in diabetic mechanisms and a novel therapy for diabetes.
μ 阿片受体在许多生理功能中发挥重要作用。芬太尼是一种广泛用于镇痛的阿片受体激动剂。本研究通过测定芬太尼是否影响新鲜分离的大鼠胰岛胰岛素释放，以及胰岛中小干扰 RNA（siRNA）是否可以敲低μ阿片受体，来检测μ阿片受体在胰岛素释放中的作用。
采用胆总管胶原酶 V 消化和不连续 Ficoll 密度梯度离心法从成熟 SD 大鼠胰腺中分离胰岛，采用半定量实时 PCR 和 Western blot 分析胰岛细胞中 μ 阿片受体 mRNA 和蛋白的 siRNA 敲低。siRNA 转染 48 小时后，将胰岛与芬太尼共培养，浓度分别为 0ng/ml、3ng/ml 和 30ng/ml，共培养 48 小时，然后进行葡萄糖刺激的胰岛素释放。作为对照，未经 siRNA 转染的胰岛在与芬太尼共培养 48 小时后，也进行胰岛素释放分析。
转染 48 小时后，针对 μ 阿片受体的特异性 siRNA 产生了 μ 阿片受体 mRNA 和蛋白的显著降低（P<0.01）。芬太尼呈浓度依赖性显著抑制胰岛的葡萄糖刺激的胰岛素释放（P<0.01）。但转染 48 小时后，葡萄糖刺激的胰岛素释放的抑制作用被逆转（P<0.01）。
RNA 干扰特异性降低 μ 阿片受体 mRNA 和蛋白表达，导致芬太尼诱导的大鼠胰岛葡萄糖刺激的胰岛素释放抑制作用逆转。芬太尼诱导的阿片受体激活作用抑制胰岛素释放。RNAi 的应用为糖尿病机制的未来研究提供了一种有前途的工具，并为糖尿病的治疗提供了一种新方法。
