Li Ping-feng, Hao Yan-sheng, Zhang Feng-xue, Liu Xin-hua, Liu Shu-lin, Li Gang
Department of Biochemistry and Molecular Biology, Peking University Health Science Center, Beijing 100083, China.
Int Immunopharmacol. 2004 Jan;4(1):79-90. doi: 10.1016/j.intimp.2003.10.014.
Methionine enkephalin, the endogenous opioid peptide, has a diversity of effects on the immune system. Although the biological effects of the pentapeptide have been well documented, little is known about the intracellular events involved in the effects of opioids on human immunodeficiency virus (HIV) infected immune cells. In the present investigation, the possible mechanism of apoptosis alleviated by exposure of methionine enkephalin at 1 micromol/l to CEM x 174 cells, the hybrid lymphocytes, infected with simian immunodeficiency virus (SIV) in vitro is elucidated. Apoptosis and cell cycle analysis is carried out by flow cytometry, the phosphorylation of mitogen-activated protein kinases (MAPK) ERK1 and ERK2 is detected by Western blotting assay, and changes of calcium concentration were analyzed using the calcium-sensitive dye Fluo-3 AM. The results exhibit that methionine enkephalin at the concentrations of 1 micromol/l increase remarkably the proportion of vital cells and decrease the apoptotic cells based on annexin V binding assay. In response to the treatment with methionine enkephalin, SIV-infected cells display a prolonged survival and are accumulated in G1 phase. Methionine enkephalin increase obviously the content of intracellular calcium in normal cells within 1-2 min and maintains a high level within monitoring time. However, the intracellular calcium reaches the highest level at 1 min and subsequently decline to background in SIV infected group. In addition, methionine enkephalin also elevates the levels of protein kinase C (PKC) activity and phosphorylated extracellular signal-regulated kinase (ERK) 1/2. It is proposed that calcium-PKC-MAPK cascade is involved in methionine enkephalin-prolonged survival of SIV-infected cells in the early stages of virus infection. The results provide a further evidence for potential use of methionine enkephalin on the therapy of Acquired Immunodeficiency Syndrome (AIDS).
甲硫氨酸脑啡肽作为内源性阿片肽，对免疫系统具有多种作用。尽管该五肽的生物学效应已有充分记载，但对于阿片类物质对人类免疫缺陷病毒（HIV）感染的免疫细胞产生影响所涉及的细胞内事件却知之甚少。在本研究中，阐明了在体外将1微摩尔/升的甲硫氨酸脑啡肽作用于感染猿猴免疫缺陷病毒（SIV）的杂交淋巴细胞CEM x 174细胞后，其减轻细胞凋亡的可能机制。通过流式细胞术进行细胞凋亡和细胞周期分析，采用蛋白质印迹法检测丝裂原活化蛋白激酶（MAPK）ERK1和ERK2的磷酸化情况，并使用钙敏染料Fluo-3 AM分析钙浓度的变化。结果显示，基于膜联蛋白V结合试验，1微摩尔/升浓度的甲硫氨酸脑啡肽显著增加了活细胞比例并减少了凋亡细胞。在用甲硫氨酸脑啡肽处理后，SIV感染的细胞显示出延长的存活时间并积累于G1期。甲硫氨酸脑啡肽在1 - 2分钟内明显增加正常细胞内的钙含量，并在监测时间内维持在高水平。然而，在SIV感染组中，细胞内钙在1分钟时达到最高水平，随后降至基线水平。此外，甲硫氨酸脑啡肽还提高了蛋白激酶C（PKC）活性和磷酸化细胞外信号调节激酶（ERK）1/2的水平。研究表明，钙 - PKC - MAPK级联反应在病毒感染早期参与了甲硫氨酸脑啡肽延长SIV感染细胞存活时间的过程。这些结果为甲硫氨酸脑啡肽在获得性免疫缺陷综合征（AIDS）治疗中的潜在应用提供了进一步的证据。
