Wan Jing, Ma Jing, Anand Vidhu, Ramakrishnan Sundaram, Roy Sabita
Department of Surgery, University of Minnesota, Minneapolis, MN 55455.
Department of Pharmacology, University of Minnesota, Minneapolis, MN 55455.
Acta Physiol (Oxf). 2015 Jun;214(2):189-199. doi: 10.1111/apha.12506. Epub 2015 Apr 21.
Opioids are the most prescribed analgesics for moderate and severe pain management; however, chronic use impairs host innate immune response and increases susceptibility to infection. Recently, autophagy has been shown to be an innate defence mechanism against bacterial infection. The effect of autophagy-induced bacterial clearance following morphine treatment has not been previously investigated.
Autophagosomes were visualized by confocal microscopy following GFP-LC3 transfection and also by transmission electron microscopy. The relative protein levels were analysed by Western blot. Macrophages were transfected with GFP-mcherry-LC3 simultaneously to monitor autolysosome formation and subsequent events that lead to degradation.
Morphine treatment potentiated LPS-induced vesicular translocation of GFP-LC3 with a concurrent increase in LC3-II levels. In addition, morphine upregulated LPS-induced Beclin1 level, but downregulated Bcl-2 level. We further show that p38 MAP kinase signalling is required for autophagy activation. In contrast, morphine inhibited LPS-induced autophagosome maturation and autophagolysosomal fusion as indicated by the failure to recruit LAMP1 into autophagosome and reduced degradation of SQSTM1/p62 protein level. Morphine modulation of LPS-induced autophagosome maturation visualized using co-localization of GFP-mcherry-LC3 was TLR4 independent, but mediated through μ opioid receptor signalling. Correspondingly, morphine and LPS co-treatment significantly increased Streptococcus pneumoniae load, when compared with LPS treatment alone.
These observations imply that although morphine treatment facilitates LPS-induced autophagy, it inhibits autophagolysosomal fusion leading to decreased bacterial clearance and increased bacterial load. These observations support the increased susceptibility to infection and the prevalence of persistent infection in the drug abuse population.
阿片类药物是用于中度和重度疼痛管理的最常用镇痛药；然而，长期使用会损害宿主的固有免疫反应并增加感染易感性。最近，自噬已被证明是一种抵抗细菌感染的固有防御机制。吗啡治疗后自噬诱导的细菌清除作用此前尚未得到研究。
通过共聚焦显微镜在转染GFP-LC3后观察自噬体，也通过透射电子显微镜观察。通过蛋白质印迹分析相对蛋白水平。巨噬细胞同时转染GFP-mcherry-LC3以监测自溶酶体形成及随后导致降解的事件。
吗啡治疗增强了脂多糖（LPS）诱导的GFP-LC3囊泡易位，同时LC3-II水平升高。此外，吗啡上调了LPS诱导的Beclin1水平，但下调了Bcl-2水平。我们进一步表明，p38丝裂原活化蛋白激酶信号传导是自噬激活所必需的。相反，吗啡抑制了LPS诱导的自噬体成熟和自噬溶酶体融合，这表现为未能将溶酶体相关膜蛋白1（LAMP1）募集到自噬体中以及SQSTM1/p62蛋白水平的降解减少。使用GFP-mcherry-LC3共定位观察到的吗啡对LPS诱导的自噬体成熟的调节与Toll样受体4（TLR4）无关，但通过μ阿片受体信号传导介导。相应地，与单独的LPS治疗相比，吗啡和LPS联合治疗显著增加了肺炎链球菌载量。
这些观察结果表明，尽管吗啡治疗促进了LPS诱导的自噬，但它抑制了自噬溶酶体融合，导致细菌清除减少和细菌载量增加。这些观察结果支持了药物滥用人群中感染易感性增加和持续性感染流行的现象。
