Role Of Spinal Mitophagic Dysfunction In Morphine Tolerance

ObjectiveTo explore the role and mechanism of spinal autophagic dysfunction in the develop-ment of morphine tolerance,thus providing a potential target for the treatment of morphine tolerance.Methods(1)Western blotting,immunofluorescence,transmission electron microscope,DCFH-DA and MDA analysis were used to examine mitochondria damage and morphology change in morphine tolerance.(2)Western blotting were used to investigate the multiple autophagy-related protein levels in different time points in morphine tolerance;Immunofluorescence was used to study the change of LC3 and p62 in neuron,microglia and astrocyte in morphine tolerance.(3)The level of ROS and mitochondrial membrane potential were detected after morphine treatment in SH-SY5Y cells.Western blotting was employed to investigate whether mitochondria damage induced by morphine is opioid receptor dependent.(4)To determine the stage of autophagic dysfunction induced by morphine,we used the methods of fluorescent probe transfection and Adenovirus infection.(5)The effects of morphine on Parkin,PINK1 and ubiquitination level of mitochondria were tested by western blotting in SH-SY5Y cells.Results(1)Chronic morphine treatment observably up-regulated the level of MAPK family and ROS in mice spinal cord;Damaged mitochondria fuction and normal shape induced by morphine were proved by increasing BAX/Bcl2 ratio and TEM results.(2)Chronic morphine treatment could up-regulate the levels of LC3 and p62 in spinal cord,and it is mainly located in neuron and partially in microglia and astrocyte;Chronic morphine treatment up-regulated the level of Atg5,but no effect on Beclin 1.(3)Morphine could markedly increase the production of ROS,meanwhile reduced the mitochondrial membrane potential in SH-SY5Y cells;Morphine up-regulated the levels of LC3 and p62 by opioid receptor-dependent way.(4)Morphine interrupted the degradation of LC3 induced by rapamycin and starvation,but had no effect on the lysosome function;Western blotting results also confirmed that chronic administra-tion of morphine interrupted the autophagy flux by inhibiting p62 degradation which similar to the effect of chloroquine;Meanwhile,morphine suppressed mitochondria-autophagosome fusion and the transportation of damaged mitochondria to lysosome.(5)Morphine suppressed recruitment of Parkin lead to of mitochondrial ubiquitination by suppressing phosphorylation of Parkin s65,and accumulation of PINK1.Conclusion(1)Morphine caused the accumulation of damaged mitochondria by inducing mitophagic dysfunction lead to a mount of ROS.(2)Morphine supressed accumulation of PINK1 and stresse induced phosphorylation of Parkin s65.It inhibited the recruitment of Parkin to mitochondria,result in reduced ubiquitination level of mitochondria.Therefore damaged mitochondria can not be identified and degraded.