Involvement of Mu Opioid Receptor Signaling in the Protective Effect of Opioid against 6-Hydroxydopamine-Induced SH-SY5Y Human Neuroblastoma Cells Apoptosis

Abstract

Introduction: The neuroprotective role of opioid morphine against 6-hydroxydopamine-induced cell death has been demonstrated. However, the exact mechanism(s) underlying such neuroprotection, especially the role of subtype receptors, has not yet been fully clarified.

Methods: Here, we investigated the effects of different opioid agonists on 6-OHDA-induced neurotoxicity in human neuroblastoma SH-SY5Y cell line as an in vitro model of Parkinson's disease. Cell damage was induced by 150 μM 6-OHDA and the cells viability was examined by MTT assay. Intracellular calcium, reactive oxygen species and mitochondrial membrane potential were assessed by fluorescence spectrophotometry method. Immunoblot technique was used to evaluate cytochrome-c and activated caspase-3 as biochemical markers of apoptosis induction.

Results: The data showed that 6-OHDA caused significant cell damage, loss of mitochondrial membrane potential and increase in intracellular reactive oxygen species and calcium levels as well as activated caspase-3 and cytochrome-c release. Incubation of SH-SY5Y cells with μ-opioid agonists, morphine and DAMGO, but not with δ-opioid agonist, DADLE, elicited protective effect and reduced biochemical markers of cell damage and death.

Discussion: The results suggest that μ-opioid receptors signaling participate in the opioid neuroprotective effects against 6-OHDA-induced neurotoxicity.

Keywords: 6-hydroxydopamine; Apoptosis; DAMGO; Morphine; SH-SY5Y cells; μ-opioid receptors.

Figure 1.

Effects of 150 μM 6-hydroxydopamine (6-OHDA) alone or accompanied with different concentration of morphine (A) and DAMGO (B) on SH-SY5Y cells viability. Data are expressed as mean±SEM; n=5–6 wells for each group; ^**P<0.01 and ^***P<0.001 significantly different versus control cells. +P<0.05 and +++P<0.001 versus 6-OHDA-treated cells.

Figure 2.

The effects of DADLE on 6-OHDA-induced SH-SY5Y cell damage. The cells were treated with 6-OHDA (150 μM) and different doses of DADLE for a 24 h period. Data are expressed as mean±SEM; n=5-6 wells for each group.

Figure 3.

The effects of μ opioid agonists morphine and DAMGO on 6-OHDA-induced intracellular ROS elevation. 24 h 6-OHDA treatment dramatically increased the intracellular level of ROS and morphine (50 μM) as well as DAMGO (0.5 and 1 μM) could suppress ROS production. Data are expressed as mean±SEM; n=5–6 wells for each group. ^*P<0.05 and ^***P<0.001 versus control cells. +++P<0.001 versus 6-OHDA-treated cells.

Figure 4.

The effects of morphine and DAMGO on 6-OHDA-induced intracellular calcium elevation. Data are expressed as mean±SEM; n=5–6 wells for each group. ^**P<0.01 and ^***P<0.001 versus control cells. ++P<0.01 versus 6-OHDA treated cells.

Figure 5.

The effects of morphine and DAMGO on 6-OHDA-induced depression of the mitochondrial membrane potential. Data are expressed as mean±SEM; n=5–6 wells for each group. ^*P<0.05 and ^***P<0.001 versus control cells. ++P<0.01 and +++P<0.001 versus 6-OHDA treated cells.

Figure 6.

Effect of morphine and DAMGO on the 6-OHDA-induced cytochrome-c release in SH-SY5Y cells. Each value represents the mean±SEM band density ratio for each group. β-actin was used as an internal control. ^**P<0.01 and ^***P<0.001 significantly different versus control group. ++P<0.001 versus 6-OHDA-treated cells.

Figure 7.

The activation of caspase-3 protein in SH-SY5Y cells exposed to 150 μM 6-OHDA and 6-OHDA plus protective doses of morphine for 24 h. Each value represents the mean±SEM band density ratio for each group. β-actin was used as an internal control. ^***P<0.001 significantly different versus control cells. +++P<0.001 versus 6-OHDA treated cells.