Sinomenine Protects Against Morphine Dependence through the NMDAR1/CAMKII/CREB Pathway: A Possible Role of Astrocyte-Derived Exosomes

Abstract

Sinomenine is a nonaddictive alkaloid used to prevent morphine dependence, even thoughits mechanism isnot fully understood. Astrocytes aggravate the pathological process in their neighboring cellsthrough exosomes in central nervous system diseases. However, the effect of sinomenine on astrocyte-derived exosomes for the amelioration of morphine dependence has not been reported yet. In this study, we found that sinomenine prevented the morphine-induced conditionedplace preference in mice. Sinomenine reduced the levels of cAMP and intracellular Ca²⁺ in morphine-treated SH-SY5Y cells. Moreover, sinomenine inhibited the expressions of p-NMDAR1/NMDAR1, p-CAMKII/CAMKII, and p-CREB/CREB in the hippocampusof morphine-dependent mice and SH-SY5Y cells. Furthermore, we found that sinomenine inhibitedthe morphine-induced activation of astrocytesin vivo and in vitro. Afterwards, exosomes were isolated from cultured primary astrocytes treated with phosphate buffer saline (PBS, ctl-exo), morphine (mor-exo), or morphine and sinomenine (Sino-exo). Subsequently, morphine-treated SH-SY5Y cells were treated with ctl-exo, mor-exo, and Sino-exo. Results showed that Sino-exo reduced the level of cAMP, intracellular Ca²⁺, and the expression of p-CAMKII/CAMKII and p-CREB/CREB in morphine-treated SH-SY5Y cells. In conclusion, we demonstrated that sinomenine exhibited protective effects against morphine dependencein vivo and in vitro through theNMDAR1/CAMKII/CREB pathway. Sinomenine-induced alterationof the function of astrocyte-derived exosomes may contribute to the antidependence effects of sinomenine in morphine dependence.

Keywords: CAMKII; CREB; NMDAR1; exosomes; morphine; sinomenine.

Figure 1

Sinomenine inhibited morphine-induced conditioned place preference in mice. (A) Schematic protocol of the conditioned place preference testing; (B) The time spent in the white compartment in preconditioning and postconditioning phases (n = 8). ^## p < 0.01 vs. control, ^ΔΔ p < 0.01 vs. mor, * p < 0.05 vs. mor + Sino. Mor, morphine; Sino, sinomenine; NMDA, n-methyl-d-aspartate.

Figure 2

Sinomenine inhibited the expressions of p-NMDAR1/NMDAR1, p-CAMKII/CAMKII, and p-CREB/CREB in the hippocampus of morphine-dependent mice. (A–D) Immunohistochemistry analysis was used to quantify the expression levels of p-NMDAR1 and NMDAR1 in the hippocampus of morphine-dependent mice (n = 3); (E–J) Western blotting analysis was used to quantify the expression levels of p-NMDAR1/NMDAR1, p-CAMKII/CAMKII, and p-CREB/CREB in the hippocampus (n = 3). ^## p < 0.01 vs. control; ^Δ p < 0.05, ^ΔΔ p < 0.01 vs. mor; * p < 0.05 vs. Sino.

Figure 3

Intervention of sinomenine on morphine-treated SH-SY5Y cells. (A) Sinomenine decreased the level of cAMP in the morphine-treated SH-SY5Y cells (n = 3). SH-SY5Y cells were pretreated with 100 μMsinomenine for 12 h and then incubated with 100 μM morphine for 48 h. NMDAR agonist was incubated with SH-SY5Y cells treated with morphine and sinomenine for 12 h. Naloxone was incubated with SH-SY5Y cells treated with morphine to allow withdrawal for 20 min before evaluation. mor: morphine; nal: naloxone; (B) Sinomenine decreased the level of intracellular Ca²⁺ in the morphine-treated SH-SY5Y cells (n = 3); (C–E) Sinomenine suppressed the expression of p-NMDAR1/NMDAR1, p-CAMKII/CAMKII, and p-CREB/CREB, as detected by Western blotting in the morphine-treated SH-SY5Y cells (n = 3); (F–H) NMDAR agonist reversed the inhibitory effect of sinomenine on p-NMDAR1/NMDAR1, p-CAMKII/CAMKII, and p-CREB/CREB (n = 3). ^# p < 0.05, ^## p < 0.01 vs. control; ^Δ p < 0.05, ^ΔΔ p < 0.01 vs. morphine; * p < 0.05, ** p < 0.01 vs. Sino.

Figure 4

Sinomenine inhibited the activation of morphine-treated astrocytes. (A,B) Immunohistochemistry analysis was used to quantify the expression level of GFAP in the hippocampus of morphine-dependent mice (n = 3); (C,D) Western blotting analysis was used to quantify the expression level of GFAP in cultured primary astrocytes (n = 3). Cultured primary astrocytes were pretreated with phosphate buffer saline (PBS) or 200 μM sinomenine for 12 h and then incubated with 100 μM morphine for 48 h. ^## p< 0.01 vs. control; ^ΔΔ p < 0.01 vs. morphine.

Figure 5

Astrocyte-derived exosomes can be taken up by neuronal SH-SY5Y cells. (A) Exosomes observed by transmission electron microscope; (B,C) Nanoparticle tracking analysis of exosomes; (B) The signal of scattered light by dynamic nanoparticles; the bright white dot indicates one moving particle; (C) Particle size distribution of nanoparticles; (D) Western blotting was used to detect TSG101, CD63, andCD81 proteins of exosomes. Ctl-exo: exosomes extracted from cultured primary astrocytes; mor-exo: exosomes extracted from cultured primary astrocytes treated with 100 μM morphine for 48 h; Sino-exo: exosomes extracted from cultured primary astrocytes treated with 100 μM morphine and 200 μMsinomenine for 48 h; (E) Astrocyte-derived exosomes were labeled by PKH67 and were taken up by neuronal SH-SY5Y cells.

Figure 6

Effects of astrocyte-derived exosomes on morphine-treated SH-SY5Y cells and subsequent intervention by sinomenine. (A) Sino-exo decreased the level of cAMP induced by mor-exo in the morphine-treated SH-SY5Y cells (n = 3); (B) Sino-exodecreased the level of intracellular Ca²⁺ induced by mor-exo in the morphine-treated SH-SY5Y cells (n = 3); (C–E) Effects of Sino-exo on p-NMDAR1/NMDAR1, p-CAMKII/CAMKII, and p-CREB/CREB induced by mor-exo in the morphine-treated SH-SY5Y cells (n = 3); (F–H) NMDAR agonist reversed the inhibitory effect of Sino-exo on p-NMDAR1/NMDAR1, p-CAMKII/CAMKII, and p-CREB/CREB (n = 3). Ctl-exo: exosomes extracted from cultured primary astrocytes; mor-exo: exosomes extracted from cultured primary astrocytes treated with 100 μM morphine for 48 h; Sino-exo: exosomes extracted from cultured primary astrocytes treated with 100 μM morphine and 200 μMsinomenine for 48 h. ^# p < 0.05, ^## p < 0.01 vs. ctl-exo; ^Δ p < 0.05, ^ΔΔ p < 0.01 vs. mor-exo; * p < 0.05, ** p < 0.01 vs. Sino-exo.