Morphine preconditioning reduces lipopolysaccharide and interferon-gamma-induced mouse microglial cell injury via delta 1 opioid receptor activation

Abstract

Microglial cells play an important role in the inflammatory response of a broad range of brain diseases including stroke, brain infection and neurodegenerative diseases. However, there is very little information regarding how to protect microglial cells. Here, we showed that incubation of the C8-B4 mouse microglial cells with lipopolysaccharide (LPS) plus interferon-gamma (IFN gamma) induced cytotoxicity as assessed by the amount of lactate dehydrogenase (LDH) released from the cells. Preconditioning the cells with morphine for 30 min concentration-dependently reduced LPS plus IFN gamma-induced cell injury. This morphine preconditioning effect was abolished by naloxone, a general opioid receptor antagonist, by naltrindole, a selective delta opioid receptor antagonist and by 7-benzylidenenaltrexone maleate, a selective delta(1) opioid receptor antagonist. However, this protective effect was not affected by beta-funaltrexamine, a selective mu opioid receptor antagonist, nor-binaltorphimine, a selective kappa opioid receptor antagonist or naltriben, a selective delta(2) opioid receptor antagonist. LPS plus IFN gamma induced the expression of inducible nitric oxide synthase (iNOS), which was not affected by morphine preconditioning. Our results suggest that morphine induced a preconditioning effect in microglial cells. This effect may be mediated by delta 1 opioid receptors and may not be through inhibiting the expression of iNOS, a potentially harmful protein.

Fig. 1. Morphine preconditioning-induced protection in microglial cells

(A) The mouse C8-B4 microglial cells were exposed to various concentrations of LPS and 10 U/ml IFNγ for 24 h. (B) The mouse C8-B4 microglial cells were pretreated with various concentrations of morphine for 30 min before they were exposed to 100 ng/ml LPS plus 10 U/ml IFNγ for 24 h. Results are mean ± SD (n = 17 for panel A and 21 for panel B). * P < 0.05 compared with control. ^ P < 0.05 compared with LPS plus IFNγ only.

Fig. 2. The effects of opioid receptor antagonists on morphine preconditioning-induced protection

The mouse C8-B4 microglial cells were pretreated with 3 μM morphine (Mor) in the presence or absence of 50 μM naloxone (Nalo), 10 μM s-funaltrexamine (FNA), 10 μM nor-binaltorphimine (BNI) or 10 μM naltrindole (NTI). Results are mean ± SD (n = 30 – 35). * P < 0.05 compared with control. ^ P < 0.05 compared with LPS plus IFNγ only. # P < 0.05 compared with morphine preconditioning and LPS plus IFNγ.

Fig. 3. The effects of δ opioid receptor antagonists on morphine preconditioning-induced protection

The mouse C8-B4 microglial cells were pretreated with 3 μM morphine (Mor) in the presence or absence of 0.5 μM BNTX or 0.5 μM naltriben (NTB). Results are mean ± SD (n = 30 – 35). * P < 0.05 compared with control. ^ P < 0.05 compared with LPS plus IFNγ only. # P < 0.05 compared with morphine preconditioning and LPS plus IFNγ.

Fig. 4. Effects of morphine preconditioning on inducible nitric oxide synthase (iNOS) expression

The mouse C8-B4 microglial cells were pretreated with 3 μM morphine (Mor) in the presence or absence of 50 μM naloxone (Nalo) for 30 min. The cells were then incubated with 100 ng/ml LPS plus 10 U/ml IFNγ for 24 h and harvested for Western blotting. Results are mean ± S.D. (n = 12).