Protective role of fentanyl in lipopolysaccharide-induced neuroinflammation in BV-2 cells

Abstract

Neurosurgery always results in neuroinflammation, which may activate microglial cells. Previous studies have demonstrated that fentanyl could be used for the induction or maintenance of anesthesia prior to surgery. However, it is unknown if fentanyl attenuates neuroinflammation prophylactically. Cell viability in groups that were treated with different concentrations of fentanyl (0.01, 0.1, 1 or 5 µmol/l) was analyzed by an MTT assay. BV-2 microglial cells were treated with lipopolysaccharide (LPS) at a concentration of 1 µg/ml to mimic neuroinflammation in vitro. BV-2 cells were pretreated with 5 µmol/l fentanyl prior to stimulation by LPS. The protein levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-10 in the culture medium were assessed by ELISA. The mRNA level of toll-like receptor (TLR)4 was evaluated by reverse transcription-quantitative polymerase chain reaction analysis. The protein levels of TLR4, glycogen synthase kinase (GSK)-3β and phosphorylated (p)-GSK-3β in BV-2 cells were assessed by western blot analysis. The MTT assay demonstrated that low concentrations of fentanyl (0.01, 0.1 or 1 µmol/l) did not affect the cell viability of BV-2 cells, while 5 µmol/l fentanyl significantly reduced BV-2 cell viability. The results of ELISA revealed that LPS significantly upregulated the release of TNF-α, IL-1β and IL-10, which were repressed by fentanyl pretreatment. Fentanyl pretreatment significantly reduced the LPS-induced elevation of TLR4 at mRNA and protein levels as well as p-GSK-3β protein levels in BV-2 cells. In conclusion, fentanyl pretreatment protects BV-2 cells from LPS-induced neuroinflammation by inhibiting TLR4 expression and GSK-3β activation. Neuroinflammation induced by surgery serves an important role in the development of postoperative cognitive dysfunction (POCD) and targeting the TLR4 and GSK-3β signaling pathway may provide a novel therapeutic approach for the treatment of POCD.

Keywords: BV-2 cells; fentanyl; glycogen synthase kinase-3β; neuroinflammation; toll-like receptor 4.

Figure 1.

Low concentrations of fentanyl did not induce cytotoxicity in BV-2 cells. The cell viability of BV-2 cells that were treated with 0.01, 0.1, 1 and 5 µmol/l fentanyl was measured using an MTT assay. *P<0.05 vs. control group. Ctrl, control.

Figure 2.

Fentanyl pretreatment suppressed the LPS-induced elevation of IL-1β, TNF-α and IL-10 in BV-2 cells. IL-1β, TNF-α and IL-10 protein levels in BV-2 cells were assessed using ELISA kits. Cells were either untreated, or treated with 5 µmol/l fentanyl, 1 µg/ml LPS or fentanyl followed by LPS. ***P<0.001 vs. control group; ^##P<0.01 vs. LPS group. Ctrl, control; IL, interleukin; TNF, tumor necrosis factor; LPS, lipopolysaccharide.

Figure 3.

LPS-induced upregulation of TLR4 mRNA level was suppressed by fentanyl. TLR4 mRNA levels were assessed using reverse transcription-quantitative polymerase chain reaction analysis. GAPDH was used as the endogenous control. BV-2 cells were either untreated, or treated with 5 µmol/l fentanyl, 1 µg/ml LPS or fentanyl followed by LPS. **P<0.01 vs. control group; ^#P<0.05 vs. LPS group. Ctrl, control; TLR, toll-like receptor; LPS, lipopolysaccharide.

Figure 4.

LPS-induced upregulation of the TLR4 protein level was repressed by fentanyl. BV-2 cells were either untreated, or treated with 5 µmol/l fentanyl, 1 µg/ml LPS or fentanyl followed by LPS. β-actin was used as the endogenous control. TLR4 protein levels were (A) measured by western blotting, then (B) quantified and statistically analyzed. **P<0.01 vs. control group; ^#P<0.05 vs. LPS group. Ctrl, control; TLR, toll-like receptor; LPS, lipopolysaccharide.

Figure 5.

Fentanyl pretreatment further promoted LPS-induced inactivation of GSK-3β. BV-2 cells were either untreated, or treated with 5 µmol/l fentanyl, 1 µg/ml LPS or fentanyl followed by LPS. GSK-3β and p-GSK-3β protein levels were (A) measured by western blotting, then (B) quantified and statistically analyzed. **P<0.01 vs. control group; ^#P<0.05 vs. LPS group. Ctrl, control; GSK, glycogen synthase kinase; p-, phosphorylated; LPS, lipopolysaccharide.