Ampicillin/Sulbactam Treatment Modulates NMDA Receptor NR2B Subunit and Attenuates Neuroinflammation and Alcohol Intake in Male High Alcohol Drinking Rats

Abstract

Exposure to ethanol commonly manifests neuroinflammation. Beta (β)-lactam antibiotics attenuate ethanol drinking through upregulation of astroglial glutamate transporters, especially glutamate transporter-1 (GLT-1), in the mesocorticolimbic brain regions, including the nucleus accumbens (Acb). However, the effect of β-lactam antibiotics on neuroinflammation in animals chronically exposed to ethanol has not been fully investigated. In this study, we evaluated the effects of ampicillin/sulbactam (AMP/SUL, 100 and 200 mg/kg, i.p.) on ethanol consumption in high alcohol drinking (HAD1) rats. Additionally, we investigated the effects of AMP/SUL on GLT-1 and N-methyl-d-aspartate (NMDA) receptor subtypes (NR2A and NR2B) in the Acb core (AcbCo) and Acb shell (AcbSh). We found that AMP/SUL at both doses attenuated ethanol consumption and restored ethanol-decreased GLT-1 and NR2B expression in the AcbSh and AcbCo, respectively. Moreover, AMP/SUL (200 mg/kg, i.p.) reduced ethanol-increased high mobility group box 1 (HMGB1) and receptor for advanced glycation end-products (RAGE) expression in the AcbSh. Moreover, both doses of AMP/SUL attenuated ethanol-elevated tumor necrosis factor-alpha (TNF-α) in the AcbSh. Our results suggest that AMP/SUL attenuates ethanol drinking and modulates NMDA receptor NR2B subunits and HMGB1-associated pathways.

Keywords: AMP/SUL; GLT-1; NMDA; ethanol; neuroinflammation.

Figure 1

Effects of ampicillin/sulbactam (AMP/SUL) treatments (100 mg/kg and 200 mg/kg, i.p.) for five consecutive days on (A) Ethanol consumption (g/kg of average body weight/day), (B) Water intake (mL/day), (C) Body weight, and (D) Food intake. Statistical analyses revealed that AMP/SUL consistently reduced ethanol intake with a concomitant increase in water intake. However, there was an increase in water intake on Day 5 as compared to baseline (p < 0.01) in ethanol-control group. While food intake was transiently reduced on the 1st day of treatment, there were no other significant effects of AMP/SUL. In addition, AMP/SUL did not affect body weight. The values are expressed as mean ± SEM (n = 9/group), (* p < 0.05 and ** p < 0.01, $ p < 0.001 and # p < 0.0001).

Figure 2

Effects of AMP/SUL treatments (100 mg/kg and 200 mg/kg, i.p.) for five days on the expression of GLT-1 in the AcbCo and AcbSh. (A) Immunoblot of GLT-1 and β-tubulin in the AcbCo. (B) Quantitative analysis using one-way ANOVA followed by Newman-Keuls test indicated that there were no significant differences in the expression of GLT-1 among water-control, ethanol-control, ethanol-AMP/SUL (100 mg/kg), and ethanol-AMP/SUL (200 mg/kg) in the AcbCo. (C) Immunoblot of GLT-1 and β-tubulin in the AcbSh. (D) Quantitative analysis using one-way ANOVA followed by Newman-Keuls test indicated that there was a significant decrease in GLT-1 expression in the ethanol control group compared to water-control group, while post-treatments with AMP/SUL (100 and 200 mg/kg) upregulated GLT-1 expression compared to ethanol-control group in the AcbSh. Ethanol-AMP/SUL (100 and 200 mg/kg) groups had lower GLT-1 expression compared to water-control group. The symbol of statistical significance between any group and water-control group is shown on the bar of the group. Water-control group data were represented as 100% (relative to water-control). The values are expressed as mean ± SEM (n = 5/group), (* p < 0.05, ** p < 0.01 and $ p < 0.001).

Figure 3

Effects of AMP/SUL treatments (100 mg/kg and 200 mg/kg, i.p.) for five days on the expression of NR2B and NR2A in the AcbCo and AcbSh. (A) Immunoblot of NR2B and β-tubulin in the AcbCo and AcbSh. (B) Quantitative analysis using one-way ANOVA followed by Newman-Keuls test indicated that there was a significant decrease in the NR2B expression in the ethanol-control group compared to the water-control group, while post-treatments with AMP/SUL (100 and 200 mg/kg) for five days normalized NR2B expression in the AcbCo. Quantitative analysis using one-way ANOVA followed by Newman-Keuls test indicated that there were no significant differences in the expression of NR2B among the water-control, ethanol-control, ethanol-AMP/SUL (100 mg/kg), and ethanol-AMP/SUL (200 mg/kg) in the AcbSh. (C) Immunoblot of NR2A and β-tubulin in the AcbCo and AcbSh. (D) Quantitative analysis using one-way ANOVA followed by Newman-Keuls test indicated that there were no significant differences in the expression of NR2A between water-control, ethanol-control, ethanol-AMP/SUL (100 mg/kg), and ethanol-AMP/SUL (200 mg/kg) in the AcbCo. Quantitative analysis using one-way ANOVA followed by Newman-Keuls test indicated that there were also no significant differences in the expression of NR2A among water-control, ethanol-control, ethanol-AMP/SUL (100 mg/kg), and ethanol-AMP/SUL (200 mg/kg) rats in the AcbSh. The symbol of statistical significance between any group and water-control group is shown on the bar of the group. Water-control group data were represented as 100% (relative to water-control). The values are expressed as mean ± SEM (n = 5/group), (** p < 0.01).

Figure 4

Effects of AMP/SUL treatments (100 mg/kg and 200 mg/kg, i.p.) for five days on the expression of HMGB1 in the AcbCo and AcbSh. (A) Immunoblot of HMGB1 and β-tubulin in the AcbCo and AcbSh. (B) Quantitative analysis using one-way ANOVA followed by Newman-Keuls test indicated that there were no significant differences in the expression of HMGB1 among the water-control, ethanol-control, ethanol-AMP/SUL (100 mg/kg), and ethanol-AMP/SUL (200 mg/kg) groups in the AcbCo. Quantitative analysis using one-way ANOVA followed by Newman-Keuls test indicated that there was a significant increase in HMGB1 expression in the ethanol-control group compared to water-control group, while post-treatment with AMP/SUL (200 mg/kg, but not 100 mg/kg) for five days normalized HMGB1 expression in the AcbSh. Ethanol-AMP/SUL (100 and 200 mg/kg) groups had higher HMGB1 expression compared to water-control group. (C) Immunoblot of RAGE and β-tubulin in the AcbCo and AcbSh. (D) Quantitative analysis using one-way ANOVA followed by Newman-Keuls test indicated that there were no significant differences in the expression of RAGE among the water-control, ethanol-control, ethanol-AMP/SUL (100 mg/kg), and ethanol-AMP/SUL (200 mg/kg) groups in the AcbCo. Quantitative analysis using one-way ANOVA followed by Newman-Keuls test indicated that there was a significant increase in RAGE expression in the ethanol-control group compared to the water-control group, while post-treatment with AMP/SUL (200 mg/kg, but not 100 mg/kg) for five days decreased RAGE expression in the AcbSh. Ethanol-AMP/SUL (100 and 200 mg/kg) groups had higher RAGE expression compared to water-control group. The symbol of statistical significance between any group and water-control group is shown on the bar of the group. Water-control group data were represented as 100% (relative to water-control). The values are expressed as mean ± SEM (n = 5/group), (* p < 0.05, ** p < 0.01 and $ p < 0.001).

Figure 5

Effects of AMP/SUL treatments (100 mg/kg and 200 mg/kg, i.p.) for five days on the expression of TNF-α in the AcbCo and AcbSh. (A) Immunoblot of TNF-α and β-tubulin in the AcbCo. (B) Quantitative analysis using one-way ANOVA followed by Newman-Keuls test indicated that there were no significant differences in the expression of TNF-α among water-control, ethanol-control, ethanol-AMP/SUL (100 mg/kg), and ethanol-AMP/SUL (200 mg/kg) rats in the AcbCo. (C) Immunoblot of TNF-α and β-tubulin in the AcbSh. (D) Quantitative analysis using one-way ANOVA followed by Newman-Keuls test indicated that there was a significant increase in the TNF-α expression in the ethanol control group compared to the water-control group, while post-treatment with AMP/SUL (100 and 200 mg/kg) for five days decreased TNF-α expression in the AcbSh. Ethanol-AMP/SUL (100 and 200 mg/kg) groups had higher TNF-α expression compared to water-control group. The symbol of statistical significance between any group and water-control group is shown on the bar of the group. Water-control group data were represented as 100% (relative to water-control). The values are expressed as mean ± SEM (n = 5/group), (** p < 0.01 and $ p < 0.001).

Figure 6

Effects of AMP/SUL treatments (200 mg/kg, i.p.) for five days on the expression of mGluR5 in the AcbCo and AcbSh. (A) Immunoblot of mGluR5 and β-tubulin in the AcbCo. (B) Quantitative analysis using one-way ANOVA followed by Newman-Keuls test indicated that there were no significant differences in the expression of mGluR5 among water-control, ethanol-control, and ethanol-AMP/SUL (200 mg/kg) groups in the AcbCo. (C) Immunoblot of mGluR5 and β-tubulin in the AcbSh. (D) Quantitative analysis using one-way ANOVA followed by Newman-Keuls test indicated that there was a significant decrease in the mGluR5 expression in the ethanol-control group compared to the water-control group, while post-treatment with AMP/SUL (200 mg/kg) for five days increased mGluR5 expression in the AcbSh. The symbol of statistical significance between any group and water-control group is shown on the bar of the group. Water-control group data were represented as 100% (relative to water-control). The values are expressed as mean ± SEM (n = 5/group), (** p < 0.01).