Xylazine Activates Adenosine Monophosphate-Activated Protein Kinase Pathway in the Central Nervous System of Rats

<div><p>Xylazine is a potent analgesic extensively used in veterinary and animal experimentation. Evidence exists that the analgesic effect can be inhibited using adenosine 5’-monophosphate activated protein kinase (AMPK) inhibitors. Considering this idea, the aim of this study was to investigate whether the AMPK signaling pathway is involved in the central analgesic mechanism of xylazine in the rat. Xylazine was administrated via the intraperitoneal route. Sprague-Dawley rats were sacrificed and the cerebral cortex, cerebellum, hippocampus, thalamus and brainstem were collected for determination of liver kinase B1 (LKB1) and AMPKα mRNA expression using quantitative real-time polymerase chain reaction (qPCR), and phosphorylated LKB1 and AMPKα levels using western blot. The results of our study showed that compared with the control group, xylazine induced significant increases in AMPK activity in the cerebral cortex, hippocampus, thalamus and cerebellum after rats received xylazine (<i>P</i> < 0.01). Increased AMPK activities were accompanied with increased phosphorylation levels of LKB1 in corresponding regions of rats. The protein levels of phosphorylated LKB1 and AMPKα in these regions returned or tended to return to control group levels. However, in the brainstem, phosphorylated LKB1 and AMPKα protein levels were decreased by xylazine compared with the control (<i>P</i> < 0.05). In conclusion, our data indicates that xylazine alters the activities of LKB1 and AMPK in the central nervous system of rats, which suggests that xylazine affects the regulatory signaling pathway of the analgesic mechanism in the rat brain.</p></div

Effect of xylazine administration on the mRNA levels of LKB1 in rats.

<p>(A) Cerebral cortex, (B) Hippocampus, (C) Thalamus, (D) Cerebellum and (E) Brainstem. Rats received saline (0.5 mL) or xylazine (5.2 mg/kg) intraperitoneally and then were sacrificed 10, 10, 20, 40 or 60 min later for control, Xyl1, Xyl2, Xyl3 or Xyl4, respectively. Total RNA was isolated and subjected to real-time PCR analysis. Each value of the expression levels of LKB1 was normalized to the expression levels of β-actin, and the control value was set to one. Data are presented as the means ± SEM, n = 6. * P < 0.05, ** P < 0.01 vs control.</p

Effect of xylazine administration on the levels of phosphorylated LKB1 in rats.

<p>(A) Cerebral cortex, (B) Hippocampus, (C) Thalamus, (D) Cerebellum and (E) Brainstem. Rats received saline (0.5 mL) or xylazine (5.2 mg/kg) intraperitoneally and then were sacrificed 10, 10, 20, 40 or 60 min later for control, Xyl1, Xyl2, Xyl3 or Xyl4, respectively. Western blot analyses were performed with anti-LKB1 and anti-phospho-LKB1 (Ser428). Data for densitometry represent the mean ± SEM obtained from six independent series of Western blotting for each animal group and time point after the procedure. * P < 0.05, ** P < 0.01 vs control. Representative blots are shown below graph.</p

Effect of xylazine administration on the mRNA levels of AMPKα2 in rats.

<p>(A) Cerebral cortex, (B) Hippocampus, (C) Thalamus, (D) Cerebellum and (E) Brainstem. Rats received saline (0.5 mL) or xylazine (5.2 mg/kg) intraperitoneally and then were sacrificed 10, 10, 20, 40 or 60 min later for control, Xyl1, Xyl2, Xyl3 or Xyl4, respectively. Total RNA was isolated and subjected to real-time PCR analysis. Each value of the expression levels of AMPKα2 was normalized to the expression levels of β-actin, and the control value was set to one. Data are presented as the means ± SEM, n = 6. * P < 0.05, ** P < 0.01 vs control.</p

Effect of xylazine administration on the levels of phosphorylated AMPKα in rats.

<p>(A) Cerebral cortex, (B) Hippocampus, (C) Thalamus, (D) Cerebellum and (E) Brainstem. Rats received saline (0.5 mL) or xylazine (5.2 mg/kg) intraperitoneally and then were sacrificed 10, 10, 20, 40 or 60 min later for control, Xyl1, Xyl2, Xyl3 or Xyl4, respectively. Western blot analyses were performed with anti-AMPKα and anti-phosphor-AMPKα (Thr172). Data for densitometry represent the mean ± SEM obtained from six independent series of Western blotting for each animal group and time point after the procedure. * P < 0.05, ** P < 0.01 vs control. Representative blots are shown below graph.</p