Korean J Physiol Pharmacol.  2017 Sep;21(5):495-507. 10.4196/kjpp.2017.21.5.495.

The activation of α₂-adrenergic receptor in the spinal cord lowers sepsis-induced mortality

• Kim SS
• Park SH
• Lee JR
• Jung JS
• Suh HW

• KMID: 2388695
• DOI: http://doi.org/10.4196/kjpp.2017.21.5.495

Abstract

The effect of clonidine administered intrathecally (i.t.) on the mortality and the blood glucose level induced by sepsis was examined in mice. To produce sepsis, the mixture of D-galactosamine (GaLN; 0.6 g/10 ml)/lipopolysaccharide (LPS; 27 µg/27 µl) was treated intraperitoneally (i.p.). The i.t. pretreatment with clonidine (5 µg/5 µl) increased the blood glucose level and attenuated mortality induced by sepsis in a dose-dependent manner. The i.t. post-treatment with clonidine up to 3 h caused an elevation of the blood glucose level and protected sepsis-induced mortality, whereas clonidine post-treated at 6, 9, or 12 h did not affect. The pre-treatment with oral D-glucose for 30 min prior to i.t. post-treatment (6 h) with clonidine did not rescue sepsis-induced mortality. In addition, i.t. pretreatment with pertussis toxin (PTX) reduced clonidine-induced protection against mortality and clonidine-induced hyperglycemia, suggesting that protective effect against sepsis-induced mortality seems to be mediated via activating PTX-sensitive G-proteins in the spinal cord. Moreover, pretreatment with clonidine attenuated the plasma tumor necrosis factor α (TNF-α) induced by sepsis. Clonidine administered i.t. or i.p. increased p-AMPKα1 and p-AMPKα2, but decreased p-Tyk2 and p-mTOR levels in both control and sepsis groups, suggesting that the up-regulations of p-AMPKα1 and p-AMPKα2, or down-regulations of p-mTOR and p-Tyk2 may play critical roles for the protective effect of clonidine against sepsis-induced mortality.

Keyword

Blood glucose; Clonidine; p-AMPKα1; Sepsis; TNF-α; α₂-adrenergic receptor

MeSH Terms

Animals
Blood Glucose
Clonidine
Glucose
GTP-Binding Proteins
Hyperglycemia
Mice
Mortality*
Pertussis Toxin
Plasma
Sepsis
Spinal Cord*
Tumor Necrosis Factor-alpha
Blood Glucose
Clonidine
GTP-Binding Proteins
Glucose
Pertussis Toxin
Tumor Necrosis Factor-alpha

Figure

• 

  Fig. 1 Dose-dependent effects of clonidine on mortality and hypoglycemia induced by sepsis. Mice were treated i.t. with various doses (0.1~5 µg) with clonidine 10 minutes prior to GaLN (0.6 g/10 ml)/LPS (27 µg/27 µl) administration. The mortality rate (A) was measured at 12, 24, 36, 48, 72, 84 and 96 h after GaLN/LPS injection. The blood glucose level (B) was measured at various times after GaLN/LPS injection. The number of animals used for each group was 10. The vertical bars indicate the standard error of the mean (***p<0.001; compared with saline+sepsis group, **p<0.01; compared with saline+sepsis group).

• 

  Fig. 2 Effect of intrathecally (i.t.) administered clonidine on the plasma insulin and the blood glucose levels in control and sepsis groups. Mice were treated i.t. with 5 µg of clonidine 10 minutes prior to GaLN (0.6 g/10 ml)/LPS (27 µg/27 µl) administration. Plasma insulin level was measured 1 (A) and 3 h (B) after i.p. injection with GaLN/LPS injection. The number of animals used for each group was 10. The vertical bars indicate the standard error of the mean (***p<0.001; compared with saline group, +++p<0.001; compared with saline+clonidine group).

• 

  Fig. 3 Effect of intrathecally (i.t.) administered clonidine on plasma corticosterone level in control and sepsis groups. Mice were treated i.t. with 5 µg of clonidine 10 minutes prior to GaLN (0.6 g/10 ml)/LPS (27 µg/27 µl) administration. Plasma corticosterone level was measured 1 (A) and 3 h (B) after i.p. injection with GaLN/LPS injection. The number of animals used for each group was 5. The vertical bars indicate the standard error of the mean (***p<0.001; compared with saline group, **p<0.01; compared with saline group, *p<0.05; compared with saline group).

• 

  Fig. 4 Effect of clonidine post-treated spinally on sepsis-induced mortality. The mice were post-treated i.t. with either clonidine (5 µg/5 µl) or saline at 1 (A), 3 (B), 6 (C), 9 (D) and 12 (E) hr after GaLN (0.6 g/10 ml)/LPS (27 µg/27 µl) administration. The mortality rate was counted at 12, 24, 36, 48, 72, 84 and 96 h after GaLN/LPS injection. The number of animals used for each group was 10.

• 

  Fig. 5 Effect of clonidine post-treated spinally on sepsis-induced hypoglycemia. The mice were post-treated i.t. with clonidine (5 µg/5 µl) or saline at 1 (A), 3 (B), 6 (C), 9 (D) and 12 (E) h after D-galactosamine (0.6 g/10 ml)/LPS (27 µg/27 µl) administration. The blood glucose level was measured at various times after GaLN/LPS injection. The number of animals used for each group was 10 (***p<0.001; compared with saline+sepsis group, **p<0.01; compared with saline+sepsis group, *p<0.05; compared with saline+sepsis group).

• 

  Fig. 6 Effect of D-glucose feeding on clonidine-regulated blood glucose level and survival rate in sepsis group. Mice were post-treated orally with either saline or D-glucose (8 g/kg) at 5.5 h and clonidine was post-treated i.t. at 6 h after GaLN (0.6 g/10 ml)/LPS (27 µg/27 µl) administration. The mortality rate (A) was counted at 12, 24, 36, 48, 72, 84 and 96 h after GaLN/LPS injection. The blood glucose level (B) was measured at various times after GaLN/LPS injection. The number of animals used for each group was 10.

• 

  Fig. 7 The profiles of blood glucose level changes of clonidine intrathecally (i.t.) administered repeatedly in control and sepsis groups. Mice were first pretreated i.t. with either saline or clonidine (5 µg/5 µl) 10 minutes prior to administration with saline or GaLN (0.6 g/10 ml)/LPS (27 µg/27 µl). Clonidine was post-treated i.t. 6 h after the induction of sepsis. The blood glucose level was measured at various times after GaLN/LPS injection. The number of animals used for each group was 10 (***p<0.001; compared with saline+clonidine/normal group, +++p<0.001; compared with saline+clonidine/sepsis group, ###p<0.001; compared with clonidine+clonidine/normal group).

• 

  Fig. 8 Effect of intrathecal pretreatment with PTX on clonidine-induced hyperglycemia and protective effects against sepsis. Mice were pre-treated i.t. once with either saline or PTX (0.1 µg/0.5 µl) for 6 days and clonidine (5 µg/5 µl) was administered i.t. 10 min prior to GaLN/LPS injection. The mortality (A) was measured at 12, 24, 36, 48, 72, 84 and 96 h after GaLN/LPS injection. The blood glucose level (B) was measured at various times after GaLN/LPS injection. The number of animals used for each group was 10 (***p<0.001; compared with PTX+clonidine/sepsis group, **p<0.01; compared with PTX+clonidine/sepsis group).

• 

  Fig. 9 Effect of clonidine administered spinally on sepsis-induced plasma TNF-α level. Mice were treated i.t. with 5 mg of clonidine 10 minutes prior to GaLN (0.6 g/10 ml)/LPS (27 µg/27 µl) administration. Plasma TNF-α level was measured 1 (A) and 3 h (B) after i.p. injection with GaLN/LPS injection. The number of animals used for each group was 6. The vertical bars indicate the standard error of the mean (***p<0.001; compared with saline+sepsis group, *p<0.05; compared with salin+sepsis group).

• 

  Fig. 10 Effect of clonidine administered spinally on liver p-AMPKα1, p-AMPKα2, p-mTOR, p-Tyk2 expression in control and sepsis groups. Mice were treated i.t. with 5 µg of clonidine 10 minutes prior to GaLN (0.6 g/10 ml)/LPS (27 µg/27 µl) administration. The proteins of liver were extracted at 1 and 3 h after sepsis-induction and the Western blot for p-AMPKα1 (A, B), p-AMPKα2 (C, D), p-mTOR (E, F), and p-Tyk2 (G, H) expression was carried out. Fifty µg of proteins were used for the detecting p-AMPKα1, p-AMPKα2, p-mTOR, and p-Tyk2 expression. The number of animals used for each group was 6~12 (**p<0.01; compared with control group, *p<0.05; compared with control group, +p<0.05; compared with sepsis group).

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  Fig. 11 Effect of clonidine administered spinally on liver AMPKα1, AMPKα2, mTOR, Tyk2 expression in control and sepsis groups. Mice were treated i.t. with 5 µg of clonidine 10 minutes prior to GaLN (0.6 g/10 ml)/LPS (27 µg/27 µl) administration. The proteins of liver were extracted at 1 and 3 h after sepsis-induction and the Western blot for AMPKα1 (A, B), AMPKα2 (C, D), mTOR (E, F), and Tyk2 (G, H) expression was carried out. Fifty µg of proteins were used for the detecting AMPKα1, AMPKα2, mTOR, and Tyk2 expression. The number of animals used for each group was 6.

• 

  Fig. 12 Effect of clonidine administered intraperitoneally on liver p-AMPKα1, p-AMPKα2, p-mTOR, and p-Tyk2 expression in control and sepsis groups. Mice were treated i.t. with 5 µg of clonidine 10 min prior to GaLN (0.6 g/10 ml)/LPS (27 µg/27 µl) administration. The proteins of liver were extracted at 3 h after sepsis-induction and the Western blot for p-AMPKα1 (A), p-AMPKα2 (B), p-mTOR (C), and p-Tyk2 (D) expression was carried out. Fifty µg of proteins were used for the detecting p-AMPKα1, p-AMPKα2, p-mTOR, and p-Tyk2 expression. The number of animals used for each group was 6~12 (*p<0.05; compared with control group, +p<0.05; compared with sepsis group).

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