The synergistic anti-asthmatic effects of glycyrrhizin and salbutamol

Abstract

Aim: To investigate the efficacy of glycyrrhizin (GL) combined with salbutamol (SA) as an anti-asthma therapy.

Methods: Rat lung beta2-adrenergic receptor (beta(2)-AR) mRNA level was measured by real-time RT PCR. Intracellular cAMP accumulation was evaluated with a reporter gene assay. An in vitro acetylcholine-induced guinea pig tracheal strip contraction model was used to test the relaxing effects of GL and SA. The anti-inflammatory effects of GL and SA were tested using tumor necrosis factor-alpha-induced NF-kappaB transcriptional activation reporter assay, I-kappaB Western blotting and interleukin-8 ELISA. An in vivo guinea pig asthma model was used to prove further the synergistic effect of GL and SA.

Results: GL (0.3 micromol/L) increased mRNA levels of beta(2)-AR in vivo and the accumulation of cAMP in vitro. The combination of GL and SA also resulted in significant complementary anti-inflammatory effects via inhibition of NF-kappaB activation, degradation of I-kappaB and production of interleukin-8. A significant synergistic effect of the combination was detected both in vitro and in vivo in a guinea pig mode.

Conclusion: The results demonstrate that GL and SA have synergistic anti-asthmatic effects and offer the possibility of a therapeutic application of GL in combination with beta(2)-AR agonists in the treatment of asthma.

Figure 1

Effects of GL on increasing β₂-AR mRNA levels. Animals inhaled atomized physiological saline (control), 0.1 μmol/L salbutamol (SA), 0.3 μmol/L glycyrrhizin (GL), or 0.1 μmol/L SA and 0.3 μmol/L GL (SA+GL) twice a day, for 7 d. Mean±SEM. n=3. ^cP<0.01 vs the saline control group. ^fP<0.01 vs the 0.3 μmol/L GL group.

Figure 2

Effects of GL on increasing salbutamol (SA)-stimulated cAMP accumulation. (A) Dose-response curve of CHO-β₂-AR-CRE-GFP cells stimulated by different concentrations of GL or SA alone, or 0.3 μmol/L GL administered in concert with different SA concentrations (SA+GL). (B) The time course of 0.01 μmol/L SA and 0.3 μmol/L GL stimulated reporter gene expression in CHO-β₂-AR-CRE-GFP cells. Mean±SEM. n=3. ^cP<0.01 vs 0.01 μmol/L SA treated group.

Figure 3

Effects of GL on enhancing the relaxing effect of β₂-AR agonists in isolated guinea pig tracheal strips. Tension is expressed as a percentage of the peak contractile response, which was assigned as 100%. Mean±SEM. n=3.

Figure 4

Effects of GL and SA on inhibiting TNF-α-induced NF-κB activation. (A) After transfected with Igκ2-IFN-LUC and pRL-TK plasmids, HEK293 cells in different groups were treated with 10 ng/mL TNF-α, 0.01 μmol/L SA or 0.01 μmol/L GL for 6 h. The luciferase activity was expressed as the ratio of firefly to renilla luminescence. Mean±SEM. n=3. ^cP<0.01 vs control without any stimulation. ^fP<0.01 vs the TNF-α-activated group. (B) NIH-3T3 cells, treated with 0.01 μmol/L GL, 0.01 μmol/L SA or the combination, were then stimulated by 10 ng/mL TNF-α. The time course of I-κBα protein degradation was analyzed by Western blot. Three separate experiments confirmed observed changes.

Figure 5

Effects of GL and SA on inhibiting TNF-α mediated IL-8 production. A549 cells (A) or human bronchial smooth muscle cells (HBSMC). (B) were incubated with 0.1 mmol/L SA, 0.1 mmol/L GL, 0.01 mmol/L dexamethasone (DEX) or their combinations for 1 h, in concert with 10 ng/mL TNF-α incubation for 6 h. IL-8 levels in culture medium were assessed by ELISA. Each bar represents the mean±SEM. n=3. ^cP<0.01 vs control without any stimulation. ^fP<0.01 vs the TNF-α-activated group. ⁱP<0.01 vs the 0.1 mmol/L GL group.

Figure 6

Synergistic anti-asthmatic effects of salbutamol (SA) and glycyrrhizin (GL) in the atomized histamine asthma induced guinea pig model. Animals were exposed to atomized 0.1% histamine solutions for 20 s. After a two day interval exposure to 0.1 μmol/L SA, 1.0 μmol/L SA, 0.3 μmol/L GL, or 0.3 μmol/L GL and 0.1 μmol/L SA occurred immediately prior to the second 0.1% histamine solution exposure. Asthma latency period increasing rate of each animal was presented as the ratio of increased time to the original latency time. Each bar represents the mean±SEM. n=7. ^cP<0.01 vs control.