Therapeutic Effects Of Glycyrrhizic Acid On Airway Inflammation In A Murine Model Of Asthma And Its Mechanism

Part Ⅰ Therapeutic effects of glycyrrhizic acid on airway inflammation in a murine model of asthmaObjective: To explore the therapeutic effects and mechanism of glycyrrhizic acid(GA) on airway inflammation in a murine model of asthma.Methods: We established and treated a murine model of asthma with three different concentrations of GA(25, 50 and 100 mg/kg). Within 24 hours after the last OVA challenge, airway responsiveness was measured and expressed as lung resistance(RL) and dynamic compliance(Cdyn) and bronchoalveolar lavage fluid(BALF) was collected for counting total cells and eosinophils(Eos), lymphocytes(Lym), neutrophils(Neu), and macrophages(Mac). Ig E and OVA-specific Ig E in serum and IL-12p70、IL-10、IL-4、IL-5、IL-13 and IFN-γ in BALF were detected by ELISA. Histological studies of lung were conducted with the hematoxylin and eosin staining(H&E) and the expression of Eotaxin, MCP-1 and CINC-1 was detected with immunohistochemical staining.Results:(1) The mice of asthma group showed the typical symptoms of acute asthma when exposed to aerosolized OVA, the symptoms were lighter in asthmatic mice treated with GA.(2) The based values of RL and Cdyn were close among each group, the RL was increased and the Cdyn was decreased along with the rising doses of methacholine. When challenged with the intravenous methacholine at the dose of 0.050, 0.100, 0.200 mg/kg, the RL was significantly elevated and the Cdyn was decreased in asthma group compared with those in control group(all P <0.05). The RL was significantly lower and the Cdyn was higher in asthma group treated with 50 and 100 mg/kg concentrations of GA at the dose of 0.100, 0.200 mg/kg of methacholine compared with those in asthma group(all P <0.05).(3) The concentrations of Ig E and OVA-specific Ig E in serum in asthma group were higher than those in control group(all P <0.05). The concentrations of Ig E and OVA-specific Ig E in serum in asthma group treated with GA were lower than those in asthma group, especially in 50 and 100 mg/kg concentrations of GA groups(all P <0.05).(4) Compared with the control group, the numbers of the total cells and eosinophils, lymphocytes and neutrophils were higher in BALF in asthma group, while the values were decreased in 100 mg/kg GA treated asthmatic group(all P <0.05).(5) Although the level of IL-12p70 in BALF was lower in asthma group than that in control group, there is no significant difference between the groups(P >0.05). The levels of IL-12p70 were higher in 50 mg/kg and 100 mg/kg GA treated asthmatic group than those in asthma group(all P <0.05). The levels of IL-4, IL-5 and IL-13 in BALF in asthmatic mice were much higher than those in control group, whereas the levels of IL-4, IL-5 and IL-13 were much lower in 50 mg/kg and 100 mg/kg GA treated asthmatic group than those in asthma group(all P <0.05). The expression of IL-10 in BALF was higher than that in control group(t =3.373,P <0.05), but the expression of IL-10 in BALF were much higher in three different concentrations GA treated group than those in asthma group(F =54.187, P <0.05). Compared with the control group, the level of IFN-γ and IFN-γ/IL-4 ratio were decreased in BALF in asthma group, and the values were increased by the treatment of GA in a dose of 50 mg/kg or 100 mg/kg(all P <0.05).(6) OVA induced infiltration of inflammatory cells around airways and blood vessels in the asthmatic mice. Administration of GA significantly reduced the infiltration of inflammatory cells in the peribronchial areas compared with the asthmatic mice especially at a dose of 50 mg/kg, 100 mg/kg.(7) The protein expression of Eotaxin, MCP-1 and CINC-1 in immunohistochemistry was increased significantly in lungs from the asthma group compared with those in the control group(all P <0.05). Treating mice with GA reduced the expression of Eotaxin, MCP-1 and CINC-1 in the asthma group in a dose-dependent manner.Conclusions:(1) GA could decrease the airway hyperresponsiveness, reduce the numbers of total cells and Eos, Lyms, Neus in BALF, lower the concentrations of Ig E and OVA-specific Ig E in serum, increase the levels of IL-10, IL-12p70 and IFN-γ and decrease the levels of IL-4, IL-15 and IL-13 in BALF in a murine model of asthma.(2) GA could downregulate the expression of Eotaxin, MCP-1 and CINC-1 in lungs in asthma group.(3) GA may effectively ameliorate the airway inflammation of asthma via affecting the balance of Th1/Th2. Part Ⅱ Effects of glycyrrhizic acid on OX40/OX40L/p38 MAPK signaling pathway in a murine model of asthmaObjective: To explore the effects of glycyrrhizic acid(GA) on OX40/OX40L/p38 MAPK signaling pathway in a murine model of asthma.Methods:(the first part of experiment) We established and treated a murine model of asthma with three different concentrations of GA(25, 50 and 100 mg/kg). The m RNA expression of OX40 and OX40 L in lung tissues was detected by RT-PCR. The expression of CD86, MHC-Ⅱ, CD40 and OX40 L on CD11c+ dendritic cells(DCs), the expression of OX40 on CD4+ T cells and CD8+ T cells and the expression of OX40 L on CD11b+ monocytes and CD19+ B cells in spleens were evaluated with flow cytometry. The protein expression of OX40, OX40 L, p38 MAPK and p-p38 MAPK was detected with immunohistochemical staining and the expression of p38 MAPK and p-p38 MAPK was also measured by Western Blotting.(the second part of experiment) We established and treated a murine model of asthma with 100 mg/kg concentration of GA, anti-OX40 L m Ab and p38 MAPK inhibitor(SB203580), respectively. Ig G2 b or DMSO was given as a isotype control. Within 24 hours after the last OVA challenge, the symptoms, airway responsiveness, total cells and eosinophils, lymphocytes, neutrophils and macrophages in BALF were observed. Ig E and OVA-specific Ig E in serum and IL-4, IL-5, IL-13 and IFN-γ in BALF were detected by ELISA. Histological studies of lung were conducted with the H&E. The protein expression of p38 MAPK and p-p38 MAPK was detected with immunohistochemical staining and Western Blotting.Results:(the first part of experiment)(1) The OX40 and OX40 L m RNA levels of lungs were significantly increased in the asthma group compared to those in control group, whereas GA markedly attenuated OVA-induced OX40 and OX40 L m RNA expression in a dose-dependent manner(all P <0.05).(2) The expression of CD86, MHC-Ⅱ, CD40, OX40 L on CD11c+ DCs in spleens was increased significantly compared with the control group(all P <0.05). Treating mice with GA reduced the expression of MHC-Ⅱ, CD40, OX40 L on CD11c+ DCs in the asthma group in a dose-dependent manner(all P <0.05).(3) OVA induced a significant increase of OX40 expression in CD4+ T cells, and OX40 L expression in CD11b+ monocytes and CD19+ B cells in spleens of asthmatic mice compared to those treated with saline. Treatment with GA dramatically decreased the levels of OX40 and OX40 L expression in spleens of asthmatic mice, especially those treated with high dosage of GA(all P <0.05).(4) The protein expression of OX40 and OX40 L in immunohistochemistry was upregulated in the lungs of the asthma group as compared with control group(all P <0.05). Treatment with GA substantially reduced the upregulation of OX40 and OX40 L and the efficacy was seen significantly at dosage of 50 mg/kg, 100 mg/kg(all P <0.05). OVA induced a significant increase of phosphorylation of p38 MAPK in lung tissues of asthmatic mice compared to those treated with saline. Treatment with GA at 50, 100 mg/kg concentrations inhibited OVA-induced phosphorylation of p38 MAPK in allergic mice(P <0.05).(the second part of experiment)(1) The mice of asthma group showed the typical symptoms of acute asthma when exposed to aerosolized OVA, the symptoms were lighter in asthmatic mice treated with GA, anti-OX40 L m Ab or SB203580.(2) The RL was significantly lower and the Cdyn was higher in asthma group treated with GA, anti-OX40 L m Ab or SB203580 at the dose of 0.050, 0.100, 0.200 mg/kg of methacholine compared with those in asthma group(all P <0.05).(3) The concentrations of Ig E and OVA-specific Ig E in serum were decreased in asthma group treated with GA, anti-OX40 L m Ab or SB203580 compared with those in asthma group(all P <0.05).(4) Compared with the asthma group, the numbers of the total cells and eosinophils, lymphocytes and neutrophils were decreased in BALF in 100 mg/kg GA, anti-OX40 L m Ab or SB203580 treated asthmatic group(all P <0.05).(5) The levels of IL-4, IL-5 and IL-13 were much lower and the levels of IFN-γ were higher in BALF in asthma group treated with GA, anti-OX40 L m Ab or SB203580 than those in asthma group(all P <0.05).(6) Administration of GA, anti-OX40 L m Ab or SB203580 significantly reduced the infiltration of inflammatory cells in the peribronchial areas compared with the asthmatic mice.(7) Treatment with GA with dose of 100 mg/kg or anti-OX40 L m Ab or SB203580 effectively inhibited OVA-induced phosphorylation of p38 MAPK in allergic mice(all P <0.05). No difference can be seen in the expression of p38 MAPK among these groups(P>0.05).Conclusions:(1) GA could inhibit the OX40 and OX40 L m RNA expression in lungs and attenuate the OX40 and OX40 L protein expression in both lungs and spleens in a dose-dependent manner.(2) GA may inhibit the Th2 responses through modulating the expression of CD86, MHC-Ⅱ, CD40, OX40 L on CD11c+ DCs.(3) High dosage of GA, anti-OX40 L m Ab or p38 MAPK inhibitor(SB203580)could relieve the symptoms of asthma attack, decrease the airway hyperresponsiveness, reduce the numbers of total cells and Eos, Lyms, Neus in BALF, lower the concentrations of Ig E and OVA-specific Ig E in serum, increase the levels of IFN-γ and decrease the levels of IL-4, IL-15 and IL-13 in BALF, lessen the inflammatory cells infiltration in lungs in a murine model of asthma.(4) Treatment with GA with dose of 100 mg/kg or anti-OX40 L m Ab or SB203580 could effectively inhibit OVA-induced phosphorylation of p38 MAPK in allergic mice.(5) GA may exert a therapeutic effect on OVA-induced experimental asthma partly by regulating the Th1/Th2 balance via OX40/OX40 L signaling and p38 MAPK pathway.Part Ⅲ The effect of Glycyrrhizic acid(GA) on the proliferation of CD4+T cells and OX40/OX40L/p38 MAPK signaling pathwayObjective: To investigate the effect of Glycyrrhizic acid(GA) on the proliferation of CD4+T cells and OX40/OX40L/p38 MAPK signaling pathway.Methods: CD4+ T cells were purified from spleens of OVA-sensitized and challenged mice by using the Mouse CD4 Cell Positive Isolation Kit and incubated with anti-CD3 m Ab(1 μg/ml) and anti-OX40 m Ab(2.5 μg/ml) or with anti-CD3 m Ab alone in the presence of various concentrations of GA(5, 10, and 100 μg/ml) or p38 MAPK inhibitor(SB203580)(10 μM). After 72 h of incubation, the proliferation of CD4+T cells was measured by CCK8 kit and the levels of IL-4, IL-5, IL-13 and IFN-γ in cultural supernatant were detected by ELISA. The expression of p38 and p-p38 MAPK of CD4+T cells was detected by Western Blotting.Results:(1) The optiacal density(OD) values were higher in splenic CD4+ T cells from OVA-induced asthmatic mice stimulated with anti-CD3 m Ab than those in control group(P <0.01), while the OD values were decreased after incubated with different concentrations of GA in group CD3, especially in 10μg/ml and 100μg/ml concentrations of GA( all P <0.01). After stimulated with both anti-CD3 m Ab and anti-OX40 m Ab, the proliferated capacity of CD4+ T cells were elevated. The OD values were much higher in group CD3+OX40 than those in group CD3+Ig G(P <0.01). The OD values were lower after intervened with different concentrations of GA compared with group CD3+OX40+DMSO, especially in 10μg/ml and 100μg/ml concentrations of GA( all P <0.01). The same effect can be seen after intervened with p38 MAPK inhibitor(SB203580)(P <0.01).(2) The levels of IL-4, IL-5 and IL-13 were significantly higher in splenic CD4+ T cells cultural supernatant from OVA-induced asthmatic mice stimulated with anti-CD3 m Ab and anti-OX40 m Ab compared with those in control group(all P <0.01). Both GA and SB203580 could inhibit the secretion of IL-4, IL-5 and IL-13. Compared with group CD3+OX40+DMSO, the levels of IL-4 and IL-13 in 10μg/ml and 100μg/ml concentrations of GA and SB203580 were much lower, while the same effect could be seen only in 100μg/ml concentration of GA and SB203580 for the secretion of IL-5(all P <0.01). The levels of IFN-γ were significantly higher in splenic CD4+ T cells from OVA-induced asthmatic mice stimulated with anti-CD3 m Ab compared with those in control group(P <0.01). While after costimulated with anti-OX40 m Ab, the levels of IFN-γ were lower compared with group CD3+Ig G(P <0.01). The levels of IFN-γ in 10μg/ml and 100μg/ml concentrations of GA and group SB203580 were much higher compared with group CD3+OX40+DMSO(all P <0.01).(3) There is no significant difference in the expression of p38 MAPK in CD4+ T cells after 72 h incubation among groups CD3, CD3+OX40, CD3+OX40+DMSO, CD3+OX40+100μg/ml GA and CD3+OX40+SB203580(all P>0.01). The expression of p-p38 MAPK in CD4+ T cells costimulated with anti-CD3 m Ab and anti-OX40 m Ab was higher than those stimulated with anti-CD3 m Ab alone(P <0.01). The expression of p-p38 MAPK was much lower in 100μg/ml concentrations of GA and group SB203580 compared with that in group CD3+OX40+DMSO(F=27.579,P=0.000).Conclusions:(1) The OX40/OX40 L costimulatory signals played an important role in CD4+ T cells proliferation and secretion of cytokines.(2) The inhibitor of p38 MAPK could block the proliferation effect mediated by the OX40/OX40 L costimulatory signaling pathway and modulate the secretion of cytokines.(3) GA could inhibit the proliferation of CD4+ T cells stimulated by anti-OX40 m Ab, modulate the production of Th1 and Th2 cytokines and down regulate the phosphorylation of p38 MAPK, which suggests that GA could inhibit CD4+ T cells to differentiate to Th2 cells by directly blocking OX40/OX40L/p38 MAPK signaling pathway.