Inhibition Of Airway Inflammation And Hyperresponsiveness By Tetrandrine Via Downregulation Of Nuclear Factor κB And Inducible Nitric Oxide Synthase In Asthmatic Mice

Objective(1) To investigate whether tetrandrine possesses the inhibitory effect on airway inflammation and airway hyperresponsiveness in a murine model of ovalbumin(OVA)-induced asthma.(2) Whether the effect of tetrandrine on asthmatic murine relates to the downregulation of classical nuclear factor κB( NF-κB) pathway, thus inhibiting the expression of inducible nitric oxide synthase( i NOS) and inflammatory cytokines(IL-4 and IL-13).Methods36 BALB/c mice were randomly divided into 6 groups, including control group(N group),asthma group(OVA group),dexamethasone group(Dex group),high-dose tetrandrine group(TH group),low-dose tetrandrine group(TL group)and solvent control group(HCl group). The mice were sensitized on days 0,7 and 14 by intraperitoneal injection of 200 μL OVA(100 μg OVA emulsified in 4 mg aluminum hydroxide gel). On days 21 to 25 after initial sensitization, the mice were challenged for 30 min with an aerosol of 5%(wt/vol) OVA in phosphate-buffered saline( PBS) using an ultrasonic nebulizer. Dexamethasone(1 mg/kg),tetrandrine(50 and 25 mg/kg)and HCl( p H 6.5~7.0) were given by intraperitoneal injection 30 min before nebulizing OVA per day up to 5 days. Control mice were sensitized and challenged through the same protocol only using PBS. 24 h after the last challenge, airway resistance was measured by pulmonary function detector.Hematoxylin & eosin(HE)staining was used to observe the airway inflammatory cells infiltration. Levels of total Ig E and OVA-specific Ig E(OVA-s Ig E)in serum and Th2 cytokines(IL-4 and IL-13)in bronchoalveolar lavage fluid(BALF)were detected by enzyme-linked immunosorbent assay(ELISA).Total number of inflammatory cells in BALF was counted with a hemocytometer. Smears of BALF cells were stained with Wright’s staining for eosinophils differential count. The protein expression of NF-κB pathway(phospho-p65, cytoplasm p65 and nucleus p65)and i NOS were determined by western blot analysis.Results(1) Airway resistance : The airway resistance in the control group was only slightly increased, whereas an obvious increase in a dose-dependent manner following the concentration of acetylcholine(Ach)could be detected in the OVA and HCl groups.The OVA and HCl groups had significantly greater airway resistance than that of the control mice. Administration of tetrandrine or dexamethasone led to a sharp decrease in airway resistance compared with the OVA group(P﹤0.05).(2) H&E staining : The OVA and HCl group mice exhibited an obvious peribronchial and perivascular inflammatory cell infiltration, revealing an enhanced airway eosinophilia as compared to the control group. Conversely,pretreatment with tetrandrine or dexamethasone protected mice from developing the OVA-induced airway inflammatory cells infiltration( P ﹤0.05).(3) ELISA :Compared to the control group,total Ig E and OVA-s Ig E in serum,Th2 cytokines(IL-4 and IL-13)in BALF were apparently elevated in the OVA and HCl group. Treatment with tetrandrine or dexamethasone markedly reduced the levels of serum immunoglobulins and Th2 cytokines in BALF compared with those in OVA group(P﹤0.05).(4) Cell count : The OVA and HCl groups showed a marked influx of inflammatory cells into the airways, including eosinophils, lymphocytes,macrophages and neutrophils compared to the control group. While treatment with tetrandrine or dexamethasone could remarkably prevent these increases(P﹤0.05).(5) Western Blot : OVA challenge could induce degradation of IκBα and phosphorylation of p65 subunit, elevate the level of p65 subunit in the nuclear extraction of the lung tissue, while reduce the level of p65 in the cytoplasm extract. In contrast, pretreatment with tetrandrine or dexamethasone augmented pulmonary IκBα expression, suppressed the phosphorylation and nuclear translocation of p65 subunit as well as the expression of i NOS protein(P﹤0.05).Conclusions(1) Tetrandrine could inhibit airway inflammation and airway hyperresponsiveness in a murine model of asthma.(2) The effect of tetrandrine on asthmatic murine may be related to the downregulation of classical NF-κB pathway, thus inhibiting the expression of i NOS and inflammatory cytokines IL-4 and IL-13.