Establishment Of A New Asthma Model In Noninvasive And Conscious Brown Norway Rat Treated By Glucosides Of Tripterygium Wilfordii

ObjectiveTo establish a noninvasive and conscious Brown Norway rat model in which both the early-phase asthmatic response (EAR) and the late-phase asthmatic response (LAR) can be observed after sensitization and subsequent inhalation challenge with ovalbumin (OVA). And then study the effect and possible mechanism of glucosides of Tripterygium Wilfordii (GTW) on EAR and LAR by using this asthma animal model.MethodPart one:establishment of asthma modelSixty-six Brown Norway rats were divided into eleven equal groups. All groups of rats except normal control group were injected subcutaneously with 0.4mL (sc in back,2 sites,0.2 mL/site) OVA or OVA+A1(OH)3 solution on day 0 and day 5. Four groups were given OVA only at the dose of 0.01,0.1,1 and 10 mg/rat, five groups were given OVA+A1(OH)3 powder at the dose of 0.1+100,1+100,10+100, 1+52 and 1+4 mg/rat, one group was given OVA+A1(OH)3 gel at the dose of 10+4 mg/rat. Normal control group was injected subcutaneously with the same volume of saline. All the groups were challenged for 10 minutes with 5% OVA aerosol on day 37. Enhanced pause (Penh) was recorded for 16 hours in a whole-body plethysmography system after challenge. Then draw the Penh curves and calculate EAR Penh area under the curve (AUC), LAR AUC, EAR peak, LAR peak, LAR duration and the counts of EAR and LAR. Specific IgE of the sera samples on day 0, 7,14,21,28,35,38 were measured by the method of ELISA. Pulmonary pathological changes were measured by the method of HE staining.Part two:treatment of the asthma model with GTWThirty Brown Norway rats were divided into five equal groups (one normal control group, one model group and three GTW groups). To establish the allergic asthma model in noninvasive and conscious Brown Norway rat and observe whole period of asthma attack after challenge, rats were sensitized with OVA, Penh was recorded for 16 hours with whole-body plethysmography after challenge with OVA. And calculate EAR AUC, LAR AUC and LAR duration. Three GTW groups rats were dosed intragastrically with GTW at 10,50 and 200 mg·kg-1, respectively,2 ml per day for 8 days from Day 30 to Day 37. The normal control group and the model group were dosed intragastrically with the same volume of saline. Lung, liver and kidney pathological changes were measured following HE staining. The hepatic and renal toxicity were also evaluated by the test of glutamic oxalacetic transaminase (AST), glutamic-pyruvic transaminase (ALT), creatinine (CR) and urea nitrogen (BUN). Real-Time PCR (RT2 Profiler PCR Array Rat Allergy & Asthma) was performed to screen differentially expressed genes.ResultPart one:establishment of asthma modelCompared with normal control group, immunized rats except the group given 0.01mg of OVA produced specific IgE (P<0.05). And the content of IgE increased sharply after 7 days, and always keep ascending until day 35 (OVA 10+AI(OH)3100 group as the representative). The whole course of asthma exacerbation was recorded successfully. Rats developed EAR and/or LAR within 16 hours following OVA challenge. Especially the group injected with OVA 10mg and Al(OH)3 100mg or Al(OH)3 gel 4 mg showed steady pattern of biphasic airway responses and their EAR peak, LAR peak, LAR duration, EAR AUC and LAR AUC increased significantly compared with normal control group (P< 0.05). Eosinophils featured inflammation was observed in the lung of model group (OVA 10+Al(OH)3100 group as the representative).Part two:treatment of the asthma model with GTWAfter the treatment of GTW 50mg·kg-1, the specific IgE content were significantly lower than model group (P<0.05). After the treatment of GTW 10,50 and 200 mg·kg-1, the EAR AUC, LAR AUC and LAR duration were significantly lower than model group (P<0.05); the biochemical indexes of ALT was higher than model group (P<0.05), and with the increase of drug dose, ALT values increase; CR was higher than model group (P<0.05); two indexes of AST and BUN did not change significantly after GTW. GTW did not cause obvious toxicity of liver and kidney pathological changes. GTW reduced pulmonary eosinophil infiltration and the number of eosinophil decrease with the increase of GTW dose. GTW change the expression of abnormal genes of asthma. The gene of Mmp9 changes obviously most, up-regulate significantly after GTW, and the trend of up-regulate intensity has a dose dependent increase. After the treatment of GTW 10,50 and 200 mg·kg-1, the gene of Mmp9 down-regulate 3.62,7.67,38.15 times (P<0.05).Conclusion In this work we successfully developed a new model using noninvasive and conscious rats, and the whole time course of asthma exacerbation in this model can be observed after OVA challenge. OVA 10+A1(OH)3 100 group is the best combination for sensitization to establish this model and show EAR and LAR steadily. GTW can attenuate the EAR and LAR of asthmatic attack and may through down-regulate the expression of Mmp9 to achieve this goal. And the treatment effect of GTW tested with this asthma animal model proved it maybe a useful tool for asthma research.