Effects Of Rho Kinase Inhibitor Fasudil On Allergic Asthma

Objective: To observe the effects of fasudil,a selective Rho-associated kinase inhibitor,and Th17 cells on house dust mites(HDM)-induced allergic asthma.Methods: Twenty-four female C57BL/6 mice were randomly divided into four groups: asthma group,fasudil-therapy asthma group,control group,fasudil-treated control group.The first two groups were sensitized and challenged with HDM to establish asthmatic models in 16 days.Mice were inoculated intratracheally(i.t.)with 15 g of HDM in 30 ml PBS on day 0,3,5,7 days,and challenged for the last 3 days.The two control mice were inoculated i.t.with 30 ml PBS in meantime.Fasudil-therapy group and fasudil-treated control group were injected intraperitoneally(i.p.)with fasudil daily at the dosage of 30 mg/kg body weight.Asthma group and control group were injected i.p.with equal volumn of PBS.Twenty-four hours after the last challenge,bronchoalveolar lavage(BAL)fluids were collocated for cell counting and Kwik-Diff staining.For lung histology,paraffin sections of lung tissues were processed for hematoxylin/eosin(H&E)and periodic acid-schiff(PAS)staining to observe the pulmonary infiltrating and mucus secretion in the bronchi.The BAL fluids from each mouse were centrifuged for the determination of inflammatory cytokines by ELISA,such as IFN-g,IL-4,IL-5,IL-17 and eotaxin.Cytokines m RNA levels were measured by real-time quantitative PCR.The percentages of Th1,Th2,Th17,and Treg cells in the spleens in each group were analyzed by flow cytometer.Results: 1.The total number of BAL cells and the number of eosinophils,neutrophils,monocytes,and lymphocytes increased significantly in two asthma groups as compared with the control ones(P<0.05).Compared between the two asthma groups,fasudil treatment markedly reduced the total number of BAL cells and the number of eosinophils,neutrophils,and monocytes(P <0.05).2.Lung histology showed that inflammation and mucus secretion was significantly alleviated in the fasudiltherapy group as compared to untreated asthma group,although both were much higher than control groups.Lung tissue structure in the two control groups was intact without inflammatory changes.3.In fasudil-therapy group,the levels of IL-4,IL-5,IL-17 and eotaxin in BAL fluids were lower than those in the untreated asthma group.The two control groups had the lowest levels(P <0.05).The IFN-g level among the four groups had no statistically significant difference(P >0.05).4.Real-time quantitative PCR showed similar results that the m RNA levels of IL-4,IL-17,eotaxin and Gob-5 in the fasudil-therapy group were lower than those in the untreated asthma group,but no statistically significant in IFN-g m RNA level.5.The percentages of Th2 and Th17 cells in the spleens were markedly reduced by fasudil therapy compared to untreated asthma group(P <0.05),and the percentage of Treg cells was increased(P <0.05).However,this did not happen for Th1 cells(P >0.05).6.There was no significant difference between the two control groups(P >0.05).Conclusion: 1.New HDM-induced asthma model had the advantages of simply to operate,short experiment time and repeatability in our study.2.Fasudil can alleviate HDM-induced allergic airway inflammation by inhibiting infiltrating cells and inflammatory cytokines and chemokines,and mucus secretion in the airway and lung tissue.Fasudil at the dosage of 30 mg/kg body weight had no significant side effects observed.Fasudil might be developed as a novel therapeutic agent for allergic asthma,especially severe asthma.3.Inhibition of Rho A-ROCK signal pathway by fasudil was followed by the downregulation of Th17 cells and IL-17 in the airways.This suggests that Rho AROCK-Th17 axis may play important role in the airway inflammation.Our study provided new ideas and insights for the pathogenesis and therapy of allergic asthma and perhaps other inflammatory disorders.