法舒地尔对支气管哮喘小鼠肺组织Rho激酶-1表达及气道炎症的影响

目的 观察Rho激酶-1抑制剂法舒地尔对支气管哮喘(简称哮喘)小鼠肺组织Rho激酶-1表达及气道炎症的影响,探讨Rho激酶-1在哮喘气道炎症中的作用机制.方法 将24只BalB/c小鼠采用随机数字表法分为对照组、哮喘组和干预组,每组8只.哮喘组、干预组小鼠分别给予卵清白蛋白(OVA)致敏和激发.每次雾化前1 h,干预组给予法舒地尔(10 mg/kg)腹腔注射.末次激发后收集BalF,离心后计数细胞总数及嗜酸粒细胞(EOS)数量.ELISA法测定BalF上清液中嗜酸粒细胞趋化因子(Eotaxin)、白细胞介素(IL)-5和IL-13水平.肺组织HE染色.采用逆转录PCR和免疫组织化学测定各组小鼠肺组织中Rho激酶-1 mRNA和蛋白的表达水平.结果 (1)哮喘组BalF中细胞总数及EOS数量分别为(1.45±0.12)× 10~9/L和(0.52 ±0.06)× 10~9/L,明显高于对照组[分别为(0.58±0.06)×10~9/L和(0.01±0.01)×10~9/L](q值分别为25.909和35.002,均P＜0.01)和干预组[分别为(0.89 ±0.09)×10~9/L和(0.20±0.04)×10~9/L](q值分别为16.676和21.537,均P＜0.01).(2)哮喘组Eotaxin、IL-5及IL-13水平分别为(45±8)ng/L、(157 ±23)ng/L和(429±46)ng/L,明显高于对照组[分别为(10 ±3)ng/L、(26±6)ng/L和(126 ±20)ng/L](q值分别为18.246、23.009、25.826,均P＜0.01);干预组分别为(20±5)ng/L、(57 ±14)ng/L和(254±28)ng/L,明显低于哮喘组(q值分别为13.119、17.503、8.449,均P＜0.01).(3)对照组小鼠气道周围无炎症细胞浸润,哮喘组小鼠气道黏膜水肿,气道壁及管周有大量以EOS为主的炎症细胞浸润,干预组气道炎症反应较哮喘组减轻.(4)哮喘组肺组织Rho激酶-1 mRNA和蛋白的表达水平明显高于对照组(q值分别为25.614和8.156,均P＜0.01),干预组Rho激酶-1 mRNA和蛋白表达水平低于哮喘组(q值分别为20.379和4.135,均P＜0.01).(5)Rho激酶-1 mRNA表达量与BalF中EOS数量、Eotaxin、IL-5和IL-13水平呈正相关(r值分别为0.709、0.600、0.613、0.650,均P＜0.01).结论 Rho激酶-1参与过敏原诱导的哮喘小鼠气道炎症的发生,应用法舒地尔抑制其表达和活性可能改善哮喘气道炎症.

Effects of fasudil on the expression of Rho kinase-1 and airway inflammation in a mouse model of asthma

Objective To explore the role of Rho kinase-1(ROCK-1)in airway inflammation of asthma by obsenrving the effects of fasudil,a specific inhibitor of ROCK-1,on the expression of Rho kinasel and airway inflammation in a mouse model of asthma.Methods Twenty-four female BalB/c mice were randomly divided into 3 groups(n=8 each):a control group,an asthmatic group and a treatment group.Mice in the asthmatic and the treatment groups were sensitized by intraperitoneal injection of OVA(25 μg)precipitated with 1 mg of alum in 200 μ1 of saline on days 1 and 15,and subsequently challenged by nebulization of 2%OVA on davs 22-26.Mice in the control group were sensitized with al(OH)3 saline and challenged with saline instead of OVA.Mice of the treatment group were iniected intraperitoneally with fasudil(10 mg/kg)1 h before each OVA challenge.all the mice were killed 24 h after the final challenge,and bronchoalveolar lavage fluid(BalF)was collected for counting total inflammatory cells and eosinophils (EOS).Cytokines and chemokines in BalF were measured by ELISA.The lung tissue slides were examined histologically.The protein and mRNA expression of ROCK-1 were measured by immunohistochemistry and RT-PCR respectively. Results (1) OVA challenge in mice of the asthmatic group caused a marked increase in the number of the total cells and eosinophils in BalF ( q = 25.909,35. 002, respectively, all P＜0. 01 ) . When fasudil was applied, both the total cell counts and the eosinophil numbers were significantly decreased. The total cell number was decreased from (1.45 ±0. 12)× 10~9/L to (0. 89 ± 0. 09 ) × 10~9/L ( q = 16. 676, P＜0. 01 ), and the number of eosinophils was decreased from (0.52 ±0.06)×10~9/L to (0.20 ±0.04) ×10~9/L (q =21.537, P＜0.01). (2)Compared with the control group, OVA challenge in mice of the asthmatic group induced eotaxin, IL-5 and IL-13 release into BalF (q = 18. 246, 23. 009, 25. 826, respectively, all P ＜0. 01 ). The eotaxin, IL-5and IL-13 levels in BalF after OVA challenge were (45± 8) ng/L, (157±23) ng/L and (429 ±46) ng/L, respectively. Application of fasudil resulted in inhibition of the augmented levels of eotaxin, IL-5 and IL-13 in BalF, decreased to (20±5) ng/L, (57 ± 14) ng/L and (254 ±28) ng/L, respectively(q=13. 119, 17.503, 8.449, respectively, all P ＜0.01). (3) Mice in the control group showed no detectable inflammatory response in the lung, whereas OVA-challenged mice induced infiltration of inflammatory cells around airways and blood vessels. The majority of the infiltrated inflammatory cells wereeesinophils. Application of fasudil significantly reduced the infiltration of inflammatory cells in theperibronchial areas compared with the asthmatic mice. (4) The expression levels of ROCK-1 mRNA and protein in mice of the asthmatic group (0. 67 ±0. 05 and 1.09 ± 0. 06) were much higher than those of the control group (0. 26 ± 0. 05 and 0. 87 ± 0. 09 ) ( q = 25. 614, 8. 156, all P ＜ 0. 01 ). When fasudil was administered, the expression levels of ROCK-1 mRNA and protein were significantly attenuated to 0. 35 ±0. 04and 0. 98 ±0. 08, compared with those of the asthmatic group (q =20. 379, 4. 135, all P ＜0. 01 ). (5) Theexpression level of ROCK-1 mRNA was positively correlated with the number of eosinophils and the levels of eotaxin, IL-5 and IL-13 in BalF (r = 0.709, 0.600, 0.613, 0.650, all P ＜ 0.01 ). Conclusion Airway inflammation of bronchial asthma was improved by inhibiting expression and activity of ROCK-1 by fasudil, suggesting that ROCK-1 may be involved in asthmatic airway inflammation induced by OVA challenge.