咪喹莫特治疗支气管哮喘作用机制的实验研究

The effects of imiquimod on an animal model of asthma

目的研究新型免疫调节剂咪喹莫特对支气管哮喘(简称哮喘)的治疗作用,探讨其可能的作用机制。方法 (1)40只 BALB/c 小鼠(每组10只)和48只 SD 大鼠(每组12只)分为对照组、哮喘组、咪喹莫特组和地塞米松组,建立大鼠和小鼠哮喘模型。用卵清白蛋白激发前吸入0.15%咪喹莫特混悬液5 ml。末次激发后24 h 观察肺组织炎症及测定气道反应性;用逆转录-聚合酶链反应(RT-PCR)法测定肺组织中白细胞介素4(IL-4)、γ干扰素(IFN-γ)、嗜酸粒细胞活化趋化因子(eotaxin)、巨噬细胞衍生趋化因子(MDC)、胸腺和活化调节趋化因子(TARC)、T-bet、GATA-3、信息转导转录激活子6(STAT_6)mRNA 的表达;用酶联免疫吸附试验(ELISA)法测定血清中 eotaxin、MDC、TARC 及支气管肺泡灌洗液中 IL-4和 IFN-γ浓度;用 Western blot 法测定肺组织中 T-bet、GATA-3、STAT_6蛋白水平;(2)分离和培养致敏大鼠气管旁淋巴结细胞(PBLN),分为空白对照组、阳性对照组、地塞米松组和药物1～3组,测定不同时间点各组细胞上清液中 IL-4、IFN-γ蛋白和细胞的 mRNA表达;(3)分离和培养致敏小鼠的脾脏 T 淋巴细胞,用流式细胞仪测定经咪喹莫特刺激后不同时间点细胞内 IL-4、IFN-γ水平;(4)培养致敏大鼠 CD_4^+T 淋巴细胞,测定经咪喹莫特干预后 T-bet、GATA-3mRNA 和蛋白的表达水平。结果用同等剂量氯化乙酰胆碱(20、40、80、160μg/ml)激发时平均呼气阻力哮喘组分别为(6.26±0.85)、(11.55±3.09)、(28.74±5.94)、(3710.83±197.49)cm H_2O·ml^(-1)·s^(-1)(1 cm H_2O=0.098 kPa),咪喹莫特组分别为(1.34±0.16)、(3.47±0.49)、(9.29±1.27)、(25.22±5.44)cm H_2O·ml^(-1)·s^(-1),两组相同剂量比较差异有统计学意义(D 值分别为88.98、56.00、45.00、108.00,P 均<0.01);哮喘组大鼠气道壁和肺组织中嗜酸粒细胞(EOS)、淋巴细胞、管壁面积/支气管管腔内周长(WA/Pi)、支气管平滑肌面积/支气管管腔内周长(ASM/Pi)分别为(26.0±1.6)、(45.2±3.2)个/mm^2、12.0±1.4、6.7±0.6,咪喹莫特组分别为(12.4±2.9)、(24.2±3.7)个/mm^2、9.2±0.6、4.0±0.5,两组比较差异有统计学意义(D 和 q 值分别为193.00、16.92、185.50、7.66,P 均<0.01);哮喘组肺组织中 T-bet mRNA 和蛋白表达量分别为0.08±0.12、0.18±0.06,咪喹莫特组分别为0.48±0.08、0.48±0.17,两组比较差异有统计学意义(D 值分别为120.96、177.98,P 均<0.01);哮喘组肺组织中 GATA-3 mRNA 和蛋白表达量分别为1.56±0.28、2.25±0.74,咪喹莫特组分别为0.54±0.14、0.50±0.14,两组比较差异有统计学意义(D 值分别为166.96、310.97,P 均<0.01);空白对照组24、48 h 时 PBLN 细胞培养液中 IL-4、IFN-γ分别为(0±0、0±0、22±5、31±5)pg/ml。随着培养时间的延长,药物2组24、48 h 时 IL-4、IFN-γ水平分别为(23±5)、(39±11)、(149±31)、(154±28)pg/ml,药物3组24、48 h 时IL-4、IFN-γ水平分别为(25±6)、(40±12)、(166±30)、(158±31)pg/ml,与阳性对照组[(43±13)、(56±12)、(100±22)、(112±33)pg/ml)]比较差异有统计学意义(药物2、3组24、48 h 时 IL-4的 D 值分别为9.90、8.79、8.80、8.10;药物2、3组24、48 h 时 IFN-γ的 q 值分别为4.80、6.40、3.95、4.31,P 均<0.05);哮喘组小鼠 BALF 中细胞总数及 EOS 分别为(8.7±1.4)×10~6/L、(29.80±7.00)%,咪喹莫特组为(4.1±1.3)×10~6/L、(8.90±2.40)%,两组比较差异有统计学意义(q 值分别为16.40、7.40,P 均<0.05);哮喘组小鼠血清 eotaxin、MDC 和 TARC 水平分别为(593±41)、(170±20)、(221±25)pg/ml,咪喹莫特组分别为(501±76)、(84±13)、(163±35)pg/ml,两组比较差异有统计学意义(q 值分别为3.70、9.80、4.50,P均<0.05);(4)咪喹莫特组小鼠肺组织 eotaxin、MDC、TARC mRNA 表达分别为0.65±0.17、0.66±0.12、0.66±0.34,哮喘组分别为0.85±0.11、0.96±0.10、0.94±0.28,对照组分别为0.45±0.08、0.39±0.09、0.24±0.08,哮喘组与咪喹莫特组比较差异有统计学意义(q 值分别为1.50、8.10、2.40, P 均<0.05);哮喘组与对照组比较差异有统计学意义(q 值分别为3.00、15.40、5.90,P 均<0.05)。结论咪喹莫特可能通过提高转录因子 T-bet mRNA\蛋白的表达和抑制转录因子 GATA-3和 STAT_6mRNA 和蛋白表达,使失衡的 Th1/Th2细胞得以纠正,从而抑制哮喘气道炎症。

Objective To study the mechanism of imiquimod on asthma animals. Methods （ 1 ） 40 mice and 48 rats were divided into 4 groups： control, asthma, dexamethasone and imiquimod groups. The asthma model was established. The mice and rats in the imiquimod group were exposed to an aerosol of 0. 15% imiquimod. Lung inflammation and airway responsiveness were measured 24 h after the last ovalbumin （ OVA ） challenge. The expression of Interleukin-4 （ IL-4 ）, interferon γ （ IFN-γ ）, eotaxin, macrophage-derived chemokine （ MDC ）, thymus and activation-regulated chemokine （ TARC ）, T-bet, GATA-3, STAT6 mRNA in the lung were determind by reverse transcription polymerase chain reaction （ RT- PCR）. The levels of eotaxin, MDC, and TARC in sera were tested by enzymelinked immunosorbentassy （ELISA）. The expression of T-bet, GATA-3 and STAT6 ptoteins in the lung were measured by Western blot. （2）Parabronchial lymphnodes （PBLN） were isolated and cultured. The PBLN cells were divided into blank control, positive control, dexamethasone and drug groups （ 1 - 3 subgroups）, cultured for different hours, and the expressions of IL4 and IFN-γ in supernatants were determined by ELISA, The mRNA expressions of the cytokines in cells weredetected by RT-PCR. （3） Flow cytometry was used to detect intracellular IL4 and IFN-γ production in spleen T lymphocytes. （4） CD4^＋ T cell of spleen pellets were subject to assessment of T-bet and GATA-3 protein and mRNA expression respectively. Results The expiration resistance was determined before and after injection of acetylcholine chloride （20 - 160 μg/ml）, and expiration resistances of the asthmatic group （ 6.26 ± 0.85 ）, （ 11.55 ± 3.09 ）, （ 28.74 ± 5.94 ）, （3710.83 ± 197.49） cm H2O· ml^-1 · s^-1 , were significantly elevated compared with those of the control group（1.34±0.16）, （3.47±0.49）, （9.29±1.27）, （25.22±5.44）cmH2O· ml^-1·s^-1, D= 88.98,56.00,45. 00,108.00, all P 〈0. 01 ）. The numbers of eosinophils and lymphoeytes, the thicknesses of WA/Pi and ASM/Pi in the asthmatic group [ （26.0 ± 1.6）/mm^2, （45.2 ± 3.2）/mm^2, 12.0 ± 1.4, 6.7 ± 0.6 ] were all significantly higher than those of the imiquimod group [ （ 12.4 ± 2.9 ）/mm^2, （ 24.2 ± 3.7）/mm^2,9.2 ±0.6,4.0±0.5, D or q=193.00, 16.92,185.50, 7.66, all P〈0.01]. In the imiquimod group, the mRNA and protein expressions of T-bet （ 0. 48 ± 0. 08, 0. 48 ± 0. 17 ） were significantly increased compared with those of the asthmatic group （0.08 ± 0. 12, 0. 18 ± 0. 06, D = 120. 96, 177.98 ,all P 〈0. 01 ）, the mRNA and protein expressions of GATA-3 in the imiquimod group were both significantly decreased compared with those of the asthmatic group （D = 166.96,310.97, all P 〈 0. 01 ）. In the control group, only low concentrations of IFN-γ [ （22±5,31±5） pg/ml] were detected in PBLN cell cultures. After 24 or 48 h stimulation, the concentrations of IFN-γ in drug 2 subgroup [ （149 ± 31）,（154 ± 28） pg/ml] and drug 3 subgroup [（166 ± 30）, （158 ± 31） pg/ml] were increased significantly ; Levels of IL-4 [ druug 2 subgroup ： （ 23 ± 5 ）, （ 39 ± 11 ） pg/ml, drug 3 subgroup ： （43 ± 13 ）, （ 56 ± 12） pg/ml] were increased slowly compared with those in the OVA group （ drug 2 subgroup 24 h IL-4, D = 9.90; drug 3 subgroup 24 h IL-4, D = 8.79, drug 2 subgroup 48 h IL-4, D = 8.80, drug 3 subgroup 48 h IL-4, D =8. 10, drug 2 subgroup 24 h IFN-γ, q =4.80, drug 3 subgroup 24 h IFN-γ, q =6.40, drug 2 subgroup 48 h IFN-γ, q =3.95, drug 3 subgroup 48 h IFN-γ, q =4. 31, all P 〈0.05）. After imiquimod treatment, the mRNA and protein levels of T-bet in imiquimod group CD4^＋ T cells were increased significantly compared with those in OVA group, and the mRNA and protein levels of GATA-3 were decreased significantly in CD4^＋ T cells of imiquimod group compared with those in OVA group. The eotaxin, MDC and TARC levels of serum in asthma group [ （593 ± 41 ） pg/ml, （ 170 ± 20） pg/ml, （221 ± 25 ） pg/ml] were significant different from those in control group [ （288 ± 66） pg/ml, （100 ± 33 ） pg/ml, （84 ± 49） pg/ml ], （ eotaxin ： q = 12.20, MDC ： q = 8.00, TARC ： q = 10.50, all P 〈 0.01 ）. MDC and TARC levels of serum in imiquimod group [ （84 ± 13） pg/ml, （163±35） pg/ml] decreased as compared with those in asthma group （ MDC：q =9.80, TARC：q =4.50, all P 〈0.01 ） and MDC levels in imiquimod group were no different with normal group （ q = 1.80, P 〉 0.05 ）. eotaxin levels of serum in imiquimod group [ （501±76） pg/ml] increased as compared with those from normal group （q =8.50, P 〈0.01 ）, and decreased as compared with those from asthma group （ q = 3.70, P 〈 0. 05 ）. （ 4 ） The expression of eoaxin, MDC, TARC and STAT6 on the bronchial epithelium in imiquimod group was decreased as compared with asthma group, but increased as compared with normal group. The eotaxin, MDC and TARC mRNA expression of the lung in asthma group（0.85±0.11,0.96±0.10, 0.94±0.28） had significant differences from those in the control group （0.45 ± 0.08, 0.39 ± 0.09, 0.24 ± 0.08, eotaxin ： q = 3.00, MDC ： q =15.40, TARC ： q = 5.90, all P 〈 0.01 ） and those in imiquimod group （ 0.65 ± 0.17, 0.66 ± 0.12, 0.66 ± 0.34, eotaxin：q=1.50, MDC：q =8. 10, TARC：q =2.40, all P〈0.05）. Conclusion These findings suggested that imiquimod can inhibit the airway inflammation of asthma animals by reducing GATA-3 mRNA and protein expression and increasing T-bet, STATs mRNA and protein expression.