阿魏酸对慢性阻塞性肺疾病小鼠肺功能的影响及作用机制研究

Effect of ferulic acid on pulmonary function in mice with chronic obstructive pulmonary disease and its mechanism

目的探讨阿魏酸对慢性阻塞性肺疾病(COPD)小鼠肺功能的保护作用及其可能机制.方法选择60只小鼠,按随机数字表法分为正常对照组、COPD模型组、罗氟司特组和阿魏酸高、中、低剂量组,每组10只,给药过程中模型组死亡1只而剔除.采用烟熏法复制COPD模型;正常对照组不进行任何处理.制模30 d开始给药,COPD模型组和正常对照组给予生理盐水;罗氟司特组给予罗氟司特65 μg/kg;阿魏酸高、中、低剂量组分别给予阿魏酸160、80、40 mg/kg;连续给药90 d后测定指标.观察各小鼠吸气峰流速(PIF)、呼气峰流速(PEF)、每分通气量(MV)、内衬间隔(MLI)、肺泡破坏指数(DI)、血清和支气管肺泡灌洗液(BALF)中白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)水平以及肺组织丝裂素活化蛋白激酶(MAPK)信号通路中p38MAPK、细胞外信号调节激酶(ERK)、c-Jun氨基末端激酶(JNK)的蛋白表达及磷酸化水平的变化.结果COPD模型组PIF、PEF、MV均较正常对照组明显降低〔PIF(mL/s):2.32±0.18比3.41±0.12,PEF(mL/s):2.31±0.22比2.90±0.15,MV(mL/s):26.20±2.70比35.18±2.30〕;罗氟司特和各剂量阿魏酸均可使PIF、PEF、MV升高,以阿魏酸高量组的升高程度较阿魏酸中、低量组更显著〔PIF(mL/s):3.24±0.13比2.88±0.15、2.51±0.10,PEF(mL/s):2.81±0.16 比 2.66±0.11、2.58±0.17,MV(mL/s):31.18±1.20 比 28.25±2.20、27.09±1.10〕;但罗氟司特组和阿魏酸组比较差异均无统计学意义(均P>0.05).COPD模型组MLI、DI和血清与BALF中炎症因子水平,以及肺组织中p38MAPK、ERK、JNK蛋白表达及磷酸化水平均较正常对照组明显升高〔MLI(μm):52.10±0.26比21.90±0.14,DI :(60.78±3.32)%比(22.47±1.05)%;血清中IL-6(ng/L):22.34±4.51比3.50±1.55,TNF-α(ng/L):27.11±3.99比4.66±1.76;BALF中IL-6(ng/L):142.92±20.10比18.77±4.17,TNF-α(ng/L):150.16±20.77比22.01±4.15,P-ERK/ERK(灰度值):0.59±0.03比0.38±0.05, P-p38MAPK/p38MAPK(灰度值):0.52±0.02比0.31±0.05,P-JNK/JNK(灰度值):0.56±0.03比0.25±0.01,均P<0.05〕;罗氟司特和各剂量阿魏酸均可使MLI、DI、炎症因子水平,以及p38MAPK、ERK、JNK蛋白表达及磷酸化水平降低,以阿魏酸高剂量组的降低程度较阿魏酸中、低剂量组更显著〔MLI(μm):25.00±0.19比30.10±0.29、38.80±0.41,DI :(26.32±3.05)%比(29.75±6.17)%、(40.56±5.81)%,血清中IL-6(ng/L):9.20±1.87 比 12.35±2.16、18.95±3.12,TNF-α(ng/L):13.37±2.73 比 18.02±2.62、21.31±3.75,BALF 中IL-6(ng/L):64.27±11.72 比 99.33±13.02、120.31±18.02,TNF-α(ng/L):58.20±10.28 比 93.83±16.26、122.68±14.85,P-ERK/ERK(灰度值):0.43±0.04比0.46±0.04、0.52±0.02,P-p38MAPK/p38MAPK(灰度值):0.33±0.03比0.34±0.03、0.38±0.02,P-JNK/JNK(灰度值):0.32±0.04比0.38±0.05、0.47±0.06〕.表明阿魏酸可改善COPD小鼠炎症细胞浸润情况.结论阿魏酸能改善COPD模型大鼠肺内炎症反应,其机制与抑制MAPK信号通路中p38MAPK、ERK、JNK的蛋白表达及磷酸化水平有关.

Objective To investigate the protective effect of ferulic acid on lung function in mice with chronic obstructive pulmonary disease(COPD)and its possible mechanism.Methods Sixty mice were randomly divided into normal control group,COPD model group,Rofloast group and ferulic acid high,medium and low dose groups,each group with 10 rats,and during administration one rat died in the mode group and was eliminated.The COPD model was duplicated by smoking method;the mice in normal control group were fed normally without any treatment.After modeling for 30 days,normal saline begun to be given to the COPD model group and normal control group;the mice in Rofluast group were given Rofluast 65μg/kg;ferulic acid 160,80,40 mg/kg were given to high,middle and low dose groups respectively.The indexes were determined after consecutive 90 days of treatment,the changes of peak inspiratory flow(PIF)rate,peak expiratory flow(PEF)rate and ventilation volume per minute(MV),mean lining interval(MLI),alveolar destruction index(DI),interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)levels in serum and bronchoalveolar lavage fluid(BALF),the protein expressions and phosphorylation levels of p38 mitogen-activated protein kinases(p38MAPK),extracellular signal-regulated kinase(ERK)and c-Jun amino terminal kinase(JNK)in the pulmonary tissue MAPK signaling pathway were observed in each mouse of various mice groups.Results In the COPD model group,the PIF,PEF,and MV were all significantly lower than those in the normal control group[PIF(mL/s):2.32±0.18 vs.3.41±0.12,PEF(mL/s):2.31±0.22 vs.2.90±0.15,MV(mL/s):26.20±2.70 vs.35.18±2.30);Luofusite and all doses of ferulic acid can increase PIF,PEF,and MV,and the degree of increase in the high dose ferulic acid group was more significant than those in the moderate and low dose ferulic acid groups[PIF(mL/s):3.24±0.13 vs.2.88±0.15,2.51±0.10,PEF(mL/s):2.81±0.16 vs.2.66±0.11,2.58±0.17,MV(mL/s):31.18±1.20 vs.28.25±2.20,27.09±1.10];however,there was no statistical significant difference between the Rofluas group and the ferulic acid groups(all P>0.05).The levels of the MLI,DI,and inflammatory factors in serum and BALF,and the protein expressions and phosphorylation levels of p38MAPK,ERK,JNK in lung tissue in model group were all significantly higher than those in normal control group[MLI(μm):52.10±0.26 vs.21.90±0.14,DI:(60.78±3.32)%vs.(22.47±1.05)%,IL-6 in serum(ng/L):22.34±4.51 vs.3.50±1.55,TNF-αin serum(ng/L):27.11±3.99 vs.4.66±1.76,IL-6(ng/L)in BALF:142.92±20.10 vs.18.77±4.17,TNF-α(ng/L):150.16±20.77 vs.22.01±4.15,P-ERK/ERK(gray value):0.59±0.03 vs.0.38±0.05,P-p38MAPK/p38MAPK(gray value):0.52±0.02 vs.0.31±0.05,P-JNK/JNK(gray value):0.56±0.03 vs.0.25±0.01,all P<0.05].The levels of MLI,DI,and inflammatory factors in serum and BALF,p38MAPK,ERK,JNK protein expression and phosphorylation in lung tissue were reduced by Rofluas and various doses of ferulic acid,the reduction levels in the high dose group of ferulic acid were more significant than those in the middle and low dose groups of ferulic acid[MLI(μm):25.00±0.19 vs.30.10±0.29,38.80±0.41,DI:(26.32±3.05)%vs.(29.75±6.17)%,(40.56±5.81)%,IL-6 in serum(ng/L):9.20±1.87 vs.12.35±2.16,18.95±3.12,TNF-α(ng/L):13.37±2.73 vs.18.02±2.62,21.31±3.75,IL-6(ng/L)in BALF:64.27±11.72 vs.99.33±13.02,120.31±18.02,TNF-α(ng/L):58.20±10.28 vs.93.83±16.26,122.68±14.85,P-ERK/ERK(gray value):0.43±0.04 vs.0.46±0.04,0.52±0.02,P-p38MAPK/p38MAPK(gray value):0.33±0.03 vs.0.34±0.03,0.38±0.02,P-JNK/JNK(gray value):0.32±0.04 vs.0.38±0.05,0.47±0.06).The ferulic acid could improve the inflammatory cell infiltration situation in mice with COPD.Conclusions Ferulic acid can improve pulmonary inflammation in COPD rats.The effective mechanism is possibly related to the inhibition of the protein expressions and phosphorylation levels of the key proteins such as p38MAPK,ERK and JNK in the MAPK signaling pathway.