基于MAPK信号通路探讨运脾泻肺化痰汤对幼龄哮喘大鼠肺组织的影响

Effect of Yunpi Xiefei Huatan decoction on lung tissue of young asthmatic rats based on MAPK signaling pathway

目的 探讨运脾泻肺化痰汤对幼龄哮喘大鼠肺组织p38丝裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)蛋白相对表达量、肺组织中细胞外信号调节激酶(extracellular regulated protein kinases, ERK)、c-Jun氨基端激酶(c-jun N-terminal kinase, JNK)基因表达水平及羟脯氨酸(hydroxyproline, HYP)水平的影响,同时对四者进行相关性分析,从而探索运脾泻肺化痰汤调控MAPK信号通路及改善哮喘症状的机制。方法 将幼龄SD大鼠随机分为空白组、模型组、中药高、中、低剂量组、西药组,每组10只;以卵清白蛋白加氢氧化铝佐剂建立幼龄哮喘大鼠模型,分别于第1天与第8天腹腔注射卵清白蛋白加氢氧化铝佐剂混合液1 mL,第15天起以1%卵清白蛋白雾化激发,每次30分钟,并隔日1次;第16天开始进行药物治疗,中药高、中、低剂量组每只大鼠依次灌胃运脾泻肺化痰汤生药剂量40 g/kg、20 g/kg、10g/kg,西药组以0.01%布地奈德雾化吸入20分钟,模型组和空白组灌胃0.9%生理盐水2 mL,每天一次定时,连续四周,第44天进行取材;以蛋白质印记法测定肺组织中p38MAPK的相对蛋白表达量;以实时荧光定量PCR测定肺组织中ERK、JNK的基因表达水平;以酶联免疫法测定肺组织中HYP浓度;同时分析各组大鼠肺组织p38MAPK蛋白相对表达量、肺组织中ERK、JNK基因表达水平及HYP水平之间的相关性。结果 模型组与空白组相比,肺组织中p38MAPK的相对蛋白表达量、ERK及JNK基因表达水平和HYP浓度均明显升高,差异有统计学意义(P<0.01);与模型组相比,中药各剂量组和西药组肺组织中p38MAPK的相对蛋白表达量、ERK及JNK基因表达水平和HYP浓度均下降,差异有统计学意义(P<0.01,P<0.05);肺组织中p38MAPK的相对蛋白表达量、ERK及JNK基因表达水平和HYP浓度两两之间均呈正相关(P<0.01)。结论 运脾泻肺化痰汤可减轻幼龄哮喘大鼠肺部炎症反应,其作用机理之一可能是抑制了MAPK信号通路相关因子的表达。

Objective To investigate the effect of Yunpi Xiefei Huatan decoction on the relative expression of p38MAPK protein in lung tissue,the expression of ERK,JNK gene and HYP level in lung tissue of rats in each group,and to analyze the correlation among these group,so as to explore the mechanism of Yunpi Xiefei Huatan decoction in regulating MAPK signaling pathway and improving asthma symptoms.Methods Young SD rats were randomly divided into blank group,model group,Chinese medicine high,medium and low dose group,western medicine group,10 rats in each group.The young asthma rat model was established by ovalbumin plus aluminum hydroxide adjuvant.On the first day and the eighth day,1 mL of ovalbumin plus aluminum hydroxide adjuvant mixture was injected intraperitoneally.From the 15th day,1%OVA was atomized for 30 minutes each time,once every other day.On the 16th day,drug treatment was started.Each rat in the high,medium and low dose groups of traditional Chinese medicine was intragastrically administered with 40 g/kg,20 g/kg and 10 g/kg of Yunpi Xiefei Huatan decoction.The western medicine group was atomized with 0.01%budesonide for 20 min.The model group and the blank group were intragastrically administered with 2mL of 0.9%normal saline once a day for four weeks;the relative protein expression of p38MAPK in lung tissue was detected by Western-Blot.Real-time fluorescent quantitative PCR(RT-PCR)was used to detect the gene expression levels of ERK and JNK in lung tissue;HYP concentration in lung tissue was detected by ELISA.At the same time,the correlation between the relative expression of p38 MAPK protein in lung tissue,the expression levels of ERK and JNK genes in lung tissue and the level of HYP was analyzed.Results Compared with the blank group,the relative protein expression of p38MAPK,ERK and JNK gene expression levels and HYP concentration in the lung tissue of the model group were significantly increased(P<0.01).Compared with the model group,the relative protein expression of p38MAPK,the expression levels of ERK and JNK genes and the concentration of HYP in the lung tissues of the TCM group and the western medicine group were decreased,and the differences were statistically significant(P<0.01,P<0.05).The relative protein expression of p38MAPK,ERK and JNK gene expression levels and HYP concentration in lung tissue were positively correlated(P<0.01).Conclusion Yunpi Xiefei Huatan decoction can alleviate pulmonary inflammation in young asthmatic model rats,one of the mechanisms may be related to inhibit the expression of MAPK signaling pathway related factors.