虫草活力素对大鼠慢性支气管哮喘模型气道重塑作用初探

Role of cordycepin on transforming growth factor-β_1 in a rat model of chronic bronchial asthma

目的对虫草活力素(简称虫草)在大鼠支气管哮喘(简称哮喘)模型气道重塑中的作用及相关机制进行初步探讨。方法 50只Wister大鼠[6～8周龄,(200±20)g],随机数字表法分为阴性对照组(A)、慢性哮喘单纯模型组(B)(卵清白蛋白系统致敏和反复激发)、慢性哮喘+虫草50mg/kg治疗8周组(C)、慢性哮喘+布地奈德(BUD)8周治疗组(D)及慢性哮喘+BUD联合虫草8周治疗组(E)。肺组织切片HE染色观察病理变化并检测气道管壁厚度,采用免疫组织化学法检测气道转化生长因子β1(TGF-β1)表达水平,应用逆转录PCR法检测肺组织A2a腺苷受体(A2aAR)mRNA表达。数据统计采用ANOVA检验进行组间分析,Mann-Whitney检验进行组内两两比较,Spearman等级相关分析。结果①HE染色显示所有药物干预组较单纯模型组炎症细胞浸润、平滑肌肥厚及黏膜肺组织水肿等炎症表现明显减轻,以D和E组最为明显;②5组气道管壁厚度依次分别为(9.89±2.09)、(21.72±3.16)、(14.94±1.96)、(12.29±2.75)和(12.25±2.32)μm2/μm,F=31.37,P<0.0001,所有药物干预组气道壁厚度均高于对照组(C组、D组和E组与A组比较后的P值分别为0.0001、0.0524、0.0524),且低于单纯模型组(C组、D组和E组与B组比较后的P值均<0.0001),差异有统计学意义,C组气道壁较D组和E组增厚,差异有统计学意义(P值分别为0.0232和0.0147);③5组TGF-β1表达强阳性率分别为(2.08±1.63)%、(29.37±5.08)%、(12.30±3.26)%、(10.78±3.35)%及(7.43±2.84)%,F=86.45,P<0.0001,所有药物干预组TGF-β1蛋白表达均高于对照组(C组、D组和E组与A组比较后的P值均<0.0001),且低于单纯模型组(C组、D组和E组与B组比较后的P值均<0.0001),差异有统计学意义;E组较C组的蛋白表达有统计学意义下降(P=0.0052);④5组肺组织A2aARmRNA表达相对强度依次为(0.35±0.06)、(0.27±0.10)、(0.52±0.07)、(0.24±0.05)及(0.47±0.08),F=26.46,P<0.0001,C组和E组A2aARmRNA表达高于A组(P值分别为0.0001及0.0007)、B组(P值分别为0.0002及0.0003)和D组(P值均<0.0001),差异有统计学意义,D组表达低于A组,差异有统计学意义(P=0.0003);⑤所有大鼠支气管壁厚度与气道TGF-β1蛋白表达存在正相关(r=0.80,P<0.01),而C组大鼠肺组织A2aARmRNA表达和TGF-β1蛋白表达呈负相关(r=-0.68,P=0.03)。结论虫草对哮喘模型的气道重塑具有一定的改善作用,其机制可能通过增加慢性哮喘模型大鼠肺组织A2aARmRNA转录,抑制气道TGF-β1的生成,从而减轻气道重塑,且虫草与糖皮质激素联合应用对于降低TGF-β1的表达具有潜在协同作用。

Objective To investigate the potential suppression role of cordycepin in airway remodeling by observing the expression of transforming growth factor-β1（TGF-β1）and A2a adenosine receptor（A2aAR）on a rat model of chronic bronchial asthma（asthma）.Methods 50 rats were randomized into control group（A）,asthma group（B）,50 mg/kg cordycepin treatment group（C）,budesonide（BUD）treatment group（D）and combination treatment group（BUD ＋ cordycepin 50 mg/kg）（E）.After 8 weeks therapy on ovalbumin challenged asthma model,histologic examination were performed to observe the general pathologic alteration and analyze the thickness of airway wall.The protein expressions of TGF-β1 were calculated by immunohistochemistry,and the transcriptions of A2aAR mRNA in the lung tissues were measured by reverse transcriptase-polymerase chain reaction（RT-PCR）.Data were evaluated using One-way ANOVA followed by Turkey＇s test and Mann-Whitney test.Results ① HE staining showed that compared with the treatment groups,there were a large number of inflammatory cells infiltration,heavier smooth muscle hypertrophy and mucous membrane hyperemia in the asthma group.② The thickness of airway in each group was（9.89±2.09）,（21.72±3.16）,（14.94±1.96）,（12.29±2.75）and（12.25±2.32）μm2/μm respectively,F=31.37,P0.000 1,airway thickness in group C,D and E were all significantly higher than group A（P=0.000 1,0.052 4 and 0.052 4 respectively）,and lower than group B（P0.000 1）.Airway in group C also showed remarkably thicker than group D and E（P=0.023 and 0.015 respectively）.③ The expressions of TGF-β1 in each group was（2.08±1.63）%,（29.37±5.08）%,（12.30±3.26）%,（10.78±3.35）% and（7.43±2.84）% respectively,F=86.45,P0.000 1,protein expressions in group C,D and E were all higher than group A（P0.000 1,0.000 1 and =0.000 1 respectively）,and lower than group B significantly（P0.000 1）,expression in group E also decreased remarkably than group C（P=0.005 2）.④ The transcription level of A2aAR mRNA in each group was（0.35±0.06）,（0.27±0.10）,（0.52±0.07）,（0.24±0.05）and（0.47±0.08）,respectively,F=26.46,P0.000 1.Compared to Group A,B and D,the levels of A2aAR mRNA in group C and E were significantly higher（P0.001）,while expressions in group D were decreased remarkably than group A（P=0.000 3）.⑤The protein levels of TGF-β1 were positively correlated with airway thickness in all rats（r=0.80,P0.01）,while negative correlation were detected with thranscription of A2aAR in group C（r=-0.68,P=0.03）.Conclusions Cordycepin may potentially inhabit the airway remodeling of asthma by up regulating the thranscription of A2aAR mRNA and down regulating the synthesis of TGF-β1,and may have synergistic effect in inhibiting the synthesis of TGF-β1 with corticosteroid.