CCR5第二胞外环的拮抗短肽对哮喘小鼠肺组织炎症细胞浸润和TNF-α表达的影响

目的:研究与CC趋化因子受体5(CC chemokine receptor 5,CCR5)第二胞外环特异性结合的拮抗短肽对哮喘小鼠肺组织炎症细胞浸润和TNF-α表达的影响。方法:筛选最适宜的卵白蛋白(ovalbumin,OVA)致敏浓度,构建OVA诱导的BALB/c小鼠哮喘模型。模型构建成功后通过尾静脉注射不同浓度拮抗短肽干预模型小鼠,HE染色对肺组织进行病理细胞学分析及炎症分级,real-time PCR与Western blot实验分别检测小鼠肺组织TNF-α的mRNA和蛋白表达水平。结果:筛选出的OVA最佳致敏浓度为500 mg/L(0.1 mL)。尾静脉注射0.2m L不同浓度(1.5 g/L、2.5 g/L和3.5 g/L)拮抗短肽干预哮喘小鼠,能减轻哮喘小鼠肺组织炎症程度,抑制TNF-α的表达,以2.5 g/L浓度效果最佳,较模型组炎症程度减轻2级,炎症细胞数明显减少,TNF-α的mRNA表达量较模型组下降近90%,蛋白表达水平下降约70%。同时该短肽(2.5 g/L)也起到了一定的预防作用,炎症程度改善1级,TNF-α的mRNA及蛋白水平较模型组下降约50%左右。结论:CCR5第二胞外环的拮抗短肽能有效减轻哮喘小鼠肺组织炎症程度,抑制TNF-α的表达。

Effects of antagonistic peptide specifically binding to second extracellular loop of CCR5 on inflammatory cell infiltration and TNF-α expression in lung tissues of asthmatic mice induced by OVA

AIM: To investigate the effects of antagonistic peptide specifically binding to the second extracellular loop of CC chemokine receptor 5 (CCR5) on inflammatory cell infiltration and TNF-α expression in lung tissues of asthmatic mice. METHODS: The asthmatic model of BALB/c mice was induced by ovalbumin (OVA) and the optimal sensitization concentration of OVA was screened. After modeling, the mice were intervened by gradual concentrations of antagonistic peptide via tail-vein injection. The pathocytological analysis and grading were performed in the lung tissues with HE staining. The expression of TNF-α at mRNA and protein levels in the lung tissues was determined by real-time PCR and Western blot. RESULTS: The optimal concentration of OVA was 500 mg/L (0.1 mL) as this concentration of OVA stably induced moderate degree of inflammation in the BALB/c mice. Treatment with different concentrations (1.5 g/L, 2.5 g/L and 3.5 g/L) of antagonistic peptide at 0.2 mL through tail-vein injection inhibited the expression of TNF-α, and markedly reduced the degree of inflammation in the lung tissues. The optimal concentration of antagonistic peptide was 2.5 g/L as the lung inflammation degree in 2.5 g/L group alleviated by 2 grades, and the number of inflammatory cells was also significantly reduced. Moreover, the mRNA expression abundance of TNF-α was nearly decreased by 90%, and the protein expression of TNF-α was decreased by 70% compared with model group. Meanwhile, the use of antagonistic peptide at 2.5 g/L before OVA stimulation confirmed the preventive function to some degree. In this group, the lung inflammation degree alleviated by 1 grade, and the expression of TNF-α at both mRNA and protein levels decreased by nearly 50%. CONCLUSION: The antagonistic peptide of CCR5 effectively inhibits the expression of TNF-α and relieves the inflammation in the asthmatic mouse lung tissues in a concentration-dependent manner.