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目的观察激素抵抗型支气管哮喘(简称SR哮喘)患者外周血维生素D水平,探讨维生素D对SR哮喘患者T淋巴细胞JNK/AP-1和糖皮质激素受体的影响。方法收集2014年至2015年期间门诊和住院急性发作的哮喘患者62例,其中激素敏感型支气管哮喘(简称SS哮喘)26例,SR哮喘36例;选取同期健康体检正常者25例作为对照。收集入组者临床资料,取外周静脉血检测25-羟维生素D[25-(OH)D]水平并分离T淋巴细胞。T淋巴细胞培养分组如下:健康对照组(A组),SS哮喘对照组(B组),SR哮喘对照组(C组),SR哮喘JNK抑制剂(SP600125)+1,25-(OH)2D3组(D组),SR哮喘JNK抑制剂组(SP600125)(E组),以及SR哮喘1,25-(OH)2D3组(F组)。T淋巴细胞共培养48 h,培养结束后采用Western blot方法检测T淋巴细胞中磷酸化JNK(p-JNK)和磷酸化糖皮质激素受体(p-GR)的表达,RT-PCR法检测c-Jun m RNA的表达。结果 B组和C组血清25-(OH)D水平均低于A组(P<0.05),且C组低于B组(P<0.05)。B组和C组T淋巴细胞中p-JNK蛋白水平高于A组(P<0.05),且C组高于B组(P<0.05),E组和F组均低于C组(P<0.05),D组低于F组(P<0.05)。C组T淋巴细胞中p-GR蛋白低于A组和B组(P<0.05),E组和F组均高于C组(P<0.05),D组高于F组(P<0.05)。RT-PCR法检测B组和C组T淋巴细胞中c-Jun m RNA水平高于A组(P<0.05),C组高于B组(P<0.05),E组和F组均低于C组(P<0.05),D组低于F组(P<0.05)。C组患者25-(OH)D水平与p-JNK和c-Jun m RNA水平呈负相关(r=–0.69,r=–0.65,P<0.05),与p-GR水平呈正相关(r=0.72,P<0.05)。结论维生素D缺乏在SR哮喘患者中患病率高。1,25-(OH)2D3可通过抑制SR哮喘T淋巴细胞JNK/AP-1信号通路而促进p-GR的表达,这可能是维生素D改善SR哮喘患者的糖皮质激素抵抗的机制之一。
Objective To observe the level of vitamin D in peripheral blood of patients with steroid-resistant bronchial asthma (SR asthma) and to investigate the effect of vitamin D on JNK / AP-1 and glucocorticoid receptor of T lymphocytes in patients with SR asthma. Methods Totally 62 outpatients and acute exacerbations of asthma were collected from 2014 to 2015. Among them, 26 were steroid-sensitive bronchial asthma (SS asthma) and 36 were asthma (SR). Twenty-five healthy controls were enrolled in this study. The clinical data of the patients were collected. Peripheral venous blood was taken to detect the level of 25-hydroxyvitamin D [25- (OH) D] and T lymphocytes were isolated. The T lymphocyte culture groups were as follows: healthy control group (group A), SS asthma control group (group B), SR asthma control group (group C), SR asthma JNK inhibitor (SP600125) + 1,25- Group D (group D), SR asthma JNK inhibitor group (group E600) (group E), and Group SR asthma 1,25- (OH) 2D3 (group F). T lymphocytes were co-cultured for 48 h. Western blot was used to detect the expression of phosphorylated p-JNK and phospho-glucocorticoid receptor (p-GR) in T lymphocytes after culture. The expression of c Expression of Jun m RNA. Results Serum levels of 25- (OH) D in group B and group C were lower than those in group A (P <0.05), and those in group C were lower than those in group B (P <0.05). The protein levels of p-JNK in group B and C were higher than those in group A (P <0.05), and those in group C were higher than those in group B (P <0.05) 0.05), D group was lower than F group (P <0.05). The protein expression of p-GR in group C was lower than that in group A and group B (P <0.05), group E and F were higher than group C (P <0.05), group D was higher than group F (P <0.05) . The mRNA level of c-Jun in T lymphocytes of group B and group C was higher than that of group A (P <0.05), that of group C was significantly higher than that of group B (P <0.05), while group E and F C group (P <0.05), D group was lower than F group (P <0.05). The level of 25- (OH) D in group C was negatively correlated with the level of p-JNK and c-Jun m RNA (r = -0.69, r = -0.65, P <0.05) 0.72, P <0.05). Conclusion The prevalence of vitamin D deficiency in SR asthma patients is high. 1,25- (OH) 2D3 can promote the expression of p-GR by inhibiting the JNK / AP-1 signaling pathway in SR asthmatic T lymphocytes, which may be one of the mechanisms by which vitamin D improves glucocorticoid resistance in SR asthmatic patients.