摘要
目的 探讨左旋紫草素对变应性鼻炎(AR)小鼠治疗效应及其相关抑制机制.方法 采用卵清蛋白(OVA)致敏建立小鼠AR模型,按随机数字表法将25只C57BL/6小鼠随机分为正常组、AR模型组、左旋紫草素低剂量和高剂量组,以及标准对照组,每组5只.除正常组外,其他各组完成AR模型构建:在第1、5、9、12日使用OVA+氢氧化铝凝胶混合制剂进行致敏,第13日开始连续7 d,使用OVA每日进行一次激发.在造模第28日起,左旋紫草素组小鼠灌胃2、8 mg/(kg·d)左旋紫草素.标准对照组小鼠灌胃给予地塞米松2.5 mg/(kg·d),正常组和AR模型组动物灌胃等剂量的二甲基亚砜,共灌胃4周,末次灌胃30 min内观察各组小鼠鼻部症状并予以评分.采用流式细胞术检测小鼠外周血辅助性T细胞(Th)1和Th2细胞亚群表达水平;ELISA检测小鼠鼻腔灌洗液白细胞介素4(IL-4)、免疫球蛋白E(IgE)、γ-干扰素(IFN-γ)水平;HE染色检测鼻黏膜病理情况.qRT-PCR法检测鼻黏膜组织中T盒子转录因子(T-bet)、GATA结合蛋白3(GATA3)、信号传导子和转录活化子1(STAT1)、STAT6 mRNA表达.结果 给药4周后,与正常组相比,AR模型组的行为学评分、Th1细胞比例、IL-4和IgE水平、GATA3和STAT6 mRNA表达显著增加(P＜0.05),Th2细胞比例、IFN-γ水平、T-bet和STAT1 mRNA表达显著降低(P＜0.05).与AR模型组相比,左旋紫草素低剂量组动物的行为学评分,Th1和Th2细胞比例,IL-4、IgE和IFN-γ水平,T-bet mRNA表达几乎无改变(P＞0.05),STAT1 mRNA表达显著提高(P＜0.05),而GATA3和STAT6 mRNA表达显著降低(P＜0.05);与AR模型组相比,左旋紫草素高剂量组动物行为学评分、Th1细胞比例、IL-4和IgE水平、GATA3和STAT6 mRNA表达显著下降(P＜0.05),Th2细胞比例、IFN-γ水平、T-bet和STAT1 mRNA表达显著增加(P＜0.05).与左旋紫草素低剂量组相比,左旋紫草素高剂量组外周血中Th1细胞比例、IL-4和IgE水平、GATA3和STAT6 mRNA表达显著下降(P＜0.05),Th2细胞比例、IFN-γ水平、T-bet和STAT1 mRNA表达显著增加(P＜0.05).结论 高剂量左旋紫草素对小鼠AR有一定的改善作用,其减轻炎症的主要潜在机制可能是通过JAK/T-bet/STAT1和JAK/GATA3/STAT6途径调节Th1/Th2失衡.
Abstract
Objective To investigate the therapeutic effect of L-shikonin on mice with allergic rhinitis(AR)and its related inhibitory mechanism.Methods Ovalbumin(OVA)-sensitized AR mouse model was established.According to random number table method,25 C57BL/6 mice were randomly divided into normal group,AR model group,low-dose and high-dose L-shikonin groups,and standard control group,with 5 mice in each group.In addition to the normal group,the other groups completed AR model construction:OVA+aluminum hydroxide gel mixed preparation was used for sensitization on the 1st,5th,9th and 12th day,and OVA was used for stimulation once a day for seven consecutive days starting from the 13th day.Starting from the 28th day of modeling,the mice in the L-shikonin group were administered with 2 and 8 mg/(kg·d)L-shikonin by gavage.The mice in the standard control group were given 2.5 mg/(kg·d)of dexamethasone by gavage,while the normal group and AR model group were given an equal dose of dimethyl sulfoxide by gavage for a total of 4 weeks.Within 30 min of the last gavage,nasal symptoms of each group of mice were observed and scored.Flow cytometry was used to detect the expression levels of T helper(Th)1 and Th2 cell subsets in peripheral blood of mice.ELISA was used to detect the levels of interleukin-4(IL-4),immunoglobulin E(IgE),and γ-interferon(IFN-γ)in the nasal lavage fluid of mice.HE staining was used to detect pathological changes in the nasal mucosa.qRT-PCR was used to detect the mRNA expression of T-box transcription factor(T-bet),GATA binding protein 3(GATA3),signal transducer and activator of transcription 1(STAT1),and STAT6 in nasal mucosal tissue.Results After 4 weeks of administration,compared with normal group,behavioral scores,Th1 cell proportion,IL-4 and IgE levels,and GATA3 and STAT6 mRNA expressions were significantly increased in AR model group(P＜0.05),while Th2 cell proportion,IFN-γ level,and T-bet and STAT1 mRNA expressions were significantly decreased in AR model group(P＜0.05).Compared with AR model group,behavioral scores,Th1 and Th2 cell proportions,IL-4,IgE and IFN-γ levels,and T-bet mRNA expression of animals in low-dose L-shikonin group had almost no changes(P＞0.05),while STAT1 mRNA expression was significantly increased(P＜0.05),and GATA3 and STAT6 mRNA expressions were significantly decreased(P＜0.05).Compared with AR model group,behavioral scores,Th1 cell proportion,IL-4 and IgE levels,and GATA3 and STAT6 mRNA expressions in high-dose L-shikonin group were significantly decreased(P＜0.05),and Th2 cell proportion,IFN-γ level,and T-bet and STAT1 mRNA expressions were significantly increased(P＜0.05).Compared with low-dose L-shikonin group,Th1 cell proportion,IL-4 and IgE levels,and GATA3 and STAT6 mRNA expressions in peripheral blood of high-dose L-shikonin group were significantly decreased(P＜0.05),and Th2 cell proportion,IFN-γ level,and T-bet and STAT1 mRNA expressions were significantly increased(P＜0.05).Conclusion High-dose L-shikonin has a certain improvement effect on AR in mice,and its main potential mechanism for reducing inflammation may be to regulate Th1/Th2 imbalance through JAK/T-bet/STAT1 and JAK/GATA3/STAT6 pathways.
维普
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