白术多糖通过Toll样受体4/核因子-κB信号通路调控雏鸡脾脏淋巴细胞免疫功能

Polysaccharide of Atractylodes macrocephala Koidz Regulates Lymphocyte Immune Function in Chick Spleen by Toll-Like Receptor 4/Nuclear Factor-Kappa B Signal Pathway

本试验旨在研究白术多糖(PAMK)对雏鸡脾脏淋巴细胞免疫功能的影响及可能的调控机制。体外分离培养21日龄雏鸡脾脏淋巴细胞,以siRNA引物转染淋巴细胞干扰Toll样受体4(TLR4)基因的表达,在此基础上用浓度为12.5 mg/L的PAMK处理转染和非转染细胞,36 h后检测TLR4及其信号通路下游基因[髓样分化因子88(MyD88)、核因子-κB(NF-κB)抑制物的激酶(IKK)、NF-κB抑制蛋白(IκB)]和细胞因子[白细胞介素-2(IL-2)、白细胞介素-4(IL-4)、γ-干扰素(IFN-γ)、脂多糖诱导的肿瘤坏死因子-α(LITAF)]的mRNA表达量,并以凝胶迁移试验(EMSA)方法检测淋巴细胞核蛋白中NF-κB的含量。结果显示:PAMK可显著提高淋巴细胞中TLR4及其信号通路下游基因IKK、IκB与细胞因子IL-2、LITAF的mRNA表达量(P<0.05),同时可显著提高转染淋巴细胞中TLR4的mRNA表达量(P<0.05);此外,PAMK还可不同程度地促进转染淋巴细胞中1型辅助性T细胞(Th1)分泌的细胞因子[IL-2(P>0.05)、IFN-γ(P<0.05)、LITAF(P>0.05)]的表达,对2型辅助性T细胞(Th2)分泌的细胞因子(IL-4)的表达无显著促进作用(P>0.05);EMSA结果显示PAMK能促进转染和非转染淋巴细胞内NF-κB入核,使核蛋白中NF-κB的含量显著升高(P<0.05)。综上所述,PAMK可活化TLR4/NF-κB信号通路,促进相关基因表达,最终使NF-κB进入细胞核调控细胞因子的转录水平,进而调控雏鸡脾脏淋巴细胞免疫功能。

The aim of this study was to investigate the effects of polysaccharide of Atractylodes macrocephala Koidz(PAMK)on lymphocyte immune function in chick spleen and possible regulatory mechanisms.The spleen lymphocytes of 21-day-old chicks were isolated and cultured in vitro,the lymphocytes were transfected by siRNA primers and the expression of Toll-like receptor 4(TLR4)was interfered.On that basis,PAMK with the concentration of 12.5 mg/L was used to treat transfected and non-transfected lymphocytes.After 36 h,the mRNA expression levels of TLR4 and its signaling pathway downstream genes[such as myeloid differentiation factor 88(MyD88),nuclear factor-kappa B(NF-κB)inhibitor kinase(IKK),NF-κB inhibitory protein(IκB)]and cytokines[such as interleukin-2(IL-2),interleukin-4(IL-4),interferon-γ(IFN-γ),lipopolysaccharide induced tumor necrosis factor-α(LITAF)]were detected.In addition,the content of NF-κB in lymphocyte nuclear protein was detected by electrophoretic mobility shift assay(EMSA)method.The results showed that PAMK could significantly increase the mRNA expression levels of TLR4 and its signaling pathway downstream genes IKK,IκB,cytokines IL-2 and LITAF in non-transfected lymphocytes(P<0.05),and significantly increase the mRNA expression levels of TLR4 in transfected lymphocytes(P<0.05).In addition,PAMK could also promote the expression of cytokines[IL-2(P>0.05),IFN-γ(P<0.05),LITAF(P>0.05)]secreted by type 1 helper T cells(Th1)in transfected lymphocytes to different degrees,there was no significant promoting effect on the expression of cytokine(IL-4)secreted by type 2 helper T cells(Th2)(P>0.05).EMSA results showed that PAMK could promote NF-κB into the nuclear in transfected and non-transfected lymphocytes,and significantly increase the content of NF-κB in nuclear protein(P<0.05).In summary,PAMK activates TLR4/NF-κB signaling pathway to promote the expression of related genes,and finally promotes NF-κB into the nuclear to regulate the transcription level of cytokines,and then regulates lymphocyte immune function in chick spleen.