小干扰RNA沉默Toll样受体4表达对慢性温和应激ApoE^(-/-)小鼠腹腔巨噬细胞TLR 4/NF-κB途径基因表达的影响

Effects of Silencing Toll-Like Receptor 4 Expression on the Expression of Toll-Like Receptor 4/Nuclear Factor-κB Signaling Pathway in Peritoneal Macrophages of Chronic Mild Stress ApoE^(-/-)Mice

目的探讨小干扰RNA(siRNA)沉默Toll样受体4(TLR4)基因对慢性温和应激ApoE-/-小鼠腹腔巨噬细胞促炎症细胞因子表达的影响。方法针对小鼠TLR4基因设计2条siRNA和一条无关序列,再据每一序列合成两条互补并含siRNA正反义链的DNA,退火后与含GFP的载体pRNAT-H1.1/Adeno相连,转染293A细胞,实时荧光PCR筛选有效的一条siRNA,经腺病毒颗粒包装与病毒扩增并感染293A细胞,G418筛选后,挑取单个阳性克隆放大培养,检测病毒滴度。120只雄性ApoE-/-小鼠随机分为三组:(1)慢性温和应激组;(2)慢性温和应激+siRNA组(10μL/只,尾静脉注射,每5日一次);(3)慢性温和应激+腺病毒空载体组。每组ApoE-/-小鼠40只,实验动物都以高脂、高胆固醇饲料喂养,分别在接受慢性温和应激0、4、12周后3个时间点处死动物,收集小鼠腹腔单核巨噬细胞,以W estern B lotting方法检测其TLR4和核因子κB(NF-κB)蛋白表达,ELISA法检测腹腔单核巨噬细胞培养上清液白细胞介素1β(IL-1β)和肿瘤坏死因子α表达。结果转染siRNA1和siRNA2后的292A细胞中TLR4 mRNA表达水平较空白对照组分别下降56%和67%,逐孔稀释滴度测定法测定的病毒滴度为3.4×1014TU/L;遭受4、12周慢性温和应激后,ApoE-/-小鼠血浆中皮质醇激素显著升高,但在同一时间点内,各组ApoE-/-小鼠血浆皮质酮激素浓度相比,差异没有统计学意义(P>0.05);与同一时期慢性温和应激组相比,siRNA组腹腔巨噬细胞TLR4和核因子κB蛋白表达水平明显降低(均P<0.05),其培养细胞上清液中的白细胞介素1β和肿瘤坏死因子α含量显著减少(P<0.01)。结论 siRNA沉默TLR4基因能有效抑制TLR4/NF-κB-IL-1β和肿瘤坏死因子α的表达,提示TLR4/NF-κB途径在慢性温和应激诱导的慢性炎症应答中可能有着重要作用。

Aim To examine the effect of Toll-like receptor 4（TLR4） gene silencing by small interfering RNA（siRNA） on the expression of cytokines produced by peritoneal macrophages in chronic mild stress（CMS） ApoE-/-mice. Methods Two siRNA sequences and one negative control sequence were designed targeting the mouse TLR4 gene.Two complementary single-strand DNA were designed and synthesized based on siRNA sequences.The DNA fragments were annealed and ligated to the GFP expression vector pRNAT-H1.1/Adeno.One siRNA with higher interference efficiency than the other was found after siRNA plasmid transfection into 293A cells mediated by liposome.After adenovirus partical packaging and production,the 293A cells were infected,and the single cell clone was acquired and cultured to establish the stable cell strain.The titer of concentrated virus was detected by hole-by-dilution titer assay.One hundred twenty male ApoE-/-mice were divided into groups of CMS control,CMS ＋ empty vector and CMS ＋ siRNA（tail vein injection,10 μL/mouse;n=40 per group）.All mice were fed a high-fat（5%,wt/w）,high-cholesterol（1%,wt/wt） diet and subjected to daily CMS for 0,4,12 weeks,respectively.Peritoneal macrophages were prepared from ApoE-/-mice and then total proteins from cells were extracted.Western Blotting was used to determine the expressions of TLR4 and nuclear factor-κB（NF-κB） of peritoneal macrophages.The supernatants of cultured peritoneal macrophages were collected and then used for detection of interleukin-1β（IL-1β） and tumor necrosis factor-α（TNF-α） levels by ELISA. Results Compared with the blank control group,real-time PCR showed that the expression of TLR4 mRNA in 293A cells was decreased by 56% and 67% after 48 h transfection with siRNA1 and siRNA2,respectively.The hole-by-dilution titer assay showed that viral titer was 3.4×1014 TU/L.After exposure to CMS for 4 and 12 weeks,there was no difference in serum corticosterone levels between siRNA group and the CMS control group（P 0.05）,and siRNA group mice demonstrated markedly decrease in TLR4（P0.05） and NF-κB（P0.05） levels of peritoneal macrophages compared with the corresponding control group,respectively.IL-1β（P0.01） and TNF-α（P0.01） levels in supernatants of cultured peritoneal macrophages of siRNA group were significantly lower than those of the CMS group. Conclusion These results showed that siRNA effectively inhibited the expressions of TLR4/NF-κB-IL-1β and TNF-α of peritoneal macrophages in CMS ApoE-/-mice,which suggested that the activation of TLR4 pathway of macrophages might play an important role in chronic inflammation response induced by CMS.