siRNA沉默STAT6抑制嗜酸粒细胞性慢性鼻窦炎产生的实验性研究

Silencing STAT6 with siRNA prevents development of eosinophilic chronic rhinosinusitis:an experimental study

目的探讨用小干扰RNA(siRNA)沉默信号传导和转录激活因子6(signal transducer and activator of transcription 6,STAT6),能否抑制小鼠嗜酸粒细胞性慢性鼻窦炎(eosinophilic chronic rhinosinusitis,ECRS)的产生。方法2022年3—9月期间,48只BALB/c雌性小鼠随机分为Control(对照)组、Vehicle(转染试剂)组、Scramble siRNA(对照siRNA)组和STAT6 siRNA组,每组12只。用卵清蛋白(OVA)联合金黄色葡萄球菌肠毒素B(staphylococcal enterotoxin B,SEB)诱导小鼠ECRS,并用siRNA鼻腔滴注进行干预。随后收集外周血以及鼻黏膜组织标本。血细胞分析仪检测外周血嗜酸粒细胞(Eos)的数目及百分比;酶联免疫吸附实验检测外周血总免疫球蛋白E(IgE)和OVA-特异性IgE(OVA-sIgE)水平,以及鼻黏膜组织中干扰素γ(IFN-γ)、白细胞介素(IL)-5、IL-17A和嗜酸粒细胞趋化因子1(Eotaxin-1)的表达量;用苏木精-伊红(HE)染色鼻黏膜组织切片,在高倍视野(HPF)下对Eos进行计数;蛋白免疫印迹检测鼻黏膜中STAT6和磷酸化STAT6(p-STAT6)蛋白水平;免疫荧光染色对鼻黏膜中p-STAT6表达进行定位。采用SPSS 18.0软件进行统计学分析。结果STAT6 siRNA组小鼠外周血Eos计数、Eos百分比、总IgE以及OVA-sIgE水平[(0.318±0.045)×10^(3)/μl、(3.667±0.479)%、(102.070±13.205)ng/ml和(38.870±7.352)ng/ml]明显低于Vehicle组[(0.532±0.049)×10^(3)/μl、(6.710±1.061)%、(203.102±29.653)ng/ml和(74.575±6.432)ng/ml,Z值分别为-2.56、-2.24、-2.40、-2.56,P值均<0.05]和Scramble siRNA组[(0.493±0.036)×10^(3)/μl、(5.858±0.872)%、(189.964±30.042)ng/ml和(80.935±8.358)ng/ml,Z值分别为-2.17、-2.08、-2.24、-2.72,P值均<0.05],且鼻黏膜组织中IL-5和Eotaxin-1表达量分别为(312.279±34.281)pg/ml和(25.297±4.323)pg/ml,也显著低于Vehicle组[(689.667±31.905)pg/ml、(68.278±6.485)pg/ml,Z值分别为-2.73、-2.88,P值均<0.01]和Scramble siRNA组[(661.783±42.094)pg/ml、(63.015±7.416)pg/ml,Z值分别为-2.72、-2.81,P值均<0.01]。STAT6 siRNA组小鼠鼻黏膜组织Eos计数为(29.132±4.163)/HPF,明显低于Vehicle组[(78.050±7.912)/HPF,Z=-2.88,P<0.01]和Scramble siRNA组[(73.864±8.671)/HPF,Z=-2.72,P<0.01]。STAT6 siRNA组小鼠鼻黏膜组织中STAT6蛋白相对表达水平为0.105±0.021,与Control、Vehicle及Scramble siRNA组(0.232±0.037、0.243±0.039、0.228±0.032)相比显著降低(Z值分别为-2.25、-2.49、-2.56,P值均<0.05)。与Vehicle组(0.613±0.046)和Scramble siRNA组(0.641±0.050)相比,STAT6 siRNA组p-STAT6蛋白相对水平(0.292±0.038)明显下降(Z值分别为-2.81、-2.88,P值均<0.01)。免疫荧光染色结果显示,p-STAT6主要位于鼻黏膜上皮细胞及炎性细胞的细胞核中,STAT6 siRNA组小鼠鼻黏膜中表达p-STAT6的绿色荧光弱于Vehicle及Scramble siRNA组。结论经鼻滴入STAT6 siRNA可下调鼻黏膜STAT6表达,降低p-STAT6水平,抑制ECRS产生。

Objective To investigate whether silencing signal transducer and activator of transcription 6(STAT6)with siRNA can inhibit eosinophilic inflammation of sinonasal mucosa in a mouse model of eosinophilic chronic rhinosinusitis(ECRS).Methods The study was conducted from March to September in 2022.Forty-eight female BALB/c mice were randomly divided into four groups:the control group,the Vehicle(transfection reagent)-treated group,the Scramble siRNA(Control siRNA)-treated group,and the STAT6 siRNA-treated group,with twelve mice in each group.An ovalbumin(OVA)-staphylococcal enterotoxin B(SEB)-induced ECRS murine model was established.SiRNA prepared in Lipofectamine was locally administered to the nasal cavity.After administration,samples of the peripheral blood and sinonasal mucosa were collected.Eosinophils in peripheral blood were detected by hematology analyzer.Total and OVA-specific IgE(OVA-sIgE)in serum were detected by enzyme-linked immunosorbent assay(ELISA).Mucosal levels of cytokines and chemokines,including interleukin(IL)-5,IL-17A,interferon-γ(IFN-γ)and eotaxin-1,were also measured using ELISA.Mucosal histological changes of eosinophil infiltration were examined using hematoxylin,and eosin staining,and tissue eosinophil count was performed using a microscope under a high-power field(HPF).Tissue expression of STAT6 and phosphorylated STAT6(p-STAT6)was detected with the western blot method.Immunofluorescence staining was used to localize the expression of p-STAT6 in sinonasal mucosa.Statistical analysis was conducted using SPSS 18.0 software.Results Peripheral blood eosinophil count,percentage of peripheral blood eosinophil,total serum IgE level,and serum OVA-sIgE level in the STAT6 siRNA-treated group[(0.318±0.045)×10^(3)/μl,(3.667±0.479)%,(102.070±13.205)ng/ml,and(38.870±7.352)ng/ml]were significantly different from those of the Vehicle-treated group[(0.532±0.049)×10^(3)/μl,(6.710±1.061)%,(203.102±29.653)ng/ml,and(74.575±6.432)ng/ml,Z value was-2.56,-2.24,-2.40,and-2.56,respectively,all P<0.05]and Scramble siRNA-treated group[(0.493±0.036)×10^(3)/μl,(5.858±0.872)%,(189.964±30.042)ng/ml,and(80.935±8.358)ng/ml,Z value was-2.17,-2.08,-2.24,and-2.72,respectively,all P<0.05].Besides,IL-5 and eotaxin-1 levels in the STAT6 siRNA-treated group[(312.279±34.281)pg/ml and(25.297±4.323)pg/ml]were significantly lower than those in the Vehicle-treated group[(689.667±31.905)pg/ml and(68.278±6.485)pg/ml,Z value was-2.73 and-2.88,respectively,both P<0.01]and Scramble siRNA-treated group[(661.783±42.094)pg/ml and(63.015±7.416)pg/ml,Z value was-2.72 and-2.81,respectively,both P<0.01].Tissue eosinophil count in sinonasal mucosa was(29.132±4.163)/HPF in the STAT6 siRNA-treated group,and were significantly less than those in the Vehicle-treated group[(78.050±7.912)/HPF,Z=-2.88,P<0.01]and Scramble siRNA-treated group[(73.864±8.671)/HPF,Z=-2.72,P<0.01].The expression level of STAT6 protein(0.105±0.021)was significantly decreased in the mice treated with STAT6 siRNA compared with PBS,Vehicle,and Scramble siRNA(0.232±0.037,0.243±0.039,and 0.228±0.032,Z value was-2.25,-2.49,and-2.56,respectively,all P<0.05).Corresponding,p-STAT6 protein level(0.292±0.038)was markedly decreased by the introduction of STAT6 siRNA,the difference was statistically significant as compared with the Vehicle-and Scramble siRNA-treated groups(0.613±0.046 and 0.641±0.050,Z value was-2.81 and-2.88,respectively,both P<0.01).Immunofluorescence staining showed that p-STAT6 was mainly located in the nucleus of nasal epithelial cells and inflammatory cells.The green fluorescence of p-STAT6 expression in sinonasal mucosa in the STAT6 siRNA-treated group was weaker than that in the Vehicle-and Scramble siRNA-treated groups.Conclusion Intranasal administration of STAT6 siRNA can significantly downregulate STAT6 expression,decrease p-STAT6 level,and prohibit the development of Th2-skewed ECRS.