敷和汤对特应性皮炎小鼠Toll样受体4/核因子-κB通路调控辅助性T细胞1/辅助性T细胞2的作用及机制研究

Effect and mechanism of Fuhe decoction on TLR4/NF-κB pathway regulating Th1/Th2 in atopic dermatitis mice

目的基于Toll样受体4(TLR4)/核因子-κB(NF-κB)信号通路探讨敷和汤对特应性皮炎(AD)小鼠模型的作用及辅助性T细胞1(Th1)/辅助性T细胞2(Th2)免疫平衡的机制。方法将40只BALB/c小鼠采用随机数字表法分为四组:对照组、模型组、地氯雷他定组、敷和汤组,每组10只,除正常组外,其余各组均用2,4-二硝基氟苯(DNFB)刺激。在DNFB刺激后的第14天各组给予相应药物治疗14 d。治疗后观察各组小鼠皮损变化;记录10 min内小鼠搔抓次数;测量胸腺、脾脏指数;苏木精-伊红(HE)染色观察皮肤组织病理形态学改变;免疫组化检测TLR4阳性蛋白;酶联免疫吸附测定法(ELISA)检测血清中免疫球蛋白E(IgE)、白细胞介素4(IL-4)、白细胞介素6(IL-6)和瘤坏死因子-α(TNF-α)水平变化;实时荧光定量逆转录聚合酶链反应(qRT-PCR)法检测小鼠皮肤组织中核因子-κB P65(NF-κB P65)、TLR4、白细胞介素2(IL-2)、白细胞介素10(IL-10)、白细胞介素1β(IL-1β)、干扰素-γ(IFN-γ)的mRNA水平变化。结果与正常组相比,模型组小鼠皮损严重、表皮明显增生、角化过度、大量炎性细胞浸润;小鼠搔抓次数(12.80±1.32)次/10分钟和皮损评分(4.00±0.67)分、脾脏和胸腺指数分别为42.53±2.04和35.76±2.01,免疫组化检测TLR4阳性蛋白为97.20±12.19;血清IgE、IL-4、IL-6和TNF-α水平分别为(302.39±46.60)μg/L、(239.54±29.23)ng/L、(249.94±33.62)ng/L、(310.01±50.09)ng/L,皮损NF-κB P65、TLR4、IL-2、IL-10、IFN-γ、IL-1β的mRNA表达分别为(6.70±0.38、7.45±0.30、4.03±0.57、3.35±0.35、5.31±0.12、41.21±1.17)显著升高(均P<0.05);敷和汤组给药14 d后小鼠搔抓次数(4.30±0.67)次/10分钟和皮损评分(1.40±0.52)分、脾脏和胸腺指数分别为29.11±1.61和22.02±1.15,免疫组化检测TLR4阳性蛋白为70.85±7.76;血清IgE、IL-4、IL-6和TNF-α水平分别含量为(183.89±11.74)μg/L、(112.67±9.233)ng/L、(121.88±10.84)ng/L、(153.24±12.78)ng/L,皮损NF-κB P65、TLR4、IL-2、IL-10、IFN-γ、IL-1β的mRNA表达分别为(2.87±0.15、2.23±0.31、2.57±0.46、2.39±0.19、2.35±0.21、23.27±2.44)低于模型组(均P<0.05);地氯雷他定组给药14 d后小鼠搔抓次数(5.30±0.67)次/10分钟和皮损评分(1.80±0.63)分、脾脏和胸腺指数分别为27.23±1.19和20.10±1.20,免疫组化检测TLR4阳性蛋白为77.28±5.77;血清IgE、IL-4、IL-6和TNF-α水平含量分别为(175.97±13.12)μg/L、(116.07±9.48)ng/L、(112.11±8.89)ng/L、(143.90±10.83)ng/L,皮损NF-κB P65、TLR4、IL-2、IL-10、IFN-γ、IL-1β的mRNA表达分别为(2.53±0.08、3.46±0.28、2.03±0.11、1.52±0.22、2.15±0.19、23.52±1.24)低于敷和汤组(均P<0.05)。结论敷和汤通过NF-κB通路的激活抑制相关炎症因子反应以及调节Th1/Th2发挥免疫稳定的作用,促进皮损修复,从而达到治疗的作用。

Objective To explore the effect of Fuhe decoction on atopic dermatitis(AD)mouse model and the mechanism of immune balance of helper T cell 1(Th1)/helper T cell 2(Th2)based on Toll-like receptor 4(TLR4)/nuclear factor-κB(NF-κB)signaling pathway.Methods Forty BALB/c mice were randomly divided into 4 groups:control group,model group,desloratadine group,Fuhe decoction group,with 10 mice in each group.Except for the normal group,the other groups were stimulated with 2,4-dinitrofluorobenzene(DNFB).On the 14th day after DNFB stimulation,each group was treated with corresponding drugs for 14 days.After treatment,the changes of skin lesions in each group were observed.The scratching times of mice within 10 min were recorded.The thymus and spleen index was measured;the histopathological changes of skin tissue were observed by hematoxylin-eosin(HE)staining.TLR4 positive protein was detected by immunohistochemistry.The levels of immunoglobulin E(IgE),interleukin 4(IL-4),interleukin 6(IL-6)and tumor necrosis factor-α(TNF-α)in serum were detected by enzyme-linked immunosorbent assay(ELISA).Real-time fluorescence quantitative reverse transcription polymerase chain reaction(qRT-PCR)was used to detect the mRNA levels of nuclear factor-κB P65(NF-κB P65),TLR4,interleukin-2(IL-2),interleukin-10(IL-10),interleukin-1β(IL-1β)and interferon-γ(IFN-γ)in mouse skin tissues.Results Compared with the normal group,the mice in the model group had severe skin lesions,obvious epidermal hyperplasia,hyperkeratosis,and a large number of inflammatory cell infiltration.The scratching times of mice were(12.80±1.32)times/10 min,the skin lesion score was(4.00±0.67)points,the spleen and thymus indexes were 42.53±2.04 and 35.76±2.01,respectively,and TLR4 was(97.20±12.19)positive protein detected by immunohistochemistry.The levels of serum IgE,IL-4,IL-6 and TNF-αwere(302.39±46.60)μg/L,(239.54±29.23)ng/L,(249.94±33.62)ng/L,(310.01±50.09)ng/L and the mRNA expressions of NF-κB P65,TLR4,IL-2,IL-10,IFN-γand IL-1βin skin lesions were 6.70±0.38,7.45±0.30,4.03±0.57,3.35±0.35,5.31±0.12,41.21±1.17 significantly increased(all P<0.05);After 14 days of administration,the scratching frequency of mice in the Fuhe Decoction group was(4.30±0.67)times/10 min,the skin lesion score was(1.40±0.52)points,the spleen and thymus indexes were 29.11±1.61 and 22.02±1.15,respectively,and the TLR4 was(70.85±7.76)positive protein detected by immunohistochemistry.The levels of serum IgE,IL-4,IL-6 and TNF-α(183.89±11.74)μg/L,(112.67±9.233)ng/L,(121.88±10.84)ng/L,(153.24±12.78)ng/L and the mRNA expression of NF-κB P65,TLR4,IL-2,IL-10,IFN-γand IL-1βin skin lesions 2.87±0.15,2.23±0.31,2.57±0.46,2.39±0.19,2.35±0.21,23.27±2.44 were lower than those in the model group(all P<0.05);After 14 days of administration,the scratching times of mice in desloratadine group were(5.30±0.67)times/10 min,the skin lesion score was(1.80±0.63)points,the spleen and thymus indexes were 27.23±1.19 and 20.10±1.20,respectively,and TLR4 was(77.28±5.77)positive protein detected by immunohistochemistry.The levels of serum IgE,IL-4,IL-6 and TNF-αwere(175.97±13.12)μg/L,(116.07±9.48)ng/L,(112.11±8.89)ng/L,(143.90±10.83)ng/L and the mRNA expressions of NF-κB P65,TLR4,IL-2,IL-10,IFN-γand IL-1βwere 2.53±0.08,3.46±0.28,2.03±0.11,1.52±0.22,2.15±0.19,23.52±1.24 were lower than those in Fuhe decoction group(all P<0.05).Conclusion Fuhe decoction can inhibit the reaction of related inflammatory factors by blocking the activation of NF-κB pathway and regulate Th1/Th2 to play a role in immune stability and promote skin repair.