白芍总苷治疗特应性皮炎小鼠的作用机制

Mechanism of total glucosides of paeony in the treatment of atopic dermatitis in mice

目的基于微小RNA-223/核苷酸结合寡聚化结构域样受体蛋白3(miR-223/NLRP3)轴探究白芍总苷对特应性皮炎(AD)的治疗作用。方法将72只BALB/c小鼠随机分成6组:空白对照组、模型组、miR-223 antagomiR组、miR-223 antagomiR阴性对照组、白芍总苷组、白芍总苷+miR-223 antagomiR组,每组12只。除空白对照组外,其余各组小鼠用2,4-二硝基氯苯(DNCB)诱导构建小鼠AD模型,并给予相应药物处理。观察各组小鼠皮肤损伤程度并给予皮损评分;记录10 min内各组小鼠抓挠次数;检测各组小鼠右侧耳朵厚度;以酶联免疫吸附(ELISA)法检测血清中免疫球蛋白E(IgE)和白细胞介素-6(IL-6)水平;以苏木精-伊红(HE)染色法观察各组小鼠背部皮肤组织病理变化;以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miR-223水平;以蛋白质印迹(Western blot)法检测NOD样受体蛋白3(NLRP3)、白细胞介素-1β(IL-1β)蛋白表达水平。结果空白对照组小鼠皮肤结构正常,模型组小鼠镜下可见背部皮肤结构损伤严重,损伤表皮增生明显,角化过度,内皮细胞水肿,有大量炎性细胞和肥大细胞浸润。空白对照组、模型组、miR-223 antagomiR组、miR-223 antagomiR阴性对照组、白芍总苷组、白芍总苷+miR-223 antagomiR组的IgE水平分别为(7.35±0.62),(36.46±3.31),(13.19±1.26),(37.80±3.59),(12.43±1.28),(9.01±0.94)ng·mL^(-1);IL-6水平为(0.52±0.11),(9.23±2.15),(2.35±1.12),(9.17±2.09),(2.13±0.25),(0.92±0.20)ng·mL^(-1);模型组与空白对照组相比,白芍总苷组、白芍总苷+miR-223 antagomiR组与模型组相比,miR-223 antagomiR组与miR-223 antagomiR阴性对照组相比,皮损程度评分、右耳厚度、抓挠次数、IgE、IL-6、miR-223、NLRP3、IL-1β的差异均有统计学意义(均P<0.05)。结论白芍总苷可下调miR-223表达,抑制NLRP3活化,减轻AD小鼠的炎症反应,促进皮肤修复。

ObjectiveTo explore the therapeutic effect of total glucosides of paeony (TGP) on atopic dermatitis (AD) based on microRNA-223/nucleotide binding oligomerization domain-like receptor protein 3 (miR-223/NLRP3) axis.Methods Seventy-two BALB/c mice were randomly divided into 5 groups:control group,model group,miR-223 antagomiR group,miR-223 antagomiR negative control group,TGP group,and TGP+miR-223 antagomiR group,with twelve mice in each group.In addition to control group,other groups of mice were induced by 2,4-dinitrochlorobenzene (DNCB) to establish AD model,and given the corresponding drug treatment.The degree of skin injury was observed and the skin lesion score was given;the scratching times of each group were recorded within 10 min;the thickness of right ear was measured;the serum levels of immunoglobulin E (IgE) and interleukin-6 (IL-6) were detected by enzyme-linked immunosorbent assay(ELISA);hematoxylin-eosin (HE) staining was used to observe the pathological changes of the back skin in each group;the expression level of miR-223 was detected by quantitative real-time polymerase chain reaction (qRT-PCR);the protein expression levels of NOD-like receptor protein 3 (NLRP3) and interleukin-1β(IL-1β) were detected by Western blot.Results The skin structure of the control group was normal,and the back skin structure of the model group was seriously damaged,with obvious hyperplasia of the damaged epidermis,hyperkeratosis,edema of endothelial cells,and infiltration of a large number of inflammatory cells and mast cells.The IgE levels in the control group,model group,miR-223 antagomiR group,miR-223 antagomir negative control group,total glucosides of paeony group,total glucosides of paeony+miR-223 antagomiR group were (7.35±0.62),(36.46±3.31),(13.19±1.26),(37.80±3.59),(12.43±1.28),(9.01±0.94) ng·m L;,IL-6 levels were (0.52±0.11),(9.23±2.15),(2.35±1.12),(9.17±2.09),(2.13±0.25),(0.92±0.20) ng·m L;.Control group compared with model group,the total glucosides of paeony group and the total glucosides of paeony+miR-223antagomiR group compared with the model group,the miR-223 antagomiR group and the miR-223 antagomiR negative control group,the difference of skin lesion degree score,right ear thickness,number of scratching,IgE,IL-6,miR-223,NLRP3,IL-1βwere statistically significant(all P<0.05).Conclusion TGP can down-regulate the expression of miR-223,inhibit the activation of NLRP3,reduce the inflammatory reaction and promote skin repair of AD mice.