玉屏风散通过ROS/NLRP3/Caspase-1信号通路抗变应性鼻炎的作用机制

Mechanism of Yupingfeng San Against Allergic Rhinitis Through ROS/NLRP3/Caspase-1 Signaling Pathway

目的:探讨玉屏风散对变应性鼻炎(AR)的治疗作用及对活性氧(ROS)/核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)/胱天蛋白酶-1(Caspase-1)通路的影响。方法:将SPF级小鼠随机分为正常组、模型组、氯雷他定组(0.9 mg·kg^(-1))、玉屏风散低、中、高剂量组(6、12、24 mg·kg^(-1)),每组10只。正常组给予常规饲养,其余各组腹腔注射[卵清蛋白(OVA)+Al(OH)_(3)+磷酸盐缓冲液(PBS)]混悬液,隔日1次,连续7次。7 d后,10%OVA溶液滴鼻,2次/d,连续7 d。造模成功后,各给药组小鼠腹腔注射不同剂量的玉屏风散汤液,正常组和模型组腹腔注射等体积生理盐水,每日记录各组小鼠喷嚏、抓鼻和流涕症状进行评分;采用酶联免疫吸附测定法(ELISA)检测小鼠鼻灌洗液和血清中卵清蛋白特异性免疫球蛋白E(OVA-sIgE)、组胺(Histamine)、嗜酸性粒细胞阳离子蛋白(ECP)、前列腺素D2(PGD2)、白细胞介素-1β(IL-1β)、IL-18、IL-4、γ干扰素(IFN-γ)水平;苏木素-伊红(HE)染色法观察小鼠鼻腔黏膜损伤状态,过碘酸雪夫(PAS)染色法观察小鼠鼻腔黏膜杯状细胞数目,免疫组化法检测小鼠鼻腔黏膜NLRP3蛋白表达;蛋白免疫印迹法(Western blot)检测鼻腔黏膜NLRP3、剪切(cleaved)Caspase-1和cleaved Gasdermin-D(GSDMD)蛋白的表达;试剂盒检测各组小鼠鼻腔ROS变化。结果:与正常组比较,模型组小鼠鼻部症状明显加重,血清及鼻灌洗液中炎症因子OVA-sIgE、Histamine、ECP、PGD2、IL-1β、IL-18、IL-4水平明显升高(P<0.05,P<0.01),IFN-γ水平明显降低(P<0.05,P<0.01);组织病理学评分、杯状细胞数目和ROS含量显著升高(P<0.01),焦亡相关蛋白NLRP3、cleaved Caspase-1、cleaved GSDMD表达升高(P<0.01)。与模型组比较,氯雷他定组和玉屏风散组小鼠鼻部症状明显减轻,血清及鼻灌洗液中炎症因子OVA-sIgE、Histamine、ECP、PGD2、IL-1β、IL-18、IL-4水平降低(P<0.05,P<0.01),IFN-γ水平升高(P<0.05、0.01);组织病理学评分、杯状细胞数目和ROS含量显著减少(P<0.05,P<0.01),焦亡相关蛋白NLRP3、cleaved Caspase-1、cleavedGSDMD表达降低(P<0.05,P<0.01)。与氯雷他定组比较,玉屏风散高剂量组疗效进一步增高(P<0.05,P<0.01)。结论:玉屏风散对变应性鼻炎具有治疗效果,其具体的作用可能与其抑制ROS/NLRP3/Caspase-1引起的细胞焦亡有关。

Objective:To investigate the therapeutic effect of Yupingfeng San on allergic rhinitis(AR)and its effect on Reactive oxygen species(ROS)/NOD-like receptor thermal protein domain associated protein 3(NLRP3)/cysteinyl aspartate specific proteinase-1(Caspase-1)pathway.Method:SPF mice were randomly divided into control group,model group,loratadine group(0.9 mg·kg^(−1)),and low,medium,and high dose Yupingfeng San groups(6,12,24 mg·kg^(−1)),with 10 mice in each group.The control group was given routine feeding,and the other groups were intraperitoneally injected with[ovalbumin(OVA)+Al(OH)3+phosphate buffer solution(PBS)]suspension once every other day for seven consecutive times.After seven days,10%OVA solution was instilled in the nose,two times each day for seven consecutive days.After successful modeling,each administration group was intraperitoneally injected with different doses of Yupingfeng San Decoction,and the control group and model group were intraperitoneally injected with an equal volume of normal saline.Symptoms of sneezing,scratching,and runny nose were recorded and scored daily.The levels of ovalbumin specific immunoglobulin E(OVA-sIgE),histamine,eosinophil cationic protein(ECP),prostaglandin D2(PGD2),interleukin-1β(IL-1β),interleukin-18(IL-18),interleukin-4(IL-4),andγinterferon(IFN-γ)in nasal lavage solution and serum of mice were detected by enzyme-linked immunosorbent assay(ELISA).The damage status of the nasal mucosa was observed by hematoxylin-eosin(HE)staining.The number of goblet cells in the nasal mucosa of mice was observed by periodic acid-Schiff(PAS)staining.The expression of NLRP3 protein in the nasal mucosa of mice was detected by immunohistochemistry.Western blot was used to detect the expressions of NLRP3,cleaved Caspase-1,and cleaved gasdermin D(GSDMD)proteins in the nasal mucosa.The test kit was used to detect the changes in ROS in the nasal cavity of mice in each group.Result:Compared with the control group,the nasal symptoms of the model group were significantly aggravated,and the levels of inflammatory factors OVA-sIgE,histamine,ECP,PGD2,IL-1β,IL-18,and IL-4 in serum and nasal lavage solution were increased(P<0.05,P<0.01).The levels of IFN-γwere decreased(P<0.05,P<0.01).The histopathological score,goblet cell number,and ROS content were significantly increased(P<0.01),and the expressions of pyrodeath-related proteins NLRP3,cleaved Caspase-1,and cleaved GSDMD were increased(P<0.01).Compared with the model group,the nasal symptoms of the loratadine group and Yupingfeng San groups were significantly relieved,and the levels of inflammatory factors OVA-sIgE,histamine,ECP,PGD2,IL-1β,IL-18,and IL-4 in serum and nasal lavage solution were decreased(P<0.05,P<0.01).The levels of IFN-γwere increased(P<0.05,P<0.01).The histopathological scores,goblet cell number,and ROS content were significantly decreased(P<0.05,P<0.01),and the expressions of pyrodeath-related proteins NLRP3,cleaved Caspase-1,and cleaved GSDMD were decreased(P<0.05,P<0.01).Compared with the loratadine group,the curative effect of the high dose Yupingfeng San group was further increased(P<0.05,P<0.01).Conclusion:Yupingfeng San has a therapeutic effect on AR,and its specific effect may be related to the inhibition of ROS/NLRP3/Caspase-1-induced cell pyroptosis.