基于IL-6/JAK2/STAT3信号通路研究氯雷他定对过敏性鼻炎大鼠的保护作用

Study on the protective effect of loratadine on allergic rhinitis rats based on IL-6/JAK2/STAT3 signaling pathway

目的研究氯雷他定对过敏性鼻炎(AR)模型大鼠IL-6/JAK2/STAT3通路的影响和保护作用。方法将45只雄性SD大鼠随机均分假手术组、AR模型组、氯雷他定(LORA)组[氯雷他定1 mg/(kg·d)B.W.]、孟鲁司特(MONT)组[孟鲁司特10 mg/(kg·d)B.W.]、联合干预组[氯雷他定1 mg/(kg·d)B.W.和孟鲁司特10 mg/(kg·d)B.W.]。使用含卵清蛋白工作液(生理盐水)对各组大鼠进行体内和体外致敏处理(对照处理)构建过敏性鼻炎大鼠模型。建模完成后随即按照对应剂量进行14 d腹腔注射药物干预,假手术组和模型组代以2 mL生理盐水。对各组大鼠外鼻及部分鼻腔进行组织切片并经过碘酸希夫碱(PAS)染色检测上皮杯状细胞增生情况,酶联免疫吸附(ELISA)法测定血清白介素6(IL-6)水平,RT-qPCR测定粘膜组织中IL-6、Janus型酪氨酸激酶2(JAK2)、信号传导蛋白和转录激活因子3(STAT3)mRNA水平,Western blot检测总JAK2和STAT3水平和其磷酸化(p-JAK2、p-STAT3)水平。结果假手术组鼻上皮组织染色浅,杯状细胞无增生;模型组上皮组织染色最深,杯状细胞增生最严重,上皮细胞群排列异常;LORA组鼻上皮组织染色程度和杯状细胞增生比模型组略有减轻;MONT组鼻上皮组织杯状细胞增生好于LORA组;联合干预组上皮细胞增生程度在单个用药基础上进一步减轻,上皮组织染色程度接近假手术组。与假手术组相比,模型组、LORA组、MONT组血清中IL-6、鼻粘膜中IL-6和STAT3 mRNA水平、p-JAK2、STAT3和p-STAT3蛋白水平,联合干预组鼻粘膜中STAT3蛋白水平显著升高(P<0.05);与模型组相比,LORA组、MONT组和联合干预组血清中IL-6、鼻粘膜中IL-6 mRNA、p-JAK2蛋白水平,MONT组、联合干预组鼻粘膜中p-STAT3蛋白水平显著降低(P<0.05);与LORA组相比,联合干预组血清中IL-6水平、鼻粘膜中IL-6 mRNA、p-JAK2、p-JAK3蛋白水平,MONT组鼻粘膜中p-JAK2、p-STAT3蛋白水平显著降低(P<0.05);与MONT组相比,联合干预组血清中IL-6水平、鼻粘膜中IL-6和STAT3 mRNA水平、p-JAK2和p-STAT3蛋白水平显著降低(P<0.05)。结论氯雷他定可能通过抑制AR大鼠IL-6转录水平,从而下调IL-6/JAK2/STAT3通路,抑制鼻粘膜组织杯状细胞增生,是治疗过敏性鼻炎的机制之一。

Objective To study the effect and protective effect of loratadine on IL-6/JAK2/STAT3 pathway in allergic rhinitis model rats.Methods 45 male SD rats were randomly divided into sham operated group,model group,LORA group [loratadine 1 mg/(kg·d) B.W.],MONT group [montelukast 10 mg/(kg·d) B.W.] and combined intervention group[loratadine 1 mg/(kg·d) B.W.and montelukast 10 mg/(kg·d) B.W.].The rats in each group were sensitized in vivo and in vitro(control) with working fluid containing ovalbumin(normal saline) to establish the rat model of allergic rhinitis.After the completion of the model establishment,14 days of intraperitoneal injection of drugs were conducted according to the corresponding dose,and the sham operation group and the model group were replaced by 2 mL of normal saline.The external nose and part of the nasal cavity of each group were sectioned,the proliferation of goblet cells was detected by periodic acid-schiff(PAS) staining;the level of serum IL-6 was determined by enzyme-linked immunosorbent assay(ELISA);the levels of IL-6,Janus type tyrosine kinase 2(JAK2),signal transduction protein and STAT3 mRNAs were measured by RT-qPCR,and Western blot was used to detect the levels of total JAK2,STAT3 and phosphorylated JAK2,STAT3(p-JAK2,p-STAT3).Results In the sham operated group,the Schiff base staining of nasal epithelium was light,and there was no obvious proliferation of goblet cells.In the model group,Schiff base staining was the deepest;goblet cell proliferation was the most obvious,and the epithelial cell group showed irregular arrangement;the reduction degree of goblet cell proliferation in LORA group was better than that in model group;the goblet cells proliferated better in MONT group than in LORA group.In the combined intervention group,the degree of epithelial cell proliferation was further reduced on the basis of single medication,and the degree of Schiff base staining in epithelial tissue was similar to that in the sham operation group.Compared with the sham operation group,the levels of IL-6 in serum,IL-6 and STAT3 mRNAs,pJAK2,STAT3 and p-STAT3 proteins in nasal mucosa in the model group,LORA group and MONT group,and the level of STAT3 protein in nasal mucosa of the combined intervention group were significantly higher(P<0.05).Compared with the model group,the levels of IL-6 in serum,IL-6 mRNA and p-JAK2 protein in nasal mucosa in LORA group,MONT group and combined intervention group,and the level of p-STAT3 protein in nasal mucosa of the MONT group and combined intervention group were significantly lower(P<0.05);compared with LORA group,the levels of IL-6 in serum,IL-6 mRNA,p-JAK2 and p-JAK3 proteins in nasal mucosa of combined intervention group,and the levels of p-JAK2,p-STAT3 proteins in nasal mucosa of the MONT group were significantly lower(P<0.05);compared with the MONT group,the level of IL-6 in serum,the levels of IL-6 and STAT3 mRNAs in nasal mucosa,the levels of p-JAK2 and p-STAT3 proteins in the combined intervention group were significantly lower(P<0.05).Conclusion Loratadine might down regulate IL-6/JAK2/STAT3 pathway and inhibit goblet cell proliferation in nasal mucosa by inhibiting IL-6 transcription in AR rats,which could be one of the mechanisms of treating AR.