泽兰叶黄素通过p38 MAPK信号通路减轻分泌性中耳炎大鼠炎症

Eupafolin attenuates inflammation in rats with secretory otitis media through p38 MAPK signaling pathway

目的考察泽兰叶黄素(Eupafolin)对分泌性中耳炎(secretory otitis media,SOM)大鼠的治疗作用及其可能机制。方法Wistar大鼠首先腹腔注射卵白蛋白(OVA)和氢氧化铝凝胶然后听泡注射OVA诱导SOM动物模型。将42只成功建模的大鼠随机分为4组:模型组(1%DMSO,n=12)、低剂量组(10 mg/kg Eupafolin,n=10)、高剂量组(50 mg/kg Eupafolin,n=10)和阳性对照组(1 mg/kg甲基强的松龙,n=10)。另外选择10只未建模的大鼠作为对照组(1%DMSO)。各组大鼠均治疗10 d。通过苏木素伊红(HE)染色观察听泡组织形态,通过Wright-Giemsa染色进行中耳积液中中性粒细胞和嗜酸性粒细胞计数。通过ELISA试剂盒检测中耳积液中干扰素-γ(IFN-γ)和白介素-4(IL-4)水平。通过qRT-PCR检测中耳黏膜中IFN-γ、IL-4和水通道蛋白-1(Aquaporin,AQP-1)的mRNA水平。通过Western blot检测中耳黏膜中AQP-1、磷酸化p38 MAPK(p-p38 MAPK)和p38 MAPK的蛋白表达水平。结果与模型组相比,低剂量组、高剂量组和阳性对照组大鼠听泡组织病变减轻,中耳积液中白细胞、中性粒细胞和嗜酸性粒细胞数量均减少(P<0.05)。与模型组相比,低剂量组、高剂量组和阳性对照组大鼠中耳积液中IL-4水平均降低(P<0.05),IFN-γ/IL-4比值升高(P<0.05),中耳黏膜中IL-4 mRNA水平均降低(P<0.05)。与模型组相比,低剂量组、高剂量组和阳性对照组大鼠中耳黏膜中AQP-1 mRNA和蛋白水平均升高(P<0.05),p-p38 MAPK蛋白水平和p-p38 MAPK/p38 MAPK均降低(P<0.05)。与低剂量组相比,高剂量组大鼠中耳积液中IL-4水平均降低(P<0.05),IFN-γ/IL-4比值升高(P<0.05),中耳黏膜中IL-4 mRNA水平降低(P<0.05),AQP-1 mRNA和蛋白水平升高(P<0.05),p-p38 MAPK蛋白水平和p-p38 MAPK/p38 MAPK降低(P<0.05)。结论泽兰叶黄素可抑制SOM大鼠中耳组织中白细胞募集,纠正Th1/Th2失衡,上调AQP-1的表达,从而减轻SOM症状,其机制可能与p38 MAPK信号通路的抑制有关。

Objective To investigate the therapeutic effect of eupafolin on secretory otitis media(SOM)in rats and its possible mechanism.Methods Wistar rats were first intraperitoneally injected with ovalbumin(OVA)and aluminum hydroxide gel and then injected with OVA in auditory vesicle to induce SOM animal models.Forty-two successfully modeled rats were randomly divided into four groups:model group(1%DMSO,n=12),low dose group(10 mg/kg Eupafolin,n=10),high dose group(50 mg/kg Eupafolin,n=10)and positive control group(1 mg/kg methylprednisolone,n=10).Unmodeled rats served as control group(1%DMSO,n=10).The rats in each group were treated for 10 d.The morphology of auditory vesicles was observed by hematoxylin-eosin(HE)staining,and the numbers of neutrophils and eosinophils in middle ear effusion were counted by Wright-Giemsa staining.The levels of interferon-γ(IFN-γ)and interleukin-4(IL-4)in middle ear effusion were detected by ELISA kits.The mRNA levels of IFN-γ,IL-4 and Aquaporin-1(AQP-1)in the middle ear mucosa were detected by qRT-PCR.The protein expression levels of AQP-1,p-p38 MAPK and p38 MAPK in the middle ear mucosa were detected by Western blot.Results Compared with model group,the pathological changes of auditory vesicles were alleviated in low dose group,high dose group and positive control group,and the numbers of leukocytes,neutrophils and eosinophils in middle ear effusion were decreased(P<0.05).Compared with model group,the level of IL-4 in middle ear effusion was decreased(P<0.05),the ratio of IFN-γ/IL-4 was increased(P<0.05),and the level of IL-4 mRNA in middle ear mucosa was decreased in low dose group,high dose group and positive control group(P<0.05).Compared with model group,the levels of AQP-1 mRNA and protein in middle ear mucosa were all increased in low dose group,high dose group and positive control group(P<0.05),while the level of p-p38MAPK protein and the ratio of p-p38MAPK/p38MAPK were decreased(P<0.05).Compared with low-dose group,IL-4 level in the middle ear effusion of rats was decreased in high-dose group(P<0.05),the IFN-γ/IL-4 ratio was increased(P<0.05),the IL-4 mRNA level in the middle ear mucosa was decreased(P<0.05),the AQP-1 mRNA and protein levels were increased(P<0.05),and the protein level of p-p38MAPK and the ratio of p-p38MAPK/p38MAPK were decreased(P<0.05).Conclusion Eupafolin can inhibit the recruitment of leukocytes in the middle ear tissue of SOM rats,correct the imbalance of Th1/Th2,and up-regulate the expression of AQP-1,thereby reducing the symptoms of SOM,which may be related to the inhibition of p38 MAPK signaling pathway.