| 郭祥,宋红,陈家昊,等.加味金匮肾气丸调控MUC5AC/EGFR通路干预铁死亡改善肾阳虚证哮喘大鼠的机制研究[J].浙江中医药大学学报,2024,48(11):1334-1344. |
| 加味金匮肾气丸调控MUC5AC/EGFR通路干预铁死亡改善肾阳虚证哮喘大鼠的机制研究 |
| The Mechanism of Jiawei Jinkui Shenqi Pills Improving Asthma Rats with Kidney Yang Deficiency Syndrome by Interfering with Ferroptosis through MUC5AC/EGFR Pathway |
| DOI：10.16466/j.issn1005-5509.2024.11.002 |
| 中文关键词: 加味金匮肾气丸 MUC5AC EGFR GPX4 铁死亡 肾阳虚 支气管哮喘 机制研究 |
| 英文关键词: Jiawei Jinkui Shenqi Pill MUC5AC EGFR GPX4 ferroptosis kidney Yang deficiency bronchial asthma mechanism research |
| 基金项目:浙江省中医药管理局项目(2021ZB132)；浙江中医药大学2023年校级研究生科学研究基金项目(2023YKJ20) |
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| 中文摘要: |
| [目的] 探究加味金匮肾气丸改善肾阳虚证哮喘的可能作用机制。 [方法] 将40只SD大鼠随机分为对照组、模型组、加味金匮肾气丸组(简称中药组)、地塞米松组(简称地米组)，每组10只。除对照组外，其他组均采用氢化可的松注射液和卵清蛋白(ovalbumin，OVA)联合诱导的方法构建肾阳虚证哮喘大鼠模型。定期监测大鼠体质量，并评估喘息症状；苏木精-伊红(hematoxylin-eosin，HE)和过碘酸希夫(periodic acid-Schiff，PAS)染色观察大鼠肺组织及支气管的病理形态；酶联免疫吸附分析(enzyme-linked immunosorbent assay，ELISA)检测大鼠血清中相关指标，免疫印迹法检测黏蛋白5AC(Mucin 5AC，MUC5AC)、表皮生长因子受体(epidermal growth factor receptor，EGFR)、谷胱甘肽过氧化酶4(glutathione peroxidase 4，GPX4)蛋白表达；实时荧光定量聚合酶链式反应(Real-time quantitative polymerase chain reaction，RT-qPCR)检测MUC5AC、EGFR、GPX4的mRNA表达。培养人支气管上皮细胞BEAS-2B，设置空白对照组、柠檬酸铁胺(ferric amine citrate，FAC)组、MUC5AC组、FAC+MUC5AC组，通过红菲咯咻微板法检测非血红素铁浓度；荧光染色检测活性氧(reactive oxygen species，ROS)水平，对比分析MUC5AC通路与铁死亡之间的关联性。 [结果] 与对照组比较，模型组大鼠肺组织支气管管壁增厚，大量炎细胞浸润，杯状细胞增生；血清白细胞介素-4(interleukin-4，IL-4)、白细胞介素-5(interleukin-5，IL-5)、白细胞介素-13(interleukin-13，IL-13)水平显著升高(P<0.05)，皮质醇(cortisol，COR)水平明显下降(P<0.05)，MUC5AC、EGFR的mRNA表达升高，GPX4的mRNA表达降低(P<0.05)，MUC5AC、EGFR蛋白表达升高，GPX4蛋白表达降低(P<0.05)。与模型组比较，中药组和地米组IL-4、IL-5、IL-13水平均降低，COR水平升高(P<0.05)；MUC5AC、EGFR蛋白水平降低，GPX4蛋白水平升高(P<0.05)；MUC5AC、EGFR的mRNA表达下调，GPX4的mRNA表达上调(P<0.05)。与空白对照组比较，其他组非血红素铁浓度显著升高(P<0.05)，ROS水平增加，且FAC+MUC5AC组比单纯MUC5AC、FAC用药组指标增高更明显。 [结论] 加味金匮肾气丸通过调节MUC5AC/EGFR通路干预铁死亡，从而改善肾阳虚证哮喘，其作用机制可能是减少黏液过度分泌并降低细胞对于铁离子的吸收率，减轻铁死亡。 |
| 英文摘要: |
| [Objective] To investigate the possible mechanism of action of Jiawei Jinkui Shenqi Pill in relieving asthma with kidney Yang deficiency. [Methods] Total 40 SD rats were randomly divided into control group， model group， Jiawei Jinkui Shenqi Pill group(referred to as Chinese medicine group)， and dexamethasone group(referred to as DXM group)，10 rats in each group， and all other groups except control group were induced by the combination of hydrocortisone injection and ovalbumin(OVA) to construct the rat model of kidney Yang deficiency asthma. The body mass of the rats was monitored regularly and wheezing symptoms were evaluated. Hematoxylin-eosin(HE) and periodic acid-Schiff(PAS) staining were used to observe the pathological morphology of rat lung tissues and bronchi; enzyme-linked immunosorbent assay(ELISA) was used to detect relevant indices in rat serum; and Western blot was used to detect protein levels of Mucin 5AC(MUC5AC)， epidermal growth factor receptor(EGFR)， and glutathione peroxidase 4(GPX4); and Real-time quantitative polymerase chain reaction(RT-qPCR) to detect the expression of MUC5AC， EGFR， GPX4 mRNA. Human bronchial epithelial cells BEAS-2B were cultured and divided into blank control group， ferric amine citrate(FAC) group， MUC5AC group， FAC+MUC5AC group. The concentration of non-haem iron was detected with red feldspun microplate method; the level of reactive oxygen species(ROS) was observed with fluorescence staining， and comparative analysis of the correlation between the MUC5AC pathway and ferroptosis. [Results] Compared with control group， the lung tissue bronchial tube wall thickened， a large number of inflammatory cells infiltrated， there was goblet cell hyperplasia in rats of model group; serum levels of interleukin-4(IL-4)， interleukin-5(IL-5)， interleukin-13(IL-13) significantly increased(P<0.05)， cortisol(COR) levels were significantly decreased(P<0.05)， MUC5AC and EGFR mRNA expression was increased， GPX4 mRNA expression was decreased(P<0.05)， MUC5AC and EGFR protein content was increased and GPX4 protein content was decreased(P<0.05). Compared with model group， serum levels of IL-4， IL-5， IL-13 decreased and COR increased in Chinese medicine group and DXM group(P<0.05); MUC5AC， EGFR levels decreased and GPX4 protein level increased(P<0.05); MUC5AC and EGFR mRNA down-regulated and GPX4 mRNA expression up-regulated(P<0.05). Compared with blank control group， non-heme iron concentration significantly increased in the other groups(P<0.05)， the level of ROS increased， and the FAC + MUC5AC group is more pronounced than the MUC5AC， FAC medication group. [Conclusion] Jiawei Jinkui Shenqi Pill can intervene ferroptosis and improve asthma of kidney yang deficiency syndrome by regulating MUC5AC/EGFR pathway.The mechanism may be to reduce the excessive secretion of mucus and reduce the absorption rate of iron ions by inhibiting the overexpression of MUC5AC， and reduce ferroptosis. |
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