王文凯,常军民,马晓丽,李改茹
WANG Wen-kai,CHANG Jun-min,MA Xiao-li,LI Gai-ru

• 1:新疆医科大学药学院
• 2:新疆天然药物活性组分与释药技术重点实验室

摘要(Abstract)：

旨在探讨天山花楸水提物(water extract of Sorbus tianschanica,STE)对哮喘气道炎症的影响及机制。将小鼠随机分为对照组、模型组、阳性药地塞米松组(2 mg·kg~(-1))、STE低剂量组(1 g·kg~(-1))、STE中剂量组(2 g·kg~(-1))、STE高剂量组(4 g·kg~(-1))。除对照组外，其余各组通过卵清蛋白诱导构建哮喘小鼠模型。通过观察肺组织的病理学变化，并测定支气管肺泡灌洗液(BALF)中白细胞介素-4(IL-4)和白细胞介素-5(IL-5)水平，以评估STE的抗炎调控作用。进一步采用转录组学和蛋白质组学方法分析肺组织中差异表达的基因与蛋白及其相关信号通路。随后，通过RT-qPCR技术验证关键基因的表达变化，并结合免疫组化和蛋白免疫印迹法探讨STE对哮喘小鼠发病机制的调节作用。最后，运用AutoDock Vina软件开展分子对接，评估STE中主要活性成分与靶蛋白磷脂酰肌醇3-激酶催化亚基α(PIK3CA)、Toll样受体4(TLR4)、蛋白激酶B1(Akt1)、基质金属蛋白酶9(MMP9)的结合能力。结果显示，模型组小鼠肺组织炎性细胞浸润及纤维组织明显增生，STE干预后上述病理改变明显减轻。与对照组相比，模型组小鼠BALF中IL-4和IL-5水平显著升高，STE干预后IL-4和IL-5水平显著下降。转录组学及蛋白质组学分析筛选出与过敏性哮喘密切相关的关键基因与蛋白，包括肿瘤坏死因子(TNF)、白细胞介素-6(IL-6)、TLR4、PIK3CA、MMP9等。RT-qPCR验证结果显示，STE高剂量干预显著下调哮喘小鼠肺组织中PIK3CA、IL-6、Akt1、MMP9、白细胞介素-13(IL-13)、核因子-κB(NF-κB)、TNF、CXC趋化因子配体1(CXCL1)、TLR4 mRNA的表达，上调信号转导及转录激活因子1(STAT1)mRNA的表达。蛋白免疫印迹及免疫组化结果进一步证实，STE可显著下调哮喘小鼠肺组织中MMP9、TLR4、PIK3CA、磷酸化蛋白激酶B(p-Akt)蛋白的表达。分子对接研究表明，STE中的山柰酚-3,7-二葡萄糖苷、异槲皮苷、槲皮素-3-龙胆二糖苷、金丝桃苷可与PIK3CA、TLR4、Akt1、MMP9稳定结合，表明这些活性成分可能通过靶向调控哮喘相关信号通路，发挥抗炎作用。综上所述，STE通过抑制PIK3CA、MMP9、p-Akt和TLR4的表达，调控TLR4/PI3K/Akt/MMP9信号通路，从而发挥抗哮喘作用。
To investigate the effects and mechanisms of the water extract from Sorbus tianschanica(STE) on asthmatic airway inflammation, the mice were randomly divided into six groups, including a control group, a model group, a positive drug dexamethasone group(2 mg·kg~(-1)), a low-dose STE group(1 g·kg~(-1)), a medium-dose STE group(2 g·kg~(-1)), and a high-dose STE group(4 g·kg~(-1)). Except for the control group, all groups were subjected to ovalbumin induction to establish an asthma mouse model. The anti-inflammatory effects of STE were evaluated by examining pathological changes in lung tissue and measuring the levels of interleukin(IL)-4 and IL-5 in bronchoalveolar lavage fluid(BALF). Transcriptomic and proteomic methods were further employed to analyze differentially expressed genes and proteins, as well as their associated signaling pathways in lung tissue. Subsequently, the expression changes of key genes were verified by reverse transcription-quantitative polymerase chain reaction(RT-qPCR), and immunohistochemistry and Western blot methods were used to explore the regulatory mechanisms of STE in the pathogenesis of asthma in mice. Molecular docking was performed by using AutoDock Vina software to evaluate the binding affinity of the main active components in STE with the target proteins, including phosphatidylinositol-3-kinase catalytic subunit α(PIK3CA), Toll-like receptor 4(TLR4), protein kinase B1(Akt1), and matrix metallopeptidase 9(MMP9). The results showed significant inflammatory cell infiltration and fibrous tissue proliferation in the lung tissue of mice in the model group. However, these pathological changes were markedly reduced following STE intervention. Compared with those of the control group, the expression levels of IL-4 and IL-5 in the BALF of the model group were significantly increased but notably decreased following STE intervention. Transcriptomic and proteomic analyses identified key genes and proteins associated with allergic asthma, including tumor necrosis factor(TNF), IL-6, TLR4, PIK3CA, and MMP9. RT-qPCR validation revealed that high-dose STE intervention significantly downregulated the expressions of PIK3CA, IL-6, Akt1, MMP9, IL-13, nuclear factor-kappa B(NF-κB), TNF, CXC motif chemokine ligand 1(CXCL1), and TLR4 mRNAs and significantly upregulated the expression of signal transducer and activator of transcription 1(STAT1) mRNA. Western blot and immunohistochemical analyses confirmed that STE significantly downregulated the expressions of MMP9, TLR4, PIK3CA, and phosphorylated protein kinase B(p-Akt) in lung tissue of asthmatic mice. Moreover, molecular docking demonstrated that kaempferol-3,7-diglucoside, isoquercitrin, quercetin-3-gentiobioside, and hyperoside in STE exhibited stable binding affinities with PIK3CA, TLR4, Akt1, and MMP9, suggesting that the active components may exert anti-inflammatory effects by targeting and modulating asthma-related signaling pathways. In summary, STE exerts anti-asthmatic effects by inhibiting the expressions of PIK3CA, MMP9, p-Akt, and TLR4 and regulating the TLR4/PI3K/Akt/MMP9 signaling pathway.

关键词(KeyWords)： 天山花楸;哮喘;气道炎症;TLR4/PI3K/Akt/MMP9信号通路;分子对接
Sorbus tianschanica;asthma;airway inflammation;TLR4/PI3K/Akt/MMP9 signaling pathway;molecular docking

Abstract:

Keywords:

基金项目(Foundation): 新疆维吾尔自治区重大科技专项(2022A03007-3)

作者(Author): 王文凯,常军民,马晓丽,李改茹
WANG Wen-kai,CHANG Jun-min,MA Xiao-li,LI Gai-ru

DOI: 10.19540/j.cnki.cjcmm.20250508.401

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