基于METTL3介导的miR-29a-3p的m^(6)A修饰探讨平喘颗粒抑制气道上皮细胞泛凋亡治疗哮喘的机制研究

Mechanisms of Pingchuan granules in inhibiting PANoptosis of airway epithelial cells through METTL3-mediated m^(6)A modification of miR-29a-3p in the treatment of asthma

目的:观察平喘颗粒对METTL3介导的气道上皮细胞中miR-29a-3p的m^(6)A修饰的影响,明确其抑制哮喘气道炎症的分子机制。方法:16HBE采用LPS诱导(50 mg/L)构建细胞模型,并给予平喘颗粒含药血清和地塞米松进行干预。分别采用CCK8法检测细胞活性、ELISA法检测炎症因子(TNF-α、IL-6和IL-8)的含量和miR-29a-3p的m^(6)A修饰水平、Western blot检测METTL3与泛凋亡蛋白的表达和qRT-PCR检测METTL3与miR-29a-3p的表达。结果:与模型组相比,平喘颗粒能够显著增加16HBE的活力,抑制炎症因子TNF-α、IL-6和IL-8的含量,下调泛凋亡相关蛋白p-RIPK3、p-MLKL、cleaved Caspase-1、cleaved Caspase-3的表达和GSDMD-NT/FL-GSDMD与GSDME-NT/FL-GSDME的比值,差异均具有统计学意义(P<0.01)。此外,平喘颗粒能够显著上调细胞中miR-29a-3p和METTL3的表达水平,促进miR-29a-3p的m^(6)A修饰水平,与模型组相比差异均具有统计学意义(P<0.01)。结论:平喘颗粒主要通过METTL3增强miR-29a-3p的m^(6)A修饰水平,抑制气道上皮细胞泛凋亡,改善气道炎症。

Objective:To observe the effect of Pingchuan granules on METTL3-mediated m6A modification of miR-29a-3p in airway epithelial cells,and to clarify its molecular mechanism in inhibiting airway inflammation of asthma.Methods:16HBE was induced by LPS(50 mg/L) to construct a cell model,and Pingchuan granules-containing serum and dexamethasone were given for intervention.The cell activity was detected by CCK8 assay,the content of inflammatory factors(TNF-α,IL-6 and IL-8) and the m^(6)A modification level of miR-29a-3p were detected by ELISA,the expression of METTL3 and PAN-apoptotic protein was detected by Western blot and the expression of METTL3 and miR-29a-3p was detected by qRT-PCR,respectively.Results:Compared with the model group,Pingchuan granules could significantly increase the activity of 16HBE,inhibit the contents of inflammatory factors TNF-α,IL-6 and IL-8,and downregulate PANoptosis-related proteins p-RIPK3,p-MLKL,cleaved Caspase-1,cleaved Caspase-3 and the ratio of GSDMD-NT/FL-GSDMD and GSDME-NT/FL-GSDME,the differences were statistically significant(P<0.01).In addition,Pingchuan granules can significantly increase the expression levels of miR-29a-3p and METTL3 in 16HBE,and promote the m^(6)A modification level of miR-29a-3p when compared with the model group,the differences were statistically significant(P<0.01).Conclusion:Pingchuan granules enhance the m^(6)A modification level of miR-29a-3p through METTL3,thereby inhibiting the PANoptosis of airway epithelial cells and improving airway inflammation.