基于空间代谢组学探究川贝母抗肺纤维化的作用机制(Ⅱ)

Mechanism of Fritillariae Cirrhosae Bulbus against pulmonary fibrosis based on spatial metabolomics(Ⅱ)

目的利用空间代谢组学技术结合常规药理实验探究川贝母Fritillariae Cirrhosae Bulbus抗肺纤维化的作用机制。方法60只SD大鼠随机分为空白组、模型组及川贝母高、中、低剂量组,每组12只。除空白组外,均采用气管注射博莱霉素建立大鼠肺纤维化模型,川贝母高、中、低剂量组连续28 d ig 0.36、0.18、0.09 g/kg川贝母粉的水溶液,空白组和模型组ig等体积蒸馏水。末次给药后对大鼠肺组织样本进行苏木素-伊红和Masson染色病理分析,并采用空气动力辅助解吸电喷雾离子化质谱成像技术对空白组和川贝母中剂量组大鼠肺组织纤维化区域进行空间代谢组学分析,基于代谢组学分析结果结合生物信息学筛选出相关性强的信号通路,并通过检测常规药理指标对川贝母在上皮-间质转化方面的作用机制进行验证。采用ELISA法测定血清中转化生长因子-β1(transforming growth factor-β1,TGF-β1)的含量,采用羟脯氨酸测定试剂盒测定肺组织中羟脯氨酸含量,采用Western blotting法测定肺组织中Smad 2/3、p-Smad 2/3、E-钙黏蛋白(E-cadherin)、α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、蛋白激酶B(protein kinase B,Akt)、p-Akt表达。结果空间代谢组学分析出71个差异代谢物及其空间信息,并富集出精氨酸脯氨酸、哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,m TOR)信号通路等8条代谢途径。与模型组相比,给药川贝母后,大鼠肺组织中炎症细胞浸润和纤维组织增生现象减少,胶原蛋白纤维下降;血清中TGF-β1和肺组织中羟脯氨酸含量降低(P<0.05),肺组织中Smad 2/3磷酸化水平和α-SMA蛋白表达水平降低(P<0.05),E-cadherin蛋白表达水平升高(P<0.05)。结论川贝母粉能够在一定程度上改善博来霉素诱导的大鼠肺纤维化,其作用机制可能是通过调控上皮-间质转化、抑制细胞外基质沉积等相关途径实现的。

Objective To investigate the mechanism of Chuanbeimu(Fritillariae Cirrhosae Bulbus,FCB) anti-pulmonary fibrosis based on spatial metabolomics technology combined with conventional pharmacological experiments.Methods Sixty SD rats were randomly allocated into control group,model group,and high-,medium-,and low-dose FCB groups,with 12 rats in each group.Apart from the control group,all groups were subjected to tracheal injections of bleomycin to establish a rat model of pulmonary fibrosis.The high-,medium-,and low-dose FCB groups received gavage administration of 0.36,0.18,and 0.09 g/kg FCB powder solution,respectively,for 28 consecutive days.The control and model groups were administered an equivalent volume of distilled water by gavage.After the final administration,pathological analysis of rat lung tissue samples was conducted using hematoxylin-eosin and Masson staining.Furthermore,spatial metabolomics analysis of the fibrotic areas in rat lung tissues from both the control group and the medium-dose FCB group was performed using aerosol-assisted desorption electrospray ionization mass spectrometry imaging.Based on the results of the metabolomics analysis,signal pathways with strong relevance were identified through bioinformatics screening.The mechanism of FCB on epithelial-mesenchymal transition was then validated by measuring standard pharmacological indices.The content of transforming growth factor-β1(TGF-β1) in serum was measured using ELISA method.The content of hydroxyproline in lung tissues was determined using a hydroxyproline assay kit.Additionally,the expression levels of proteins such as Smad 2/3,p-Smad 2/3,E-cadherin,α-smooth muscle actin(α-SMA),protein kinase B(Akt) and p-Akt in lung tissues were quantified using Western blotting analysis.Results In the spatial metabolomic analysis,a total of 71 differential metabolites were identified along with their spatial information,which were enriched in eight metabolic pathways including arginine-proline metabolism and mammalian target of rapamycin(mTOR) signaling pathway.Compared to the model group,after administration of FCB,there was a reduction in inflammatory cell infiltration and fibrotic tissue proliferation in rat lung tissues,as well as a decline in collagen fiber content.The contents of TGF-β1 in serum and hydroxyproline in lung tissues were decreased(P < 0.05).Moreover,there was a decrease in the phosphorylation level of Smad2/3 and expression level of α-SMA in lung tissues(P < 0.05),while the expression level of Ecadherin was increased(P < 0.05).Conclusion FCB can improve bleomycin-induced pulmonary fibrosis in rats to some extent.The possible mechanism might be realized through regulating pathways associated with epithelial-mesenchymal transitions and inhibiting the deposition of extracellular matrix.