摘要
目的:基于空间代谢组学技术结合药理指标,解析川贝母粉改善博莱霉素诱导的大鼠肺纤维化的作用机制。方法:将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,其他组大鼠均采用气管注射博莱霉素建立肺纤维化模型。术后川贝母高、中、低剂量组分别给予0.36、0.18、0.09 g·kg^(-1)川贝母粉的水溶液,连续给药28 d,空白组和模型组灌胃给予等量蒸馏水。末次给药后收集肺组织及血液,通过苏木素-伊红(HE)、马松(Masson)染色综合评估大鼠肺组织病理状况,利用空气动力辅助解吸电喷雾离子化质谱成像技术对不同实验组大鼠肺组织进行质谱成像分析,依据HE染色图选择模型组和川贝母高剂量组肺组织纤维化区域进行空间代谢组学分析,采用有监督的正交偏最小二乘法-判别分析(OPLS-DA)获得的变量重要性投影(VIP)值>1、t检验P<0.05及变异倍数分析来筛选组间差异代谢产物;采用京都基因与基因组百科全书(KEGG)数据库对筛选出的差异代谢物进行代谢通路分析。采用蛋白免疫印迹法(Western blot)检测大鼠肺组织中核转录因子-κB p65(NF-κB p65)和血红素加氧酶-1(HO-1)蛋白表达;生化检测大鼠肺组织中超氧化物歧化酶(SOD)、丙二醛(MDA)和谷胱甘肽(GSH)水平;采用酶联免疫吸附测定法(ELISA)检测大鼠血清中白细胞介素(IL)-1β、IL-6、核因子E2相关因子2(Nrf2)、肿瘤坏死因子-α(TNF-α)水平,对筛选出的部分相关性强的信号通路进行验证。结果:质谱成像实验结果显示,肺纤维化大鼠在灌胃川贝母粉28 d后,其肺组织纤维化区域的L-精氨酸含量与模型组大鼠比较具有明显差异,磷脂酰胆碱含量低于模型组大鼠肺纤维化区域中磷脂酰胆碱含量。Western blot结果表明,与模型组比较,在灌胃给药川贝母粉28 d后,可以抑制肺纤维化大鼠肺组织中NF-κB p65蛋白表达量上升,高剂量川贝母粉灌胃给药后能够明显抑制HO-1表达量降低(P<0.05)。细胞因子检测结果显示,与模型组比较,川贝母高、中、低剂量组大鼠血清中IL-1β、IL-6及TNF-α的含量均降低,高剂量川贝母粉灌胃后能够明显抑制由博莱霉素引起的SOD、GSH、Nrf2含量降低及MDA含量升高的趋势。结论:川贝母粉灌胃给药可一定程度改善博莱霉素诱导的大鼠肺纤维化,其作用机制可能与调控炎症(NF-κB p65)、氧化应激(Nrf2/HO-1)等相关途径有关。
Objective:Based on spatial metabolomics technology combined with pharmacological indexes,to analyze the mechanism of Fritillariae Cirrhosae Bulbus(FCB)powder in improving bleomycininduced pulmonary fibrosis in rats.Method:Sixty SD rats were randomly divided into five groups,including the blank group,the model group,and high,medium,low dosage groups of FCB.Except for the blank group,rats in all other groups were injected with bleomycin by tracheal injection to establish a pulmonary fibrosis model.Postoperatively,the high,medium and low dosage groups of FCB were administered aqueous solutions of FCB powder at doses of 0.36,0.18,0.09 g·kg^(-1),respectively,continuously for 28 d.The blank and model groups were given an equal volume of distilled water by gavage.After the last administration,lung tissues and blood samples were collected,the pathological conditions of rat lung tissues were comprehensively evaluated by hematoxylin-eosin(HE)and Masson staining,and aerodynamic assisted desorption electrospray ionization mass spectrometry imaging(AFADESI-MSI)was used for MSI of rat lung tissues from different experimental groups.Spatial metabolomics analysis was conducted on the fibrotic areas of lung tissues in the model group and the high dosage group of FCB based on HE staining images.Differential metabolites between groups were screened by orthogonal partial least squares-discriminant analysis(OPLS-DA),with variable importance in the projection(VIP)values>1,t-test P<0.05,and fold change analysis.Metabolic pathway analysis of the identified differential metabolites was performed using Kyoto Encyclopedia of Genes and Genomes(KEGG).Protein expression levels of nuclear transcription factor-κB p65(NF-κB p65)and heme oxygenase-1(HO-1)in rat lung tissues were detected by Western blot.Biochemical assessments of superoxide dismutase(SOD),malondialdehyde(MDA)and glutathione(GSH)levels in rat lung tissues were conducted.Serum levels of interleukin(IL)-1β,IL-6,nuclear factor erythroid 2 related factor 2(Nrf2),and tumor necrosis factor-α(TNF-α)were measured by enzyme linked immunosorbent assay(ELISA),and some of the screened signaling pathways with strong correlation were verified.Result:The results of MSI experiment showed that after 28 d of the administration of FCB powder to rats with pulmonary fibrosis,the content of L-arginine in the fibrotic regions of lung tissues was significantly different from that of rats in the model group,and the content of phosphatidylcholine was lower than that in the fibrotic region of lung tissues of rats in the model group.Western blot results confirmed that,in comparison to the model group,oral administration of FCB powder for 28 d could inhibit the elevated expression of NF-κB p65 protein in the lung tissues of rats with pulmonary fibrosis.Furthermore,high dose of FCB powder was able to significantly inhibit the expression of HO-1 after oral administration(P<0.05).The cytokine detection results indicated that the concentrations of IL-1β,IL-6 and TNF-αin the serum of rats from the high,medium,low dosage groups of FCB were reduced by comparing with the model group,and the high dose of Chuanbeimu powder administered by gavage could significantly inhibit the trend of decreased SOD,GSH,Nrf2 contents and increased MDA content induced by bleomycin.Conclusion:Oral administration of FCB powder has the potential to partially ameliorate bleomycin-induced pulmonary fibrosis in rats,and its mechanism may be related to the regulation of pathways associated with inflammation(NF-κB p65)and oxidative stress(Nrf2/HO-1).
作者
秦善博
谭鹏
郝露
谢俊杰
林俊芝
张磊
赵军宁
QIN Shanbo;TAN Peng;HAO Lu;XIE Junjie;LIN Junzhi;ZHANG Lei;ZHAO Junning(Key Laboratory of Biological Evaluation of Traditional Chinese Medicine(TCM)Quality of National Administration of TCM,Sichuan Academy of Chinese Medicine Sciences,Chengdu 610041,China;College of Food and Biological Engineering,Chengdu University,Chengdu 610106,China;TCM Regulating Metabolic Diseases Key Laboratory of Sichuan Province,Hospital of Chengdu University of TCM,Chengdu 610075,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2024年第13期150-159,共10页
Chinese Journal of Experimental Traditional Medical Formulae
基金
四川省科技厅重点研发项目(2022YFS0429)
中央引导地方科技发展项目(2022ZYD0102)
四川省中医药管理局项目(2023MS496)
四川省科研院所基本科研项目(2022JDKY0036)。
关键词
川贝母
肺纤维化
空间代谢组学
炎症因子
氧化应激
质谱成像
博莱霉素
Fritillariae Cirrhosae Bulbus
pulmonary fibrosis
spatial metabolomics
inflammation factor
oxidative stress
mass spectrometry imaging
bleomycin
