基于UPLC-Q-TOF-MS分析大麻二酚对肺纤维化的影响  

Effect of Cannabidiol on Pulmonary Fibrosis Analyzed by UPLC-Q-TOF-MS

作　　者：孙梦迪 张飞宇 杨慧聪 王宇[1] 陈平平[1] 卢芳[1] 刘树民[1] SUN Mengdi;ZHANG Feiyu;YANG Huicong;WANG Yu;CHEN Pingping;LU Fang;LIU Shumin(Graduate School,Institute of Traditional Chinese Medicine,Heilongjiang University of Chinese Medicine,Harbin 150040,China)

机构地区：[1]黑龙江中医药大学中医药研究院,哈尔滨150040

出　　处：《中国实验方剂学杂志》2024年第12期185-193,共9页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：黑龙江省省属本科高校中央支持地方高校改革发展资金人才培养项目(2021ZYQGLG001);黑龙江中医药大学优秀创新人才支持计划项目(2018RCL13)。

摘　　要：目的:应用超高效液相色谱-四极杆-飞行时间质谱法(UPLC-Q-TOF-MS)进行肺组织代谢组学分析,探讨大麻二酚抗肺纤维化的作用机制。方法:将48只SD大鼠随机分为空白组、模型组、泼尼松组(3.15 mg·kg^(-1))、大麻二酚低、中、高剂量组(12、36、108 mg·kg^(-1)),每组8只。除空白组外,各组均通过气管内注射5 mg·kg^(-1)博来霉素构建肺纤维化大鼠模型,造模成功后各组按剂量每天灌胃给药1次,空白组灌胃等量生理盐水,连续给药28 d。观察大鼠肺组织病理变化,酶联免疫吸附测定法(ELISA)检测血清中基质金属蛋白酶-7(MMP-7)、Ⅱ型肺泡细胞表面抗原(KL-6)、肺表面活性物质相关蛋白A(SP-A)、肺表面活性物质相关蛋白D(SP-D)的表达水平,免疫组化检测肺组织Ⅰ型胶原蛋白(Col-Ⅰ)、纤维连接蛋白(FN)的表达水平,免疫荧光检测黏蛋白5AC(MUC5AC)的表达。基于UPLC-Q-TOF-MS筛选大麻二酚治疗肺纤维化的潜在生物标志物并进行通路富集分析。结果:与空白组比较,模型组大鼠肺组织出现大量炎性细胞浸润及连续纤维化病灶;与模型组比较,泼尼松组和大麻二酚组大鼠肺组织中炎性浸润减少,蓝色胶原沉积减少。与空白组比较,模型组血清中MMP-7、KL-6、SP-A、SP-D的表达量显著升高(P<0.01);与模型组比较,泼尼松组和大麻二酚高剂量组MMP-7、KL-6、SP-A、SP-D的表达量明显降低(P<0.05,P<0.01)。与空白组比较,模型组肺组织中Col-Ⅰ和FN的表达水平显著升高,MUC5AC的荧光强度显著增加(P<0.01);与模型组比较,泼尼松组和大麻二酚高剂量组肺组织中Col-Ⅰ和FN的表达水平明显降低(P<0.05,P<0.01),MUC5AC的表达显著下降(P<0.01)。模型组较空白组共筛选出18个差异化合物,可作为潜在生物标志物,大麻二酚可回调其中16个化合物,主要涉及亚油酸代谢,苯丙氨酸、酪氨酸和色氨酸生物合成,花生四烯酸代谢,烟酸和烟酰胺代谢4种代谢途径。与Objective:To investigate the mechanism of anti-pulmonary fibrosis of cannabidiol by ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry(UPLC-Q-TOF-MS).Method:SD rats were randomly divided into control blank group,model group,prednisone group(3.15 mg·kg-1),low(12 mg·kg-1),medium(36 mg·kg-1)and high(108 mg·kg-1)dose groups,with 8 rats in each group.The rat model of pulmonary fibrosis was established by intratracheal injection of 5mg·kg-1 bleomycin,which was administered continuously for 28 days after successful modeling.The pathological changes of rat lung tissue were observed.Enzyme-linked immunosorbent assay(ELISA)was used to detect the expression levels of matrix metalloproteinase 7(MMP-7),type II alveolar cell surface antigen(KL-6),pulmonary surfactant associated protein A(SP-A)and pulmonary surfactant associated protein D(SP-D)in serum.The expression levels of type I collagen(Col-1)and fibronectin(FN)in lung tissues were detected by immunohistochemistry,and the expression of MUC5AC was detected by immunofluorescence.UPLC-Q-TOF-MS technique was used to search for potential biomarkers and related metabolic pathways.Result:Compared with the blank group,there were a large number of inflammatory cell infiltration and continuous fibrosis lesions in the lung tissue of rats in the model group.Compared with the model group,the inflammatory infiltration and blue collagen deposition in the lung tissue of rats in the prednisone and cannabidiol groups were reduced.Compared with blank group,the expressions of MMP-7,KL-6,SP-A and SP-D in serum in model group were significantly increased(P<0.01),while the expressions of MMP-7,KL-6,SP-A and SP-D in prednisone group and cannabidiol group were significantly decreased compared with model group(P<0.05,P<0.01).Compared with blank group,the expression levels of Col-1 and FN in lung tissue of model group were significantly increased.The fluorescence intensity of MUC5AC was significantly increased(P<0.01),the expression levels of Col-1 and FN

关 键 词：大麻二酚 肺纤维化 代谢组学 炎症 免疫 超高效液相色谱-四极杆-飞行时间质谱法(UPLC-Q-TOF-MS) 

分 类 号：R22[医药卫生—中医基础理论] R28[医药卫生—中医学] O657[理学—分析化学] R563[理学—化学]