摘要
目的基于非靶向代谢组学研究补气摄血方对急性脑梗死静脉溶栓后血脑屏障内皮细胞的保护作用及机制。方法将105只大鼠随机分为空白组、假手术组、模型组、阿托伐他汀组、补气摄血方的低、中、高剂量组,每组15只。建立大脑中动脉栓塞再灌注模型并静脉注射阿替普酶。通过神经功能缺损评分观察大鼠神经功能改变,苏木素—伊红(hematoxylin-eosin staining,HE)染色观察脑组织结构变化,透射电镜观察血脑屏障超微结构变化,ELISA法检测大鼠血清基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)及内皮素-1(endothelin-1,ET-1)蛋白表达,Western Blot法及免疫荧光法检测脑组织MMP-9及ET-1蛋白表达。通过LC-MS技术进行非靶向代谢组学分析补气摄血方治疗后差异代谢产物及代谢通路。结果(1)与假手术组比较,模型组神经功能缺损评分明显升高,差异有统计学意义(P<0.05);与模型组比较,阿托伐他汀组神经功能缺损评分降低(P<0.05);与模型组比较,补气摄血方各剂量组大鼠神经功能缺损评分降低(P<0.05)。(2)HE染色结果显示:与假手术组比较,模型组大鼠脑组织神经元细胞排列不规则,细胞核固缩,分离度增加;与模型组比较,阿托伐他汀组及补气摄血方各剂量组神经元细胞排列较整齐,分离度减小。(3)透射电镜观察结果显示:与假手术组比较,模型组大鼠内皮细胞肿胀,紧密连接结构出现断裂和消失;与模型组比较,阿托伐他汀组及补气摄血方组内皮细胞肿胀及紧密连接结构断裂、消失情况好转。(4)ELISA法检测结果显示:与假手术组比较,模型组血清MMP-9、ET-1蛋白表达水平显著升高(P<0.01);与模型组比较,阿托伐他汀组及补气摄血方中、高剂量组MMP-9蛋白表达水平显著降低(P<0.01);与模型组比较,阿托伐他汀组及补气摄血方高剂量组ET-1蛋白表达水平显著降低(P<0.01),补气摄血方中、低剂量组ET-1蛋白表达水平降低(P<0.05)。(5)Western Blot法检测结果显示:与假手术组相比,模型组脑组织MMP-9、ET-1蛋白表达水平显著升高(P<0.01);与模型组相比,阿托伐他汀组、补气摄血方低、中及高剂量组脑组织MMP-9、ET-1蛋白表达水平显著降低(P<0.01)。且补气摄血方低、中及高剂量组MMP-9、ET-1蛋白表达水平有显著差异(P<0.05)。(6)免疫荧光检测结果显示:与假手术组相比,模型组脑组织MMP-9、ET-1蛋白表达水平显著升高(P<0.01);与模型组相比,阿托伐他汀组、补气摄血方低、中及高剂量组脑组织MMP-9、ET-1蛋白表达水平显著降低(P<0.01)。(7)非靶向代谢组学结果显示:假手术组与模型组有392种差异代谢物,模型组与补气摄血方组共有215种差异代谢物;补气摄血方回调代谢产物有花生四烯酸、二十碳五烯酸、二十二碳五烯酸、氧化谷胱甘肽等共39种;涉及不饱和脂肪酸的生物合成、铁死亡、泛酸和辅酶A生物合成、谷胱甘肽代谢等共18条代谢途径。结论补气摄血方可能通过干预不饱和脂肪酸的生物合成、铁死亡等途径调节花生四烯酸、二十碳五烯酸及氧化谷胱甘肽等代谢物表达保护血脑屏障内皮细胞,进而改善急性脑梗死预后。
Objective To investigate the protective effects and mechanisms of Buqi Shexue Formula on blood-brain barrier endothelial cells following intravenous thrombolysis in acute cerebral infarction,based on non-targeted metabolomics.Methods In this study,a total of 105 rats were randomly divided into blank group,sham-operated group,model group,atorvastatin group,and low,medium,and high dosage groups of Buqi Shexue Formula,with 15 rats in each group.The middle cerebral artery occlusion and reperfusion model was established,followed by intravenous injection of alteplase.Neurological deficits were assessed using the neurological deficit score to evaluate the neurologic changes in rats.Hematoxylin-eosin(HE)staining was employed to observe the structural changes in brain tissue.Transmission electron microscopy was used to examine the ultrastructural alterations of the blood-brain barrier.The expression levels of matrix metalloproteinase-9(MMP-9)and endothelin-1(ET-1)in rat serum were measured using enzyme-linked immunosorbent assay(ELISA).Western blotting and immunofluorescence were applied to detect the protein expression of MMP-9 and ET-1 in brain tissue.Differential metabolites and metabolic pathways were identified through non-targeted metabolomics analysis using LC-MS technology following treatment with Buqi Shexue Formula.Results(1)Compared with the sham operation group,the neurological deficit score in the model group was significantly increased(P<0.05).Compared with the model group,the neurological deficit score in the atorvastatin group was decreased(P<0.05).Similarly,compared with the model group,the neurological deficit score in the Buqi Shexue Formula group was also decreased(P<0.05).(2)Examination of brain tissue using HE staining revealed that,compared with the sham operation group,the model group exhibited irregular neuronal arrangement,condensed nuclei,and increased cell separation.In contrast,both the atorvastatin group and the Buqi Shexue Formula group showed more regular neuronal arrangement and decreased cell separation.(3)Transmission electron microscopy observation of brain tissue revealed that compared with the sham operation group,endothelial cells in the model group exhibited swelling,with disruption and disappearance of tight junction structures.In contrast,both the atorvastatin group and the Buqi Shexue Formula group showed improved endothelial cell swelling and reduced disruption and disappearance of tight junction structures.(4)ELISA analysis demonstrated that,compared with the sham operation group,the expression levels of MMP-9 and ET-1 in the serum were significantly elevated in the model group(P<0.01).Compared with the model group,the expression levels of MMP-9 were significantly reduced in the atorvastatin group and the medium-and high-dose groups of Buqi Shexue Formula(P<0.01).Compared with the model group,the expression levels of ET-1 were significantly decreased in the atorvastatin group and the high-dose group of Buqi Shexue Formula(P<0.01),while the expression levels of ET-1 were decreased in the medium-and low-dose groups of Buqi Shexue Formula(P<0.05).(5)Western Blot analysis revealed that,compared with the sham operation group,the expression levels of MMP-9 and ET-1 in brain tissue were significantly elevated in the model group(P<0.01).Compared with the model group,the expression levels of MMP-9 and ET-1 in brain tissue were significantly reduced in the atorvastatin group and the low-dose,medium-dose,and high-dose groups of Buqi Shexue Formula(P<0.01).Moreover,significant differences in the expression levels of MMP-9 and ET-1 were observed among the low-dose,medium-dose,and high-dose groups of Buqi Shexue Formula(P<0.05).(6)Immunofluorescence analysis demonstrated that,compared with the sham operation group,the expression levels of MMP-9 and ET-1 were significantly increased in the model group(P<0.01).Compared with the model group,the expression levels of MMP-9 and ET-1 were significantly decreased in the atorvastatin group and the low-dose,medium-dose,and high-dose groups of Buqi Shexue Formula(P<0.01).(7)Non-targeted metabolomics analysis revealed that 392 differential metabolites were identified between the sham operation group and the model group,while 215 differential metabolites were shared between the model group and the Buqi Shexue Formula group.The metabolites regulated by Buqi Shexue Formula included arachidonic acid,eicosapentaenoic acid,docosapentaenoic acid,and oxidized glutathione,among 39 others.These metabolites were associated with 18 metabolic pathways,including unsaturated fatty acid biosynthesis,ferroptosis,pantothenate and CoA biosynthesis,and glutathione metabolism.Conclusion Buqi Shexue Formula may protect blood-brain barrier endothelial cells and improve the prognosis of acute cerebral infarction by modulating the expression of metabolites such as arachidonic acid,eicosapentaenoic acid,and oxidized glutathione through the intervention of pathways involved in unsaturated fatty acid biosynthesis and ferroptosis.
作者
赵聪
王革生
马涛
李文京
刘乙兴
袁健
ZHAO Cong;WANG Gesheng;MA Tao;LI Wenjing;LIU Yixing;YUAN Jian(The Second Clinical School of Beijing University of Chinese Medicine,Beijing 100029,China)
出处
《环球中医药》
2025年第8期1553-1566,共14页
Global Traditional Chinese Medicine
基金
国家自然科学基金面上项目(82374275)
国家重点研发计划(2022YFC3501102)。
