怀菊花中咖啡酰基奎宁酸类化合物通过调节ERK/MAPK信号通路改善LPS诱导的HUVEC血管内皮细胞损伤
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摘要
初步研究怀菊花总提物对脂多糖(lipopolysaccharide, LPS)诱导的急性肺损伤小鼠模型的影响,并进一步探究从怀菊花中分离得到的3种咖啡酰基奎宁酸类化合物对血管内皮细胞损伤模型的干预作用及其机制。本文所有动物实验饲养及操作严格遵守国家动物福利伦理与保护相关规定。采用鼻腔滴注LPS的方法建立急性肺损伤小鼠模型,经菊花总提物灌胃给药6天后,检测小鼠肺湿重/干重比及小鼠肺泡灌洗液中肿瘤坏死因子-α(tumour necrosis factor-α, TNF-α)、白介素-6 (interleukin-6, IL-6)、白介素-1β(interleukin-1β, IL-1β);使用人脐静脉内皮细胞(human umbilical vein endothelial cells, HUVEC)饥饿处理12 h后,以2.5μg·mL-1 LPS诱导24 h建立血管内皮细胞损伤模型,经菊花中提取到的3种咖啡酰基奎宁酸类化合物处理24 h后,采用ELISA法测定细胞培养上清液中TNF-α、IL-6、IL-1β、血管细胞黏附分子-1 (vascular cell adhesion molecule-1, VCAM-1)、内皮素-1 (endothelin-1, ET-1)水平, TBA法检测丙二醛(malondialdehyde, MDA)水平,羟胺法检测超氧化物歧化酶(superoxide dismutase, SOD)水平,一步法检测一氧化氮(nitric oxide, NO)水平, Western blot法检测细胞内丝裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)信号通路p-MEK1/2、MEK1/2、p-ERK1/2、ERK1/2、p-JNK、JNK、p-P38、P38蛋白表达。结果显示,怀菊花总提物可显著降低小鼠肺湿重/干重比,降低肺泡灌洗液TNF-α、IL-6、IL-1β水平;怀菊花中咖啡酰基奎宁酸类化合物可显著升高SOD、NO水平,降低TNF-α、IL-6、IL-1β、VCAM-1、ET-1、MDA水平,并显著下调p-MEK1/2、p-ERK1/2的表达。综上,怀菊花总提物对急性肺损伤小鼠具有一定的保护作用,怀菊花中咖啡酰基奎宁酸类化合物可能是通过调节ERK/MAPK信号通路,抑制炎症因子、调节氧化应激水平,以及调节细胞间黏附分子和血管舒缩因子,从而改善LPS诱导的血管内皮细胞损伤。
To explore the effect of total extract of Chrysanthemum morifolium on lipopolysaccharide(LPS)-induced acute lung injury in mice, we studied the effects of three caffeoyl quinic acids isolated from Chrysanthemum morifolium on vascular endothelial cell injury and their mechanisms of action. All animal experiments were carried out strictly in accordance with the National Animal Welfare Ethics and Protection Regulations. A mouse model of acute lung injury was established by intranasal instillation of LPS. After 6 days of oral administration of chrysan‐themum extract, the lung wet weight/dry weight ratio, tumour necrosis factor-α(TNF-α), interleukin-6(IL-6), and interleukin-1β(IL-1β) were measured in mouse bronchoalveolar lavage fluid. Human umbilical vein endothelial cells(HUVEC) were serum starved for 12 h and treated with 2.5 μg · mL-1 LPS for 24 h to establish the in vitro model of vascular endothelial cell injury. After 24 h of treatment of caffeoyl quinic acids from Chrysanthemum morifolium, the levels of TNF-α, IL-6, IL-1β, vascular cell adhesion molecule-1(VCAM-1) and endothelin-1(ET-1)were measured by ELISA in the cell culture supernatant, the malondialdehyde(MDA) level was detected by TBA method, the superoxide dismutase(SOD) level was determined by hydroxylamine method, and the nitric oxide(NO) level was assayed by a one-step method. The levels of p-MEK1/2, MEK1/2, p-ERK1/2, ERK1/2, p-JNK,JNK, p-P38 and P38 of mitogen-activated protein kinase(MAPK) signaling pathway were detected by Western blot. The total extract of Chrysanthemum morifolium can significantly reduce the wet weight/dry weight ratio of lung in mice and the levels of TNF-α, IL-6 and IL-1β in alveolar lavage fluid. The caffeoyl quinic acids from Chry‐santhemum morifolium significantly increased the levels of SOD and NO, decreased the levels of TNF-α, IL-6,IL-1β, VCAM-1, ET-1 and MDA, and significantly reduced the levels of p-MEK1/2, p-ERK1/2. In conclusion, total extracts of Chrysanthemum morifolium exhibit certain protective effect on mice with acute lung injury, and three caffeoyl quinic acids from Chrysanthemum morifolium may improve LPS-induced vascular endothelial cell injury by inhibiting inflammatory cytokines and oxidative stress, and regulating inter-cellular adhesion molecule and vasomotor factors through ERK/MAPK signaling pathway.
To explore the effect of total extract of Chrysanthemum morifolium on lipopolysaccharide(LPS)-induced acute lung injury in mice, we studied the effects of three caffeoyl quinic acids isolated from Chrysanthemum morifolium on vascular endothelial cell injury and their mechanisms of action. All animal experiments were carried out strictly in accordance with the National Animal Welfare Ethics and Protection Regulations. A mouse model of acute lung injury was established by intranasal instillation of LPS. After 6 days of oral administration of chrysan‐themum extract, the lung wet weight/dry weight ratio, tumour necrosis factor-α(TNF-α), interleukin-6(IL-6), and interleukin-1β(IL-1β) were measured in mouse bronchoalveolar lavage fluid. Human umbilical vein endothelial cells(HUVEC) were serum starved for 12 h and treated with 2.5 μg · mL-1 LPS for 24 h to establish the in vitro model of vascular endothelial cell injury. After 24 h of treatment of caffeoyl quinic acids from Chrysanthemum morifolium, the levels of TNF-α, IL-6, IL-1β, vascular cell adhesion molecule-1(VCAM-1) and endothelin-1(ET-1)were measured by ELISA in the cell culture supernatant, the malondialdehyde(MDA) level was detected by TBA method, the superoxide dismutase(SOD) level was determined by hydroxylamine method, and the nitric oxide(NO) level was assayed by a one-step method. The levels of p-MEK1/2, MEK1/2, p-ERK1/2, ERK1/2, p-JNK,JNK, p-P38 and P38 of mitogen-activated protein kinase(MAPK) signaling pathway were detected by Western blot. The total extract of Chrysanthemum morifolium can significantly reduce the wet weight/dry weight ratio of lung in mice and the levels of TNF-α, IL-6 and IL-1β in alveolar lavage fluid. The caffeoyl quinic acids from Chry‐santhemum morifolium significantly increased the levels of SOD and NO, decreased the levels of TNF-α, IL-6,IL-1β, VCAM-1, ET-1 and MDA, and significantly reduced the levels of p-MEK1/2, p-ERK1/2. In conclusion, total extracts of Chrysanthemum morifolium exhibit certain protective effect on mice with acute lung injury, and three caffeoyl quinic acids from Chrysanthemum morifolium may improve LPS-induced vascular endothelial cell injury by inhibiting inflammatory cytokines and oxidative stress, and regulating inter-cellular adhesion molecule and vasomotor factors through ERK/MAPK signaling pathway.
引文
[1]Zhang XH.Study on the Changes of Chemical Composition,Anti-inflammatory Efficacy and Mechanism of Different Cultivars of Fios Chrysanthemi(不同来源菊花化学成分、抗炎作用及其机理的研究)[D].Nanjing:Nanjing University of Chinese Medicine,2014.
[2]Tao JH,Duan JA,Zhang W,et al.Polysaccharides from Chrysan‐themum morifolium Ramat ameliorate colitis rats via regulation of the metabolic profiling and NF-κB/TLR4 and IL-6/JAK2/STAT3 signaling pathways[J].Front Pharmacol,2018,9:746.
[3]Cheng XL.The Protective Effect of Chrysanthemum Extract on Acute Lung Injury in Mice(菊花提取物对小鼠急性肺损伤的保护作用)[D].Henan:Henan University,2015.
[4]Huang Y,Huang YL,Su Y,et al.Effect of the extraction of Chrysanthemum indicum on the protein expression of TNF-α,IL-6 and pathological morphological changes of lung in chronic bronchitis model of rats[J].Chin J Gerontol(中国老年学杂志),2010,30:2614-2616.
[5]Feng WS,Chen WJ,Li M,et al.Glycosides from the flowers of Chrysanthemum morifolium[J].J Chin Med Mater(中药材),2018,41:338-341.
[6]Feng WS,Chen WJ,Zheng XK,et al.Flavonoids from the flowers of Chrysanthemum morifolium Ramat.[J].Chin Pharm J(中国药学杂志),2017,52:1497-1502.
[7]Qiu J,Yu L,Zhang X,et al.Asiaticoside attenuates lipopolysac‐charide-induced acute lung injury via down-regulation of NF-κBsignaling pathway[J].Int Immunopharmacol,2015,26:181-187.
[8]Liu XX,Yu DD,Chen MJ,et al.Hesperidin ameliorates lipo‐polysaccharide-induced acute lung injury in mice by inhibiting HMGB1 release[J].Int Immunopharmacol,2015,25:370-376.
[9]Qiushi W,Guanghua L,Guangquan X.Acanthoic acid amelio‐rates lipopolysaccharide-induced acute lung injury[J].Eur JPharmacol,2015,750:32-38.
[10]Feng WS,Fan H,Xu RH,et al.The mechanism of the improve‐ment of vascular endothelial cell injury induced by eriodictyol-7-O-β-D-glucoside[J].J Chin Med Mater(中药材),2018,41:1177-1181.
[11]Yu ZR,Du GH.Advances in the treatment of acute lung injury caused by endotoxin[J].Chin J New Drug(中国新药杂志),2017,26:1510-1515
[12]Cheng X,Yang YL,Li WH,et al.Effect of esculin on acute lung injury induced by LPS in mice and possible mechanism[J].Chin J New Drug(中国新药杂志),2018,27:1849-1854.
[13]Lin Y,Lai WF,Su YQ,et al.Effect of emodin on inflammatory response of astrocytes induce by LPS[J].Chin Pharmacol Bull(中国药理学通报),2018,34:1528-1533.
[14]Zheng ZL,Qiu Y,Huang JW.Study of optimum dosage and application approach of ketamine for inflammation in acute lung injury of Wistar rat[J].Prog Anat Sci(解剖科学进展),2018,24:472-475.
[15]Sheng Y.Study the therapeutic effect of glucocorticoid on acute lung injury[J].Guide China Med(中国医药指南),2017,15:50-51.
[16]Yang YX,Li GY.Progression of lipopolysaccharide signal path‐way[J].J Central South Univ(Med Ed)(中南大学学报医学版),2006,31:141-145.
[17]Wu LN.Role of P38 MAPK and ERK1/2 Signaling Pathway in the Regulation of Heme Oxygenase-1 Expression in the Lung Tissue during Endotoxic Shock Induced ALI in Rats(P38MAPK和ERK1/2信号通路在内毒素性休克诱发急性肺损伤大鼠肺组织HO-1表达过程中的作用)[D].Tianjin:Tianjin Medical University,2013.
[2]Tao JH,Duan JA,Zhang W,et al.Polysaccharides from Chrysan‐themum morifolium Ramat ameliorate colitis rats via regulation of the metabolic profiling and NF-κB/TLR4 and IL-6/JAK2/STAT3 signaling pathways[J].Front Pharmacol,2018,9:746.
[3]Cheng XL.The Protective Effect of Chrysanthemum Extract on Acute Lung Injury in Mice(菊花提取物对小鼠急性肺损伤的保护作用)[D].Henan:Henan University,2015.
[4]Huang Y,Huang YL,Su Y,et al.Effect of the extraction of Chrysanthemum indicum on the protein expression of TNF-α,IL-6 and pathological morphological changes of lung in chronic bronchitis model of rats[J].Chin J Gerontol(中国老年学杂志),2010,30:2614-2616.
[5]Feng WS,Chen WJ,Li M,et al.Glycosides from the flowers of Chrysanthemum morifolium[J].J Chin Med Mater(中药材),2018,41:338-341.
[6]Feng WS,Chen WJ,Zheng XK,et al.Flavonoids from the flowers of Chrysanthemum morifolium Ramat.[J].Chin Pharm J(中国药学杂志),2017,52:1497-1502.
[7]Qiu J,Yu L,Zhang X,et al.Asiaticoside attenuates lipopolysac‐charide-induced acute lung injury via down-regulation of NF-κBsignaling pathway[J].Int Immunopharmacol,2015,26:181-187.
[8]Liu XX,Yu DD,Chen MJ,et al.Hesperidin ameliorates lipo‐polysaccharide-induced acute lung injury in mice by inhibiting HMGB1 release[J].Int Immunopharmacol,2015,25:370-376.
[9]Qiushi W,Guanghua L,Guangquan X.Acanthoic acid amelio‐rates lipopolysaccharide-induced acute lung injury[J].Eur JPharmacol,2015,750:32-38.
[10]Feng WS,Fan H,Xu RH,et al.The mechanism of the improve‐ment of vascular endothelial cell injury induced by eriodictyol-7-O-β-D-glucoside[J].J Chin Med Mater(中药材),2018,41:1177-1181.
[11]Yu ZR,Du GH.Advances in the treatment of acute lung injury caused by endotoxin[J].Chin J New Drug(中国新药杂志),2017,26:1510-1515
[12]Cheng X,Yang YL,Li WH,et al.Effect of esculin on acute lung injury induced by LPS in mice and possible mechanism[J].Chin J New Drug(中国新药杂志),2018,27:1849-1854.
[13]Lin Y,Lai WF,Su YQ,et al.Effect of emodin on inflammatory response of astrocytes induce by LPS[J].Chin Pharmacol Bull(中国药理学通报),2018,34:1528-1533.
[14]Zheng ZL,Qiu Y,Huang JW.Study of optimum dosage and application approach of ketamine for inflammation in acute lung injury of Wistar rat[J].Prog Anat Sci(解剖科学进展),2018,24:472-475.
[15]Sheng Y.Study the therapeutic effect of glucocorticoid on acute lung injury[J].Guide China Med(中国医药指南),2017,15:50-51.
[16]Yang YX,Li GY.Progression of lipopolysaccharide signal path‐way[J].J Central South Univ(Med Ed)(中南大学学报医学版),2006,31:141-145.
[17]Wu LN.Role of P38 MAPK and ERK1/2 Signaling Pathway in the Regulation of Heme Oxygenase-1 Expression in the Lung Tissue during Endotoxic Shock Induced ALI in Rats(P38MAPK和ERK1/2信号通路在内毒素性休克诱发急性肺损伤大鼠肺组织HO-1表达过程中的作用)[D].Tianjin:Tianjin Medical University,2013.