陈皮总黄酮干预血管平滑肌细胞糖胺聚糖代谢的机制研究
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摘要
目的研究陈皮总黄酮(CPTF)干预血管平滑肌细胞(VSMC)糖胺聚糖(GAGs)代谢的作用机制。方法MOVAS细胞分为正常组、模型组、CPTF组(100μg·mL-1);采用氧化低密度脂蛋白(ox-LDL)诱导建立VSMC增殖模型。采用硫酸咔唑法测定GAGs总量;以HPLC法检测半乳糖胺含量;以流式细胞仪检测成纤维细胞生长因子-硫酸乙酰肝素-成纤维细胞生长因子受体1c(bFGF-HS-FGFR1c)三元复合物的荧光信号;高通量测序检测GAGs转录组基因表达。结果与正常组比较,模型组的GAGs、硫酸软骨素(CS)含量增多(P <0.05),HS含量降低(P <0.05),Cspg5(调节CS合成)、Hs3st3b1(调节HS、肝素合成)基因表达明显上调,B3gnt3(调节鞘糖脂生物合成-乳酸和新乳酸系列)、Has3(调节透明质酸合成)、Fut1(调节鞘糖脂生物合成-globo系列)基因表达下调。与模型组比较,CPTF组的GAGs、CS含量降低(P <0.01),HS含量增多(P <0.01),B3gnt3、Has3、Fut1、Hs3st3b1基因表达上调,Cspg5基因表达明显下调。结论 CPTF可能通过调节Cspg5基因的表达及HS的合成来干预VSMC的GAGs代谢,从而发挥一定的抗动脉粥样硬化的作用。
Objective To study the mechanism of Chenpi total flavonoids(CPTF)on the metabolism of glycosaminoglycans(GAGs)in vascular smooth muscle cells(VSMC).Methods VSMC were cultured in vitro and divided into normal group,model group,and CPTF group(100μg·mL~(-1));VSMC proliferation model was established by ox-LDL induction.Total amount of GAGs was measured by carbazole sulfate method.The fluorescence signal of fibroblast growth factor-heparan sulfate-fibroblast growth factor receptor 1c(b FGF-HS-FGFR1c)ternary complex was detected by flow cytometry.The content of galactosamine was detected by HPLC.Gene expression level of GAGs in transcriptome was detected by high-throughput sequencing.Results Compared with the normal group,GAGs and CS content in the model group increased(P<0.05);HS content decreased(P<0.05);the genes expression of Cspg5(regulating CS synthesis),Hs3st3b1(regulating HS,heparin synthesis)were up-regulated significantly,and the genes expression of B3gnt3(regulating glycosphingolipid biosynthesis-lactic acid and new lactic acid series),Has3(regulating HA synthesis),Fut1(regulating glycosphingolipid biosynthesis-globo series)were down-regulated.Compared with the model group,the contents of GAGs and CS in CPTF group decreased(P<0.01);the content of HS increased(P<0.01);the genes expression of B3gnt3,Has3,Fut1,Hs3st3b1were up-regulated,and the gene expression of Cspg5 was down-regulated significantly.Conclusion CPTF may interfere with the metabolism of GAGs in VSMC by regulating the expression of Cspg5 gene and the synthesis of HS,thereby play a role in anti-atherosclerosis.
Objective To study the mechanism of Chenpi total flavonoids(CPTF)on the metabolism of glycosaminoglycans(GAGs)in vascular smooth muscle cells(VSMC).Methods VSMC were cultured in vitro and divided into normal group,model group,and CPTF group(100μg·mL~(-1));VSMC proliferation model was established by ox-LDL induction.Total amount of GAGs was measured by carbazole sulfate method.The fluorescence signal of fibroblast growth factor-heparan sulfate-fibroblast growth factor receptor 1c(b FGF-HS-FGFR1c)ternary complex was detected by flow cytometry.The content of galactosamine was detected by HPLC.Gene expression level of GAGs in transcriptome was detected by high-throughput sequencing.Results Compared with the normal group,GAGs and CS content in the model group increased(P<0.05);HS content decreased(P<0.05);the genes expression of Cspg5(regulating CS synthesis),Hs3st3b1(regulating HS,heparin synthesis)were up-regulated significantly,and the genes expression of B3gnt3(regulating glycosphingolipid biosynthesis-lactic acid and new lactic acid series),Has3(regulating HA synthesis),Fut1(regulating glycosphingolipid biosynthesis-globo series)were down-regulated.Compared with the model group,the contents of GAGs and CS in CPTF group decreased(P<0.01);the content of HS increased(P<0.01);the genes expression of B3gnt3,Has3,Fut1,Hs3st3b1were up-regulated,and the gene expression of Cspg5 was down-regulated significantly.Conclusion CPTF may interfere with the metabolism of GAGs in VSMC by regulating the expression of Cspg5 gene and the synthesis of HS,thereby play a role in anti-atherosclerosis.
引文
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[7]崔怡迪.5-羟基-6,7,8,4’,5’-五甲氧基黄酮对肿瘤细胞内糖胺聚糖组成与结构的影响[D].青岛:中国海洋大学,2014.
[8]任珍芸,陈晓航,王玺,等.硫酸-咔唑微孔板法检测肺炎链球菌荚膜多糖中糖醛酸含量[J].微生物学免疫学进展,2017,45(2):36-41.
[9]罗立,王娜,张玉.柱前衍生化HPLC-FD法测定大鼠体内当归多糖组分ASP1的含量[J].中国药师,2017,20(3):438-442.
[10]徐玲玲.嘧啶类姜黄素类似物抗结肠癌活性及机制研究[D].青岛:中国海洋大学,2014.
[11]KIM D,PERTEA G,TRAPNELL C,et al.TopHat2:accurate alignment of transcriptomes in the presence of insertions,deletions and gene fusions[J].Genome Biol,2013,14(4):R36.
[12]NOBLE M I,DRAKE-HOLLAND A J,VINK H,et al.Hypothesis:arterial glycocalyx disfunction is the first step in the atherothrombotic process[J].Qjm-Monthly Journal of the Association of Physicians,2008,101(7):513-518.
[13]GONZALES J C,GORDTS P L,FOLEY E M,et al.Apolipoproteins E and AV mediate lipoprotein clearance by hepatic proteoglycans[J].The Journal of Clinical Investigation,2013,123(6):2742-2751.
[14]O’BRIEN K D,LEWIS K,FISCHER J W,et al.Smooth muscle cell biglycan overexpression results in increased lipoprotein retention on extracellular matrix:implications for the retention of lipoproteins in atherosclerosis[J].Atherosclerosis,2004,177(1):29-35.
[15]NEUFELD E B,ZADROZNY L M,PHILLIPS D,et al.Decorin and biglycan retain LDL in disease-prone valvular and aortic subendothelial intimal matrix[J].Atherosclerosis,2014,233(1):113-121.
[2]HANSSON G K.Mechanisms of disease:Inflammation,atherosclerosis,and coronary artery disease[J].The New England Journal of Medicine,2005,352(16):1685-1695.
[3]吴慧君,欧金龙,池晓玲,等.陈皮药理作用研究概述[J].实用中医内科杂志,2013,25(9):91-92.
[4]姜涛.基于“痰”致病理论研究橙皮苷对动脉粥样硬化相关蛋白聚糖的作用[D].广州:广州中医药大学,2016.
[5]吴迪.陈皮总黄酮的提取工艺优化及其抗过敏活性研究[D].长春:吉林大学,2016.
[6]胡彦武,李海涛,刘凯,等.淫羊藿苷通过影响MAPK信号通路抑制ox-LDL诱导的血管平滑肌细胞增殖作用研究[J].中国中药杂志,2016,41(19):3655-3660.
[7]崔怡迪.5-羟基-6,7,8,4’,5’-五甲氧基黄酮对肿瘤细胞内糖胺聚糖组成与结构的影响[D].青岛:中国海洋大学,2014.
[8]任珍芸,陈晓航,王玺,等.硫酸-咔唑微孔板法检测肺炎链球菌荚膜多糖中糖醛酸含量[J].微生物学免疫学进展,2017,45(2):36-41.
[9]罗立,王娜,张玉.柱前衍生化HPLC-FD法测定大鼠体内当归多糖组分ASP1的含量[J].中国药师,2017,20(3):438-442.
[10]徐玲玲.嘧啶类姜黄素类似物抗结肠癌活性及机制研究[D].青岛:中国海洋大学,2014.
[11]KIM D,PERTEA G,TRAPNELL C,et al.TopHat2:accurate alignment of transcriptomes in the presence of insertions,deletions and gene fusions[J].Genome Biol,2013,14(4):R36.
[12]NOBLE M I,DRAKE-HOLLAND A J,VINK H,et al.Hypothesis:arterial glycocalyx disfunction is the first step in the atherothrombotic process[J].Qjm-Monthly Journal of the Association of Physicians,2008,101(7):513-518.
[13]GONZALES J C,GORDTS P L,FOLEY E M,et al.Apolipoproteins E and AV mediate lipoprotein clearance by hepatic proteoglycans[J].The Journal of Clinical Investigation,2013,123(6):2742-2751.
[14]O’BRIEN K D,LEWIS K,FISCHER J W,et al.Smooth muscle cell biglycan overexpression results in increased lipoprotein retention on extracellular matrix:implications for the retention of lipoproteins in atherosclerosis[J].Atherosclerosis,2004,177(1):29-35.
[15]NEUFELD E B,ZADROZNY L M,PHILLIPS D,et al.Decorin and biglycan retain LDL in disease-prone valvular and aortic subendothelial intimal matrix[J].Atherosclerosis,2014,233(1):113-121.