摘要
为揭示3-芳基香豆素衍生物3-(4′-羟基苯基)-6-羟基香豆素(SJ-6)对抗血管钙化的作用机制,采用晚期糖基化终产物(AGEs)对人体主动脉血管平滑肌细胞(HCASMCs)进行钙化诱导并通过茜素红染色及定量进行钙化鉴定。分别检测了化合物SJ-6对碱性磷酸酶(ALP)活力、细胞增殖率、钙含量、总活性氧(ROS)、超氧化物歧化酶(SOD)、AGEs、肿瘤坏死因子-α(TNF-α)、白细胞介素6(1L-6)、白细胞介素β(1L-β)、成骨相关转录因子2 mRNA(Runx2 mRNA)、晚期糖基化终产物受体(RAGE)、核因子κB(NF-κB)、NADPH氧化酶-1(NOX-1)、蛋白激酶C(PKC)、蛋白激酶B(AKT)、p38丝裂原活化蛋白激酶(p38 MAPK)、α-平滑肌肌动蛋白(SMA-α)蛋白表达的影响。根据研究结果发现化合物SJ-6可明显降低钙化细胞模型中AGEs含量、ALP活性、细胞内钙离子含量、ROS含量、Runx2 mRNA以及炎症因子TNF-α、1L-6和1L-β的含量(P < 0.05),并升高SOD的含量(P < 0.01),这些作用与阳性对照药氨基胍盐酸盐(AGH)相似。进一步对化合物SJ-6进行深入的药理机制研究,发现化合物SJ-6能够明显抑制钙化细胞模型中关键信号蛋白RAGE、NF-κB、NOX-1、PKC、Akt、p-p38等蛋白的表达(P < 0.01),并增加平滑肌动蛋白SMA-α的表达(P < 0.01),通过抑制氧化应激和AGEs/RAGE、Akt/PKC及NF-κB信号通路的表达抑制血管钙化,提示化合物SJ-6有望成为治疗血管钙化的新型药物。
关键词
血管钙化是一种类似于骨形成的过程,具有高度可调节性和主动
近年来,越来越多的研究表明,与单一靶点药物相比,多靶点的天然药物在心血管的保护中具有不可替代的优势,尤其是香豆素类化合物具有良好的AGEs生成抑制活性,异黄酮类化合物具有良好的抗氧化、抗炎活性,近年来备受关

Figure 1 Synthetic route of compound SJ-6
SJ-6(纯度:99.07%)由山东第一医科大学药学与制药科学学院孙捷课题组提供。0.2%茜素红S染液、MTT、超氧化物歧化酶(SOD)检测试剂盒、总RNA提取试剂盒、40%多聚甲醛(北京Solarbio公司);胎牛血清、DMEM培养基、(美国Gibco公司);十六烷基氯化吡啶(美国Sigma公司);碱性磷酸酶活力检测试剂盒(上海Beyotime公司);WBKLS0100(美国Millipore公司);钙离子检测试剂盒(北京Leagene公司);p-p38抗体、t-p38抗体、PKC抗体、NF-κB抗体(上海Cell Signaling Technology公司);Akt抗体、RAGE抗体(美国Immunoway公司);NOX-1抗体、β-actin抗体、辣根标记山羊抗小鼠gG(H+L)(美国Proteintech公司);辣根标记山羊抗兔lgG(H+L)(北京Zsbio公司);IL-β酶联免疫试剂盒、IL-6酶联免疫试剂盒、TNF-α酶联免疫试剂盒、AGEs酶联免疫试剂盒(上海朗顿生物科技有限公司);总蛋白提取试剂盒(上海Bestbio公司);Real Time q-PCR试剂盒(日本TaKaRa公司);其他试剂均为市售分析纯;引物委托Biosune 生物技术公司设计合成引物序列(基因:RUNX2;序列:5′-TCTCCAACCCACGAATGCAC-3′,5′-ATACCGAGGGACATGCCTGA-3′;引物长度:76 bp)。
3111型CO2培养箱、Evolution 220紫外分光光度计(美国Thermo公司);Eclipse TE 2000-S荧光显微镜(日本Nikon公司);Infinite M200 PRO型酶标仪(法国Tecan公司);CFX96 TOUCK荧光定量PCR仪、Trans-Blot SD半干转印系统(美国Bio-Rad公司);Azure cSeries 200型化学发光成像仪(美国Azure Biosystems公司)。
取生长状态良好的第2 ~ 5代HCASMCs,按照每皿1 × 1
取生长状态良好的第2 ~ 5代HCASMCs,按照每皿1 × 1
将细胞以每孔5.0 × 1
取生长状态良好的第2 ~ 5代HCASMCs,按照每皿1 × 1
取生长状态良好的第2 ~ 5代HCASMCs,按照每皿1 × 1
取生长状态良好的第2 ~ 5代HCASMCs,按照每皿1 × 1
取生长状态良好的第2 ~ 5代HCASMCs,按照每皿1 × 1
取生长状态良好的第2 ~ 5代HCASMCs,按照每皿1 × 1
取生长状态良好的第2 ~ 5代HCASMCs,按照每皿1 × 1
取生长状态良好的第2 ~ 5代HCASMCs,按照每皿1 × 1
取生长状态良好的第2 ~ 5代HCASMCs,按照每皿1 × 1
MTT测定的结果表明,AGEs和化合物SJ-6均对细胞存活没有显著影响(P > 0.05)(

Figure 2 Effects of different concentrations of compound SJ-6 on cell viability () 1: Normal; 2: DMSO; 3: AGEs; 4: AGEs+DMSO; 5: AGH; 6: 0.025 mmol/L SJ-6; 7: 0.012 mmol/L SJ-6; 8: 0.006 mmol/L SJ-6
AGEs:Advanced glycation end products;AGH:Aminoguanidine hydrochloride
AGEs能促进血管平滑肌细胞的钙

Figure 3 Calcified nodules of smooth muscle cells stained with alizarin red 1: Normal; 2: DMSO; 3: AGEs; 4: AGEs+DMSO; 5: AGH; 6: 0.025 mmol/L SJ-6; 7: 0.012 mmol/L SJ-6; 8: 0.006 mmol/L SJ-6
A: Alizarin red stained calcium nodules under microscope(100 μm); B: Alizarin red staining quantitative results (
研究表明,ALP与主动脉钙化密切相
∆∆P < 0.01 vs normal group;
与对照组相比,AGEs组的细胞内AGEs含量显著增加(P < 0.01)(

Figure 4 Effects of different concentrations of compound SJ-6 on AGEs content () 1: Normal; 2: DMSO; 3: AGEs; 4: AGEs+DMSO; 5: AGH; 6: 0.025 mmol/L SJ-6; 7: 0.012 mmol/L SJ-6; 8: 0.006 mmol/L SJ-6
相比对照组,AGEs组细胞内钙含量明显增加(P < 0.01),说明AGEs可以诱导HCASMCs钙化。与AGEs组相比,不同浓度的SJ-6干预组细胞内钙含量明显降低(P < 0.01),在不同浓度组之间,随着化合物的浓度增加,细胞内钙含量逐渐下降,说明化合物SJ-6可以抑制AGEs诱导的HCASMCs钙化,且细胞内钙含量减少与化合物SJ-6呈浓度依赖性(

Figure 5 Effects of different concentrations of SJ-6 on intracellular calcium content () 1: Normal; 2: DMSO; 3: AGEs; 4: AGEs+DMSO; 5: AGH; 6: 0.025 mmol/L SJ-6; 7: 0.012 mmol/L SJ-6; 8: 0.006 mmol/L SJ-6
∆∆P < 0.01 vs normal group;
氧化应激可以诱发心血管疾

Figure 6 Effects of compound SJ-6 on ROS. The induction of AGEs increases the ROS content in vascular smooth muscle cells, and each compound group can reduce the ROS contentA: Effects of different concentrations of SJ-6 on ROS fluorescence intensity of each group(100 μm); B: Quantitative results of ROS fluorescence intensity of cells in each group with different concentrations of SJ-6 () 1: Normal; 2: DMSO; 3: AGEs; 4: AGEs+DMSO; 5: AGH; 6: 0.025 mmol/L SJ-6; 7: 0.012 mmol/L SJ-6; 8: 0.006 mmol/L SJ-6
∆∆P < 0.01 vs normal group;
与对照组相比,AGEs组细胞的抗氧化指标SOD的活性显著降低(P < 0.01)。相比AGEs组不同浓度化合物SJ-6干扰组细胞内SOD的活性明显升高(P < 0.01),且不同浓度组之间,SOD的活性随化合物浓度增加而逐渐升高,说明化合物SJ-6可以增强细胞的抗氧化能力,且抗氧能力的增强与化合物SJ-6呈浓度依赖性(

Figure 7 Effect of different concentrations of compound SJ-6 on SOD expression () 1: Normal; 2: DMSO; 3: AGEs; 4: AGEs+DMSO; 5: AGH; 6: 0.025 mmol/L SJ-6; 7: 0.012 mmol/L SJ-6; 8: 0.006 mmol/L SJ-6
∆∆P < 0.01 vs normal control group;
与对照组相比,AGEs组细胞的炎性细胞因子TNF-α,1L-6和1L-β显着增加(P < 0.01),表明AGEs可以诱导HCASMCs发生炎症反应。相比AGEs组不同浓度SJ-6干扰组细胞内炎症因子TNF-α、1L-6、1L-β的表达明显降低(P < 0.01),且不同浓度组之间,炎症因子表达量随化合物浓度增加而逐渐下降,说明化合物SJ-6可以抑制AGEs诱导的HCASMCs的炎症反应,且炎症因子表达量的减少与化合物SJ-6呈浓度依赖性(

Figure 8 Influence of different concentrations of compound SJ-6 on the expression of inflammatory factors TNF-α(A), 1L-6(B), 1L-β(C). The induction of AGEs increased the content of TNF-α, 1L-6, 1L-β () 1: Normal; 2: DMSO; 3: AGEs; 4: AGEs+DMSO; 5: AGH; 6: 0.025 mmol/L SJ-6; 7: 0.012 mmol/L SJ-6; 8: 0.006 mmol/L SJ-6
∆∆P < 0.01 vs normal control group;
Runx2 mRNA是正常骨骼形成所必需的,AGEs显著增加了Runx2 mRNA(P < 0.01),表明AGEs可以诱导HCASMCs的成骨转化。与AGEs组相比,化合物SJ-6的浓度升高时,细胞中Runx2 mRNA的表达逐渐降低(P < 0.01),提示化合物SJ-6可以抑制AGEs诱导的HCASMCs的成骨转化且Runx 2 mRNA表达量的减少与化合物SJ-6呈浓度依赖性(

Figure 9 Effect of different concentrations of compound SJ-6 on the expression of Runx2 mRNA (). The induction of AGEs increased the Runx2 mRNA content of the osteogenic transcription factor in vascular smooth muscle cells, and each compound group could reduce the Runx2 mRNA content 1: Normal; 2: DMSO; 3: AGEs; 4: AGEs+DMSO; 5: AGH; 6: 0.025 mmol/L SJ-6; 7: 0.012 mmol/L SJ-6; 8: 0.006 mmol/L SJ-6
∆∆P < 0.01 vs normal control group;
与对照组相比,AGEs组RAGE, NF-κB,p-p38,Akt,PKC,NOX-1蛋白达量明显增高(P < 0.01),平滑肌动蛋白SMA-α的蛋白表达量明显降低(P < 0.01),与AGEs组相比,化合物SJ-6处理组中RAGE,NF-κB,p-p38,Akt,PKC,NOX-1蛋白达量表达量明显降低(P < 0.01),SMA-α的蛋白表达量明显增多(P < 0.01),且表达量随化合物浓度增加而逐渐降低,说明化合物SJ-6可以抑制AGEs诱导的HCASMCs向成骨细胞分化的相关通路,且这种抑制具有浓度依赖性(

Figure 10 Effects of compound SJ-6 at different concentrations on RAGE, SMA-α, NF-κB, p38, Akt, PKC, NOX-1 protein expression induced by AGEs ()
A: Protein expression trends of RAGE, SMA-α, NF-κB, p38, Akt, PKC, NOX-1 at different concentrations of SJ-6 were detected by western blot; B:Gray analysis of the relative gray value of RAGE and SMA-α protein bands; C: Gray analysis of the relative gray value of NOX-1 and NF-κB protein bands; D: Gray analysis of the relative gray value of p-p38 and t-p38 protein bands; E: Gray analysis of the relative gray value of Akt and PKC protein bands
血管钙化是糖尿病、动脉粥样硬化、高血压、慢性肾脏病等多种疾病的共同病理表现,而目前血管钙化的发病机制尚不十分清
化合物SJ-6在不影响细胞的正常增殖的情况下呈剂量依赖性地降低血管平滑肌细胞的钙化程度,包括促进AGEs的裂解、降低胞内的钙离子浓度、减弱ALP酶活力、抑制成骨相关转录因子以及相关信号蛋白的表达并增强平滑肌动蛋白的表达,同时伴随着氧化应激以及部分炎症反应的减弱和抗氧化能力的增强。因此推测化合物SJ-6对于钙化的保护作用机制可能与其抗氧化特性以及对AGEs/RAGE通路的抑制有关。
化合物SJ-6降低了ROS、OX-1、NF-κB、p38、Akt、PKC蛋白和炎症因子TNF-α、1L-6、1L-β的总量,同时增加了细胞内SOD的含量,推测该化合物可抑制氧化应激过程,从而抑制NF-κB和PKC信号通路并减少β细胞的炎症反应,最终减轻钙化过程。
AGEs的刺激增加了RAGE的表达,并且AGEs/RAGE途径与血管钙化密切相
氨基胍同样能够缓解钙化对平滑肌细胞的损害,氨基胍的保护作用与0.01
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