丹皮酚减轻SIRT6/PARP1介导的DNA损伤抑制血管平滑肌细胞衰老的作用  

Inhibitory Effect of Paeonol on Vascular Smooth Muscle Cell Senescence by Reducing SIRT6/PARP1-mediated DNA Damage

作　　者：蒋婷婷 刘雅蓉 施晓艳 杨宇龙 戴敏 JIANG Tingting;LIU Yarong;SHI Xiaoyan;YANG Yulong;DAI Min(Key Laboratory of Research and Development of Traditional Chinese Medicine in Anhui Province,School of Pharmacy,Anhui University of Chinese Medicine,Hefei 230012,China)

机构地区：[1]安徽中医药大学药学院,安徽省中药研究与开发重点实验室,合肥230012

出　　处：《中国实验方剂学杂志》2023年第10期83-92,共10页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(82174014);安徽省自然科学基金项目(2108085QH375)。

摘　　要：目的:探讨丹皮酚(Pae)对血管紧张素Ⅱ(AngⅡ)诱导的血管平滑肌细胞(VSMCs)衰老的作用及对沉默调节信息因子6(SIRT6)/腺苷二磷酸核糖聚合酶1(PARP1)信号通路的影响。方法:建立AngⅡ(100 nmol·L^(-1))诱导的VSMCs应激性衰老模型,实验分为正常组、模型组、Pae低、中、高(30、60、120μmol·L^(-1))浓度组。采用β-半乳糖苷酶(SA-β-Gal)染色法检测细胞衰老阳性率;细胞增殖与活性检测(CCK-8)法检测细胞增殖能力;蛋白免疫印迹法(Western blot)检测SIRT6、PARP1、衰老相关基因p16、p21、p53、增殖细胞核抗原(PCNA)、脱氧核糖核酸(DNA)损伤蛋白磷酸化的组蛋白H2AX(即p-H2AX或γ-H2AX)的表达并采用Ed U染色法检测VSMCs增殖变化;si RNA-SIRT6转染制备沉默的VSMCs,观察SIRT6沉默的VSMCs中Pae对SIRT6、PARP1及p16、γ-H2AX蛋白的表达。结果:SA-β-Gal染色结果显示,与正常组比较,模型组SA-β-Gal染色衰老阳性率增加(P<0.01);与模型组比较,Pae给药组均有效降低SA-β-Gal染色阳性率(P<0.05,P<0.01)。Western blot结果显示,与正常组比较,模型组PCNA、SIRT6、PARP1表达下调、衰老相关蛋白p16、p21、p53、γ-H2AX的表达上调(P<0.05,P<0.01);与模型组比较,Pae给药干预后,各浓度组促进了PCNA、SIRT6、PARP1的蛋白表达,抑制了p16、p21、p53、γ-H2AX的蛋白表达,且呈剂量依赖性(P<0.05,P<0.01)。Ed U染色结果显示,与正常组比较,模型组Ed U阳性细胞数减少(P<0.01);与模型组比较,Pae给药组Ed U阳性细胞数明显增加(P<0.05,P<0.01)。SIRT6沉默后,Pae促进SIRT6、PARP1及抑制p16的作用被逆转(P<0.05,P<0.01)。此外加入SIRT6抑制剂IN-1可促进AngⅡ诱导细胞衰老的发生(P<0.05,P<0.01)。结论:Pae能够有效抑制VSMCs的衰老,其机制作用可能与调控SIRT6/PARP1信号通路相关。Objective:To investigate whether the effects of paeonol(Pae)on angiotensinⅡ(AngⅡ)-induced senescence in vascular smooth muscle cells(VSMCs)were related to angiotensinogen of silencing regulatory information factor 6(SIRT6)/adenosine diphosphate ribose polymerase 1(PARP1)signaling pathway in VSMCs.Method:The model of VSMC-stress aging induced by AngⅡ(100 nmol·L^(-1))was established.The rats were divided into normal group,model group,low,medium,and high-concentration Pae groups(30,60,120μmol·L^(-1)).The positive rate of cell senescence was detected by SA-β-Gal staining,the ability of cell proliferation was detected by the cell counting kit-8(CCK-8)method,the expression of SIRT6,PARP1,p16,p21,p53,proliferating cell nuclear antigen(PCNA),deoxyribonucleic acid(DNA)-damaged proteinγ-H2AX was detected by Western blot,and VSMC proliferation was detected by EdU staining.The silenced VSMCs were prepared by siRNA-SIRT6 transfection,and the protein expressions of SIRT6,PARP1,p16,andγ-H2AX in VSMCs silenced by SIRT6 were observed.Result:The results of SA-β-Gal staining showed that the senescence positive rate of SA-β-Gal staining in the model group was higher than that in the normal group(P<0.01),and the positive rate of SA-β-Gal staining in the Pae group was significantly lower than that in the model group(P<0.05,P<0.01).The results of Western blot showed that as compared with the normal group,the expression of PCNA,SIRT6,and PARP1 in the model group was down-regulated,and the expression of aging-related proteins p16,p21,p53,andγ-H2AX was up-regulated in the model group(P<0.05,P<0.01).Compared with the model group,Pae promoted the protein expression of PCNA,SIRT6,and PARP1 and inhibited the protein expression of p16,p21,p53,andγ-H2AX in a dose-dependent manner(P<0.05,P<0.01).The results of EdU staining showed that the number of EdU positive cells in the model group was lower than that in the normal group(P<0.01),and the number of EdU positive cells in Pae groups was significantly higher than that in the mode

关 键 词：丹皮酚 沉默调节信息因子6(SIRT6) 腺苷二磷酸核糖聚合酶1(PARP1) 血管平滑肌细胞(VSMCs) 衰老 

分 类 号：R2-0[医药卫生—中医学] R22R285.5R289R33