人参三七川芎提取物对高糖高脂诱导的衰老血管内皮细胞自噬的影响
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摘要
目的观察人参三七川芎提取物对血管内皮细胞自噬流的影响,探讨其延缓高糖高脂诱导血管内皮细胞衰老的作用机制。方法采用40 mmol/L葡萄糖和100μmol/L棕榈酸钠造模,实验分为空白对照组、衰老模型组和中药组(200 mg/L),干预48 h。采用CCK-8检测细胞增殖能力;β-半乳糖苷酶染色检测细胞衰老程度;Western blot检测细胞p16、p21、微管相关蛋白1轻链3-Ⅱ(LC3B-Ⅱ)和p62蛋白表达;免疫荧光检测自噬流变化。结果与空白对照组比较,衰老模型组细胞增殖能力和LC3B-Ⅱ表达降低,β-半乳糖苷酶蓝染细胞数量和p16、p21、p62蛋白表达增加,并存在自噬流阻滞;与衰老模型组比较,中药组细胞增殖能力增强,LC3B-Ⅱ表达升高,β-半乳糖苷酶蓝染细胞数量减少,p16、p21和p62表达降低,自噬流畅通。结论人参三七川芎提取物可延缓高糖高脂引起的血管内皮细胞衰老,其机制可能与增强细胞自噬活性有关。
Objective To explore the effects of extracts of Ginseng Radix et Rhizoma, Notoginseng Radix et Rhizoma and Chuanxiong Rhizoma on autophagy of vascular endothelial cells; To discuss its mechanism for delaying high glucose and high fat-induced vascular endothelial cell senescence. Methods 40 mmol/L glucose and 100 μmol/L palmitate were used to establish model. The experiment was divided into control group, senescence model group and TCM group(200 mg/L) for 48 h. Cell proliferation was detected by CCK-8; cell senescence was detected by β-galactosidase staining; p16, p21, LC3 B-Ⅱand p62 protein expression levels were detected by Western blot; autophagy flux was detected by immunofluorescence. Results Compared with the control group, the cell proliferation ability and LC3 B-expression decreased, the number of SAⅡ-β-gal blue-stained cells and expression of p16, p21 and p62 increased, and autophagic flux blocked in the senescence model group. Compared with the senescence model group, extracts of Ginseng Radix et Rhizoma, Notoginseng Radix et Rhizoma and Chuanxiong Rhizoma could increase cell proliferation and LC3 B-Ⅱexpression levels, reduce the number of SA-β-gal blue-stained cells and reduce the expression levels of p16, p21 and p62 and maintain the autophagic flux unobstructed. Conclusion Extracts of Ginseng Radix et Rhizoma, Notoginseng Radix et Rhizoma and Chuanxiong Rhizoma can delay endothelial cellular senescence induced by high glucose and high fat, and its mechanism may be related to up-regulating cellular autophagy activity.
Objective To explore the effects of extracts of Ginseng Radix et Rhizoma, Notoginseng Radix et Rhizoma and Chuanxiong Rhizoma on autophagy of vascular endothelial cells; To discuss its mechanism for delaying high glucose and high fat-induced vascular endothelial cell senescence. Methods 40 mmol/L glucose and 100 μmol/L palmitate were used to establish model. The experiment was divided into control group, senescence model group and TCM group(200 mg/L) for 48 h. Cell proliferation was detected by CCK-8; cell senescence was detected by β-galactosidase staining; p16, p21, LC3 B-Ⅱand p62 protein expression levels were detected by Western blot; autophagy flux was detected by immunofluorescence. Results Compared with the control group, the cell proliferation ability and LC3 B-expression decreased, the number of SAⅡ-β-gal blue-stained cells and expression of p16, p21 and p62 increased, and autophagic flux blocked in the senescence model group. Compared with the senescence model group, extracts of Ginseng Radix et Rhizoma, Notoginseng Radix et Rhizoma and Chuanxiong Rhizoma could increase cell proliferation and LC3 B-Ⅱexpression levels, reduce the number of SA-β-gal blue-stained cells and reduce the expression levels of p16, p21 and p62 and maintain the autophagic flux unobstructed. Conclusion Extracts of Ginseng Radix et Rhizoma, Notoginseng Radix et Rhizoma and Chuanxiong Rhizoma can delay endothelial cellular senescence induced by high glucose and high fat, and its mechanism may be related to up-regulating cellular autophagy activity.
引文
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[3]LIAO P,YANG D,LIU D,et al.GLP-1 and ghrelin attenuate high glucose/high lipid-induced apoptosis and senescence of human microvascular endothelial cells[J].Cellular Physiology&Biochemistry International Journal of Experimental Cellular Physiology Biochemistry&Pharmacology,2017,44(5):1842.
[4]HAYASHI T,MATSUIHIRAI H,MIYAZAKIAKITA A,et al.Endothelial cellular senescence is inhibited by nitric oxide:implications in atherosclerosis associated with menopause and diabetes[J].Proc Natl Acad Sci USA,2006,103(45):17018-17023.
[5]LEVINE E,PACKER M,CODOGNO P.Development of autophagy inducers in clinical medicine[J].Journal of Clinical Investigation,2015,125(1):14-24.
[6]蔡世忠,王亚平.细胞衰老与细胞自噬的生物学关联及其意义[J].生命科学,2011,23(4):335-341.
[7]HARRISON D E,STRONG R,SHARP Z D,et al.Rapamycin fed late in life extends lifespan in genetically heterogeneous mice[J].Nature,2009,460:392-395.
[8]MADEO F,TAVERNARAKIS N,KROEMER G.Can autophagy promote longevity?[J].Nat Cell Biol,2010,12(9):842-846.
[9]王强,修成奎,杨静,等.人参三七川芎醇提物对衰老人心脏微血管内皮细胞自噬的影响[J].中医杂志,2017,58(6):516-519.
[10]修成奎,雷燕,王强,等.自然衰老大鼠血管老化中细胞骨架的改变及人参三七川芎提取物的干预作用[J].中国中西医结合杂志,2017,37(8):968-972.
[11]王雪,修成奎,杨静,等.人参-三七-川芎提取物对高糖高脂诱导血管内皮细胞衰老的影响[J].中国实验方剂学杂志,2019,25(1):124-129.
[12]范芳,陈娇,徐勇,等.高糖通过调控FBW7表达影响自噬的研究[J].中国糖尿病杂志,2017,25(6):546-551.
[13]MIZUSHIMA N,LEVINE B,CUERVO A M,et al.Autophagy fights disease through cellular self-digestion[J].Nature,2008,451:1069.
[14]KLIONSKY D J,ABDELMOHSEN K,ABE A,et al.Guidelines for the use and interpretation of assays for monitoring autophagy(3rd edition)[J].Autophagy,2016,12(1):1-222.
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[16]HO T T,WARR M R,ADELMAN E R,et al.Autophagy maintains the metabolism and function of young and old stem cells[J].Nature,2017,543(7644):205-210.
[17]CHEN F,CHEN B,XIAO F Q,et al.Autophagy protects against senescence and apoptosis via the RAS-mitochondria in highglucose-induced endothelial Cells[J].Cellular Physiology&Biochemistry,2014,33(4):1058-1074.
[18]WEIKEL K A,CACICEDO J M,RUDERMAN N B,et al.Glucose and palmitate uncouple AMPK from autophagy in human aortic endothelial cells[J].American Journal of Physiology Cell Physiology,2015,308(3):249-263.
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[20]VOGHEL G,THORINTRESCASES N,FARHAT N,et al.Cellular senescence in endothelial cells from atherosclerotic patients is accelerated by oxidative stress associated with cardiovascular risk factors[J].Mechanisms of Ageing&Development,2007,128(11):662-671.
[21]ZHANG Y,XU X,RRE J.MTOR overactivation and interrupted autophagy flux in obese hearts:a dicey assembly?[J].Autophagy,2013,9(6):939-941.