水解南珠液能促进人微血管内皮细胞的增殖和迁移
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摘要
背景:珍珠有镇静安神的药效,临床可应用于高血压病,但水解珍珠液对高血压病的防治作用及相关机制研究目前报道尚少。目的:观察水解南珠液对人微血管内皮细胞生物学行为和分泌功能的影响。方法:人微血管内皮细胞培养,传代。实验分为水解南珠液高、中、低剂量组:分别加入含质量浓度为120,60,30 mg/L水解南珠液的培养基200μL;对照组:只加200μL的培养基。CCK8法检测人微血管内皮细胞增殖情况,流式细胞术检测细胞的周期分布,TUNEL法了解细胞的凋亡情况,Transwell法检测细胞迁移情况,硝酸还原酶法检测细胞的NO分泌量及化学荧光法检测细胞活性氧的分泌情况。结果与结论:(1)与对照组相比,低、中、高剂量水解南珠液对人微血管内皮细胞均有促增殖作用,且浓度越高,对细胞的促增殖能力越强(P<0.05),因此确定120 mg/L水解南珠液为最佳作用浓度;(2)与对照组相比,水解南珠液组细胞S期和G2期百分比增加,而G1期的细胞比例则减少(P<0.05),提示水解南珠液可以促进血管内皮细胞的周期进展;(3)各组均未发现有细胞核被染成绿色的凋亡细胞;(4)水解南珠液组的迁移细胞数比对照组显著增加(P<0.05);(5)水解南珠液组细胞NO分泌量明显高于对照组(P<0.05),活性氧量显著低于对照组(P<0.05);(6)结果表明,水解南珠液能促进体外培养的人微血管内皮细胞的增殖和迁移,并具有促进其分泌NO及抑制其活性氧分泌的功能,在保护内皮细胞功能方面可能有积极的作用。
BACKGROUND: Pearl has the tranquilizing effect, and it can be applied in the treatment of hypertension. However, there is little report on the prevention and cure effect and mechanism of hydrolyzed pearl liquid on hypertension. OBJECTIVE: To observe the effect of hydrolysis of Hepu pearl(hydrolyzed Nanzhu fluid) on the biological behavior and secretion of human microvascular endothelial cells. METHODS: Human microvascular endothelial cells were cultured and passaged. There were four groups, and the microvascular endothelial cells were incubated in the 200 μL culture medium containing nothing(control group), and 120, 60 and 30 mg/L hydrolysis of Nanzhu fluid. The cell proliferation and migration was detected by cell conuting kit-8 assay and Transwell assay respectively; the cell cycle distribution was tested by flow cytometry; the cell apoptosis was assayed by TUNEL method; the secretion of nitric oxide and reactive oxygen species was tested by nitrale reduetase and chemical fluorescence method, respectively. RESULTS AND CONCLUSION: Compared with the control group, hydrolysis of Nanzhu fluid significantly promoted the proliferation of microvascular endothelial cells in a manner-dependent manner(P < 0.05), suggesting the optimal concentration was 120 mg/L. Compared with the control group, the percentage of cells in S and G2 phase was significantly increased, and the percentage of cells in the G1 phase was significantly reduced in the hydrolysis of Nanzhu fluid group(P < 0.05), indicating hydrolysis of Nanzhu fluid could promote cell cycle progression. Apoptotic cells with green-stained nucleus were invisible in both groups. The number of cell migration in the hydrolysis of Nanzhu fluid group was significantly more than that in the control group(P < 0.05). Compared with the control group, there was a significant increase in the nitric oxide secretion, and a significant decrease in the production of reactive oxygen species in the hydrolysis of Nanzhu fluid group(P < 0.05). To conclude, hydrolysis of Nanzhu fluid can promote the proliferation and migration of human microvascular endothelial cells in vitro, and has the function of promoting the secretion of nitric oxide and inhibiting reactive oxygen species secretion, implying its positive role in the protection of endothelial cell function.
BACKGROUND: Pearl has the tranquilizing effect, and it can be applied in the treatment of hypertension. However, there is little report on the prevention and cure effect and mechanism of hydrolyzed pearl liquid on hypertension. OBJECTIVE: To observe the effect of hydrolysis of Hepu pearl(hydrolyzed Nanzhu fluid) on the biological behavior and secretion of human microvascular endothelial cells. METHODS: Human microvascular endothelial cells were cultured and passaged. There were four groups, and the microvascular endothelial cells were incubated in the 200 μL culture medium containing nothing(control group), and 120, 60 and 30 mg/L hydrolysis of Nanzhu fluid. The cell proliferation and migration was detected by cell conuting kit-8 assay and Transwell assay respectively; the cell cycle distribution was tested by flow cytometry; the cell apoptosis was assayed by TUNEL method; the secretion of nitric oxide and reactive oxygen species was tested by nitrale reduetase and chemical fluorescence method, respectively. RESULTS AND CONCLUSION: Compared with the control group, hydrolysis of Nanzhu fluid significantly promoted the proliferation of microvascular endothelial cells in a manner-dependent manner(P < 0.05), suggesting the optimal concentration was 120 mg/L. Compared with the control group, the percentage of cells in S and G2 phase was significantly increased, and the percentage of cells in the G1 phase was significantly reduced in the hydrolysis of Nanzhu fluid group(P < 0.05), indicating hydrolysis of Nanzhu fluid could promote cell cycle progression. Apoptotic cells with green-stained nucleus were invisible in both groups. The number of cell migration in the hydrolysis of Nanzhu fluid group was significantly more than that in the control group(P < 0.05). Compared with the control group, there was a significant increase in the nitric oxide secretion, and a significant decrease in the production of reactive oxygen species in the hydrolysis of Nanzhu fluid group(P < 0.05). To conclude, hydrolysis of Nanzhu fluid can promote the proliferation and migration of human microvascular endothelial cells in vitro, and has the function of promoting the secretion of nitric oxide and inhibiting reactive oxygen species secretion, implying its positive role in the protection of endothelial cell function.
引文
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[23]Li M,Tejada T,Lambert JP,et al.Angiotensin type 2-receptor(AT2R)activation induces hypotension in apolipoprotein E-deficient mice by activating peroxisome proliferator-activated receptor-γ.Am J Cardiovasc Dis.2016;6(3):118-128.
[24]刘勉.PGE2对NR8383巨噬细胞株促进HUVEC细胞迁移和成管能力调控作用的实验研究[M].南方医科大学,2016.
[25]Parsons J,Horwitz AR,Schwartz MA,et al.Cell adhesion:integrating cytoskeletal dynamics and cellular tension.Nat Rev Mol Cell Biol.2010;11(9):633-643.
[26]罗晓佳,陈晓平.血管内皮细胞损伤与高血压[J].心血管病学进展,2010,31(4):573-577.
[27]Joubert DP,Granados JZ,Oliver JM,et al.An Acute Bout of Aquatic Treadmill Exercise Induces Greater Improvements in Endothelial Function and Post-Exercise Hypotension than Land Treadmill Exercise:A Crossover Study.Am J Phys Med Rehabil.2018.doi:10.1097/PHM.0000000000000923.
[28]Taverne YJ,de Wijs-Meijler D,Te Lintel Hekkert M,et al.Normalization of hemoglobin-based oxygen carrier-201 induced vasoconstriction:targeting nitric oxide and endothelin.J Appl Physiol(1985).2017;122(5):1227-1237.
[29]Ritchie RH,Drummond GR,Sobey CG,et al.The opposing roles of NO and oxidative stress in cardiovascular disease.Pharmacol Res.2017;116:57-69.
[30]Altabas,V.Diabetes,Endothelial Dysfunction,and Vascular Repair:What should a Diabetologist Keep His Eye on?Int J Endocrinol.2015;2015:848272.
[31]Sena CM.Pereira AM,Seica R.Endothelial dysfunction-a major mediator of diabetic vascular disease.Biochim Biophys Acta.2013;1832(12):2216-2231.
[2]龚康敏,郑源庞,胡利平.珍珠粉胶囊治疗中老年高血压90例[J].浙江中医杂志,2000,10:456.
[3]武双平,李彦霞,肖红,等.复方珍珠降压胶囊治疗高血压前期疗效观察[J].上海中医药杂志,2014,48(9):44-46.
[4]赵明文,赵志元,鄢欢.复方珍珠降压胶囊对老年高血压患者的疗效分析[J].深圳中西医结合杂志,2015,25(16):149-150.
[5]马洪田.珍珠降压包合物治疗高血压病的实验研究[M].青岛大学,2013.
[6]蒲月华,邓旗,何锦锋,等.水解珍珠体外抗氧化活性的研究[J].化学研究与应用,2017,29(4):555-559.
[7]邓云云,王笑月,李才广,等.珍珠水解液的美白作用机理研究[J].中国现代中药,2017,19(7):992-994.
[8]Modena MG,Bonetti L,Coppi F,et al.Prognostic role of reversible endothelial dysfunction in hypertensive postmenopausal women.J Am Coll Cardiol.2002;40(3):505-510.
[9]Francesco P,Raffaele M,Giovanni T,et al.Endothelial dysfunction and mild renal insufficiency in essential hypertension.Circulation.2004;110:821-825
[10]Nitenberg A,Chemla D,Antony I.Epicardial coronary artery constriction to cold pressor test is predictive of cardiovascular events in hypertensive patients with angiographically normal coronary arteries and without other majorcoronary risk factor.Atherosclerosis.2004;173(1):115-123.
[11]Wallace SM,Yasmin L,Mc Eniery CM,et al.Isolated systolic hypertension is characterized by increased aortic stiffness and endothelial dysfunction.Hypertension.2007;50:228-233.
[12]Johnson FK,Johnson RA,Peyton KJ,et al.Arginase inhibition restores arteriolar endothelial function in Dahl rats with salt-induced hypertension.Am J Physiol Regul Integr Comp Physiol.2005;288(4):1057-1062.
[13]Spencer CG,Martin SC,Felmeden DC,et al.Relationship of homocysteine to markers of platelet and endothelial activation in"highrisk"hypertensives:a substudy of the Anglo-Scandinavian Cardiac Outcomes Trial.Int JCardiol.2004;94(2-3):293-300.
[14]Nitenberg A.Hypertension,endothelial dysfunction and cardiovascular risk.Arch Mal Coeur Vaiss.2006;99:915-921.
[15]Vinod M AA.Cardiovascular effects of asymmetric dimethy larginine.Circulation.2004;109(25):327.
[16]Modena MG,Bonetti L,Coppi F,et al.Prognostic role of reversible endothelial dysfunction in hypertensive postmenopausal women.J Am Coll Cardiol.2002;40(3):505-510.
[17]Vieira Dias J,Gloaguen C,Kereselidze D,et al.Gamma Low-Dose-Rate Ionizing Radiation Stimulates Adaptive Functional and Molecular Response in Human Aortic Endothelial Cells in a Threshold-,Dose-,and Dose Rate-Dependent Manner.Dose Response.2018;16(1):1559325818755238.
[18]何梦钰,左祥荣,解卫平.内质网应激与血管内皮细胞功能障碍关系的研究进展[J].中国医药导报,2015,12(27):30-34.
[19]马一铭,李丽,蔡红雁,等.体外震波对人脐静脉内皮细胞增殖、细胞周期及细胞间黏附因子-1表达的影响[J].中国循环杂志,2016,31(10):1013-1016.
[20]李悦,杨向红.生长抑制因子4对内皮细胞增殖、迁移及血管形成相关因子表达的影响[J].中国医科大学学报,2017,46(2):160-168.
[21]Knipe RS,Probst CK,Lagares D,et al.The Rho Kinase Isoforms ROCK1 and ROCK2 Each Contribute to the Development of Experimental Pulmonary Fibrosis.Am J Respir Cell Mol Biol.2017.doi:10.1165/rcmb.2017-0075OC.
[22]Sinharoy P,Bratz IN,Sinha S,et al.TRPA1 and TRPV1 contribute to propofol-mediated antagonism of U46619-induced constriction in murine coronary arteries.PLo S One.2017;12(6):e0180106.
[23]Li M,Tejada T,Lambert JP,et al.Angiotensin type 2-receptor(AT2R)activation induces hypotension in apolipoprotein E-deficient mice by activating peroxisome proliferator-activated receptor-γ.Am J Cardiovasc Dis.2016;6(3):118-128.
[24]刘勉.PGE2对NR8383巨噬细胞株促进HUVEC细胞迁移和成管能力调控作用的实验研究[M].南方医科大学,2016.
[25]Parsons J,Horwitz AR,Schwartz MA,et al.Cell adhesion:integrating cytoskeletal dynamics and cellular tension.Nat Rev Mol Cell Biol.2010;11(9):633-643.
[26]罗晓佳,陈晓平.血管内皮细胞损伤与高血压[J].心血管病学进展,2010,31(4):573-577.
[27]Joubert DP,Granados JZ,Oliver JM,et al.An Acute Bout of Aquatic Treadmill Exercise Induces Greater Improvements in Endothelial Function and Post-Exercise Hypotension than Land Treadmill Exercise:A Crossover Study.Am J Phys Med Rehabil.2018.doi:10.1097/PHM.0000000000000923.
[28]Taverne YJ,de Wijs-Meijler D,Te Lintel Hekkert M,et al.Normalization of hemoglobin-based oxygen carrier-201 induced vasoconstriction:targeting nitric oxide and endothelin.J Appl Physiol(1985).2017;122(5):1227-1237.
[29]Ritchie RH,Drummond GR,Sobey CG,et al.The opposing roles of NO and oxidative stress in cardiovascular disease.Pharmacol Res.2017;116:57-69.
[30]Altabas,V.Diabetes,Endothelial Dysfunction,and Vascular Repair:What should a Diabetologist Keep His Eye on?Int J Endocrinol.2015;2015:848272.
[31]Sena CM.Pereira AM,Seica R.Endothelial dysfunction-a major mediator of diabetic vascular disease.Biochim Biophys Acta.2013;1832(12):2216-2231.