摘要
背景与目的:异位钙化即非骨组织发生矿物质沉积,主要是钙盐沉积,血管组织钙化是其中的一种,分为中膜钙化和内膜钙化。后者与动脉粥样硬化密切相关,早期参与的细胞主要是巨噬细胞和肥大细胞。硫化氢在心血管系统中具有重要的生理与病理作用,本实验拟利用L-半胱氨酸作为硫化氢的内源性供体,在β-甘油磷酸钠(β-sodium glycerophosphate,β-GP)和氧化低密度脂蛋白(oxidized low density lipoprotein,oxLDL)诱导的巨噬细胞钙盐沉积的细胞模型上研究内源性硫化氢对巨噬细胞吞噬脂质过程中钙化和骨形成蛋白(Bone morphogenetic protein,BMP)表达的影响及其机制,为进一步探讨其对内膜钙化的作用提供实验依据。
方法:
实验一钙化细胞模型的建立
小鼠单核巨噬细胞RAW264.7用5mmol/L的β-GP和40mg/mL的oxLDL孵育3天,von Kossa染色形态学观察细胞钙盐沉积,原子吸收分光光度法测定细胞钙含量。
实验二不同浓度L-半胱氨酸对细胞钙化和BMP表达的影响
1、正常对照组:含10%胎牛血清RPMI-1640培养基。
2、钙化组:40mg/mL oxLDL和5mmol/L的β-GP培养基孵育细胞。
3、不同浓度L-半胱氨酸组(50,100,200mmol/L):钙化组细胞培养基的基础上加入相应浓度L-半胱氨酸(50,100,200mmol/L),孵育细胞4天。
实验三L-半胱氨酸处理不同时间对细胞钙化和BMP表达的影响
1、正常对照组:含10%胎牛血清RPMI-1640培养基。
2、不同时间处理组(0,2,4,6d):最佳浓度L-半胱氨酸处理钙化组细胞不同时间(0,2,4,6d)。
实验四PPG对细胞钙化和BMP表达的影响
1、正常对照组:10%胎牛血清RPMI-1640培养基。
2、不同浓度PPG处理钙化细胞组(0,2,4,8mmol/L):不同浓度PPG孵育钙化细胞组12h。
结果:
钙化组细胞von Kossa染色见细胞内有大量褐色颗粒聚集,钙含量高于对照组2.1倍(P<0.01), BMP mRNA和蛋白表达分别是正常对照组的1.6倍和1.8倍(P<0.05),L-半胱氨酸组较钙化组细胞,细胞von Kossa染色褐色颗粒减少,钙含量降低,BMP蛋白和mRNA表达也呈下降趋势,特别是50mmol/L的L-半胱氨酸组作用最显著von Kossa染色几乎未见褐色颗粒,其BMP mRNA和蛋白表达低于钙化组1.6倍和2.2倍(P<0.05),接近正常对照组。PPG组结果相反,即随着PPG浓度的升高,细胞钙化程度加重,von Kossa染色可见大量褐色颗粒,BMP mRNA和蛋白的表达也随浓度升高而增加。
结论:
1、内源性硫化氢可以减少巨噬细胞吞噬脂质过程中的细胞内钙含量和钙沉积;
2、内源性硫化氢可以下调巨噬细胞吞噬脂质过程中BMP的表达水平。
Objective:
Heterotopic calcification, namely, the mineral deposition occurred in the nonosteonal tissue, mainly refers to the deposition of calcium salt, and one kind, the calcification of vascular tissue, is classified into tunica medial calcification, and tunica intimal calcification. The tunica intimal calcification is closely relevant to artherosclerosis, and its early involvements primarily include macrophages and clasmatoblast. The solfurated hydrogen possesses important physiological action and pathological action of cardiovascular. With L-cysteine as the endogenous donor of solfurated hydrogen, based on the model of calcium salt deposition in macrophages which is guided byβ-GP and oxLDL, this research attempts to study the effects of endogenous sulfureted hydrogen on calcification during macrophages lipid loading and the factors behind them, providing some experimental evidence for further exploration of its effects on the tunica intimal calcification.
Methods:
Expriment 1: Establish the model of the calcification: Incubate RAW264.7 with 40mg/ml oxLDL and 5mmol/Lβ-GP for three days; obseve calcium deposition by von Kossa staining and determine cumulation content by atomic absorption spectrophotometry.
Expriment 2: Effect on the cell calcification of endogenous sulfureted hydrogen with different concentration:
1.Control group: cell incubated in RPMI -1640 with 10% tetal caif serum .
2.Calfication and 0mmol/L L-lysine groups:cell incubated with oxLDL,β-GP and 0mmol/L L-lysine.
3. Calfication and 50mmol/L L-lysine groups:cell incubarted with oxLDL,β-GP and 50mmol/L L-lysine; calfication and 100mmol/L L-lysine groups:cell incubarted with oxLDL,β-GP and 100mmol/L L-lysine; calfication and 200mmol/L L-lysine groups:cell incubarted with oxLDL,β-GP and 200mmol/L L-lysine.
Expriment 3: Effect of endogenous sulfureted hydrogen on the cell calcification in different periods of time:
1. Control group: cell incubated in RPMI -1640 with 10% tetal calf serum.
2. 0d :cell incubated with oxLDL,β-GP and bestL-lysine contration for 0 day; 2d: cell incubated with cell were incubated with oxLDL,β-GP and best L-lysine contration for 2 days; 4d: cell incubated with oxLDL,β-GP and best L-lysine contration for 4 days; 6d: cell incubated with oxLDL,β-GP and best L-lysine contration for 6 days.
Expriment 4: Effects of PPG on cell calcification:
RAW264.7 was incubated with oxLD,β-GP and PPG at different concentrations(0,2,4,8mmol/L) for 4 days.then we detected calcium content, calcium deposition and OPN mRNA and protein eapression were determined by RT-PCR and Westen blot, respectively. Raw264.7 were divided into five groups as followings:1) control group:cell incubated in RPMI -1640 with 10% tetal caif serum . 2) calfication and 0mmol/L PPG group:cell incubarted with oxLDL,β-GP and 0mmol/L PPG. 3) calfication and 2 mmol/L PPG group:cell incubarted with oxLDL,β-GP and 2mmol/L PPG. 4) calfication and 4mmol/L PPG group:cell incubated with oxLDL,β-GP and5 mmol/LPPG. 5) calfication and 8mmol/L PPG group:cell incubarted with oxLDL,β-GP and 8mmol/L PPG.
Results:
Compared with the control group, macrophages incubated with oxLDl andβ-glicerophosplale evidently showed a numbers of black particles, oxLDl andβ-glicerophosplale caused recrease the content of calium by 210%, and they also can recrease BMP mRNA and protein expression by 180% and 160% respectively. Compared with the calcification group, calcification macrophages incubated with L-cysteine macrophages showed less black particles,and also the contend of the calium. BMP mRNA and protein expression decreased too.expecially at the 50mmol/L level. L-cysteine caused a deceased BMP mRNA and protein expression by 161% and 220% respectively compared with calcification cell. Calcification cell incubated with PPG showed the opposite results compared with the cell incubated with L-cysteine..
Conclusion:
1.Endogenous sulfureted hydrogen can decrease the calcium content and calcium deposition during the macrophages lipid loading.
2.Endogenous sulfureted hydrogen can down-regulate BMP expression during the macrophages lipid loading.
引文
[1] Hunt JL, Fairman R, Mitchell ME, Carpenter JP, Golden M, Khalapyan T, Wolfe M, Neschis D, Milner R, Scoll B, Cusack A, Mohler ER 3rd. Bone formation in carotid plaques: a clinicopathological study [J]. Stroke. 2002,33:1214–1219.
[2] Mohler ER 3rd, Gannon F, Reynolds C, Zimmerman R, Keane MG,Kaplan FS. Bone formation and inflammation in cardiac valves [J]. Circulation, 2001, 103: 1522–1528.
[3] Kodo O, Sabokbar A, Itonaga I, et al. Interleukin6 and interleukin11 support human osteoclast formation by a RANKL independent mechanism[J].Bone, 2003,32(1):1-7.
[4] Kobayashi K, Takahashi N, Jimi E, et al. Tumor necrosis factors alpha stimulates osteoclast differentiation by a mechanism independent of the ODF/RANKLRANK interaction[J]. J Exp Med, 2000,191(2):275-286.
[5] Doeller JE, Isbell TS, Benavides G, Koenitzer J, Patel H, Patel RP, Lancaster JR Jr: Polarographic measurement of hydrogen sulfide production and consumption by mammalian tissues[J]. Anal Biochem, 2005, 341, 40–51.
[6] Kashiba M, Kajimura M, Goda N,et al. From O2 to H2S:a landscape view of gas biology[J]. Keio J Med, 2002,51(1):1-10.
[7] Khan AA, Schuler MM, Prior MG,et al. Effects of hydrogen sulfide exposure on lung mitochondrial respiratory chain enzymes in rats[J].Toxicol Appl Pharmacol, 1990,103(3):482-490.
[8] Hosok R, Matsuki N, Kimura H. The possible role of hydrogen sulfide as an endogenous smooth muscle relaxant in synergy with nitric oxide[J]. Biochem Biophys Res Commun, 1997,237(3):527-531.
[9] Zhao W, Zhang J, Lu Y,et al. The vasorelaxant effect of H2S as a novel endogenous gaseous K(ATP) channel opener[J].The EMBO Journal, 2001,20: 6008-6016.
[10] Geng B, Yang J, Qi Y, et al. H2S generated by heart in rat and its effects on cardiac function[J]. Biochem Biophy Res Commun, 2004,313(2):362-368.
[11] Oh GS, Pae HO, Lee BS, et al. Hydrogen sulfide inhibits nitric oxide production and nuclear factor-κB via heme oxygenase-1 expression in RAW264.7 macrophagesstimulated with lipopolysaccharide[J]. Radic Bio Med, 2006,41(1):106-119.
[12] Wang R.Two's company,three's a crowd:can H2S be the third endogenous gaseous transmitter? FASEB J, 2002,16(13):1792-1798.
[13] Zhong G, Chen F, Cheng Y, et al. The role of hydrogen sulfide generation in the pathogenesis of hypertension in rats induced by inhibition of nitric oxide synthase[J]. Hypertens, 2003,21(10):1879-1885.
[14] Geng B, Yang J, Qi Y, et al.H2S generated by heart in rat and its effects on cardiac function[J].Biochem Biophys Res Commun, 2004,313(2): 362-370.
[15] Geng B, Chang L, Pan C, et al.Endogenous hydrogen sulfide regulation of myocardial injury induced by isoproterenol[J].Biochem Biophys Res Commun,2004,318(3):756-63.
[16] Zhu YZ, Wang ZJ, Ho P, et al.Hydrogen sulfide and its possible roles in myocardial ischemia in experimental rats[J].J Appl Physiol, 2007, 102(1):261-269.
[17] Bian JS, Yong QC, Pan TT, et al.Role of hydrogen sulfide in the cardioprotection caused by ischemic preconditioning in the rat heart and cardiac myocytes[J].J Pharmacol Exp Ther,2006,316(2):670-678.
[18] Hsu HH, Artigues A, Villar MT. Induction of calcification by serum depletion in cell culture: a model for focal calcification in aortas related to atherosclerosis [J]. Lipids Health Dis, 2008, 7: 2.
[19] Hsu JJ, Tintut Y, Demer LL. Murine Models of Atherosclerotic calcification[J]. Current Drug Targets, 2008, 9(3): 224-228.
[20] Schmermund A, M?hlenkamp S, Erbel R. The latest on the calcium story[J]. Am J Cardiol.,2002, 90(10C): 12L-14L.
[21] Shioi A, Mori K, Jono S et al. Mechanism of atherosclerotic calcification[J]. Z Kardiol, 2000, 89(2): 75-84.
[22] Bobryshev YV, Killingsworth MC, Lord RS et al. Matrix vesicles in the fibrous cap of atherosclerotic plaque:Possible contribution to plaque rupture[J]. J Cell Mol Med, 2008,4:67-70.
[23] Fischer JW, Steitz SA, Johnson PY et al. Decorin Promotes Aortic Smooth Muscle Cell Calcification and Colocalizes to calcified Regions in Human AtheroscleroticLesions[J]. Arterioscler. Arterioscler Thromb Vasc Biol. 2004, 24(12): 391-396.
[24] Budoff MJ, Achenbach S, Blumenthal RS et al. Assessment of coronary artery disease by cardiac computed tomography: a scientific statement from the American Heart Association Committee on Cardiovascular Imaging and Intervention, Council on Cardiovascular Radiology and Intervention, and Committee on Cardiac Imaging, Council on Clinical Cardiology [J]. Circulation, 2006, 114(16): 1761-1791.
[25] Bobryshev YV.Transdifferentiation of smooth muscle cells in atherosclerotic arteries in situ:implications for diffuse intimal calcification[J]. J Pathol,2005,205(5):641-650.
[26] Bennett BJ, Scatena M, Kirk EA et al. Osteoprotegerin Inactivation Accelerates Advanced Atherosclerotic Lesion Progression and Calcification in Older ApoE–/– Mice[J]. Arterioscler Thromb Vasc Biol. 2006, 26(9): 2117-2124.
[27] Tintut Y, Demer L. Role of osteoprotegerin and its ligands and competing receptors in atherosclerotic calcification[J].J Investig Med,2006,54(7): 395-401.
[28] Mathew S, Tustison KS, Sugatani T et al. The mechanism of phosphorus as a cardiovascular risk factor in CKD[J].J Am Soc Nephrol.2008,19(6): 1092-1105.
[29] Byon CH, Javed A, Dai Q et al. Oxidative Stress Induces Vascular Calcification through Modulation of the Osteogenic Transcription Factor Runx2 by AKT Signaling[J]. J Biol Chem,2008, 283(22): 15319-15327.
[30] Li JJ, Zhu CG, Yu B et al. The role of inflammation in coronary artery calcification[J]. Ageing Res Rev, 2007, 6(4): 263-270.
[31] Bear M, Butcher M, Shaughnessy SG. Oxidized low-density lipoprotein acts synergistically with beta-glycerophosphate to induce osteoblast differentiation in primary cultures of vascular smooth muscle cells[J]. J Cell Biochem,2008,105(1):185-193.
[32] Chen NX,Duan D,O'Noill KD et al. High glucose increases the expression of Cbfal and BMP-2 and enhances the calcification of vascular smooth muscle cells[J]. Nephrology Dialysis Transplantion, 2006, 21(12): 3435-3442.
[33]翟永志,晏沐阳.动脉粥样硬化钙化与易损斑块[J].心血管康复杂志,2007,16(6):598-600.
[34] G?ssl M, Versari D, Hildebrandt H et al.Vulnerable plaque: detection and management[J]. Med Clin North Am, 2007, 91(4): 573-601.
[35] Montecucco F, Steffens S, Mach F.The immune response is involved in atherosclerotic plaque calcification: could the RANKL/RANK/OPG system be a marker of plaque instability?[J]. Clin Dev Immunol, 2007, 2007: 75-85.
[36] Nishikawa K,Kamezawa Y,et al. Time Course of Osteopontin, Osteocalcin, and Osteonectin accumulation and Calcification after Acute Vessel Wall Injury. Histochemistry and Cytochemistry [J].Circ Res, 2001, 49(1):79–86.
[37] Isoda K, Nishikawa K,Kamezawa Y, et al. Osteopontin plays an important role in the development of medial thickening and neointimal formation[J].Circ Res,2002,91:77-82.
[38] Mori S,Takemoto M, Yo kote K,et al. Hyperglycemia induced alteration of vascular smooth muscle phenotype. [J]Diabetes Complications, 2002,16:65-68.
[39] Khan SA,Lopez2Chua CA,Zhang J,et al.Soluble osteopontin inhibit s apoptosis of adherent endothelial cells deprived of growth factors[J].Cell Biochem,2002,85:728-736.
[40] Li G, Oparil S, Kelp ke SS.et al. Fibroblast growth factor receptor signaling induces osteopontin expression and vascular smooth muscle cell-dependent adventitial fibroblast migration in vitro [J].Circulation,2002,13,106:854-859.
[41]江海龙,吴宏超,李志梁,等.冠心病患者血浆中新型气体信号分子硫化氢的变化[J].第一军医大学学报,2005,25:951-954.
[42] Tzoulaki I, MurrayG, LeeA,etal. C-reactive protein, interleukin-6, and soluble adhesion molecules as predictors of progressive peripheral atherosclerosis in the general population: Edinburgh Artery Study[J]. Circulation, 2005, 112: 976-983.
[43]张春雨,杜军保,卜定芳,等.低氧性肺动脉高压大鼠肺组织硫化氢的变化[J].基础医学与临床,2004,24:145-148.
[44]王燕飞,唐朝枢,杜军保,硫化氢抑制apoE基因敲除小鼠动脉粥样硬化中ICAM-1的表达.基础医学与临床,2009,29(1):19-23.
[45] Moeen Abedin, Yin Tintut and Linda L. Demer, Vascular Calcification:Mechanisms and Clinical Ramifications[J]. Arterioscler. Thromb. Vasc. Biol, 2004,24:1161-1170.
[46] Anuar F, Whiteman M, Siau JL, Kwong SE, Bhatia M,Moore PK: Nitric oxide-releasing flurbiprofen reduces formation of proinflammatory hydrogen sulfide in lipopolysaccharide-treated rat[J]. Br J Pharmacol, 2006,147, 966–974.
[47] Distrutti E, Sediari L, Mencarelli A, Renga B, Orlandi S,Russo G, CaliendoG:5-Amino-2-hydroxybenzoicacid4-(5-thioxo-5H-[1,2]dithiol-3yl)-phenyl-ester(ATB-429), a hydrogen sulfide-releasing derivative of mesalamine, exerts anti nociceptive effects in a model of postinflammatory hypersensitivity [J]. JPharmacol Exp ther, 2006, 319: 447–458.
[1] Reiffenstein RJ , Hulbert WC ,Roth SH. Toxicology of hydrogen sul- fide [J ] . Annu Rev Pharmacol Toxicol ,1992 ,32 :1092134.
[2] Chen P , Poddar R , Tipa EV , et al . Homocysteine metabolism in cardiovascular cells and tissues : implications for hyperhomocysteinemia and cardiovascular disease [J ] . Adv Enzyme Regul ,1999 ,39 :932109.
[3] Bao L , Vlcek C , Paces V , et al . Identification and tissue distribution of human cystathionine beta-synthase mRNA isoforms[J ] . Arch Biochem Biophys ,1998 ,350 :952103.
[4] Hosoki R , Matsiki N , Kimura H. The possible role of hydrogen sulfide as an endogenous smooth muscle relaxant in synergy with nitric oxide[J] .Biochem BiophysicRes Commun ,1997 ,237 :5272531.
[5] Zhao W, Zhang J , Lu Y, et al . The vasorelaxant effect of H2S as a novel endogenous gaseous KATP channel opener [J ] . EMBO J ,2001 ,20 :600826016.
[6]Wang R. Two’s company , three’s a crowd : can H2S be the third endogenous gaseous transmitter [ J ] FASEB J , 2002 , 16 : 1792-1798.
[7]Kredich NM, Foote LJ , Keenen BS. The stoichiometry and kinetics of the inducible cysteine desulfhydrase from Salmonella typhimurium [J ] . J Biol Chem,1973 ,248 :618726197.
[8]Budoff et al. Assessment of coronary artery disease by cardiaccomputed tomography:a scientific statement from the American heart association committee on cardiovascular imaging and intervention[J]. Circulation, 2006, 114(16): 1761-1791.
[9]Bobryshev YV, Transdifferentiation of smooth muscle cells in atherosclerotic arteries in situ:implications for diffuse intimal calcification[J]. J Pathol,2005,205(5):641-691.
[10]Shioi A. Mechanism of atherosclerotic calcification[J]. Z Kardiol, 2000, 89(2): 75-84.
[11]Bobryshev YV et al. Matrix vesicles in the fibrous cap of atherosclerotic plaque:Possible contribution to plaque rupture[J]. J Cell Mol Med, 2007, 9: 35-60.
[12]Jens W et al. Decorin Promotes Aortic Smooth Muscle Cell Calcification and Colocalizes to calcified Regions in Human Atherosclerotic Lesions[J]. Arterioscler. Thromb. Vasc. Biol. 2004, 24: 2391-2396.
[13]Brian J et al. Osteoprotegerin Inactivation Accelerates Advanced Atherosclerotic Lesion Progression and Calcification in Older ApoE–/– Mice[J]. Thromb. Vasc. Biol. 2006, 26: 2117-2124.
[14]Emer L et al. Role of osteoprotegerin and its ligands and competing receptors in atherosclerotic calcification[J]. J Investig Med, 2006, 54(7): 395-401.
[15]Mathew S et al. The mechanism of phosphorus as a cardiovascular risk factor in CKD[J]. J Am Soc Nephrol. 2008, 19(6): 1092-1197.
[16]Tintut Y et al. Oxidative Stress Induces Vascular Calcification through Modulation of the Osteogenic Transcription Factor Runx2 by AKT Signaling[J]. J Biol Chem, 2008, 283(22): 15319-15327.
[17]Li,J et al. The role of inflammation in coronary artery ca;cification. Ageing Res Rev, 2007, 6(4): 263-270.
[18]Bear M et al. Oxidized low-density lipoprotein acts synergistically with beta-glycerophosphate to induce osteoblast differentiation in primary cultures of vascular smooth muscle cells[J]. J Cell Biochem,2008,1097-4644.
[19]Chen NX et al. High glucose increases the expression of Cbfal and BMP-2 and enhances the calcification of vascular smooth muscle cells[J]. Nephrology Dialysis Transplantion, 2006, 21(12): 3435-3442.
[20]翟永志等,动脉粥样硬化钙化与易损斑块[J].心血管康复杂志,2007,16(6):598-600.
[21]Versari D et al. Vulnerable plaque: detection and management[J]. Med Clin North Am,2007, 91(4): 573-601.
[22]Montecucco F et al. The immune response is involved in atherosclerotic plaque calcification: could the RANKL/RANK/OPG system be a marker of plaque instability?[J] Clin Immunol. 2007, 2007: 75805.
[23]Hsu H et al. Induction of calcification by serum depletion in cell culture: a model for focal calcification in aortas related to atherosclerosis[J]. Lipids Health Dis, 2008, 7: 2.
[24]Jeffrey J et al. Hsu1, Murine Models of Atherosclerotic Calcification. Current Drug Targets, 2008, 9: 224-228.
[25]Schmermund A et al, The latest on the calcium story[J]. Am J Cardiol. 2002, 90(10C): 12-14.