肝素对大鼠肾小球系膜细胞和核因子κB的抑制作用
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摘要
目的
在肾小球炎症和硬化过程中，系膜细胞的增殖扮演着重要的角色，通过抑制过度增殖的系膜细胞，有利于防治肾小球炎症的进行性发展。近年来有不少体外、体内试验证实肝素能抑制系膜细胞增殖，减轻肾脏病变。肝素抗增殖的机制包括：肝素能阻断细胞周期进程，抑制细胞内与增殖相关的信号传导，如抑制蛋白激酶C和有丝分裂原活化蛋白激酶的活化，调节血小板衍化生长因子、表皮生长因子等的合成及功能。核因子κB(NF-κB)是一种重要的转录因子，介导细胞内的信号传导，调控多种基因的表达。有研究表明，NF-κB与系膜细胞增殖密切相关。还有学者发现，NF-κB反义寡核苷酸可减少系膜细胞增殖，提示通过抑制NF-κB活化可抑制系膜细胞增殖。我们推测，肝素可能是通过抑制肾小球系膜细胞NF-κB的活化来抑制系膜细胞增殖。本文通过研究肝素对大鼠肾小球系膜细胞NF-κB的作用及其对系膜细胞增殖的影响，旨在探讨肝素抑制系膜细胞增殖的细胞内机制。
方法
1．大鼠肾小球系膜细胞株购自上海长征医院，常规传代培养。
2．四甲基偶氮唑蓝(MTT)方法测定细胞增殖：将系膜细胞接种于96孔板，使其贴壁，多数细胞处于静止期，分组如下：(1)对照组；(2)脂多糖组；(3)肝素组(肝素+脂多糖)：肝素的终浓度分别为3μg／ml、30μg／ml、300μg／ml，每组6个复孔。培养20小时后用MTT法观察各组中系膜细胞的增殖水平。
3．Western Blot法测定NF-κB蛋白p65的表达：将系膜细胞接种于25cm~2的培养瓶中，使其贴壁，多数细胞处于静止期，分三组：(1)对照组；(2)脂多糖组；(3)肝素组(肝素+脂多糖)：肝素的终浓度为300μg／ml，培养24小时后提取核蛋白，用紫外分光光度法测定核蛋白浓度，用Western Blot法测定NF-κB蛋白p65的表达，重复3次，结果用凝胶系统分析软件进行定
量分析。
4.统计学处理
各组数据以均数士标准差(又土s)表示，多组资料间比较采用单因素方
差分析，LSD法多重比较组间差异。以P<0.05判断为差异显著性。所有
数据在SPSS(1 2 .0版本)统计软件包上进行统计处理。
结果
1.四甲基偶氮哇蓝(MTr)方法测定细胞增殖
与对照组相比，脂多糖组明显促进系膜细胞增生(P<0.
组相比，肝素组明显抑制脂多糖诱导的系膜细胞增殖(P<0.
浓度的增加，抑制作用增强(P<0.05)。
2.Westeo Blot法测定NF一KB蛋白颐5的表达
对照组未见NF一KB p65的表达，脂多糖组可明显见到
表达，而肝素组NF一KB师5的表达明显减弱(P<0.05)。
05);与脂多糖
05)，且随肝素
NF一KB p65的
结论
1.肝素能够抑制脂多糖诱导的肾小球系膜细胞增殖，且随肝素浓度的
增加，抑制作用增强。
2.肝素能够抑制肾小球系膜细胞NF一KB两5的活化。
Preface
Glomerular mesangial cells (GMCs) proliferation play significant pathogen-esis roles in the development of glomerular injury. Some findings demonstrared that nuclear factor - kappa B ( NF - KB ) appear to be important in cell proliferation. Recently, there are increasing evidences that suggest that heparin can inhibit GMCs proliferation. We hypothesized that heparin may decrease GMCs proliferation through the inhibition of NF - kB activation. To address this issue, we studied the effects of heparin on GMCs proliferation and activation of NF -KB.
Materials and Methods
1. The rat GMCs were bought from Shanghai Changzheng Hospital and cultured using the methods described by reference.
2. GMCs were cultured in 96 well plates. Three group were established: (1) GMCs group; (2) lipopolysaccharide (LPS) group (GMCs + UPS) ; (3) heparin group (GMCs + LPS + heparin) , in which there are different concentrations of heparin (3 g/ml,30 g/ml ,300 g/ml). GMCs proliferation was detected by MTT at 24hs after cultured.
3. GMCs were cultured in 25 cm2 culture flasks. Three group were established: (1) GMCs group; (2) lipopolysaccharide (LPS) group (GMCs + LPS) ; (3) heparin group (GMCs + LPS + heparin) , the terminal concentrations of heparin is 300 g/ml. After GMCs were cultured 24 hours, a nuclear protein extract was obtained. Immunoblotting was applied to examine the expression of p65 in nuclear.
4. Statistical analysis
Results were expressed as mean SD ( x s ). Comparisions among 3 groups were analyzed by one - way analysis of variance, followed by LSD multiple comparision test to evaluate statistical difference between 2 groups (SPSS12. 0 statistical software) , p <0.05 is considered as significant.
Results
1. Effct of heparin on GMCs proliferation
In this study, heparin was found to inhibit GMCs proliferation in a dose -dependent relationship with the concentration.
2. Effct of heparin on NF - KB acivation
The result of western blot showed that the NF - KB expression was obviously higher in the LPS group than in the GMCs group, while it was obviously lower in the haparin group than that in the LPS group.
Conclusion
1. Heparin can inhibit GMCs proliferation.
2. Heparin can inhibit the activation of NF - KB.
在肾小球炎症和硬化过程中，系膜细胞的增殖扮演着重要的角色，通过抑制过度增殖的系膜细胞，有利于防治肾小球炎症的进行性发展。近年来有不少体外、体内试验证实肝素能抑制系膜细胞增殖，减轻肾脏病变。肝素抗增殖的机制包括：肝素能阻断细胞周期进程，抑制细胞内与增殖相关的信号传导，如抑制蛋白激酶C和有丝分裂原活化蛋白激酶的活化，调节血小板衍化生长因子、表皮生长因子等的合成及功能。核因子κB(NF-κB)是一种重要的转录因子，介导细胞内的信号传导，调控多种基因的表达。有研究表明，NF-κB与系膜细胞增殖密切相关。还有学者发现，NF-κB反义寡核苷酸可减少系膜细胞增殖，提示通过抑制NF-κB活化可抑制系膜细胞增殖。我们推测，肝素可能是通过抑制肾小球系膜细胞NF-κB的活化来抑制系膜细胞增殖。本文通过研究肝素对大鼠肾小球系膜细胞NF-κB的作用及其对系膜细胞增殖的影响，旨在探讨肝素抑制系膜细胞增殖的细胞内机制。
方法
1．大鼠肾小球系膜细胞株购自上海长征医院，常规传代培养。
2．四甲基偶氮唑蓝(MTT)方法测定细胞增殖：将系膜细胞接种于96孔板，使其贴壁，多数细胞处于静止期，分组如下：(1)对照组；(2)脂多糖组；(3)肝素组(肝素+脂多糖)：肝素的终浓度分别为3μg／ml、30μg／ml、300μg／ml，每组6个复孔。培养20小时后用MTT法观察各组中系膜细胞的增殖水平。
3．Western Blot法测定NF-κB蛋白p65的表达：将系膜细胞接种于25cm~2的培养瓶中，使其贴壁，多数细胞处于静止期，分三组：(1)对照组；(2)脂多糖组；(3)肝素组(肝素+脂多糖)：肝素的终浓度为300μg／ml，培养24小时后提取核蛋白，用紫外分光光度法测定核蛋白浓度，用Western Blot法测定NF-κB蛋白p65的表达，重复3次，结果用凝胶系统分析软件进行定
量分析。
4.统计学处理
各组数据以均数士标准差(又土s)表示，多组资料间比较采用单因素方
差分析，LSD法多重比较组间差异。以P<0.05判断为差异显著性。所有
数据在SPSS(1 2 .0版本)统计软件包上进行统计处理。
结果
1.四甲基偶氮哇蓝(MTr)方法测定细胞增殖
与对照组相比，脂多糖组明显促进系膜细胞增生(P<0.
组相比，肝素组明显抑制脂多糖诱导的系膜细胞增殖(P<0.
浓度的增加，抑制作用增强(P<0.05)。
2.Westeo Blot法测定NF一KB蛋白颐5的表达
对照组未见NF一KB p65的表达，脂多糖组可明显见到
表达，而肝素组NF一KB师5的表达明显减弱(P<0.05)。
05);与脂多糖
05)，且随肝素
NF一KB p65的
结论
1.肝素能够抑制脂多糖诱导的肾小球系膜细胞增殖，且随肝素浓度的
增加，抑制作用增强。
2.肝素能够抑制肾小球系膜细胞NF一KB两5的活化。
Preface
Glomerular mesangial cells (GMCs) proliferation play significant pathogen-esis roles in the development of glomerular injury. Some findings demonstrared that nuclear factor - kappa B ( NF - KB ) appear to be important in cell proliferation. Recently, there are increasing evidences that suggest that heparin can inhibit GMCs proliferation. We hypothesized that heparin may decrease GMCs proliferation through the inhibition of NF - kB activation. To address this issue, we studied the effects of heparin on GMCs proliferation and activation of NF -KB.
Materials and Methods
1. The rat GMCs were bought from Shanghai Changzheng Hospital and cultured using the methods described by reference.
2. GMCs were cultured in 96 well plates. Three group were established: (1) GMCs group; (2) lipopolysaccharide (LPS) group (GMCs + UPS) ; (3) heparin group (GMCs + LPS + heparin) , in which there are different concentrations of heparin (3 g/ml,30 g/ml ,300 g/ml). GMCs proliferation was detected by MTT at 24hs after cultured.
3. GMCs were cultured in 25 cm2 culture flasks. Three group were established: (1) GMCs group; (2) lipopolysaccharide (LPS) group (GMCs + LPS) ; (3) heparin group (GMCs + LPS + heparin) , the terminal concentrations of heparin is 300 g/ml. After GMCs were cultured 24 hours, a nuclear protein extract was obtained. Immunoblotting was applied to examine the expression of p65 in nuclear.
4. Statistical analysis
Results were expressed as mean SD ( x s ). Comparisions among 3 groups were analyzed by one - way analysis of variance, followed by LSD multiple comparision test to evaluate statistical difference between 2 groups (SPSS12. 0 statistical software) , p <0.05 is considered as significant.
Results
1. Effct of heparin on GMCs proliferation
In this study, heparin was found to inhibit GMCs proliferation in a dose -dependent relationship with the concentration.
2. Effct of heparin on NF - KB acivation
The result of western blot showed that the NF - KB expression was obviously higher in the LPS group than in the GMCs group, while it was obviously lower in the haparin group than that in the LPS group.
Conclusion
1. Heparin can inhibit GMCs proliferation.
2. Heparin can inhibit the activation of NF - KB.
引文
1. Mezzano S A, Barria M, Droguett MA, et al. Tublar NF- kappaB and AP-1 activation in human proteinuric renal disease. Kidney Int, 2001, 60 (4): 1366- 1377.
2. Mudge SJ, Paizis K, Auwardt RB, et al. Activation of a nuclear factor-kappaB by podocytes in the autologous phase of passive Heymann nephritis. Kidney Int, 2001, 59(3): 923-931.
3. Auwardt RB, Mudge SJ, Chen CG, et al. Regulation of NF - kappaB by corticesteroids in rat mesangial cells. J Am Soc Nephrol, 1998, 9 (9): 1620- 1628.
4. Massy ZA, Guijarro C, O'Donnell MP, et al. The central role of NF- kappaB in mesangial cell activation. Kidney Int, 1999, 56(Suppl 71): S76 -S79.
5. Rovin BH, Dickerson JA, Tan LC, et al. Activation of NF- kappaB correlates with MCP- 1 expression by human mesangial cell. Kidney Int, 1995, 48(4): 1263- 1271.
6. Tomita N, Morishita R, Tomita S, et al. Inhibition of TNF- alpha, induced cytokine and adhesion molecule. Expression in glomerular cells in vitro and in vivo by transcription factor decoy for NF- kappaB. Exp Nephrol, 2001, 9(3): 181-190.
7. Burg M, Ostendorf T, Mooney A, et al. Treatment of experimental mesangioproliferative glomerulonephritis with non- anticoagulant heparin: therapeutic efficacy and safety. Lab Invest, 1997, 76(4): 505-516.
8. Wang ZQ, Liang KH, Pahl MV, et al. Effect of heparin on mesangial cell growth and gene expression of matrix proteins. Nephrol Dial Transplant, 1998, 13(12): 3052 -3057.
9. Edgar S, Patrick M, Mithael M, et al. Rapid detection of octamer binding- proteins with 'mini- extracts', prepared from a small number of cells. Nucl Acid Res, 1989, 17(15): 6419.
10. Striker LJ, Doi T, Elliot S, et al. The contribution of glomerular mesangial
cells to progressive glomerulosclerosis. Semin Nephrol, 1989, 9(4): 318- 328.
11. Couser WG, Johnson RJ. Mechanisms of progressive renal disease in glomerulonphritis. Am J Kid Dis, 1994, 23 (2): 193- 198.
12. Klahr S, Schreiner G, Ichkawa I, et al. The progression of renal disease. N Engl J Med, 1988, 318(25): 1657-1666.
13. Coffey AK, Karnovsky MJ. Heparin inhibits mesangial cell proliferaion in Habu venom- induced glomerular injury. Am J Phathol, 1985, 120 (2): 248- 255.
14. Castellot JJ Jr, Hoover RL, Harper PA, et al. Heparin and glomerular epithelial ceil secreted neparin -like species inhibit mesangial cell proliferation. Am J Pathol, 1985, 120(3): 426-435.
15．王宗谦，周希静．肝素对肾小球系膜细胞和细胞外基质的影响．中华肾脏病学杂志，1996，12(5)：307-308．
16. Groggel GC, Marinides GN, Hovingh P, et al. Inhibition of rat mesangial cell growth by heparin Sulfate. Am J Physiol, 1990, 258: F259 -265.
17. Striker LJ, Peten EP, Elliot SJ, et al. Mesangial ceils turnover: effect of heparin and peptide growth factors. Lab Invest, 1991, 64(4): 446 -456.
18. Tak PP, Firestein GS. NF - κB : a key role in inflammatory disease. J Clin Invest, 2001, 107 (1): 7- 11.
19. Guijarro C, Kim Y, Kasiske BL, et al. Central role of the transcription factor NF- kappaB in mesangial ceil proeluction of chemokeines. Contrib Nephrol, 1997, 120: 210-218.
20. Kumar A, Hawkins KS, Hannan MA, et al. Activation of PKC - beta (Ⅰ) in glomerular mesangial cells is associated with specific NF -kappaB subunit transloeation. Am J Physiol Renal Physiol, 2001, 281 (4): F613-619.
21. Park CW, Kim JH, Lee JH, et al., High glucose- induced intercellular adhesion molecule- 1 expression through an osmotic effect in rat mesangial cells is PKC- NF- κB -dependent. Diabetologia, 2000, 43 (12): 1544 - 1553.
22. Ogata N, Yamamoto H, Kugiyama K, et al. Involvement of protein kinase
C in superoxide anion -induced activation of nuclear factor - kappa B in human endothelial ceils. Cardiovasc Res, 2000, 45 (2): 513 - 521.
23. Thourani VH, Brar SS, Kennedy TP, et al. Nonanticoagulant heparin inhibits NF- kappaB activation and attenuates myocardial reperfusion injury. Am J Physiol Heart Circ Physiol, 2000, 48: H2084—2093.
2. Mudge SJ, Paizis K, Auwardt RB, et al. Activation of a nuclear factor-kappaB by podocytes in the autologous phase of passive Heymann nephritis. Kidney Int, 2001, 59(3): 923-931.
3. Auwardt RB, Mudge SJ, Chen CG, et al. Regulation of NF - kappaB by corticesteroids in rat mesangial cells. J Am Soc Nephrol, 1998, 9 (9): 1620- 1628.
4. Massy ZA, Guijarro C, O'Donnell MP, et al. The central role of NF- kappaB in mesangial cell activation. Kidney Int, 1999, 56(Suppl 71): S76 -S79.
5. Rovin BH, Dickerson JA, Tan LC, et al. Activation of NF- kappaB correlates with MCP- 1 expression by human mesangial cell. Kidney Int, 1995, 48(4): 1263- 1271.
6. Tomita N, Morishita R, Tomita S, et al. Inhibition of TNF- alpha, induced cytokine and adhesion molecule. Expression in glomerular cells in vitro and in vivo by transcription factor decoy for NF- kappaB. Exp Nephrol, 2001, 9(3): 181-190.
7. Burg M, Ostendorf T, Mooney A, et al. Treatment of experimental mesangioproliferative glomerulonephritis with non- anticoagulant heparin: therapeutic efficacy and safety. Lab Invest, 1997, 76(4): 505-516.
8. Wang ZQ, Liang KH, Pahl MV, et al. Effect of heparin on mesangial cell growth and gene expression of matrix proteins. Nephrol Dial Transplant, 1998, 13(12): 3052 -3057.
9. Edgar S, Patrick M, Mithael M, et al. Rapid detection of octamer binding- proteins with 'mini- extracts', prepared from a small number of cells. Nucl Acid Res, 1989, 17(15): 6419.
10. Striker LJ, Doi T, Elliot S, et al. The contribution of glomerular mesangial
cells to progressive glomerulosclerosis. Semin Nephrol, 1989, 9(4): 318- 328.
11. Couser WG, Johnson RJ. Mechanisms of progressive renal disease in glomerulonphritis. Am J Kid Dis, 1994, 23 (2): 193- 198.
12. Klahr S, Schreiner G, Ichkawa I, et al. The progression of renal disease. N Engl J Med, 1988, 318(25): 1657-1666.
13. Coffey AK, Karnovsky MJ. Heparin inhibits mesangial cell proliferaion in Habu venom- induced glomerular injury. Am J Phathol, 1985, 120 (2): 248- 255.
14. Castellot JJ Jr, Hoover RL, Harper PA, et al. Heparin and glomerular epithelial ceil secreted neparin -like species inhibit mesangial cell proliferation. Am J Pathol, 1985, 120(3): 426-435.
15．王宗谦，周希静．肝素对肾小球系膜细胞和细胞外基质的影响．中华肾脏病学杂志，1996，12(5)：307-308．
16. Groggel GC, Marinides GN, Hovingh P, et al. Inhibition of rat mesangial cell growth by heparin Sulfate. Am J Physiol, 1990, 258: F259 -265.
17. Striker LJ, Peten EP, Elliot SJ, et al. Mesangial ceils turnover: effect of heparin and peptide growth factors. Lab Invest, 1991, 64(4): 446 -456.
18. Tak PP, Firestein GS. NF - κB : a key role in inflammatory disease. J Clin Invest, 2001, 107 (1): 7- 11.
19. Guijarro C, Kim Y, Kasiske BL, et al. Central role of the transcription factor NF- kappaB in mesangial ceil proeluction of chemokeines. Contrib Nephrol, 1997, 120: 210-218.
20. Kumar A, Hawkins KS, Hannan MA, et al. Activation of PKC - beta (Ⅰ) in glomerular mesangial cells is associated with specific NF -kappaB subunit transloeation. Am J Physiol Renal Physiol, 2001, 281 (4): F613-619.
21. Park CW, Kim JH, Lee JH, et al., High glucose- induced intercellular adhesion molecule- 1 expression through an osmotic effect in rat mesangial cells is PKC- NF- κB -dependent. Diabetologia, 2000, 43 (12): 1544 - 1553.
22. Ogata N, Yamamoto H, Kugiyama K, et al. Involvement of protein kinase
C in superoxide anion -induced activation of nuclear factor - kappa B in human endothelial ceils. Cardiovasc Res, 2000, 45 (2): 513 - 521.
23. Thourani VH, Brar SS, Kennedy TP, et al. Nonanticoagulant heparin inhibits NF- kappaB activation and attenuates myocardial reperfusion injury. Am J Physiol Heart Circ Physiol, 2000, 48: H2084—2093.
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