肾康注射液对高糖培养肾小球系膜细胞周期异常的调节作用
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摘要
目的：观察中药复方制剂肾康注射液对高糖培养大鼠肾小球系膜细胞周期异常的调节作用及其机制，探讨肾康注射液对早期DN的防治作用。
方法：将体外培养的正常大鼠肾小球系膜细胞分为7组，分别为正常组、高糖组、甘露醇组、苯那普利组和不同剂量肾康注射液治疗组，每组设3个复孔，共培养72小时。应用流式细胞术观察细胞周期分布、RNA/DNA比值、细胞大小；ELISA法测定培养细胞上清液中FN的含量；RT-PCR检测各组细胞TGF-β_1mRNA、P~(21)mRNA的表达。
结果：(1)高糖培养肾小球系膜细胞出现细胞周期分布异常，G_1期细胞比例增高，G_2期、S期比例下降，RNA/DNA比值增高，以及细胞体积增大；肾康注射液高剂量组可以使细胞周期分布、RNA/DNA比值趋于正常，并能抑制细胞体积增大。(2)高糖培养系膜细胞FN合成及分泌增多，肾康注射液高剂量组能抑制FN的过度合成及分泌。(3)高糖培养系膜细胞TGF-β_1mRNA、P~(21)mRNA表达增加，肾康注射液可以抑制TGF-β ImRNA、P~(21)mRNA过度表达，高剂量组作用优于苯那普利。
结论：(1)高糖培养肾小球系膜细胞出现细胞周期异常改变，表现为细胞停滞在G_1期，细胞肥大，FN合成及分泌增多；肾康注射液可以改善细胞周期异常改变，促使细胞周期正常运转，肥大减轻，FN异常合成及分泌减少。(2)肾康注射液在转录水平影响系膜细胞TGF-β_1以及P~(21)合成，从而促使细胞周期正常运转，可能是肾康注射液调节高糖培养肾小球系膜细胞周期异常的重要机理。
Objective: To investigate effects and mechanisms of Shenkang Injection, a compound TCM preparation, on abnormal cell cycle of glomerular mesangial cells cultured in high glucose and explore the effects of Shenkang Injection to prevent and treat diabetic nephropathy.
Methods: Cultured rat glomerular mesangial cells were diviced into 7 groups: comtrol group, High Glucose-treated group, Mannitol-treated group, Benazepril-treated group, Shenkang Injection-treated groups with different dose. Every group was trested for 72 hours. The cell cycle , RNA/DNA and cells vovulme of glomerular mesangial cells were measured by flow cytometry. The level of fibronectin in the cultured supernatant was measured by ELISA . The expression of TGF- 1 mRNA and P21 mRNA were examined by RT-PCR.
Results: (1) High glucose caused the abnormal cell cycle of glomerular mesangial cells as follows: cells proportion in Gl phase increased, cell proportion in G2 and S phase detreased, RNA/DNA up-regulated, cellular size enlarged. The high-dose Shenkang Injection could decrease cells proportion in phase Gl, increase that in phase G2 and S, and reduce RNA/DNA and cellular size. (2) High glucose increase the level of FN in glumerular measangial cella. The High-dose Shenkang Injection could ruduce the level
of FN. (3)High glucose increased the expression of TGF-31mRNA and P21mRNA , Shenkang Injection could inhibit the overexpression of TGF- PlmRNA and P21mRNA induced by high glucose. High-dose The effect of Shenkang Injection is superio to Benazepril.
Conclusion: (1) The abnormal cell cycle of high-glucose cultured glomerular mesangial cells presented retention of cell division at Gl phase, RNA/DNA increase, cells hypertrophy, FN synthesis and secretion increase; Shenkang Injection was able to improve abnormal cell cycle and regulate cell proportion of different phases. In addition, it could reduce hypertrophy of cells, FN synthesis and secretion. (2) TGF- PlmRNA and P21mRNA synthesis of mesangial cells were regulated at transcription level by Shengkang Injection. Consequently, normal cell cycle, were obtained. This may be the important regulative mechanism of Shenkang Injection on abnormal cell cycle of glomerular mesangial cells in high glucose.
方法：将体外培养的正常大鼠肾小球系膜细胞分为7组，分别为正常组、高糖组、甘露醇组、苯那普利组和不同剂量肾康注射液治疗组，每组设3个复孔，共培养72小时。应用流式细胞术观察细胞周期分布、RNA/DNA比值、细胞大小；ELISA法测定培养细胞上清液中FN的含量；RT-PCR检测各组细胞TGF-β_1mRNA、P~(21)mRNA的表达。
结果：(1)高糖培养肾小球系膜细胞出现细胞周期分布异常，G_1期细胞比例增高，G_2期、S期比例下降，RNA/DNA比值增高，以及细胞体积增大；肾康注射液高剂量组可以使细胞周期分布、RNA/DNA比值趋于正常，并能抑制细胞体积增大。(2)高糖培养系膜细胞FN合成及分泌增多，肾康注射液高剂量组能抑制FN的过度合成及分泌。(3)高糖培养系膜细胞TGF-β_1mRNA、P~(21)mRNA表达增加，肾康注射液可以抑制TGF-β ImRNA、P~(21)mRNA过度表达，高剂量组作用优于苯那普利。
结论：(1)高糖培养肾小球系膜细胞出现细胞周期异常改变，表现为细胞停滞在G_1期，细胞肥大，FN合成及分泌增多；肾康注射液可以改善细胞周期异常改变，促使细胞周期正常运转，肥大减轻，FN异常合成及分泌减少。(2)肾康注射液在转录水平影响系膜细胞TGF-β_1以及P~(21)合成，从而促使细胞周期正常运转，可能是肾康注射液调节高糖培养肾小球系膜细胞周期异常的重要机理。
Objective: To investigate effects and mechanisms of Shenkang Injection, a compound TCM preparation, on abnormal cell cycle of glomerular mesangial cells cultured in high glucose and explore the effects of Shenkang Injection to prevent and treat diabetic nephropathy.
Methods: Cultured rat glomerular mesangial cells were diviced into 7 groups: comtrol group, High Glucose-treated group, Mannitol-treated group, Benazepril-treated group, Shenkang Injection-treated groups with different dose. Every group was trested for 72 hours. The cell cycle , RNA/DNA and cells vovulme of glomerular mesangial cells were measured by flow cytometry. The level of fibronectin in the cultured supernatant was measured by ELISA . The expression of TGF- 1 mRNA and P21 mRNA were examined by RT-PCR.
Results: (1) High glucose caused the abnormal cell cycle of glomerular mesangial cells as follows: cells proportion in Gl phase increased, cell proportion in G2 and S phase detreased, RNA/DNA up-regulated, cellular size enlarged. The high-dose Shenkang Injection could decrease cells proportion in phase Gl, increase that in phase G2 and S, and reduce RNA/DNA and cellular size. (2) High glucose increase the level of FN in glumerular measangial cella. The High-dose Shenkang Injection could ruduce the level
of FN. (3)High glucose increased the expression of TGF-31mRNA and P21mRNA , Shenkang Injection could inhibit the overexpression of TGF- PlmRNA and P21mRNA induced by high glucose. High-dose The effect of Shenkang Injection is superio to Benazepril.
Conclusion: (1) The abnormal cell cycle of high-glucose cultured glomerular mesangial cells presented retention of cell division at Gl phase, RNA/DNA increase, cells hypertrophy, FN synthesis and secretion increase; Shenkang Injection was able to improve abnormal cell cycle and regulate cell proportion of different phases. In addition, it could reduce hypertrophy of cells, FN synthesis and secretion. (2) TGF- PlmRNA and P21mRNA synthesis of mesangial cells were regulated at transcription level by Shengkang Injection. Consequently, normal cell cycle, were obtained. This may be the important regulative mechanism of Shenkang Injection on abnormal cell cycle of glomerular mesangial cells in high glucose.
引文
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61 同57．
62 同9．
63 同54．
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71 同67.
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73 同68．
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77 Noble NA, Border WA. Angiotensin Ⅱ in renal fibrosis:should TGF-β_1 rather than blood pressure be the therapeutic target. Seminars in Nephrology, 1997, 5: 455-466
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79 梅小斌，高从容，崔若兰等．糖尿病大鼠肾小球周期素激酶抑制剂P27的变化及意义．肾脏病与透析肾移植杂志，2002，11(1)：17-20
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83 刘宇宁．肾康注射液防治糖尿病肾病肾小球硬化的实验研究．成都中医药大学博士学位论文，内部资料，2000
84 毛炜．中药肾康注射液防治肾小球硬化的作用机理探讨．成都中医药大学博士学位论文，内部资料，1998
85 Young BA, Johnson RJ, Alpers CE, et al. Cellular events in the evolution of experimental diabetic nephropathy. Kidney Int, 1995,47:935-944
86 Wolf G, Schroeder R, Ziyadeh FN, et al. High glucose stimulates expression of p27 in cultured mouse measangial cells: relationship to hypertrophy. Am J Physiol, 1997,273(Renal physiol, 42): 348-356
87 同85.
88 同76.
89 Sharma K, Jin Y, Guo J et al. Neutralization of TGF- β_1 by anti- TGF- β_1 antibody attenuates kidney hypertrophy and the enhanced extracellular matrix gene expression in STZ-induced diabetic mice. Diabetes, 1996,45:522-530
90 同9.
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2 刘志红．糖尿病肾病．中华肾脏病杂志，2000，16(2)：126-131．
3 贾汝汉，石明．糖尿病肾病的一体化治疗．第三届全国肾移植慢性肾衰学术研讨会专题讲座集，2002，成都
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61 同57．
62 同9．
63 同54．
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72 吴永贵，余毅，林善锬等．苯那普利对糖尿病大鼠肾组织细胞周期蛋白激酶抑制剂p~(21)蛋白表达的影响．肾脏病与透析肾移植杂志，1999，8(1)：21-23
73 同68．
74 林善锬，吴永贵，余毅等．苯那普利对糖尿病大鼠肾皮质p~(21)蛋白表达的调节及其机制．中华内分泌代谢杂志，1999，15(4)：220-224
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77 Noble NA, Border WA. Angiotensin Ⅱ in renal fibrosis:should TGF-β_1 rather than blood pressure be the therapeutic target. Seminars in Nephrology, 1997, 5: 455-466
78 Ziyadeh FN, KumarS, Mark E et al. Stimulation of collage gene expression and protein sythesis in murine meangial cells by high glucose is mediated by autocrine activation of transforming growth factor- β.J Clin Invest, 1994,93:536-542
79 梅小斌，高从容，崔若兰等．糖尿病大鼠肾小球周期素激酶抑制剂P27的变化及意义．肾脏病与透析肾移植杂志，2002，11(1)：17-20
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84 毛炜．中药肾康注射液防治肾小球硬化的作用机理探讨．成都中医药大学博士学位论文，内部资料，1998
85 Young BA, Johnson RJ, Alpers CE, et al. Cellular events in the evolution of experimental diabetic nephropathy. Kidney Int, 1995,47:935-944
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87 同85.
88 同76.
89 Sharma K, Jin Y, Guo J et al. Neutralization of TGF- β_1 by anti- TGF- β_1 antibody attenuates kidney hypertrophy and the enhanced extracellular matrix gene expression in STZ-induced diabetic mice. Diabetes, 1996,45:522-530
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