摘要
目的：探讨普伐他汀联合洛沙坦治疗对慢性环孢素A(CsA)肾毒性抗纤维化作用的分子机制。
方法：5组Sprague-Dawley大鼠分别给予橄榄油(1mg. kg-1.d-1)、CsA(15 mgkg-1.d-1皮下注射)、CsA和洛沙坦(10 mg.kg-1.d-1饮用水中)、CsA和两种药物联合治疗。检测各组大鼠的肾功能、收缩期血压、血脂水平；肾组织纤维化以三色染色观察；利用Northern杂交、RNA原位杂交、免疫印迹法分别检测转化生长因子β1(TGF-β1)和TGF-β诱导基因h3(βig-h3)的表达。
结果：与CsA肾毒性相比,普伐他汀或洛沙坦单独治疗明显减轻肾小管间质纤维化[洛沙坦：(26±10)%;普伐他汀：(31±8)%,P<0.01 Vs.(45±6)%],同时伴有TGF-β1[洛沙坦：(230±20)%;普伐他汀：(240±15)%,P<0.05]和βig-h3[洛沙坦：(178±21)%;普伐他汀：(167±10)%,P<0.05]表达下调,联合治疗进一步减轻上述指标[纤维化程度：(11±5)%;TGF-β1：(150±28)%;βig-h3：(126±9)%,P<0.05 vs.单独治疗组]。直线相关分析示,βig-h3表达与TGF-β1(r=0.787,P<0.001)或肾小管间质纤维化程度(r=0.688,P<0.001)呈正向相关。然而,各组间收缩期血压、血脂水平无显著性差异。
结论：在慢性环孢A肾毒性中,洛沙坦和普伐他汀联合治疗通过抑制βig-h3表达具有抗纤维化的协同作用,而不依赖于其降压或降血脂作用。
Objective:To explore the anti-fibrotic mechanism of pravastatin and losartan co-treatment in a rat model of chronic cyclosporine A (CsA) nephrotoxicity.
Methods:Five subgroups of Sprague-Dawley rats were given vehicle, CsA (15mg/kg s.c), CsA and losartan (10mg/kg in drinking water), CsA and pravastatin (20mg/kg in drinking water), or a combination of CsA, losartan, and pravastatin for 4 weeks. Renal function, systolic blood pressure, and lipid profiles were measured. In addition, renal histopathology (tubulointerstitial fibrosis, TIF) and expressions of transforming growth factorβ1 (TGF-β1) and TGF-βinducible gene-h3 (βig-h3) were evaluated with Northern blot, in situ hybridization, and immunoblotting.
Results:Compared with the CsA-treated rats, the pravastatin or losartan-treated rats significantly decreased TIF [losartan:(26±10)%; pravastatin:(31±8)%, P<0.01 vs. (45±6)%] in parallel with down-regulating TGF-β1 [losartan:(230±20)%; pravastatin:(240±15)%, P<0.05] andβig-h3 [losartan:(178±21)%; pravastatin:(167±10)%, P<0.05] expression, and that combined treatment with losartan and pravastatin further decreased these parameters compared with giving each drug alone [TIF:(11±5)%; TGF-(31: (150±28)%;βig-h3:(126±9)%, P<0.05]. Correlation analysis revealed thatβig-h3 expression closely associated with TGF-β1 expression (r=0.787, P<0.001) and TIF (r=0.688, P<0.001). However, there were no significant differences in systolic blood pressure or serum lipid parameters between groups.
Conclusion:Combined treatment with pravastatin and losartan provided synergistic effect on fibrotic processes by inhibitingβig-h3 expression in a rat model of chronic CsA-induced nephrotoxicity, and this effect was independent of their hypotensive and hypolipidemic actions.
引文
[1]金英顺,洪英礼,崔镇花,刘雨田,金海峰,金华,等.血管紧张素Ⅱ及其受体在慢性环孢素A肾毒性大鼠肾组织中的表达.第二军医大学学报,2010,31：1286-1290.
[2]Shihab FS, Andoh TF, Tanner AM, Noble NA, Border WA, Franceschini N, et al. Role of transforming growth factor-beta 1 in experimental chronic cyclosporine nephropathy. Kidney Int,1996,49:1141-1151.
[3]Yun SJ, Kim MO, Kim SO, Park J, Kwon YK, Kim IS, et al. Induction of TGF-beta-inducible gene-h3 (betaig-h3) by TGF-betal in astrocytes:Implications for astrocyte response to brain injury. Brain Res Mol Brain Res,2002,107:57-64.
[4]Yang CW, Ahn HJ, Kim WY, Li C, Jung JY, Yoon SA, et al. Synergistic effects of mycophenolate mofetil and losartan in a model of chronic cyclosporine nephropathy. Transplantation,2003,75:309-315.
[5]Li C, Yang CW, Park JH, Lim SW, Sun BK, Jung JY, et al. Pravastatin treatment attenuates interstitial inflammation and fibrosis in a rat model of chronic cyclosporine-induced nephropathy. Am J Physiol Renal Physiol,2004, 286:F46-F57.
[6]Lee SH, Li C, Lim SW, Ahn KO, Choi BS, Kim YS, et al. Attenuation of interstitial inflammation and fibrosis by recombinant human erythropoietin in chronic cyclosporine nephropathy. Am J Nephrol,2005,25:64-76.
[7]Li C, Sun BK, Lim SW, Song JC, Kang SW, Kim YS, et al. Combined effects of losartan and pravastatin on interstitial inflammation and fibrosis in chronic cyclosporine-induced nephropathy. Transplantation,2005,79:1522-1529.
[8]Li C, Yang CW, Park CW, Ahn HJ, Kim WY, Yoon KH, et al. Long-term treatment with ramipril attenuates renal osteopontin expression in diabetic rats. Kidney Int,2003,63:454-463.
[9]Kawai T, Masaki T, Doi S, Arakawa T, Yokoyama Y, Doi T, et al. PPAR-gamma agonist attenuates renal interstitial fibrosis and inflammation through reduction of TGF-beta. Lab Invest,2009,89:47-58.
[10]An WS, Kim HJ, Cho KH, Vaziri ND. Omega-3 fatty acid supplementation attenuates oxidative stress, inflammation, and tubulointerstitial fibrosis in the remnant kidney. Am J Physiol Renal Physiol,2009,297:F895-F903.
[11]Islam M, Burke JF Jr, McGowan TA, Zhu Y, Dunn SR, McCue P, et al. Effect of anti-transforming growth factor-beta antibodies in cyclosporine-induced renal dysfunction. Kidney Int,2001,59:498-506.
[12]Schultz-Cherry S, Chen H, Mosher DF, Misenheimer TM, Krutzsch HC, Roberts DD, et al. Regulation of transforming growth factor-beta activation by discrete sequences of thrombospondin. J Biol Chem,1995,270:7304-7310
[13]Oreffo RO, Mundy GR, Seyedin SM, Bonewald LF. Activation of the bone-derived latent TGF beta complex by isolated osteoclasts. Biochem Biophys Res Commun,1989,158:817-823.
[14]Sun BK, Li C, Lim SW, Jung JY, Lee SH, Kim IS, et al. Expression of transforming growth factor-beta-inducible gene-h3 in normal and cyclosporine-treated rat kidney. Jung JY, Lee SH, Kim IS, Kim YS, Kim J, Bang BK, Yang CW. J Lab Clin Med,2004,143:175-183.
[15]Luo JD, Zhang WW, Zhang GP, Guan JX, Chen X. Simvastatin inhibits cardiac hypertrophy and angiotensin-converting enzyme activity in rats with aortic stenosis.Clin Exp Pharmacol Physiol,1999,26:903-908.
[16]Wassmann S, Laufs U, Baumer AT, Muller K, Konkol C, Sauer H, et al. Inhibition of geranylgeranylation reduces angiotensin II-mediated free radical production in vascular smooth muscle cells:involvement of angiotensin AT1 receptor expression and Racl GTPase. Mol Pharmacol,2001,59:646-654.
[17]Shihab FS, Yi H, Bennett WM, Andoh TF. Effect of nitric oxide modulation on TGF-betal and matrix proteins in chronic cyclosporine nephrotoxicity.Kidney Int,2000,58:1174-1185.
[18]Wu J, Yang X, Zhang YF, Zhou SF, Zhang R, Dong XQ, et al. Angiotensin II upregulates Toll-like receptor 4 and enhances lipopolysaccharide-induced CD40 expression in rat peritoneal mesothelial cells. Inflamm Res,2009, 58:473-482.
[19]Lim SW, Li C, Ahn KO, Kim J, Moon IS, Ahn C, et al. Cyclosporine-induced renal injury induces toll-like receptor and maturation of dendritic cells.Transplantation,2005,80:691-69.
[20]Weitz-Schmidt G, Welzenbach K, Brinkmann V, Kamata T, Kallen J, Bruns C, et al. Statins selectively inhibit leukocyte function antigen-1 by binding to a novel regulatory integrin site. Nat Med,2001,7:687-692.
[21]Katznelson S, Wilkinson AH, Kobashigawa JA, Wang XM, Chia D, Ozawa M, et al. The effect of pravastatin on acute rejection after kidney transplantation-a pilot study. Transplantation,1996,61:1469-1474.
[22]Hodgkinson CP, Ye S. Statins inhibit toll-like receptor 4-mediated lipopolysaccharide signaling and cytokine expression. Pharmacogenet Genomics, 2008,18:803-813.
[1]Li C, Lim SW, Sun BK,, et al. Chronic cyclosporine nephrotoxicity:new insights and preventive strategies. Yonsei Med J.2004 Dec 31;45(6):1004-16.
[2]. Li C, Yang CW, Kim W Y, et al. Reversibility of chronic cyclosporine nephropathy in rats after withdrawal of cyclosporine. Am J Physiol Renal Physiol,2003,284:F389-F398.
[3]Yang CW, Ahn HJ, Kim WY, et al. Cyclosporine withdrawal and mycophenolate mofetil treatment effects on the progression of chronic cyclosporine nephrotoxicity. Kidney Int,2002,62:20-30.
[4]Madsen K, Marcussen N, Pedersen M, et al. Angiotensin Ⅱ promotes development of the renal microcirculation through AT1 receptors.J Am Soc Nephrol.2010 Mar;21(3);448-59.
[5]Medina C, Santos-Martinez MJ, Santana A,et al. Transforming growth factor-beta type 1 receptor (ALK5) and Smad proteins mediate TIMP-1 and collagen synthesis in experimental intestinal fibrosis. J Pathol.2011 Feb 3. doi: 10.1002/path.2870. [Epub ahead of print]
[6]Tache D, Bogdan F, Pisoschi C,et al. Evidence for the involvement of TGF-β1-CTGF axis in liver fibrogenesis secondary to hepatic viral infection. Rom J Morphol Embryol.2011;52(1 Suppl):409-12.
[7]Eddy AA.Molecular insights into renal interstitial fibrosis.J Am Soc Nephrol,1996,7:2495-2508.
[8]Shehata M,Cope GH,Johnson TS,et al.Cyclosporine enhances the expression of TGF-Pin the juxtaglomerular cells of the rat kidney.Kidney Int,1995.48;1487-1496.
[9]张国华,张训,侯凡凡.转化生长因子β在实验性慢性环孢素A肾中的作用.中华器官移植杂志.2000.21(4),228-230.
[10]Abdd-Wahab N, Weston BS, Roberts T, et al. Connective tissue growth factor an d regulation of the mesangial cell cycle:role in cellular hypertrophy[J]. JAm Soc Nephrol,2002,13(10):2437-2445.
[11]Yun SJ, Kim MO, Kim SO, Park J, Kwon YK, Kim IS, et al. Induction of TGF-beta-inducible gene-h3 (betaig-h3) by TGF-betal in astrocytes:Implications for astrocyte response to brain injury. Brain Res Mol Brain Res,2002,107:57-64.
[12]Cha DR, Kim IS, Kang YS,et al. Urinary concentration of transforming growth factor-beta-inducible gene-h3(beta ig-h3) in patients with Type 2 diabetes mellitus. Diabet Med.2005 Jan;22(1):14-20.
[13]Langham RG, Egan MK, Dowling JP, et al.Transforming growth factor-betal and tumor growth factor-beta-inducible gene-H3 in nonrenal transplant cyclosporine nephropathy. Transplantation.2001 Dec 15;72(11):1826-9.
[14]Langham RG, Egan MK, Dowling JP,et al. Transforming growth factor-betal and tumor growth factor-beta-inducible gene-H3 in nonrenal transplant cyclosporine nephropathy. Transplantation.2001 Dec 15;72(11):1826-9.
[15]Medina C, Santos-Martinez MJ, Santana A,et al.Transforming growth factor-beta type 1 receptor (ALK5) and Smad proteins mediate TIMP-1 and collagen synthesis in experimental intestinal fibrosis. J Pathol.2011 Feb 3. doi: 10.1002/path.2870. [Epub ahead of print]
[16]Tache D, Bogdan F, Pisoschi C,et al. Transforming growth factor-beta type 1 receptor (ALK5) and Smad proteins mediate TIMP-1 and collagen synthesis in experimental intestinal fibrosis. Rom J Morphol Embryol.2011;52(1 Suppl):409-12.
[17]于光禄.钙拮抗剂的临床新用途.亚太传统医药.2008.4(8)；39.
[18]Rodicio J L. Calcium antagonists and renal protection from cyclosporine nephrotoxicity:long-term trial in renal transplantation patients[J]. J Cardiovasa Pharmaeol,2000,35:7.
[19]Lee SK, Park JY, Yu ES, et al. Individual or combined effects of enalapril and verapamil on chronic cyclosporine nephrotoxicity in rats[J]. J Korean Med Sci,1999,14(6):653.
[20]于洪芳.钙拮抗剂的临床应用.中国社区医师.2008,23(10)；28.
[21]吴闯.环孢素肾毒A的药物防护.中国药房.2004,15(6)：372-373.
[22]高友兵刘勇刘伟.钙拮抗剂对重症急性胰腺炎肾损害保护作用的实验研究.医学临床研究.2009,26(10)：1845-1846.
[23]高友兵,汪训实,陈玉石,等.钙通道阻滞剂对重症急性胰腺炎大鼠炎性因子的影响[J].中华普通外科杂志,2002,17(9)：564-565.
[24]黄鹏钧,吴先明,李一德.三类不同降压药物对肾功能的保护作用.中华医护杂志.2007,4(3)：232-233.
[25]谢杨,饶邦复,李岗莉,等.钙通道阻滞剂的临床应用[J].药学专论,2002, 11(1)：65-66.
[26]周峰,陈香美,王小丹,等.介导血管紧张素Ⅱ上调近端肾小管上皮细胞金属蛋白酶组织抑制1表达的信号分子研究[J].中华肾脏病杂志,2003,19(3)：151.
[27]梁冰,刘章锁,程根阳.贝那普利联合氯沙坦对环孢素A致大鼠慢性肾毒性的防治作用Journal of Zhengzhou University,2007,42 (6):1055-1057.
[28]金英顺,洪英礼,崔镇花,等.血管紧张素Ⅱ及其受体在慢性环孢素A肾毒性大鼠肾组织中的表达.第二军医大学学报,2010,31：1286-1290..
[29]郭胜才,冯友根.洛沙坦的药动学特点及药物相互作用.中国医院药学杂志.1998.18(6)：278-279.
[30]王平贤,王安静,黄赤兵,等.洛沙坦预防慢性移植物肾病.重庆医学.2005：34(2),238-239.
[31]金英顺金秀男金华等,洛沙坦对环孢素A慢性肾毒性大鼠核因子KB的影响,中华肾脏病杂志,2007.23(2)；87-90.
[32]Asai T, Nakatani T, Tam ada S, et al. Activation of transcription factors AP--1 and NF—kappa B in chronic cyclosporine A nephrotoxicity:role in beneficial efects of magnesium supplementation. Transplantation,2003,75: 1040-1044
[33]Tamada S, Nakatan i T, Asai T, et al. Inhibition of nuclear factor-kappa B activation by pyrrolidine dithiocarbamate prevents chronic FK506 nephropathy. Kidney Int,2003,63:306.314
[34]Haugen EN, Croatt AJ, Nath KA, et al. Angitoensin II induces renal oxidant stress in vivo and hemoxygenase-1 in vivo and in vitro [J]. Kidney Int.2000, 58:144-152,
[35]李志辉,易著文.洛沙坦对阿霉素致大鼠慢性肾脏损害的长期保护作用.湖南医科大学学报.2002.27(4)：305-308.
[36]李志辉,易著文.洛沙坦延缓阿霉素肾病鼠慢性病理进展的实验研究.中华儿科杂志.2001,39(12)：712-717.
[37]郭志新,邱明才.洛沙坦对糖尿病大鼠肾脏转化生长因子-β及其Ⅱ型受体表达的影响及机制.中国糖尿病杂志.2003,11(6)：421-425.
[38]张淑芳,王丽,李彦华.糖尿病肾病早期TGF-13变化及洛沙坦的干预治疗.山东医药2007,47(1)：21-22.
[39]刘建军,甘华,杜晓刚.洛沙坦对糖尿病大鼠肾脏炎症反应及足细胞损伤 的影响.中华肾脏病杂志.2007,23(3)：189-193.
[40]贾玉娜.他汀类药物的非降脂作用新发展.中国冶金工业医学杂.2005,22(4)：400-401.
[41]董祝斌,张学农.他汀类药物的研究进展.中国新医药.2003,2(10)：48.
[42]邱国应.他汀类药物的临床应用新进展.中国心血管病研究杂志[J],2006,4(8)：638-940.
[43]戴笃荣.他汀类药物的免疫抑制及抗肿瘤作用[J].中国医院药学杂志,2007.27(8)：1139-1141.
[44]张承俊,朱兴雷.辛伐他汀对肿瘤坏死因子a诱导的人脐静脉内皮细胞基质金属蛋白酶P表达的影响[J].中国动脉硬化杂志,2006,14(1)：50-52.
[45]毛利东.普伐他汀的药理作用及临床应用.中国实用医药.2010,5(15)：180-181.
[46]陈碧珊,徐洁平,李冶,等.普伐他汀对血管紧张素Ⅱ诱导大鼠血管平滑肌细胞增殖的影响.汕头大学医学院学报.2009,22(1)：15-16.
[47]叶江枫,戚好文,梁军,等.维甲酸及氟伐他汀对放射肺损伤的治疗作用[J].第四军医大学学报,2003,24(18)：1707.
[48]宋国华.普伐他汀对高血压肾病患者血清TGF-β的影响.山东医药,2006,46(25)：49-50.
[49]於文丽,徐联芳,杨仁勇.普伐他汀和福辛普利对糖尿病大鼠肾小球中TGF-β1的影响.中国医师杂志.2004,6(12)：1655-1656.
[50]钱雅新,彭文,王浩.糖尿病肾病血清TGF—改变及普伐他汀的治疗作用.实用诊断与治疗杂志,2006,20(2)：117-119.
[51]戴春笋,刘志红,李恒,等.冬虫夏草防治环孢素A肾毒性的体内与体外实验研究[J].肾脏病与透析肾移植杂志,2000,9(4)：322-328
[52]张莉.冬虫夏草对大鼠糖尿病肾病的保护作用[D].南昌：南昌大学医学院,2007.
[53]许风雷,环文英,吴泰相,等.冬虫夏草治疗慢性肾病临床疗效的系统评价[J].中国循证医学杂志,2006,6(11)：814-807
[54]马清泉,王赕,宋增艳.冬虫夏草的药理与临床研究进展.现代中西医结合杂志.2009.18(19)：2353-2354.
[55]王筱霞,吴兆龙.冬虫夏草对离体人肾小球系膜增殖的影响.中国临床药学杂志,2001,10(1)：24-26.
[56]李立.冬虫夏草制剂在肾脏病中的作用机制.临床肾脏病杂.2010.10(6)： 286-287.
[57]Shi B, Wang Z, Jin H, et al. Immunoregulatory Cordyceps sinensis increases regulatory T cells to Thl7 cell ratio and delays diabetes in NOD mice. Int Immunopharmacol,2009,5:582-586.
[58]吕小波,尹鸿萍,李海涛,等.虫草多糖对顺铂诱导急性肾衰的防治作用.江苏中医药,2008,2：77-78.
[59]刘茂东,李英,迟雁青,等.冬虫夏草对马兜铃酸肾病保护作用的研究.中国中西医结合肾病杂志,2007,3：158--159.
[60]金永东,宁建平,张义雄,等.冬虫夏草对糖尿病大鼠肾小管上皮细胞ILK表达的影响.医学临床研究,2008,6：1022-1025.
[61]龚伟,黎磊石,陈丹,等.百令(冬虫夏草)对糖尿病大鼠转化生长因子p及其受体表达的影响.肾脏病与透析肾移植杂志,2006,4：329-339.
[62]李暖,陈新德,杨达胜,等.百令胶囊对大鼠肾小管间质纤维化的防治作用.中国中西医结合肾病杂志,2007,4：227--228.
[63]Li Y, Xue WJ, Tian PX, et al. Clinical application of Cordyceps sinensis on immunosuppressive therapy in renal transplantation. Transplant Proc,2009,5: 1565-1569.
[64]李淑娟.茶多酚的保健和药理作用研究进展.西北药学杂志2010,25(1)：78-79.