丹参多酚酸盐通过替代途径抑制膜性肾病大鼠肾脏RAS系统激活
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摘要
目的:观察膜性肾病(MN)大鼠肾组织组织蛋白酶D(CtsD)、糜蛋白酶(Chymase)及其诱导的肾素-血管紧张素系统(RAS)在肾脏中的表达,并评估丹参多酚酸盐干预后的影响。方法:采用尾静脉注射阳离子化牛血清白蛋白(C-BSA)的方法复制MN大鼠模型,造模完成后经肾脏病理检查以确保造模成功。造模成功的大鼠随机分为模型组、盐酸贝那普利10mg/kg组和丹参多酚酸盐16.7、33.3、66.7 mg/kg组。各组按相应药物及剂量给药,治疗结束后检测各组大鼠24小时尿蛋白定量(UTP)、总胆固醇(TC)、三酰甘油(TG)、血清白蛋白(ALB)、尿素氮(BUN)、血肌酐(Scr)及血管紧张素Ⅱ(AngⅡ)水平来评估各组大鼠治疗情况;免疫组化及RT-PCR法检测各组大鼠肾组织CtsD、Chymase和AngⅡ的蛋白和基因表达情况。结果:病理显示MN大鼠模型复制成功。经盐酸贝那普利和丹参多酚酸盐各剂量干预后UTP、TC、TG水平较模型组明显降低,ALB水平较模型组明显升高。进一步研究显示,模型组大鼠血清AngⅡ较正常组明显升高,经盐酸贝那普利和丹参多酚酸盐33.3、66.7 mg/kg干预后均较模型组显著下降,但丹参多酚酸盐33.3、66.7 mg/kg组下降程度不及盐酸贝那普利组。免疫组化及RT-PCR显示CtsD、Chymase及AngⅡ在模型组大鼠肾组织较正常组表达增强,三者mRNA表达量显著增多。丹参多酚酸盐各剂量组肾组织中CtsD、Chymase及AngⅡ三种蛋白及其mRNA表达较模型组均显著减少。结论:在MN中可能存在CtsD和Chymase诱导的RAS激活,而丹参多酚酸盐可能通过抑制CtsD和Chymase的表达从而抑制RAS激活来减轻MN大鼠临床表现。
Objective: To observe the expressions of cathepsin D( CtsD),chymotrypsin( Chymase) and its induced renin-angiotensin system( RAS) in the kidney of rats with membranous nephropathy,and evaluate the effect of Salvianolate intervention. Methods: Rat model of membranous nephropathy was established by tail intravenous injection of cationized bovine serum albumin( C-BSA). After the model was established,renal pathology was performed to ensure successful modeling. All model rats were randomly divided into the model group,10 mg/kg Benazepril hydrochloride group and Salvianolate( 16. 7,33. 3,66. 7 mg/kg) groups. Rats in each group were administered with the corresponding drugs. After the treatment,the levels of 24-hour urine total protein( UTP),total cholesterol( TC),triglyceride( TG),serum albumin( ALB),serum creatinine( Scr) and angiotensin Ⅱ( AngⅡ) were measured to evaluate the clinical treatment situation. Immunohistochemistry and RT-PCR were used to detect the protein and gene expressions of CtsD,Chymase and AngⅡ in renal tissues of each group. Results: Pathology results showed that the MN rat model was successfully replicated. The levels of UTP,TC,TG were significantly reduced,and ALB was significantly higher than that in the model group after the intervention. Further studies showed that serum AngI I in the model group was significantly higher than that in the normal group,and was significantly decreased after the intervention with Salvianolate and Benazepril,while the data in Benazepril group showed more significant difference. Compared with the normal group,results in the immunohistochemistry and RT-PCR assay showed that expression levels of CtsD,Chymase and AngⅡ were significantly increased in the renal tissue of the model group. Compared with the model group,AngⅡ and its mRNA in the renal tissue were significantly decreased after the intervention with Benazepril. The expressions of CtsD,Chymase,AngⅡ and their mRNAs in the renal tissue of Salvianolate groups were significantly lower than those in the model group,and the decreases of CtsD and Chymase were more significant in 33. 3 mg/kg and 66. 7 mg/kg groups than those in 16. 7 mg/kg group. Conclusion: CtsD and Chymase induced RAS activation may be present in membranous nephropathy,while Salvianolate may attenuate the clinical manifestations of membranous nephropathy in rats by inhibiting the expressions of CtsD and Chymase and inhibiting RAS activation.
Objective: To observe the expressions of cathepsin D( CtsD),chymotrypsin( Chymase) and its induced renin-angiotensin system( RAS) in the kidney of rats with membranous nephropathy,and evaluate the effect of Salvianolate intervention. Methods: Rat model of membranous nephropathy was established by tail intravenous injection of cationized bovine serum albumin( C-BSA). After the model was established,renal pathology was performed to ensure successful modeling. All model rats were randomly divided into the model group,10 mg/kg Benazepril hydrochloride group and Salvianolate( 16. 7,33. 3,66. 7 mg/kg) groups. Rats in each group were administered with the corresponding drugs. After the treatment,the levels of 24-hour urine total protein( UTP),total cholesterol( TC),triglyceride( TG),serum albumin( ALB),serum creatinine( Scr) and angiotensin Ⅱ( AngⅡ) were measured to evaluate the clinical treatment situation. Immunohistochemistry and RT-PCR were used to detect the protein and gene expressions of CtsD,Chymase and AngⅡ in renal tissues of each group. Results: Pathology results showed that the MN rat model was successfully replicated. The levels of UTP,TC,TG were significantly reduced,and ALB was significantly higher than that in the model group after the intervention. Further studies showed that serum AngI I in the model group was significantly higher than that in the normal group,and was significantly decreased after the intervention with Salvianolate and Benazepril,while the data in Benazepril group showed more significant difference. Compared with the normal group,results in the immunohistochemistry and RT-PCR assay showed that expression levels of CtsD,Chymase and AngⅡ were significantly increased in the renal tissue of the model group. Compared with the model group,AngⅡ and its mRNA in the renal tissue were significantly decreased after the intervention with Benazepril. The expressions of CtsD,Chymase,AngⅡ and their mRNAs in the renal tissue of Salvianolate groups were significantly lower than those in the model group,and the decreases of CtsD and Chymase were more significant in 33. 3 mg/kg and 66. 7 mg/kg groups than those in 16. 7 mg/kg group. Conclusion: CtsD and Chymase induced RAS activation may be present in membranous nephropathy,while Salvianolate may attenuate the clinical manifestations of membranous nephropathy in rats by inhibiting the expressions of CtsD and Chymase and inhibiting RAS activation.
引文
[1]Mezzano S A, Aros C A, Droguett A, et al. Renal angiotensin II up-regulation and myofibroblast activation in human membranous nephropathy . Kidney Int Suppl, 2003, (86)∶ S39～45.
[2]Press E M, Porter R R, Cebra J. The isolation and properties of a proteolytic enzyme, cathepsin D, from bovine spleen . Biochem J, 1960, doi: 10.1042/bj0740501.
[3]Graciano M L, Cavaglieri Rde C, Dellê H, et al. Intrarenal renin-angiotensin system is upregulated in experimental model of progressive renal disease induced by chronic inhibition of nitric oxide synthesis . J Am Soc Nephrol, 2004, 15(7)∶ 1805～1815.
[4]Doggrell S A, Wanstall J C. Vascular chymase: pathophysiological role and therapeutic potential of inhibition . Cardiovasc Res, 2004, 61(4)∶ 653～662.
[5]Wu C C, Chen J S, Huang C F, et al. Approaching Biomarkers of Membranous Nephropathy from a Murine Model to Human Disease . J Biomed Biotechnol, 2011, doi: 10.1155/2011/581928.
[6]Miyake-Ogawa C, Miyazaki M, Abe K, et al. Tissue-specific expression of renin-angiotensin system components in IgA nephropathy. Am J Nephrol, 2005,25(1)∶ 1～12.
[7]Ohashi N, Isobe S, Ishigaki S, et al. Intrarenal renin-angiotensin system activity is augmented after initiation of dialysis . Hypertens Res, 2017, 40 (4)∶ 364～370.
[8]陈成, 陈永忠, 邹襄谷, 等. 丹参多酚酸盐对心衰模型大鼠去甲肾上腺素、血管紧张素-II、肿瘤坏死因子的影响 . 国际中医中药杂志, 2014, 36(8)∶ 702～706.
[9]陈素枝,陈文军,张卓,等.丹参多酚酸盐对阳离子化牛血清白蛋白致膜性肾病大鼠的影响 .中草药,2018, 49(8)∶ 1877～1883.
[10]Border W A, Ward H J, Kamil E S, et al. Inducion of membranous nephropathy in rabbits by administration of an exogenous cationic antigen . J Clin Invest, 1982, 69(2)∶ 4514～4561.
[11]Emans T W, Janssen B J, Pinkham M I. et al. Exogenous and endogenous angiotensin-II decrease renal cortical oxygen tension in conscious rats by limiting renal blood flow . J Physiol, 2016, 594 (21)∶ 6287～6300.
[12]Juillerat L , Nussberger J, Ménard J, et al. Determinants of angiotensin II generation during converting enzyme inhibition . Hypertension, 1990, 16 (5)∶ 564～572.
[13]Nielsen R, Mollet G, Esquivel E L, et al. Increased lysosomal proteolysis counteracts protein accumulation in the proximal tubule during focal segmental glomerulosclerosis . Kidney Int, 2013, 84 (5)∶ 902～910.
[14]Fox C, Cocchiaro P, Oakley F, et al. Inhibition of lysosomal protease cathepsin D reduces renal fibrosis in murine chronic kidney disease . Sci Rep, 2016, doi: 10.1038/srep20101.
[15]Yamamoto-Nonaka K, Koike M, Asanuma K, et al. Cathepsin D in Podocytes Is Important in the Pathogenesis of Proteinuria and CKD . J Am Soc Nephrol, 2016, 27 (9)∶ 2685～700.
[16]Wei C C, Tian B, Perry G, et al. Differential ANG II generation in plasma and tissue of mice with decreased expression of the ACE gene . Am J Physiol Heart Circ Physiol, 2002, 282 (6)∶ 2254～2258.
[17]Takai S, Sakaguchi M, Jin D, et al. Different angiotensin II-forming pathways in human and rat vascular tissues. Clin Chim Acta,2001,305(1-2)∶191～195.
[18]Belova L A. Angiotensin II_Generating Enzymes . Biochemistry (Moscow), 2000, 65(12)∶1589～1599.
[19]Cocchiaro P, Fox C, Tregidgo NW, et al. Lysosomal protease cathepsin D: a new driver of apoptosis during acute kidney injury . Sci Rep, 2016, doi: 10.1038/srep27112.
[20]Doggrell SA, Wanstall JC. Vascular chymase: pathophysiological role and therapeutic potential of inhibition . Cardiovasc Res, 2004, 61(4)∶ 653～662.
[21]Mou Y, Zhang Y, Guo C, et al. Integrated Treatment of Prostaglandin E1 and Angiotensin-Converting Enzyme Inhibitor in Diabetic Kidney Disease Rats: Possible Role of Antiapoptosis in Renal Tubular Epithelial Cells . DNA Cell Biol, 2018, 37(2)∶ 133～141.
[2]Press E M, Porter R R, Cebra J. The isolation and properties of a proteolytic enzyme, cathepsin D, from bovine spleen . Biochem J, 1960, doi: 10.1042/bj0740501.
[3]Graciano M L, Cavaglieri Rde C, Dellê H, et al. Intrarenal renin-angiotensin system is upregulated in experimental model of progressive renal disease induced by chronic inhibition of nitric oxide synthesis . J Am Soc Nephrol, 2004, 15(7)∶ 1805～1815.
[4]Doggrell S A, Wanstall J C. Vascular chymase: pathophysiological role and therapeutic potential of inhibition . Cardiovasc Res, 2004, 61(4)∶ 653～662.
[5]Wu C C, Chen J S, Huang C F, et al. Approaching Biomarkers of Membranous Nephropathy from a Murine Model to Human Disease . J Biomed Biotechnol, 2011, doi: 10.1155/2011/581928.
[6]Miyake-Ogawa C, Miyazaki M, Abe K, et al. Tissue-specific expression of renin-angiotensin system components in IgA nephropathy. Am J Nephrol, 2005,25(1)∶ 1～12.
[7]Ohashi N, Isobe S, Ishigaki S, et al. Intrarenal renin-angiotensin system activity is augmented after initiation of dialysis . Hypertens Res, 2017, 40 (4)∶ 364～370.
[8]陈成, 陈永忠, 邹襄谷, 等. 丹参多酚酸盐对心衰模型大鼠去甲肾上腺素、血管紧张素-II、肿瘤坏死因子的影响 . 国际中医中药杂志, 2014, 36(8)∶ 702～706.
[9]陈素枝,陈文军,张卓,等.丹参多酚酸盐对阳离子化牛血清白蛋白致膜性肾病大鼠的影响 .中草药,2018, 49(8)∶ 1877～1883.
[10]Border W A, Ward H J, Kamil E S, et al. Inducion of membranous nephropathy in rabbits by administration of an exogenous cationic antigen . J Clin Invest, 1982, 69(2)∶ 4514～4561.
[11]Emans T W, Janssen B J, Pinkham M I. et al. Exogenous and endogenous angiotensin-II decrease renal cortical oxygen tension in conscious rats by limiting renal blood flow . J Physiol, 2016, 594 (21)∶ 6287～6300.
[12]Juillerat L , Nussberger J, Ménard J, et al. Determinants of angiotensin II generation during converting enzyme inhibition . Hypertension, 1990, 16 (5)∶ 564～572.
[13]Nielsen R, Mollet G, Esquivel E L, et al. Increased lysosomal proteolysis counteracts protein accumulation in the proximal tubule during focal segmental glomerulosclerosis . Kidney Int, 2013, 84 (5)∶ 902～910.
[14]Fox C, Cocchiaro P, Oakley F, et al. Inhibition of lysosomal protease cathepsin D reduces renal fibrosis in murine chronic kidney disease . Sci Rep, 2016, doi: 10.1038/srep20101.
[15]Yamamoto-Nonaka K, Koike M, Asanuma K, et al. Cathepsin D in Podocytes Is Important in the Pathogenesis of Proteinuria and CKD . J Am Soc Nephrol, 2016, 27 (9)∶ 2685～700.
[16]Wei C C, Tian B, Perry G, et al. Differential ANG II generation in plasma and tissue of mice with decreased expression of the ACE gene . Am J Physiol Heart Circ Physiol, 2002, 282 (6)∶ 2254～2258.
[17]Takai S, Sakaguchi M, Jin D, et al. Different angiotensin II-forming pathways in human and rat vascular tissues. Clin Chim Acta,2001,305(1-2)∶191～195.
[18]Belova L A. Angiotensin II_Generating Enzymes . Biochemistry (Moscow), 2000, 65(12)∶1589～1599.
[19]Cocchiaro P, Fox C, Tregidgo NW, et al. Lysosomal protease cathepsin D: a new driver of apoptosis during acute kidney injury . Sci Rep, 2016, doi: 10.1038/srep27112.
[20]Doggrell SA, Wanstall JC. Vascular chymase: pathophysiological role and therapeutic potential of inhibition . Cardiovasc Res, 2004, 61(4)∶ 653～662.
[21]Mou Y, Zhang Y, Guo C, et al. Integrated Treatment of Prostaglandin E1 and Angiotensin-Converting Enzyme Inhibitor in Diabetic Kidney Disease Rats: Possible Role of Antiapoptosis in Renal Tubular Epithelial Cells . DNA Cell Biol, 2018, 37(2)∶ 133～141.