雷公藤多苷抑制糖尿病大鼠肾组织MBL及MASP2的表达并减轻肾损伤
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摘要
目的研究雷公藤多苷对糖尿病大鼠肾组织甘露糖结合凝集素(MBL)及甘露聚糖结合凝集素丝氨酸肽酶2(MASP2)表达的影响,探讨雷公藤多苷对糖尿病大鼠肾脏损伤的保护机制。方法 45只雄性SD大鼠随机分为实验组(n=35)和正常对照组(n=10)。实验组喂以高糖高脂饲料6周后,腹腔注射链脲佐菌素(STZ)建立糖尿病肾病大鼠模型。实验组有33只大鼠造模成功,将33只大鼠随机分为糖尿病肾病模型组(n=16)和雷公藤多苷治疗组(n=17),治疗组以雷公藤多苷[10 mg/(kg·d)]灌胃8周。第14周末,比较模型组和治疗组24 h尿蛋白定量及血清生化指标;过碘酸希夫反应(PAS)观察肾组织形态学改变;荧光定量PCR检测肾组织MBL1、MASP2、核因子κB(NF-κB)、单核细胞趋化蛋白1(MCP-1)mRNA水平,Western blot法检测肾组织MBL-A、MASP2、NF-κB、MCP-1的蛋白水平,免疫组织化学染色检测MBL-A、MASP2蛋白在肾组织的表达和分布。结果与治疗组相比,模型组大鼠24 h尿蛋白定量、血清肌酐、尿素氮增高,肾组织MBL、MASP2、NF-κB及MCP-1表达明显增加;相关性分析显示,MBL与MASP2、NF-κB、MCP-1表达及24 h尿蛋白定量呈显著正相关。结论雷公藤多苷能抑制糖尿病模型大鼠肾组织MBL及MASP2的表达,减轻糖尿病大鼠肾损伤。
Objective To explore the effect of Triperygium wilfordii multiglucoside( TWM) on the expressions of mannose-binding lectin( MBL) and mannan-binding lectin serine peptidase 2( MASP2) in the kidney of diabetic rats,and discuss the protective role of TWM in diabetic nephropathy and its possible mechanism. Methods Forty-five male SD rats were randomly assigned into model group( n = 35) and normal control group( n = 10). Rats in the normal control group were fed with regular diet,while those in the model group were fed with high-fat high-sugar diet and given an intraperitoneal injection of 40 mg/kg streptozotocin( STZ) six weeks later. The successfully induced type 2 diabetic rat models( n = 33)were then randomized into DM group( n = 16) and TWM treatment group( n = 17) treated with TWM [10 mg/( kg· d)] for eight weeks. At the end of 14 th week,the levels of blood sugar,24-hour urine protein,serum creatinine and blood urea nitrogen were measured. Renal pathological changes were examined with PAS staining. MBL-A and MASP2 expressions were detected by immunohistochemistry in the renal tissues. The expressions of MBL1,MASP2,NF-κB and MCP-1 mRNAs were semi-quantified by real-time PCR. The expressions of MBL-A,MASP2,NF-κB and MCP-1 proteins were determined by Western blotting. Results Compared with the diabetic group,the levels of serum creatinine,blood urea nitrogen,24-hour urine protein decreased,renal histopathology was improved,the expressions of MBL,MASP2,NF-κB and MCP-1 mRNAs and proteins were reduced in the TWM group. Correlation analysis showed that the expression of MBL was positively correlated with MASP2,NF-κB,MCP-1 and 24-hour urine protein. Conclusion MBL and MASP2 are over-expressed in the kidney of diabetic rats. TWM can ameliorate kidney damage in diabetic rats and delay the development of diabetic nephropathy by inhibiting the expressions of MBL and MASP2.
Objective To explore the effect of Triperygium wilfordii multiglucoside( TWM) on the expressions of mannose-binding lectin( MBL) and mannan-binding lectin serine peptidase 2( MASP2) in the kidney of diabetic rats,and discuss the protective role of TWM in diabetic nephropathy and its possible mechanism. Methods Forty-five male SD rats were randomly assigned into model group( n = 35) and normal control group( n = 10). Rats in the normal control group were fed with regular diet,while those in the model group were fed with high-fat high-sugar diet and given an intraperitoneal injection of 40 mg/kg streptozotocin( STZ) six weeks later. The successfully induced type 2 diabetic rat models( n = 33)were then randomized into DM group( n = 16) and TWM treatment group( n = 17) treated with TWM [10 mg/( kg· d)] for eight weeks. At the end of 14 th week,the levels of blood sugar,24-hour urine protein,serum creatinine and blood urea nitrogen were measured. Renal pathological changes were examined with PAS staining. MBL-A and MASP2 expressions were detected by immunohistochemistry in the renal tissues. The expressions of MBL1,MASP2,NF-κB and MCP-1 mRNAs were semi-quantified by real-time PCR. The expressions of MBL-A,MASP2,NF-κB and MCP-1 proteins were determined by Western blotting. Results Compared with the diabetic group,the levels of serum creatinine,blood urea nitrogen,24-hour urine protein decreased,renal histopathology was improved,the expressions of MBL,MASP2,NF-κB and MCP-1 mRNAs and proteins were reduced in the TWM group. Correlation analysis showed that the expression of MBL was positively correlated with MASP2,NF-κB,MCP-1 and 24-hour urine protein. Conclusion MBL and MASP2 are over-expressed in the kidney of diabetic rats. TWM can ameliorate kidney damage in diabetic rats and delay the development of diabetic nephropathy by inhibiting the expressions of MBL and MASP2.
引文
[1]Axelgaard E,?stergaard J A,Thiel S,et al.Diabetes is associated with increased autoreactivity of mannan-binding lectin[J/OL].J Diabetes Res,2017,2017:6368780.doi:10.1155/2017/6368780.Epub 2017 Feb 28.
[2]?stergaard J A,Ruseva M M,Malik T H,et al.Increased autoreactivity of the complement-activating molecule mannan-binding lectin in a type 1 diabetes model[J].J Diabetes Res,2016,2016:1825738.doi:10.1155/2016/1825738.Epub 2016 Feb 10.
[3]Ghosh P,Sahoo R,Vaidya A,et al.Role of complement and complement regulatory proteins in the complications of diabetes[J].Endocr Rev,2015,36(3):272-288.
[4]Krogh S S,Holt C B,Steffensen R,et al.Plasma levels of MASP-1,MASP-3 and MAp44 in patients with type 2 diabetes-influence of glycaemic control,body composition and polymorphisms in the MASP1 gene[J].Clin Exp Immunol,2017,189(1):103-112.
[5]Ghazanfari M,Falahati M,Fattahi A,et al.Is MBL serum concentration a reliable predictor for recurrent vulvovaginal candidiasis?[J].Mycoses,2017 Nov 6.doi:10.1111/myc.12723.[Epub ahead of print].
[6]Hayashi N,Okada K,Matsui Y,et al.Glomerular mannose-binding lectin deposition in intrinsic antigen-related membranous nephropathy[J].Nephro Dial Transplant,2017 Jul 18.doi:10.1093/ndt/gfx235.[Epub ahead of print].
[7]DobóJ,Pál G,Cervenak L,et al.The emerging roles of mannose-binding lectin-associated serine proteases(MASPs)in the lectin pathway of complement and beyond[J].Immunol Rev,2016,274(1):98-111.
[8]蒯巧林.雷公藤多苷治疗糖尿病肾病的效果和安全性[J].中外医学研究,2017,15(26):10-11.
[9]Huang Q,Shang G,Deng H,et al.High mannose-binding lectin serum levels are associated with diabetic retinopathy in Chinese patients with type 2 diabetes[J/OL].PLo S One,2015,10(7):e0130665.doi:10.1371/journal.pone.0130665.e Collection 2015.
[10]季鸣,王丽嫄,金晶,等.补体系统药物研究进展[J].中国生化药物杂志,2016,36(12):7-10.
[11]常欣蓓,倪兆慧.甘露糖结合凝集素与肾脏疾病[J].肾脏病与透析肾移植杂志,2013,22(5):476-481.
[12]Hovind P,Hansen T K,Tarnow L,et al.Mannose-binding lectin as a predictor of microalbuminuria in type 1 diabetes:an inception cohort study[J].Diabetes,2005,54(5):1523-1527.
[13]Guan L Z,Tong Q,Xu J.Elevated serum levels of mannose-binding lectin and diabetic nephropathy in type 2 diabetes[J/OL].PLo SOne,2015,10(3):e 0119699.doi:10.1371/journal.pone.0119699.e Collection 2015.
[14]Zhao S Q,Hu Z.Mannose-binding lectin and diabetic nephropathy in type 1 diabetes[J].J Clin Lab Anal,2016,30(4):345-350.
[15]Flyvbjerg A.The role of the complement system in diabetic nephropathy[J].Nat Rev Nephrol,2017,13(5):311-318.
[16]Ge Y,Paisie T K,Newman J R B,et al.UBASH3A mediates risk for type 1 diabetes through inhibition of T-cell receptor-induced NF-κB signaling[J].Diabetes,2017,66(7):2033-2043.
[17]朱志红,张翔,史文伟,等.GDM患者血浆甘露糖结合凝集素水平变化及其对TLR4/通路的调节作用[J].山东医药,2017,57(12):74-76.
[18]Yi H,Peng R,Zhang L Y,et al.LincRNA-Gm4419 knockdown ameliorates NF-κB/NLRP3 inflammasome-mediated inflammation in diabetic nephropathy[J].Cell Death Dis,2017,8(2):e2583.doi:10.1038/cddis.2016.451.
[19]Malik S,Suchal K,Khan S I,et al.Apigenin ameliorates streptozotocininduced diabetic nephropathy in rats via MAPK/NF-κB/TNF-αand TGF-β1/MAPK/Fibronectin pathways[J].Am J Physiol Renal Physiol,2017,313(2):F414-F422.
[20]孙文佳,闵伟红,邱斌,等.NF-κB信号通路与糖尿病肾病治疗研究进展[J].现代生物医学进展,2016,16(18):3589-3592.
[21]Roos A,Rastaldi M P,Calvaresi N,et al.Glomerular activation of the lectin pathway of complement in Ig A nephropathy is associated with more severe renal disease[J].J Am Soc Nephrol,2006,17(6):1724-1734.
[22]赵程程,邓小云,钟燕,等.2 012-2016年中南大学湘雅二医院雷公藤多苷片应用分析[J].中国医院用药评价与分析,2017,17(3):292-296.
[23]赵瑞萍,陈卫东,杨萍.雷公藤多苷对糖尿病肾病大鼠肾组织MBL、IL-6、TNF-α表达的影响[J].中国中西医结合肾病杂志,2015(2):113-115.
[24]张继强,张燕,尹晓丽,等.雷公藤多苷对早期糖尿病肾病大鼠肾组织单核细胞趋化蛋白1及结缔组织生长因子表达的影响[J].肾脏病与透析肾移植杂志,2014,23(2):146-151.
[25]陈卫东,张继强,张燕,等.雷公藤多苷对糖尿病肾病大鼠肾基质金属蛋白酶-9和基质金属蛋白酶抑制剂-1表达的影响[J].中国临床药理学杂志,2015(12):1157-1161.
[2]?stergaard J A,Ruseva M M,Malik T H,et al.Increased autoreactivity of the complement-activating molecule mannan-binding lectin in a type 1 diabetes model[J].J Diabetes Res,2016,2016:1825738.doi:10.1155/2016/1825738.Epub 2016 Feb 10.
[3]Ghosh P,Sahoo R,Vaidya A,et al.Role of complement and complement regulatory proteins in the complications of diabetes[J].Endocr Rev,2015,36(3):272-288.
[4]Krogh S S,Holt C B,Steffensen R,et al.Plasma levels of MASP-1,MASP-3 and MAp44 in patients with type 2 diabetes-influence of glycaemic control,body composition and polymorphisms in the MASP1 gene[J].Clin Exp Immunol,2017,189(1):103-112.
[5]Ghazanfari M,Falahati M,Fattahi A,et al.Is MBL serum concentration a reliable predictor for recurrent vulvovaginal candidiasis?[J].Mycoses,2017 Nov 6.doi:10.1111/myc.12723.[Epub ahead of print].
[6]Hayashi N,Okada K,Matsui Y,et al.Glomerular mannose-binding lectin deposition in intrinsic antigen-related membranous nephropathy[J].Nephro Dial Transplant,2017 Jul 18.doi:10.1093/ndt/gfx235.[Epub ahead of print].
[7]DobóJ,Pál G,Cervenak L,et al.The emerging roles of mannose-binding lectin-associated serine proteases(MASPs)in the lectin pathway of complement and beyond[J].Immunol Rev,2016,274(1):98-111.
[8]蒯巧林.雷公藤多苷治疗糖尿病肾病的效果和安全性[J].中外医学研究,2017,15(26):10-11.
[9]Huang Q,Shang G,Deng H,et al.High mannose-binding lectin serum levels are associated with diabetic retinopathy in Chinese patients with type 2 diabetes[J/OL].PLo S One,2015,10(7):e0130665.doi:10.1371/journal.pone.0130665.e Collection 2015.
[10]季鸣,王丽嫄,金晶,等.补体系统药物研究进展[J].中国生化药物杂志,2016,36(12):7-10.
[11]常欣蓓,倪兆慧.甘露糖结合凝集素与肾脏疾病[J].肾脏病与透析肾移植杂志,2013,22(5):476-481.
[12]Hovind P,Hansen T K,Tarnow L,et al.Mannose-binding lectin as a predictor of microalbuminuria in type 1 diabetes:an inception cohort study[J].Diabetes,2005,54(5):1523-1527.
[13]Guan L Z,Tong Q,Xu J.Elevated serum levels of mannose-binding lectin and diabetic nephropathy in type 2 diabetes[J/OL].PLo SOne,2015,10(3):e 0119699.doi:10.1371/journal.pone.0119699.e Collection 2015.
[14]Zhao S Q,Hu Z.Mannose-binding lectin and diabetic nephropathy in type 1 diabetes[J].J Clin Lab Anal,2016,30(4):345-350.
[15]Flyvbjerg A.The role of the complement system in diabetic nephropathy[J].Nat Rev Nephrol,2017,13(5):311-318.
[16]Ge Y,Paisie T K,Newman J R B,et al.UBASH3A mediates risk for type 1 diabetes through inhibition of T-cell receptor-induced NF-κB signaling[J].Diabetes,2017,66(7):2033-2043.
[17]朱志红,张翔,史文伟,等.GDM患者血浆甘露糖结合凝集素水平变化及其对TLR4/通路的调节作用[J].山东医药,2017,57(12):74-76.
[18]Yi H,Peng R,Zhang L Y,et al.LincRNA-Gm4419 knockdown ameliorates NF-κB/NLRP3 inflammasome-mediated inflammation in diabetic nephropathy[J].Cell Death Dis,2017,8(2):e2583.doi:10.1038/cddis.2016.451.
[19]Malik S,Suchal K,Khan S I,et al.Apigenin ameliorates streptozotocininduced diabetic nephropathy in rats via MAPK/NF-κB/TNF-αand TGF-β1/MAPK/Fibronectin pathways[J].Am J Physiol Renal Physiol,2017,313(2):F414-F422.
[20]孙文佳,闵伟红,邱斌,等.NF-κB信号通路与糖尿病肾病治疗研究进展[J].现代生物医学进展,2016,16(18):3589-3592.
[21]Roos A,Rastaldi M P,Calvaresi N,et al.Glomerular activation of the lectin pathway of complement in Ig A nephropathy is associated with more severe renal disease[J].J Am Soc Nephrol,2006,17(6):1724-1734.
[22]赵程程,邓小云,钟燕,等.2 012-2016年中南大学湘雅二医院雷公藤多苷片应用分析[J].中国医院用药评价与分析,2017,17(3):292-296.
[23]赵瑞萍,陈卫东,杨萍.雷公藤多苷对糖尿病肾病大鼠肾组织MBL、IL-6、TNF-α表达的影响[J].中国中西医结合肾病杂志,2015(2):113-115.
[24]张继强,张燕,尹晓丽,等.雷公藤多苷对早期糖尿病肾病大鼠肾组织单核细胞趋化蛋白1及结缔组织生长因子表达的影响[J].肾脏病与透析肾移植杂志,2014,23(2):146-151.
[25]陈卫东,张继强,张燕,等.雷公藤多苷对糖尿病肾病大鼠肾基质金属蛋白酶-9和基质金属蛋白酶抑制剂-1表达的影响[J].中国临床药理学杂志,2015(12):1157-1161.