LY333531对高糖高脂下系膜细胞分泌细胞外基质的影响
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摘要
探讨PKCβ抑制剂(LY333531)对高糖高脂环境下系膜细胞分泌细胞外基质(ECM)的影响,单独培养人肾小球系膜细胞,实验分为对照组、LY333531组、高糖高脂组、高糖高脂+LY333531组.首先采用qRTPCR检测PKCβI的表达,然后通过ELISA法检测细胞上清液Col IV、Fn的含量.结果显示,高糖高脂组相较于对照组,PKCβI mRNA的表达水平明显增加(P<0.05),LY333531可以明显抑制PKCβI的表达(P<0.05);高糖高脂组相较于对照组,细胞上清液Col IV、Fn含量明显增加(P<0.05),而这种作用可被LY333531所抑制(P<0.05).表明高糖高脂可通过促进系膜细胞PKCβI表达从而诱导ECM的分泌,增加肾纤维化发生的风险,而LY333531可明显抑制这种作用.
To investigate the effect of PKCβinhibitor(LY333531)on extracellular matrix(ECM)of masangial cells in high glucose(HG)and lysophosphatidylcholine(LPC)environment,human mesangial cells(HMCs)were cultured alone and divided into 4groups:control;LY333531;HG and LPC(HG+LPC);HG,LPC and LY333531(HG+LPC+LY333531).First,we used the method of quantitative real-time PCR(qRT-PCR)to detect the expression of PKCβI;then,the levels of collagen IV and fibronectin were detected by enzyme linked immunosorbent assay(ELISA)in the supernatant.These results suggested that the mRNA expression of PKCβI in the group of HG+LPC was higher than the control group(P<0.05),and the effect could be obviously inhibited by LY333531(P<0.05).Meanwhile,compared with the control group,the level of collagen IV and fibronectin in the supernatant was significantly increased in the group of HG+LPC(P <0.05),and the effect could be also inhibited by LY333531notably(P<0.05).Therefore,we could conclude thatthe HG+LPC can aggrandize the secretion of ECM by increasing the expression of PKCβI in mesangial cells,which can increase the risk of renal fibrosis,however,LY333531 can weaken the effect obviously.
To investigate the effect of PKCβinhibitor(LY333531)on extracellular matrix(ECM)of masangial cells in high glucose(HG)and lysophosphatidylcholine(LPC)environment,human mesangial cells(HMCs)were cultured alone and divided into 4groups:control;LY333531;HG and LPC(HG+LPC);HG,LPC and LY333531(HG+LPC+LY333531).First,we used the method of quantitative real-time PCR(qRT-PCR)to detect the expression of PKCβI;then,the levels of collagen IV and fibronectin were detected by enzyme linked immunosorbent assay(ELISA)in the supernatant.These results suggested that the mRNA expression of PKCβI in the group of HG+LPC was higher than the control group(P<0.05),and the effect could be obviously inhibited by LY333531(P<0.05).Meanwhile,compared with the control group,the level of collagen IV and fibronectin in the supernatant was significantly increased in the group of HG+LPC(P <0.05),and the effect could be also inhibited by LY333531notably(P<0.05).Therefore,we could conclude thatthe HG+LPC can aggrandize the secretion of ECM by increasing the expression of PKCβI in mesangial cells,which can increase the risk of renal fibrosis,however,LY333531 can weaken the effect obviously.
引文
[1]施进宝.早期糖尿病肾病的研究进展[J].中国中医药现代远程教育,2012(1):155-158.
[2]周素娴,彭鹰,于健,等.细胞相互作用对PAF、系膜细胞paf-R基因表达的影响及阿托伐他汀的干预作用[J].四川生理科学杂志,2014(2):55-57.
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[4]AL-KHODOR S,ABU K Y.Triggering ras signalling by intracellular francisella tularensis through recruitment of PKCalpha and betaI to the SOS2/GrB2 complex is essential for bacterial proliferation in the cytosol[J].Cell Microbiol,2010,12(11):1604-1621.
[5]NOH H,KING G L.The role of protein kinase C activation in diabetic nephropathy[J].Kidney Int Suppl,2007(106):S49-S53.
[6]BRYANT D M,ROIGNOT J,DATTA A,et al.A molecular switch for the orientation of epithelial cell polarization[J].Dev Cell,2014,31(2):171-187.
[7]MEIER M,PARK J K,OVERHEU D,et al.Deletion of protein kinase C-beta isoform in vivo reduces renal hypertrophy but not albuminuria in the streptozotocin-induced diabetic mouse model[J].Diabetes,2007,56(2):346-354.
[8]MENNE J,SHUSHAKOVA N,BARTELS J,et al.Dual inhibition of classical protein kinase C-alpha and protein kinase C-beta isoforms protects against experimental murine diabetic nephropathy[J].Diabetes,2013,62(4):1167-1174.
[9]周素娴,雷闽湘,赵晋晋.D-葡萄糖对系膜细胞产生细胞外基质的时效与量效研究[J].广西医学,2009(8):1072-1073.
[10]DUAN J G,CHEN X Y,WANG L,et al.Sex differences in epidemiology and risk factors of acute coronary syndrome in Chinese patients with type 2diabetes:a long-term prospective cohort study[J].PLoS One,2015,10(4):e122031.
[11]张倩,石蒙,檀金川.TGF-β_1与糖尿病肾病的关系及中医药研究进展[J].中国中西医结合肾病杂志,2014(11):1030-1032.
[12]赵玲,张园.糖尿病肾病中医证候研究进展[J].中国中医药现代远程教育,2010(7):199-200.
[13]吕高虹,许惠琴.肾系膜细胞与糖尿病肾病变的相关性研究[J].辽宁中医药大学学报,2011(12):48-50.
[14]ROSSERT J,TERRAZ-DURASNEL C,BRIDEAU G.Growth factors,cytokines,and renal fibrosis during the course of diabetic nephropathy[J].Diabetes Metab,2000,26(Suppl 4):16-24.
[15]周素娴.高糖高脂对肾小球细胞影响的研究进展[J].新医学,2014(5):286-289.
[16]吕高虹,许惠琴,秦佩佩,等.高糖对人肾小球系膜细胞增殖及细胞外基质的影响[J].中国老年学杂志,2013(5):1066-1067.
[17]李宏光,蔡元菊,邹锦慧,等.高糖高脂饮食对兔肾小管间质纤维化的影响[J].动物学杂志,2010(1):145-150.
[18]罗霞,邓玲艳,许文娟,等.腺苷酸活化蛋白激酶通过抑制mTOR信号通路缓解糖尿病大鼠肾脏细胞外基质沉积[J].华中科技大学学报(医学版),2015(1):10-15.
[19]GERALDES P,KING G L.Activation of protein kinase C isoforms and its impact on diabetic complications[J].Circ Res,2010,106(8):1319-1331.
[20]雷少青,苏娃婷,徐金金,等.PKC-β抑制剂LY333531对糖尿病大鼠心肌功能及Caveolins蛋白的影响[J].临床和实验医学杂志,2014(16):1305-1308.
[21]KELLY D J,ZHANG Y,HEPPER C,et al.Protein kinase C beta inhibition attenuates the progression of experimental diabetic nephropathy in the presence of continued hypertension[J].Diabetes,2003,52(2):512-518.
[22]KOYA D,HANEDA M,NAKAGAWA H,et al.Amelioration of accelerated diabetic mesangial expansion by treatment with a PKC beta inhibitor in diabetic db/db mice,a rodent model for type 2diabetes[J].FASEB J,2000,14(3):439-447.
[23]SOETIKNO V,WATANABE K,SARI F R,et al.Curcumin attenuates diabetic nephropathy by inhibiting PKC-alpha and PKC-beta1activity in streptozotocin-induced type I diabetic rats[J].Mol Nutr Food Res,2011,55(11):1655-1665.
[24]SCHWARTZ S G,FLYNN H J,AIELLO L P.Ruboxistaurin mesilate hydrate for diabetic retinopathy[J].Drugs Today(Barc),2009,45(4):269-274.
[2]周素娴,彭鹰,于健,等.细胞相互作用对PAF、系膜细胞paf-R基因表达的影响及阿托伐他汀的干预作用[J].四川生理科学杂志,2014(2):55-57.
[3]周素娴,雷闽湘,赵晋晋,等.高糖高脂对系膜细胞产生细胞外基质及PAF的影响[J].中国病理生理杂志,2009(10):2056-2057.
[4]AL-KHODOR S,ABU K Y.Triggering ras signalling by intracellular francisella tularensis through recruitment of PKCalpha and betaI to the SOS2/GrB2 complex is essential for bacterial proliferation in the cytosol[J].Cell Microbiol,2010,12(11):1604-1621.
[5]NOH H,KING G L.The role of protein kinase C activation in diabetic nephropathy[J].Kidney Int Suppl,2007(106):S49-S53.
[6]BRYANT D M,ROIGNOT J,DATTA A,et al.A molecular switch for the orientation of epithelial cell polarization[J].Dev Cell,2014,31(2):171-187.
[7]MEIER M,PARK J K,OVERHEU D,et al.Deletion of protein kinase C-beta isoform in vivo reduces renal hypertrophy but not albuminuria in the streptozotocin-induced diabetic mouse model[J].Diabetes,2007,56(2):346-354.
[8]MENNE J,SHUSHAKOVA N,BARTELS J,et al.Dual inhibition of classical protein kinase C-alpha and protein kinase C-beta isoforms protects against experimental murine diabetic nephropathy[J].Diabetes,2013,62(4):1167-1174.
[9]周素娴,雷闽湘,赵晋晋.D-葡萄糖对系膜细胞产生细胞外基质的时效与量效研究[J].广西医学,2009(8):1072-1073.
[10]DUAN J G,CHEN X Y,WANG L,et al.Sex differences in epidemiology and risk factors of acute coronary syndrome in Chinese patients with type 2diabetes:a long-term prospective cohort study[J].PLoS One,2015,10(4):e122031.
[11]张倩,石蒙,檀金川.TGF-β_1与糖尿病肾病的关系及中医药研究进展[J].中国中西医结合肾病杂志,2014(11):1030-1032.
[12]赵玲,张园.糖尿病肾病中医证候研究进展[J].中国中医药现代远程教育,2010(7):199-200.
[13]吕高虹,许惠琴.肾系膜细胞与糖尿病肾病变的相关性研究[J].辽宁中医药大学学报,2011(12):48-50.
[14]ROSSERT J,TERRAZ-DURASNEL C,BRIDEAU G.Growth factors,cytokines,and renal fibrosis during the course of diabetic nephropathy[J].Diabetes Metab,2000,26(Suppl 4):16-24.
[15]周素娴.高糖高脂对肾小球细胞影响的研究进展[J].新医学,2014(5):286-289.
[16]吕高虹,许惠琴,秦佩佩,等.高糖对人肾小球系膜细胞增殖及细胞外基质的影响[J].中国老年学杂志,2013(5):1066-1067.
[17]李宏光,蔡元菊,邹锦慧,等.高糖高脂饮食对兔肾小管间质纤维化的影响[J].动物学杂志,2010(1):145-150.
[18]罗霞,邓玲艳,许文娟,等.腺苷酸活化蛋白激酶通过抑制mTOR信号通路缓解糖尿病大鼠肾脏细胞外基质沉积[J].华中科技大学学报(医学版),2015(1):10-15.
[19]GERALDES P,KING G L.Activation of protein kinase C isoforms and its impact on diabetic complications[J].Circ Res,2010,106(8):1319-1331.
[20]雷少青,苏娃婷,徐金金,等.PKC-β抑制剂LY333531对糖尿病大鼠心肌功能及Caveolins蛋白的影响[J].临床和实验医学杂志,2014(16):1305-1308.
[21]KELLY D J,ZHANG Y,HEPPER C,et al.Protein kinase C beta inhibition attenuates the progression of experimental diabetic nephropathy in the presence of continued hypertension[J].Diabetes,2003,52(2):512-518.
[22]KOYA D,HANEDA M,NAKAGAWA H,et al.Amelioration of accelerated diabetic mesangial expansion by treatment with a PKC beta inhibitor in diabetic db/db mice,a rodent model for type 2diabetes[J].FASEB J,2000,14(3):439-447.
[23]SOETIKNO V,WATANABE K,SARI F R,et al.Curcumin attenuates diabetic nephropathy by inhibiting PKC-alpha and PKC-beta1activity in streptozotocin-induced type I diabetic rats[J].Mol Nutr Food Res,2011,55(11):1655-1665.
[24]SCHWARTZ S G,FLYNN H J,AIELLO L P.Ruboxistaurin mesilate hydrate for diabetic retinopathy[J].Drugs Today(Barc),2009,45(4):269-274.