高糖高脂环境下内皮素-1在肾小球系膜细胞与内皮细胞相互关系中的作用
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摘要
第一章高糖高脂环境下系膜细胞与内皮细胞间相互作用对细胞外基质产生的影响
目的:观察不同浓度D-葡萄糖及溶血卵磷脂(LPC)在不同时间点对细胞外基质(ECM)-纤维连接蛋白(Fn)、Ⅳ型胶原(ColⅣ)产生的影响,初步探讨高糖高脂条件下系膜细胞与内皮细胞间相互作用对ECM产生的影响,从细胞间相互作用的角度探讨糖尿病肾病(DN)的发病机制。
方法:采用微孔底膜套皿法构建系膜细胞与内皮细胞共培养模型,以单独培养的系膜细胞作为对照。实验分为正常对照组(5.5mM D-葡萄糖)、高糖组(10、20、30mm D-葡萄糖)、高脂组(5、10、20mg/LLPC),各组分别培养6h、12h、24h、48h,ELISA法测定细胞上清液Fn、ColⅣ的含量。
结果
1.共培养及单培养条件下,与正常对照组相比,在相同时间点上,高浓度D-葡萄糖(10、20、30mm D-葡萄糖)呈浓度依赖方式促进细胞上清液Fn、ColⅣ产生(均P＜0.05);高浓度D-葡萄糖(10、20、30mM D-葡萄糖)作用6h、12h、24h、48h均能引起细胞上清液Fn、ColⅣ显著升高(均P＜0.05),24h达高峰,48h有所下降;但随作用时间延长,正常对照组细胞上清液Fn、ColⅣ含量无明显变化(P＞0.05)。
2.在共培养及单培养条件下,随作用时间的延长,5、10、20mg/LLPC对细胞上清液中Fn、ColⅣ的分泌均有促进作用(均P＜0.05),其中作用24h达平台期,48h与24h相比无明显统计学意义(P＞0.05);与正常对照组相比,5、10、20mg/L的LPC在各作用时间点均能引起细胞上清液Fn、ColⅣ的升高(均P＜0.05),Fn、ColⅣ含量与LPC浓度成正比。
3.正常对照组中,共培养时细胞上清液Fn、ColⅣ含量与单培养相比均无显著差异(均P＞0.05);高糖(10、20、30mM D-葡萄糖)、高脂(5、10、20mg/L LPC)条件下共培养时Fn、ColⅣ含量均较单培养时明显增高,共培养6、12、24、48h与系膜细胞单培养比较,P均＜0.05。
结论:
1.高浓度D-葡萄糖及LPC可呈浓度及时间依赖方式促进系膜细胞Fn、ColⅣ的产生。加入内皮细胞与系膜细胞共培养,细胞上清液Fn、ColⅣ含量增加更明显。
2.高糖高脂环境下,系膜细胞与内皮细胞间可能存在相互作用,促进ECM的合成。
第二章高糖高脂环境下内皮素-1在系膜细胞与内皮细胞相互关系中的作用
目的:观察共培养模型中,高糖高脂对内皮细胞内皮素-1(ET-1)表达及细胞上清液Fn、ColⅣ合成的影响,初步探讨内皮细胞生成的ET-1在系膜细胞与内皮细胞相互关系中的作用。
方法:采用系膜细胞与内皮细胞共培养方式,按照培养条件不同分为6组:正常对照组(5.5mM D-葡萄糖)、高糖组(30mM D-葡萄糖)、甘露醇组(24.5mM甘露醇)、高脂组(20mg/L LPC)、高糖高脂联合作用组(30mM D-葡萄糖+20mg/L LPC)、内皮素受体拮抗剂BQ-123干预组:BQ-123工作浓度分别为0.01μM、0.1μM、1μM。各组均培养24h。ELISA法测定细胞上清液Fn、ColⅣ含量;放免法测定上清液ET-1含量;RT-PCR法检测内皮细胞ET-1 mRNA及系膜细胞ET-1特异性受体-ETRA mRNA的表达。
结果:
1.共培养条件下,30mM D-葡萄糖及20mg/L LPC干预24h能促进细胞上清液中Fn、ColⅣ的产生(均P＜0.05);相同渗透压的甘露醇对Fn、ColⅣ的分泌均无明显影响(均P＞0.05)。高糖高脂联合作用组细胞上清液中Fn、ColⅣ的增加更为明显,与高糖组及高脂组相比较均有统计学意义(均P＜0.05)。
2.与正常对照组相比,D-葡萄糖及LPC刺激均能使内皮细胞ET-1mRNA及蛋白表达增加(P＜0.05),高糖高脂联合作用下,上述刺激作用更加明显(P＜0.05)。
3.与正常对照组比较,高糖组、高脂组均可增加系膜细胞ETRAmRNA的表达(均P＜0.05),高糖高脂联合作用组ETRA mRNA表达增加更为明显(P＜0.05),甘露醇对系膜细胞ETRA mRNA表达无显著影响(P＞0.05)。
4.在共培养模型中,经内皮素受体拮抗剂BQ-123(0.01-1μM)预处理组细胞上清液中ColⅣ及Fn的含量较高糖高脂联合作用组明显减少(均P＜0.05),且随拮抗剂浓度的增加,抑制作用愈明显(P＜0.05)。BQ-123预处理后,内皮细胞ET-1及系膜细胞ETRA mRNA的表达与高糖高脂联合作用组比较无显著差异(P＞0.05)。
结论:
1.高糖及高脂能促进系膜细胞与内皮细胞共培养模型中细胞上清液Fn、ColⅣ及内皮细胞ET-1的表达及合成,高糖高脂联合作用组Fn、ColⅣ及ET-1的表达及合成增加更明显。
2.ET-1特异性受体拮抗剂BO-123呈浓度依赖方式抑制高糖高脂所致的系膜细胞ECM合成增加,高糖高脂所致的ECM产生增加可能部分由ET-1介导。
3.高糖高脂可促进系膜细胞ETRA表达,加重ET-1对肾脏的损害。
4.高糖高脂条件下,内皮细胞分泌的ET-1可能对系膜细胞产生影响,促进ECM的合成及释放。
第三章高糖高脂环境下内皮素-1促进细胞外基质合成的作用机制及氯沙坦的干预作用
目的:通过研究高糖高脂条件下ET-1对人肾小球系膜细胞内PKCβI/PDGF-B信号通路及其下游靶基因Fn、ColⅣ的影响,进一步探讨DN发病的分子机制及氯沙坦的肾脏保护作用。
方法:
1.采用体外系膜细胞单培养方式,按培养条件分为正常对照组、ET-1组、ET-1+RG50864组、ET-1+LY333531组、高糖高脂+ET-1组、高糖高脂+ET-1+RG50864组、高糖高脂+ET-1+LY333531组、高糖高脂+ET-1+氯沙坦组;各组细胞分别培养24h。RT-PCR法检测系膜细胞PDGF-B mRNA的表达;Western-blot法检测系膜细胞PDGF-B蛋白表达水平;ELISA法检测上清液Fn、ColⅣ的蛋白含量。
2.取处于对数生长期的系膜细胞分为正常对照组、ET-1组、ET-1+LY333531组、高糖高脂+ET-1组、高糖高脂+ET-1+LY333531组、高糖高脂+ET-1+氯沙坦组;各组细胞分别培养24h。RT-PCR法检测系膜细胞PKCβI mRNA的表达;Western-blot法检测系膜细胞PKCβI蛋白表达水平;细胞免疫荧光检测系膜细胞PKCβI蛋白的表达及分布。
结果:
1.ET-1刺激能促进上清液Fn、ColⅣ的产生及系膜细胞内PDGF-B mRNA及蛋白表达(P＜0.05);高糖高脂对此有协同作用(P＜0.05)。酪氨酸蛋白激酶抑制剂RG50864可以抑制高糖高脂条件下ET-1所致的Fn、ColⅣ产生增加(P＜0.05),但其对系膜细胞PDGF-B mRNA及蛋白表达无明显影响(P＞0.05)。
2.与正常对照组相比,ET-1能上调系膜细胞内PKCβI mRNA及蛋白表达(P＜0.05),ET-1刺激后,细胞免疫荧光显示,PKCβI蛋白表达增强,且发生胞膜及胞核转位;高糖高脂对此有协同作用。PKCβI抑制剂LY333531干预能显著下调系膜细胞PKCβI、PDGF-B mRNA及蛋白的表达,减少下游靶基因Fn、ColⅣ的产生(P＜0.05)。
3.与高糖高脂+ET-1组比较,氯沙坦干预可显著下调系膜细胞PKCβI及PDGF-B mRNA和蛋白表达(P＜0.05),明显减少细胞上清液中Fn、ColⅣ的产生(P＜0.05)。
结论:
1.高糖高脂条件下,ET-1所致系膜细胞ECM产生增加可能由PKCβI信号通路依赖产生的PDGF-B部分介导。
2.高糖高脂条件下,氯沙坦可抑制高糖高脂条件下ET-1对PKCβI/PDGF-B通路的激活,从而减少下游靶基因Fn、ColⅣ的产生,发挥肾脏保护作用。
Part 1.The effect of high glucose and lysophosphatidylcholine on the role of extracellular matrix accumulation via the interaction of mesangial cells and endothelial cells
Objective:To investigate the interaction between mesangial cells and endothelial cells under different concentrations of glucose and lysophosphatidylcholine(LPC) at different time points,further explored the mechanism of diabetic nephropathy through examining concentration of fibronectin(Fn) and collagen typeⅣ(ColⅣ) in culture media.
Methods:The model of intercellular interaction between mesangial cells and endothelial cells was established using Millicell membrane culture dish.The mesangial cells were cultured separately or co-cultured with endothelial cells and divided into three guoups:control,high glucose (10,20,30mM glucose) and LPC(5,10,20mg/L LPC),each group was cultured for 6,12,24,48 hours respectively.The concentration of Fn and ColⅣin culture media was determined by enzyme-linked immunosorbent assay(ELISA).
Results:
(1) Both in monolayer and co-culture cell culture,the concentration of Fn and ColⅣhad no significant change in control group at different time points(P＞0.05).With the same cultured time,the level of Fn and ColⅣof high glucose was higher than that of control group in co-culture and monolayer cell culture(P＜0.05),and the expression intensity increased in a concentration-dependent manner;The concentration of Fn and ColⅣin culture media treated with high glucose for 6,12,24,48 hours respectively increased in a time-dependent manner(P＜0.05), arrived peak level at 24h,then decreased slightly at 48 h.
(2) Compared with control group,the concentration of Fn and ColⅣincreased in LPC group from 6h time point,the expression intensity enhanced in a time-dependent manner(P＜0.05),arrived peak level at 24 h; 5,10,20mg/L LPC could increase the secrection of Fn and ColⅣin culture media in a concentration-dependent manner(P＜0.05).
(3) In control group,the level of Fn and ColⅣbetween co-culture and monolayer cells culture had no significant difference(P＞0.05). Exposed to high glucose or LPC,the concentration of Fn and ColⅣin co-cultured cells was higher than that in monolayer cells culture at 6,12, 24,48h time points(P＜0.05).
Conclusions:
(1) Both in co-culture and monolayer cell culture,high glucose and LPC increases the concentration of Fn and ColⅣin a concentration and time-dependent manner.
(2) On the condition of high glucose and LPC,there is some kind of interaction between mesangial cells and endothelial cells which can increase the excretion of extracellular matrix.
Part 2.Effect of endothelin-1 on the interaction between human mesangial cells and endothelial cells on the condition of high glucose and LPC
Objective:To investigate the effect of endothelin-1 on the role of extracellular matrix accumulation via the interaction between mesangial cells and endothelial cells on the condition of high glucose and LPC through examining expression of endothelin-1(ET-1),Fn and ColⅣin co-cultured cell culture.
Methods:The model of intercellular interaction between mesangial cells and endothelial cells was established and divided into six groups: control,mannitol,high glucose,LPC,high glucose and LPC, BQ-123(0.01μM,0.1μM,1μM),each group was cultured for 24 hours. The concentration of Fn and ColⅣin culture media was determined by ELISA,ET-1 in supernatant was detected by radio-immunoassay,ET-1 mRNA of endothelial cells and ETRA mRNA of mesangial cells was measured by reverse transcription polymerase chain response(RT-PCR).
Results:
(1) When mesangial cells were co-cultured with endothelial cells cultured for 24 hours,the concentration of Fn and ColⅣin culture media increased in high glucose group and LPC group(P＜0.05).Compare with high glucose group and LPC group,the expression of Fn and ColⅣincreased significantly in high glucose and LPC group(P＜0.05),but there was no significant difference between mannitol group and control group(P＞0.05).
(2) Compared with control group,ET-1 increased remarkably in high glucose group and LPC group(P＜0.05),the stimulating effect was more pronounced in high glucose and LPC group(P＜0.05).
(3) In co-culture cell culture,compared with control group,ETRA mRNA expression of mesangial cells in high glucose group and LPC group increased remarkably(P＜0.05),the stimulating effect was more pronounced in high glucose and LPC group(P＜0.05).
(4) Compared with high glucose and LPC group,ET-1 receptor antagonist BQ-123 could inhibit Fn and ColⅣsecretion in a dosedependent manner in co-cultured model(P＜0.05),but BQ-123 had no effect on the expression of ET-1 and ETRA(P＞0.05).
Conclusions:
(1) The expression of Fn,ColⅣand ET-1 is found to be remarkably increased on the condition of high glucose and LPC in co-cultured mode.
(2) ET-1 receptor antagonist BQ-123 can inhibit the secretion of Fn and ColⅣin a dose-dependent manner.ET-1 may play an important role in extracellular matrix accumulation on the condition of high glucose and LPC.
(3) The expression of ETRA in mesangial cells is promoted by high glucose and LPC,which can enlarge the biological effect of ET-1.
(4) ET-1 produced by endothelial cells may have an important impact on mesangial cells,which can improve the synthesis of extracellular matrix.
Part 3.The mechanism of extracellular matrix accumulation induced by endothelin-1 on the condition of high glucose and LPC and the intervention effect of losartan
Objective:To define whether ET-1 activated protein kinase CβI/ platelet- derived growth factor-B(PKCβI/PDGF-B) signaling pathway to mediate extracellular matrix accumulation,further explored the molecular mechanism of diabetic nephropathy and the intervention effect of losartan.
Methods:
(1) Mesangial cells were cultured seperately and divided into eight groups:control,ET-1,ET-1 + RG50864,ET-1 + LY333531,high glucose and LPC+ ET-1,high glucose and LPC + ET-1 + RG50864,high glucose and LPC + ET-1 + LY333531,high glucose and LPC + ET-1 + losartan, and each group was cultured for 24 hours.Fn and ColⅣin supernatant was detected by ELISA,the expression of PDGF-B mRNA was measured by RT-PCR and PDGF-B protein expression was detected by Westernblot.
(2) The monolayer mesangial cells were divided into six groups: control,ET-1,ET-1 + LY333531,high glucose and LPC + ET-1,high glucose and LPC + ET-1 + LY333531,high glucose and LPC + ET-1 + losartan,and each group was cultured for 24 hours.Fn and ColⅣin supernatant was detected by ELISA,the expression of PKCβ1 mRNA was measured by RT-PCR,the protein expression of PKCβI was detected by Western-blot and immunofluorescence.
Results:
(1)The expression and secretion of ColⅣ,Fn and PDGF-B increased remarkably at 24h after ET-1 stimulation(P＜0.05),the stimulating effect could be enlarged by high glucose and LPC(P＜0.05). Addition of a tyrosine kinase inhibitor RG50864 to cells could prevent ET-1-induced Fn and ColⅣprotein up-secretion(P＜0.05),but RG50864 had no effect on the PDGF-B mRNA and protein expression of mesangial cells(P＞0.05).
(2)After ET-1 stimulation,HMC PKCβ1 mRNA and protein expression was up-regulated compared with control(P＜0.05),and immunofluorescence demonstrated that ET-1-induced PKCβ1 transmitting from cytoplasm to nucleus and membrane,and the stimulating effect could be enlarged by high glucose and LPC(P＜0.05). Further more,LY333531 could prevent ET-1-induced Fn and ColⅣprotein up-secretion through attenuating PKCβI and PDGF-B expression of mesangial cells(P＜0.05).
(3)Compared with high glucose and LPC + ET-1 group,losartan could decrease PKCβI and PDGF-B expression(P＜0.05),partly prevented ET-1-induced extracellular matrix secretion on the condition of high glucose and LPC(P＜0.05).
Conclusions:
(1) ET-1 stimulates human glomerular mesangial cells extracellular matrix secretion may by activating PKCβI/PDGF-B signaling on the condition of high glucose and LPC.
(2) Losartan can protect kidney by inhibitting PKCβI/PDGF-B signaling and decreasing the extracellular matrix secretion.
目的:观察不同浓度D-葡萄糖及溶血卵磷脂(LPC)在不同时间点对细胞外基质(ECM)-纤维连接蛋白(Fn)、Ⅳ型胶原(ColⅣ)产生的影响,初步探讨高糖高脂条件下系膜细胞与内皮细胞间相互作用对ECM产生的影响,从细胞间相互作用的角度探讨糖尿病肾病(DN)的发病机制。
方法:采用微孔底膜套皿法构建系膜细胞与内皮细胞共培养模型,以单独培养的系膜细胞作为对照。实验分为正常对照组(5.5mM D-葡萄糖)、高糖组(10、20、30mm D-葡萄糖)、高脂组(5、10、20mg/LLPC),各组分别培养6h、12h、24h、48h,ELISA法测定细胞上清液Fn、ColⅣ的含量。
结果
1.共培养及单培养条件下,与正常对照组相比,在相同时间点上,高浓度D-葡萄糖(10、20、30mm D-葡萄糖)呈浓度依赖方式促进细胞上清液Fn、ColⅣ产生(均P＜0.05);高浓度D-葡萄糖(10、20、30mM D-葡萄糖)作用6h、12h、24h、48h均能引起细胞上清液Fn、ColⅣ显著升高(均P＜0.05),24h达高峰,48h有所下降;但随作用时间延长,正常对照组细胞上清液Fn、ColⅣ含量无明显变化(P＞0.05)。
2.在共培养及单培养条件下,随作用时间的延长,5、10、20mg/LLPC对细胞上清液中Fn、ColⅣ的分泌均有促进作用(均P＜0.05),其中作用24h达平台期,48h与24h相比无明显统计学意义(P＞0.05);与正常对照组相比,5、10、20mg/L的LPC在各作用时间点均能引起细胞上清液Fn、ColⅣ的升高(均P＜0.05),Fn、ColⅣ含量与LPC浓度成正比。
3.正常对照组中,共培养时细胞上清液Fn、ColⅣ含量与单培养相比均无显著差异(均P＞0.05);高糖(10、20、30mM D-葡萄糖)、高脂(5、10、20mg/L LPC)条件下共培养时Fn、ColⅣ含量均较单培养时明显增高,共培养6、12、24、48h与系膜细胞单培养比较,P均＜0.05。
结论:
1.高浓度D-葡萄糖及LPC可呈浓度及时间依赖方式促进系膜细胞Fn、ColⅣ的产生。加入内皮细胞与系膜细胞共培养,细胞上清液Fn、ColⅣ含量增加更明显。
2.高糖高脂环境下,系膜细胞与内皮细胞间可能存在相互作用,促进ECM的合成。
第二章高糖高脂环境下内皮素-1在系膜细胞与内皮细胞相互关系中的作用
目的:观察共培养模型中,高糖高脂对内皮细胞内皮素-1(ET-1)表达及细胞上清液Fn、ColⅣ合成的影响,初步探讨内皮细胞生成的ET-1在系膜细胞与内皮细胞相互关系中的作用。
方法:采用系膜细胞与内皮细胞共培养方式,按照培养条件不同分为6组:正常对照组(5.5mM D-葡萄糖)、高糖组(30mM D-葡萄糖)、甘露醇组(24.5mM甘露醇)、高脂组(20mg/L LPC)、高糖高脂联合作用组(30mM D-葡萄糖+20mg/L LPC)、内皮素受体拮抗剂BQ-123干预组:BQ-123工作浓度分别为0.01μM、0.1μM、1μM。各组均培养24h。ELISA法测定细胞上清液Fn、ColⅣ含量;放免法测定上清液ET-1含量;RT-PCR法检测内皮细胞ET-1 mRNA及系膜细胞ET-1特异性受体-ETRA mRNA的表达。
结果:
1.共培养条件下,30mM D-葡萄糖及20mg/L LPC干预24h能促进细胞上清液中Fn、ColⅣ的产生(均P＜0.05);相同渗透压的甘露醇对Fn、ColⅣ的分泌均无明显影响(均P＞0.05)。高糖高脂联合作用组细胞上清液中Fn、ColⅣ的增加更为明显,与高糖组及高脂组相比较均有统计学意义(均P＜0.05)。
2.与正常对照组相比,D-葡萄糖及LPC刺激均能使内皮细胞ET-1mRNA及蛋白表达增加(P＜0.05),高糖高脂联合作用下,上述刺激作用更加明显(P＜0.05)。
3.与正常对照组比较,高糖组、高脂组均可增加系膜细胞ETRAmRNA的表达(均P＜0.05),高糖高脂联合作用组ETRA mRNA表达增加更为明显(P＜0.05),甘露醇对系膜细胞ETRA mRNA表达无显著影响(P＞0.05)。
4.在共培养模型中,经内皮素受体拮抗剂BQ-123(0.01-1μM)预处理组细胞上清液中ColⅣ及Fn的含量较高糖高脂联合作用组明显减少(均P＜0.05),且随拮抗剂浓度的增加,抑制作用愈明显(P＜0.05)。BQ-123预处理后,内皮细胞ET-1及系膜细胞ETRA mRNA的表达与高糖高脂联合作用组比较无显著差异(P＞0.05)。
结论:
1.高糖及高脂能促进系膜细胞与内皮细胞共培养模型中细胞上清液Fn、ColⅣ及内皮细胞ET-1的表达及合成,高糖高脂联合作用组Fn、ColⅣ及ET-1的表达及合成增加更明显。
2.ET-1特异性受体拮抗剂BO-123呈浓度依赖方式抑制高糖高脂所致的系膜细胞ECM合成增加,高糖高脂所致的ECM产生增加可能部分由ET-1介导。
3.高糖高脂可促进系膜细胞ETRA表达,加重ET-1对肾脏的损害。
4.高糖高脂条件下,内皮细胞分泌的ET-1可能对系膜细胞产生影响,促进ECM的合成及释放。
第三章高糖高脂环境下内皮素-1促进细胞外基质合成的作用机制及氯沙坦的干预作用
目的:通过研究高糖高脂条件下ET-1对人肾小球系膜细胞内PKCβI/PDGF-B信号通路及其下游靶基因Fn、ColⅣ的影响,进一步探讨DN发病的分子机制及氯沙坦的肾脏保护作用。
方法:
1.采用体外系膜细胞单培养方式,按培养条件分为正常对照组、ET-1组、ET-1+RG50864组、ET-1+LY333531组、高糖高脂+ET-1组、高糖高脂+ET-1+RG50864组、高糖高脂+ET-1+LY333531组、高糖高脂+ET-1+氯沙坦组;各组细胞分别培养24h。RT-PCR法检测系膜细胞PDGF-B mRNA的表达;Western-blot法检测系膜细胞PDGF-B蛋白表达水平;ELISA法检测上清液Fn、ColⅣ的蛋白含量。
2.取处于对数生长期的系膜细胞分为正常对照组、ET-1组、ET-1+LY333531组、高糖高脂+ET-1组、高糖高脂+ET-1+LY333531组、高糖高脂+ET-1+氯沙坦组;各组细胞分别培养24h。RT-PCR法检测系膜细胞PKCβI mRNA的表达;Western-blot法检测系膜细胞PKCβI蛋白表达水平;细胞免疫荧光检测系膜细胞PKCβI蛋白的表达及分布。
结果:
1.ET-1刺激能促进上清液Fn、ColⅣ的产生及系膜细胞内PDGF-B mRNA及蛋白表达(P＜0.05);高糖高脂对此有协同作用(P＜0.05)。酪氨酸蛋白激酶抑制剂RG50864可以抑制高糖高脂条件下ET-1所致的Fn、ColⅣ产生增加(P＜0.05),但其对系膜细胞PDGF-B mRNA及蛋白表达无明显影响(P＞0.05)。
2.与正常对照组相比,ET-1能上调系膜细胞内PKCβI mRNA及蛋白表达(P＜0.05),ET-1刺激后,细胞免疫荧光显示,PKCβI蛋白表达增强,且发生胞膜及胞核转位;高糖高脂对此有协同作用。PKCβI抑制剂LY333531干预能显著下调系膜细胞PKCβI、PDGF-B mRNA及蛋白的表达,减少下游靶基因Fn、ColⅣ的产生(P＜0.05)。
3.与高糖高脂+ET-1组比较,氯沙坦干预可显著下调系膜细胞PKCβI及PDGF-B mRNA和蛋白表达(P＜0.05),明显减少细胞上清液中Fn、ColⅣ的产生(P＜0.05)。
结论:
1.高糖高脂条件下,ET-1所致系膜细胞ECM产生增加可能由PKCβI信号通路依赖产生的PDGF-B部分介导。
2.高糖高脂条件下,氯沙坦可抑制高糖高脂条件下ET-1对PKCβI/PDGF-B通路的激活,从而减少下游靶基因Fn、ColⅣ的产生,发挥肾脏保护作用。
Part 1.The effect of high glucose and lysophosphatidylcholine on the role of extracellular matrix accumulation via the interaction of mesangial cells and endothelial cells
Objective:To investigate the interaction between mesangial cells and endothelial cells under different concentrations of glucose and lysophosphatidylcholine(LPC) at different time points,further explored the mechanism of diabetic nephropathy through examining concentration of fibronectin(Fn) and collagen typeⅣ(ColⅣ) in culture media.
Methods:The model of intercellular interaction between mesangial cells and endothelial cells was established using Millicell membrane culture dish.The mesangial cells were cultured separately or co-cultured with endothelial cells and divided into three guoups:control,high glucose (10,20,30mM glucose) and LPC(5,10,20mg/L LPC),each group was cultured for 6,12,24,48 hours respectively.The concentration of Fn and ColⅣin culture media was determined by enzyme-linked immunosorbent assay(ELISA).
Results:
(1) Both in monolayer and co-culture cell culture,the concentration of Fn and ColⅣhad no significant change in control group at different time points(P＞0.05).With the same cultured time,the level of Fn and ColⅣof high glucose was higher than that of control group in co-culture and monolayer cell culture(P＜0.05),and the expression intensity increased in a concentration-dependent manner;The concentration of Fn and ColⅣin culture media treated with high glucose for 6,12,24,48 hours respectively increased in a time-dependent manner(P＜0.05), arrived peak level at 24h,then decreased slightly at 48 h.
(2) Compared with control group,the concentration of Fn and ColⅣincreased in LPC group from 6h time point,the expression intensity enhanced in a time-dependent manner(P＜0.05),arrived peak level at 24 h; 5,10,20mg/L LPC could increase the secrection of Fn and ColⅣin culture media in a concentration-dependent manner(P＜0.05).
(3) In control group,the level of Fn and ColⅣbetween co-culture and monolayer cells culture had no significant difference(P＞0.05). Exposed to high glucose or LPC,the concentration of Fn and ColⅣin co-cultured cells was higher than that in monolayer cells culture at 6,12, 24,48h time points(P＜0.05).
Conclusions:
(1) Both in co-culture and monolayer cell culture,high glucose and LPC increases the concentration of Fn and ColⅣin a concentration and time-dependent manner.
(2) On the condition of high glucose and LPC,there is some kind of interaction between mesangial cells and endothelial cells which can increase the excretion of extracellular matrix.
Part 2.Effect of endothelin-1 on the interaction between human mesangial cells and endothelial cells on the condition of high glucose and LPC
Objective:To investigate the effect of endothelin-1 on the role of extracellular matrix accumulation via the interaction between mesangial cells and endothelial cells on the condition of high glucose and LPC through examining expression of endothelin-1(ET-1),Fn and ColⅣin co-cultured cell culture.
Methods:The model of intercellular interaction between mesangial cells and endothelial cells was established and divided into six groups: control,mannitol,high glucose,LPC,high glucose and LPC, BQ-123(0.01μM,0.1μM,1μM),each group was cultured for 24 hours. The concentration of Fn and ColⅣin culture media was determined by ELISA,ET-1 in supernatant was detected by radio-immunoassay,ET-1 mRNA of endothelial cells and ETRA mRNA of mesangial cells was measured by reverse transcription polymerase chain response(RT-PCR).
Results:
(1) When mesangial cells were co-cultured with endothelial cells cultured for 24 hours,the concentration of Fn and ColⅣin culture media increased in high glucose group and LPC group(P＜0.05).Compare with high glucose group and LPC group,the expression of Fn and ColⅣincreased significantly in high glucose and LPC group(P＜0.05),but there was no significant difference between mannitol group and control group(P＞0.05).
(2) Compared with control group,ET-1 increased remarkably in high glucose group and LPC group(P＜0.05),the stimulating effect was more pronounced in high glucose and LPC group(P＜0.05).
(3) In co-culture cell culture,compared with control group,ETRA mRNA expression of mesangial cells in high glucose group and LPC group increased remarkably(P＜0.05),the stimulating effect was more pronounced in high glucose and LPC group(P＜0.05).
(4) Compared with high glucose and LPC group,ET-1 receptor antagonist BQ-123 could inhibit Fn and ColⅣsecretion in a dosedependent manner in co-cultured model(P＜0.05),but BQ-123 had no effect on the expression of ET-1 and ETRA(P＞0.05).
Conclusions:
(1) The expression of Fn,ColⅣand ET-1 is found to be remarkably increased on the condition of high glucose and LPC in co-cultured mode.
(2) ET-1 receptor antagonist BQ-123 can inhibit the secretion of Fn and ColⅣin a dose-dependent manner.ET-1 may play an important role in extracellular matrix accumulation on the condition of high glucose and LPC.
(3) The expression of ETRA in mesangial cells is promoted by high glucose and LPC,which can enlarge the biological effect of ET-1.
(4) ET-1 produced by endothelial cells may have an important impact on mesangial cells,which can improve the synthesis of extracellular matrix.
Part 3.The mechanism of extracellular matrix accumulation induced by endothelin-1 on the condition of high glucose and LPC and the intervention effect of losartan
Objective:To define whether ET-1 activated protein kinase CβI/ platelet- derived growth factor-B(PKCβI/PDGF-B) signaling pathway to mediate extracellular matrix accumulation,further explored the molecular mechanism of diabetic nephropathy and the intervention effect of losartan.
Methods:
(1) Mesangial cells were cultured seperately and divided into eight groups:control,ET-1,ET-1 + RG50864,ET-1 + LY333531,high glucose and LPC+ ET-1,high glucose and LPC + ET-1 + RG50864,high glucose and LPC + ET-1 + LY333531,high glucose and LPC + ET-1 + losartan, and each group was cultured for 24 hours.Fn and ColⅣin supernatant was detected by ELISA,the expression of PDGF-B mRNA was measured by RT-PCR and PDGF-B protein expression was detected by Westernblot.
(2) The monolayer mesangial cells were divided into six groups: control,ET-1,ET-1 + LY333531,high glucose and LPC + ET-1,high glucose and LPC + ET-1 + LY333531,high glucose and LPC + ET-1 + losartan,and each group was cultured for 24 hours.Fn and ColⅣin supernatant was detected by ELISA,the expression of PKCβ1 mRNA was measured by RT-PCR,the protein expression of PKCβI was detected by Western-blot and immunofluorescence.
Results:
(1)The expression and secretion of ColⅣ,Fn and PDGF-B increased remarkably at 24h after ET-1 stimulation(P＜0.05),the stimulating effect could be enlarged by high glucose and LPC(P＜0.05). Addition of a tyrosine kinase inhibitor RG50864 to cells could prevent ET-1-induced Fn and ColⅣprotein up-secretion(P＜0.05),but RG50864 had no effect on the PDGF-B mRNA and protein expression of mesangial cells(P＞0.05).
(2)After ET-1 stimulation,HMC PKCβ1 mRNA and protein expression was up-regulated compared with control(P＜0.05),and immunofluorescence demonstrated that ET-1-induced PKCβ1 transmitting from cytoplasm to nucleus and membrane,and the stimulating effect could be enlarged by high glucose and LPC(P＜0.05). Further more,LY333531 could prevent ET-1-induced Fn and ColⅣprotein up-secretion through attenuating PKCβI and PDGF-B expression of mesangial cells(P＜0.05).
(3)Compared with high glucose and LPC + ET-1 group,losartan could decrease PKCβI and PDGF-B expression(P＜0.05),partly prevented ET-1-induced extracellular matrix secretion on the condition of high glucose and LPC(P＜0.05).
Conclusions:
(1) ET-1 stimulates human glomerular mesangial cells extracellular matrix secretion may by activating PKCβI/PDGF-B signaling on the condition of high glucose and LPC.
(2) Losartan can protect kidney by inhibitting PKCβI/PDGF-B signaling and decreasing the extracellular matrix secretion.
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