Effects Of Lignans From Eucommia Ulmoides On AngⅡ-induced Proliferation And Biosynthesis Of Extracellular Matrix In Rat Mesangial Cells

Objective:To study the effects of lignans from Eucommia ulmoides on the proliferation and the biosynthesis of extracellular matrix (ECM) in rat mesangial cell induced by Ang II, providing theoretical bases for the application of lignans from Eucommia ulmoides in renoprotection.Methods:Rat glomerular mesangial cell (HBZY-1) were cultured in vitro and divided into7groups:control group, Ang II group (10-8mol/L Ang II), epalrestat group (10-8mol/L Ang II+20μmol/L epalrestat), losartan group (10-8mol/L Ang II+20μmol/L losartan), low concentration lignans group (10-8mol/L Ang II+20mg/L lignans), middle concentration lignans group (10-8mol/L Ang II+40mg/L lignans), high concentration lignans group (10-8mol/L Ang II+80mg/L lignans). The proliferation of cell was assessed by MTT. The mRNA levels of Col I, Col III, Col IV, FN and AR in HBZY-1were determined by RT-qPCR. Western Blot was used to detect the protein levels of Col I, Col III, Col IV, FN and AR in HBZY-1.Results:(1) Ang II induced the proliferation of HBZY-1significantly, compared with control (1.76±0.06vs1.67±0.05, P=0.14). The proliferation was inhibited significantly in the presence of20μmol/L losartan (1.63±0.04vs1.76±0.06, P=0.014),20μmol/L epalrestat (1.65±0.04vs1.76±0.06, P<0.001) or different concentration of lignans (1.68±0.05,1.58±0.07,1.59±0.07vs1.76±0.06, P=0.020, P<0.001, P<0.001).(2) Ang II (10-8mol/L) increased the mRNA expression of Col IV (2.33±0.13vs1.00±0.31, P<0.01), FN (1.17±0.04vs1.00±0.08, P<0.01), Col I (1.36±0.05vs1.00±0.05, P<0.01), Col III (1.31±0.03vs1.00±0.04, P<0.01) and AR (1.35±0.04vs1.00±0.11, P<0.01) in HBZY-1significantly, compared control group.Losartan decreased the mRNA expression of Col IV (1.28±0.09vs2.33±0.13, P<0.01), FN (0.79±0.03vs1.17±0.04, P<0.01), Col I (1.07±0.11vs1.36±0.05, P<0.01), Col III (1.11±0.04vs1.31±0.03, P<0.01) and AR (1.22±0.03vs1.35±0.04, P<0.05) in HBZY-1significantly, compared Ang.II group. Different concentration of lignans dose-dependently decreased the mRNA expression of FN(0.86±0.06,0.90±0.03,0.67±0.01vs1.17±0.04, P<0.01),Col Ⅰ(0.75±0.01,0.67±0.05,0.55±0.01vs1.36±0.05,P<0.01),Col Ⅲ(0.78±0.07,0.34±0.02,0.18±0.01vs1.31±0.03,P<0.01) and AR(1.08±0.03,0.98±0.09,0.86±0.08vs1.35±0.04,P<0.01),and high dose of lignan decreased the mRNA level of Col Ⅳ significantly(1.51±0.21vs2.33±0.13,P<0.01).Epalrestat also had reductive effects on the mRNA levels of FN(0.76±0.03vs1.17±0.04,P<0.01),Col Ⅰ(1.25±0.05vs1.36±0.05,P<0.05) and Col Ⅲ(0.68±0.02vs1.31±0.03,P<0.01)significantly,compared Ang Ⅱ group.On the contrary,Epalrestat promoted Col Ⅳ(2.54±0.14vs2.33±0.13,P=0.275)and AR(3.78±0.13vs1.35±0.04,P<0.01)mRNA expresslon.(3)Ang Ⅱ(10-8mol/L)increased the protein expression of Col Ⅳ(2.18±0.26vs1.00±0.02,P<0.01),FN(2.43±0.03vs1.00±0.07,P<0.01), Col Ⅰ(1.29±0.05vs1.00±0.04,P<0.01),Col Ⅲ(5.08±0.21vs1.00±0.43,P<0.01)and AR(2.02±0.08vs1.00±0.08,P<0.01)in HBZY-1significantly,compared control group.Losartan decreased the mRNA expression of Col Ⅳ(0.47±0.15vs2.18±0.26,P<0.01),FN(0.17±0.01vs2.43±0.03,P<0.01),Col Ⅰ(0.47±0.02vs1.29±0.05,P<0.01),Col Ⅲ(0.91±0.36vs5.08±0.21,P<0.01) and AR(0.35±0.15vs2.02±0.08,P<0.01)in HBZY-1significantly, compared Ang.Ⅱ group.Different concentration of lignans decreased the protein level of Col Ⅳ (1.71±0.07,1.58±0.08,0.50±0.14vs2.18±0.26,P<0.05,P<0.01,P<0.01),FN(0.19±0.09,0.40±0.12,±.20±0.10vs2.43±0.03,P<0.01), Col Ⅰ(0.76±0.01,0.71±0.02,0.66±0.02vs1.29±0.05,P<0.01),Col Ⅲ (2.78±0.21,1.9±0.37,1.44±0.39vs5.08±0.21,P<0.01)and AR(0.52±0.17,0.66±0.22,0.45±0.23vs2.02±0.08,P<0.01)in HBZY-1.Epalrestat also had reductive effects on the mRNA levels of FN (2.07±0.06vs2.43±0.03,P<0.01),Col Ⅰ(1.07±0.02vs1.29±0.05,P<0.01) and Col Ⅲ(1.12±0.46vs5.08±0.21,P<0.01)significantly,compared Ang Ⅱ group.On the contrary,Epalrestat promoted Col Ⅳ(2.71±0.42vs 2.18±0.26, P<0.01) and AR (3.13±0.24vs2.02±0.08, P<0.01) protein expression significantly.Conclusions:(1) Ang II stimulates the biosynthesis of Col III in HBZY-1. Losartan could inhibit the stimulative effect of Ang II.(2) Lignans can inhibit the proliferation and the biosynthesis of ECM (Col IV, FN, Col I and Col III) and AR in HBZY-1induced by Ang II=(3) Epalrestat can suppress the proliferation and the biosynthesis of ECM (FN, Col I and Col III) in HBZY-1induced by Ang II.