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To study the effects of lignans from Eucommia ulmoides on the proliferation 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 into6groups:control group, Ang Ⅱ group (10-8mol/L Ang Ⅱ), epalrestat group (10-8mol/L Ang Ⅱ+20μmol/L epalrestat), 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 after24,36,48h treatment. The cell cycle and cell apoptosis were determined by flow cytometre. The mRNA and protein level of P21, P27, Bax, Bcl-2and AR in HBZY-1were determined by RT-qPCR and Western Blot. The activity of AR was detected by UV determination.Results:(1) Ang Ⅱ induced the proliferation of HBZY-1significantly compared with (?)(1.91±0.06vs1.49±0.08, P<0.001,48h). The proliferation was inhibited significantly in the presence of different concentration of lignans (1.60±0.03,1.58±0.07,1.52±0.04vs1.91±0.06; all the P values less than0.001) and epalrestat (1.60±0.04vs1.91±0.06, P<0.001) compared with the Ang Ⅱ group. It prompted that model builded successfully, and epalrestat and different concentrations of lignans can exhibit the proliferation;(2) Ang Ⅱ induced the G1phase cell decreased and S phase cell increased signifycantly compared with control (G,:69.2%±0.3%vs73.9%±0.6%, P= 0.024;22.4%±1.1%vs12.6%±0.2%, P <0.001). Different concentration of lignans (14.0%±0.6%,10.1%±0.2%,7.1%±0.7%vs22.4%±1.1%; all the P values less than0.001) and epalrestat (11.1%±0.4%vs22.4%±1.1%, P <0.001) can inhibit cell to enter S phase significantly; the G1phase cell incresded obviously only in high concentration of lignans (77.6%±0.7%vs69.2%±0.3%, P=0.009) and the effect is concentration dependent compared with the Ang Ⅱ group. It prompted that Ang Ⅱ can induce quiescent cells into the phase of proliferation, but epalrestat and lignans can prevent mesangial cells from G1to S phase, thereby inhibiting proliferation;(3) Ang Ⅱ induced the mRNA expression of P21and P27decreased significantly compared with control (P21:0.31±0.01vs1.00±0.03, P <0.001; P27:0.30±0.03vs1.00±0.02, P <0.001). Different concentration of lignans (P21:1.18±0.05,1.34±0.06,1.78±0.11vs1.00±0.03, all the P values less than0.001; P27:1.17±0.05,1.50±0.06,2.10±0.03vs1.00±0.02, all the P values less than0.001) and epalrestat (P21:1.38±0.07vs1.00±0.03, P <0.001; P27:1.45±0.05vs1.00±0.02, P <0.001) can increase the mRNA expression of P21and P27compared with the Ang Ⅱ group significantly;Ang II induced the protein expression of P21and P27decreased significantly compared with control (P21:0.52±0.06vs1.00±0.14, P=0.004; P27:0.72±0.02vs1.00±0.12, P <0.001). Different concentrations of lignans (P21:2.23±0.15,3.23±0.23,5.24±0.22vs0.52±0.06, all the P values less than0.001; P27:1.96±0.06,2.24±0.06,3.14±0.06vs0.72±0.02, P all the P values less than0.001) and epalrestat (P21:3.21±0.12vs0.52±0.06, P<0.001; P27:2.36±0.04vs0.72±0.02, P <0.001) can increase the protein expression of P21and P27significantly compared with the Ang II group. It prompted that the expression of P21and P27played an important role in the process of regulating cell cycle on HBZY-1;(4) The apoptosis rate of Ang Ⅱ group were significantly higher than the control (10.5%±0.3%vs8.7%±0.3%, P=0.001). The apoptosis rate of different concentrations of lignans (16.2%±0.3%,18.7%±0.2%,22.5%±0.9%vs10.5%±0.3%; all the P values less than0.001) and epalrestat (17.9%±0.6%vs10.5%±0.3%; P <0.001) increased significantly compared with the Ang Ⅱ group.The higher the concentration of lignan the greater the apoptosis rate compared with the Ang Ⅱ group. It prompted that epalrestat and lignans may inhibite proliferation of HBZY-1through promoting apoptosis;(5) Ang Ⅱ induced the mRNA expression of Bax and Bcl-2increased significantly compared with control (Bax:1.70±0.02vs1.00±0.08, P <0.001; Bcl-2:1.34±0.06vs l.00±0.03, P <0.001). Different concentration of lignans (1.89±0.02,2.30±0.01,3.29±0.10vs1.00±0.08; P=0.001, P <0.001, P <0.001) and epalrestat (2.25±0.02vs1.00±0.08; P <0.001) can increase the mRNA expression of Bax significantly compared with the Ang Ⅱ group, but the mRNA expression of Bcl-2was no ststistical differences between four groups and the Ang Ⅱ group (1.43±0.02,1.31±0.05,1.34±0.07,1.28±0.04vs1.34±0.06; P=0.056, P=0.435, P=0.942, P=0.167);Ang Ⅱ induced the protein expression of Bax and Bcl-2increased significa-ntly compared with control (Bax:1.81±0.04vs1.00±0.05, P <0.001; Bcl-2:2.24±0.10vs1.00±0.10, P <0.001). Different concentration of lignans (2.13±0.04,2.39±0.04,3.15±0.06vs1.81±0.04; all the P values less than0.001) and epalrestat (2.29±0.05vs1.81±0.04; P <0.001) can increase the protein expression of Bax significantly compared with the Ang Ⅱ group, but the protein expression of Bcl-2was no ststistical differentces between four groups and the Ang Ⅱ group (2.21±0.09,2.31±0.12,2.28±0.12,2.29±0.02vs2.24±0.10; P=0.769, P=0.365, P=0.617, P=0.481). It prompted that the expression of Bax and Bcl-2played an important role in the process of regulating cell apoptosis on HBZY-1;(6) Ang Ⅱ can improve the acvtity of AR significantly compared with control (0.19±0.03vs0.14±0.01, P=0.012). Different concentrations of lignans (0.11±0.01,0.11±0.01,0.12±0.03vs0.19±0.03; P<0.001, P=0.001, P=0.002) and epalrestat (0.11±0.01vs0.19±0.03; P=0.001) can decrease the acvtity significantly compared with the Ang II group. It prompted that the activation of AR may induce proliferation, inhibiting the AR activity can inhibit the proliferation of HBZY-1;(7) Ang Ⅱ induced the mRNA expression of AR increased significantly compared with control (1.35±0.04vs1.00±0.11, P<0.001). Different concentration of lignans (1.08±0.03,0.98±0.09,0.86±0.08vs1.35±0.04; P all the P values less than0.001) can decreased the mRNA expression of AR significantly compared the Ang Ⅱ group, but the mRNA expression of AR in epalrestat group (3.78±0.13vs1.35±0.04, P<0.001increased significantly compared with the Ang Ⅱ group;Ang Ⅱ induced the protein expression of AR increased significantly comp-ared with control (2.0±0.08vs1.00±0.08, P=0.003). Different concentrations of lignans (0.52±0.17,0.66±0.22,0.45±0.23vs2.02±0.08; all the P values less than0.001) can decreased the protein expression of AR significantly compared with the Ang II group, but the protein expression of AR in epalrestat group (3.13±0.24vs2.02±0.08, P=0.001) increased significantly compared with the Ang Ⅱ group. It prompted that the expression of AR played an important role in the process of epalrestat and lignans inhibiting HBZY-1proliferation.Conclusions:The HBZY-1expressed proliferative changing after Ang Ⅱ stimulation, but epalrestat and lignans can significantly inhibit the proliferation and the effect will be changed according to the incubation time and lignans concentration.Lignans and epalrestat can suppress the proliferation of HBZY-1through regulating P21, P27, Bax except Bcl-2, and AR may play a key role in this process.
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