| Objective: Pulmonary fibrosis (PF) is a kind of interstitial disorder inlung caused by many factors. Interstitial collagens in fibrotic lung are mainlysynthesed by myofibroblasts (MFb), a type of fibroblasts which ischaracterized by smooth musle actin (α-SMA) protein expression.Aminoguanidine (AG) is an inducible nitric oxide synthase (iNOS) inhibitorwhile1400W is a more selective and efficient iNOS inhibitor.The effects ofAG or1400W on CTGF expression and myofibroblast transformation of lungfibroblasts are rarely reported. RNA interference (RNAi) is a newsequence-specific gen silencing mechanism which through the specific smallinterfering RNA（siRNA）to silence the expression of aim gene. It has beenextensively applied in the research on gene function and gene therapy. Toprovide data for evaluating the role of iNOS inhibitor in the prevention andtreatment of pulmonary fibrosis, this study was to investigate the effects ofAG and1400W on the expressions of connective tissue growth factor (CTGF)protein and α-SMA protein in lung fibroblasts and to clarify the abovemechanism with CTGF siRNA in vitro.Methods: Lung fibroblasts from male SD rat (130-150g) were culturedin vitro; MTT was used to estimate the effects of AG and1400W at differentconcentrations on the survival ratio of the lung fibroblasts; Western Blot wasused to determine the expressions of CTGF and α-SMA protein in lungfibroblasts.Results:1The effects of TGF-on the expression of CTGF protein in lung fibloblastsat different time pointsLung fibroblasts of the basic state referred to the ones cultured in normal state. Compared with the respective control group, the expression of CTGFprotein was increased at24h,48h and72h(P＜0.01). Compared with the24hand72h group, the expression of CTGF protein was increased moresignificant at48h (P＜0.05). This experiment chose the48h as the best timepoint for TGF-inducing the CTGF protein expression in lung fibroblasts ofthe basic state.TGF--dealed lung fibroblasts referred to the ones stimulated by TGF-for48h. Compared with the respective control group, the expression of CTGFprotein was all up-regulated at24h,48h and72h in the TGF--dealed lungfibroblasts（P＜0.01）.2The effects of AG and1400W at different concentrations on the survivalratio of lung fibroblasts in basic state and TGF--dealed lung fibroblastsCompared with the control group, the effect of AG(1mmol/L) was of nostatistical significance on the survival ratio of lung fibroblasts in basic stateand TGF--dealed lung fibroblasts（P＞0.05）, the survival ratio with AG (5mmol/L,10mmol/L,20mmol/L,30mmol/L,40mmol/L) was up-regulatedsignifcantly in lung fibroblasts of both states（P＜0.01，P＜0.001）.Compared with the control group, the effects of1400W (20nmol/L,40nmol/L,80nmol/L,100nmol/L,160nmol/L,200nmol/L and320nmol/L) wereof no statistical significance on the survival ratio of lung fibroblasts in basicstate and TGF--dealed lung fibroblasts (P＞0.05）.3The effects of AG and1400W on the protein expressions of CTGF andα-SMA in lung fibroblasts3.1The effects of AG and1400W at different concentrations on the proteinexpressions of CTGF and α-SMA in lung fibloblasts of the basic stateCompared with control group, the protein expressions of CTGF andα-SMA in lung fibloblasts of the basic state were of no obvious difference inAG (0.1mmol/L) group（P＞0.05）, up-regulated significantly in the AG(1mmol/L) group(P＜0.01) and decreased with AG(10mmol/L,20mmol/L,30mmol/L）increasing（P＜0.05,P＜0.01）. It indicated that AG(1mmol/L) oflow concentrations can induce the protein expressions of CTGF and α-SMA in basic-state lung fibroblasts; while AG (10mmol/L,20mmol/L,30mmol/L)of high concentrations can down-regulate the protein expressions of CTGFand α-SMA.In the1400W(20nmol/L) group, the protein expressions of CTGF andα-SMA were up-regulated significantly compared with the control group (P＜0.05）. The protein expressions of CTGF and α-SMA significantly decreasedwith1400W(160mmol/L) compared with the control group（P＜0.05）. Itindicated that1400W (20nmol/L) of low concentrations can induce theprotein expressions of CTGF and α-SMA in basic-state lung fibroblasts, while1400W(160nmol/L) of high concentrations can down-regulate the proteinexpressions of CTGF and α-SMA.3.2The effects of AG and1400W at different time points on the proteinexpressions of CTGF and α-SMA in lung fibloblasts of the basic stateCompared with the respective control group, the protein expressions ofCTGF and α-SMA were of no difference at24h with the AG (1mmol/L)（P＞0.05）.At48h and72h, the CTGF protein expression was both up-regulated(P<0.001,P<0.05), and the α-SMA protein expression was significantlyup-regulated (P<0.01,P<0.05). The CTGF and α-SMA protein expressionswere up-regulated more significantly at48h than at72h（P＜0.01, P＜0.05）.Thus, this experiment chose the48h as the best time point for AG（1mmol/L）inducing the protein expressions of CTGF and α-SMA in lung fibroblasts.Compared with the respective control group, the protein expressions ofCTGF and α-SMA were both up-regulated at24h and48h with1400W(20nmol/L)(P <0.05，P＜0.01), with no difference at72h（P＞0.05）. Theincrease of CTGF and α-SMA protein expressions was more significant at48h than at24h (P <0.05).4The effects of AG and1400W at different concentrations on CTGF andα-SMA protein expressions in TGF--dealed lung fibroblastsCompared with the group of the basic-state lung fibroblasts, the proteinexpressions of CTGF and α-SMA were increased in the TGF--dealed group(P <0.01). Compared with the TGF--dealed group, the protein expressions of CTGF and α-SMA were of no significant difference in the TGF-β+AG(1mmol/L) group (P>0.05) while in the TGF-β+AG,(10,20,30mmol/L)groups, the protein expressions of CTGF and α-SMA gradually decreasedwith the concentration increasing (P＜0.05,P＜0.01）.Compared with the group of the basic-state lung fibroblasts, the proteinexpressions of CTGF and α-SMA were increased in the TGF--dealed group(P <0.01). The protein expressions of CTGF and α-SMA were of nosignificant differences between TGF--treated group and TGF-+1400W (20nmol/L) group(P>0.05). Compared with TGF--dealed group, the proteinexpressions of CTGF and α-SMA decreased in the TGF-+1400W (40,80nmol/L) group (P＜0.05,P＜0.01).5Screening of CTGF siRNACompared with the NC siRNA group, the protein expressions of CTGFand α-SMA were significantly decreased in the6737siRNA group(P＜0.01,P＜0.05）, and in the6738siRNA group(P＜0.05）. The inhibiting rate forCTGF protein expression was (425)%and (294)%for6737siRNA groupand6738siRNA group, respectively. The data indicated that6737siRNAexerted the most effective inhibiting effect.6The effect of CTGF siRNA on the protein expressions of CTGF and α-SMAin lung fibroblasts induced by AG or1400WCompared with the NC group, the protein expressions of CTGF andα-SMA were decreased in the6737siRNA group (P <0.05). Compared withthe AG(1mmol/L) group,the protein expressions of CTGF and α-SMA wereof no difference in the AG+NC group. Compared with the AG+NC group, theprotein expressions of CTGF and α-SMA were significantly decreased in theAG+6737siRNA group (P <0.01) and, in the AG+6738siRNA group, theprotein expressions of CTGF and α-SMA was downregulated (P <0.05).Itindicated that both6737siRNA and6738siRNA could downregulate theprotein expression of CTGF, inhibiting rates were (416)%and (244)%respectively,and lead to the downregulation of α-SMA protein expression.Compared with the1400W+NC group, the protein expressions of CTGF and α-SMA were significantly decreased in the1400W+6737siRNA group,(P <0.01), and in the1400W+6738siRNA group(P <0.05).Conclusion:1AG(1mmol/L) and1400W (20nmol/L) of low concentrations can inducethe protein expressions of CTGF and α-SMA in basic-state lung fibroblasts,and promote the myofibroblast transformation of pulmonary fibroblast in rat;while AG (10mmol/L,20mmol/L,30mmol/L) and1400W(160nmol/L) ofhigh concentrations can down-regulate the protein expressions of CTGF and α-SMA.2AG(1mmol/L) and1400W (20nmol/L) of low concentrations have noobvious effect on the protein expressions of CTGF and α-SMA inTGF--dealed lung fibroblasts; while AG(10mmol/L,20mmol/L,30mmol/L)of high concentrations can inhibit the expressions.3CTGF siRNA can inhibit the up-regulation of the protein expression ofCTGF in lung fibroblasts induced by AG(1mmol/L) and1400W(20nmol/L)and lead to the down-regulation of the α-SMA protein, indicating that thedown-regulation of CTGF protein expression can hinder the phenotypictransformation of rat lung fibroblasts into myofibroblasts. |
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