The Experimental Research By Peroxiredoxin Ⅰ Gene Silencing To The Expression Of The Collagen In Lung Fibroblast

Objectives The purpose of this study is to investigate whether the Reactive Oxygensubstances (ROS) is involved in transforming growth factor (TGF-β1) induced activationof c-Jun N terminal kinase (JNK), then reveal the pulmonary fibroblasts (PF) proliferationand collagen synthesis, and promote the silicosis fibrosis. Using the function ofPeroxiredoxin I (Prx-1, a new type of peroxidase) in ROS removal, Observe whether thisinhibition can suppress the effect of promoting fibrosis by TGF-β1, to provide certainexperiment evidence that Prx-1could become a new target of prevention and treatment forsilicosis.Methods Culture human embryonic lung fibroblasts Regularly, after cell synchronization,stimulated by TGF-β1for0min、30min、60min、3h、24h、48h. Extracting the cell totalprotein, Using western blot to detect the expression of Prx-1in lung fibroblasts effected byTGF-β1. Then regular culturing and synchronization cells, the lung fibroblasts weredivided into four groups randomly:control, TGF-β1group, negative control and Prx-1siRNA group. Add MEM which containing0.4%serum to the normal control group, thenTGF-β1group, negative control and Prx-1siRNA group of cells are given5μg/L TGF-β1,while the negative control and Prx-1siRNA group were transfected negative siRNA orPrx-1siRNA oligo by lipofectamine2000respectively in advance. Using laser scanningconfocal microscope (LSCM) to observe the transfection of siRNA oligo, and Real timePCR test the Prx-1mRNA expression, Groups of cells with or without the TGF-β1stimulated in different times, then testing cell proliferation of each Group by theMTT.Western blot to detect protein expression of collagenⅠ and Ⅲ, Prx-1, JNK andPhospho-JNK(p-JNK). Immunofluorescence cytochemistry method to detect theexpression of8-OHdG (specific product by DNA oxidative damage) respectively.Results1.TGF-β1stimulate expression of Prx-1protein increased in human embryoniclung fibroblasts, stimulus30min Prx-1protein expression is increased, with extension oftime the expression is also increasing apparently, peaked when stimulating3h, then dropbut still higher than the control group.2.Negative siRNA and Prx-1siRNA group of cellswere transfected by FAM-siRNA for16h, cells with successful transfection can be seengreen fluorescent expression in cells, which distributed in the cytoplasm by LSCM. On thecondition ofLipofectamine2000:siRNA=5:5, the transfection efficiency is highest around80%. And Real Time PCR results show that compared with no transfection group, all ofthree groups siRNA Prx-1[siRNA Prx-1(209), siRNA Prx-1(289) and siRNA Prx-1(453)]can reduce the Prx-1mRNA expression, while negative siRNA didn’t have this function,The Prx-1mRNA inhibition efficiency of siRNA Prx-1(209), siRNA Prx-1(289) and siRNA Prx-1(453) was86%,74%and95%respectively, choose siRNA Prx-1(453) usedin next experiments.3.Western blot results show that compared with control group, TGF-β1can stimulate Prx-1protein expression increased in human embryonic lung fibroblasts,while compared by TGF-β1group, Prx-1expression has no obvious change in negativesiRNA group, Prx-1siRNA group has no obvious stripe to explain Prx-1siRNA can makethe Prx-1gene successfully silence.4.Compared with control group, TGF-β1stimulate thehuman embryonic lung fibroblast proliferation and collagen type Ⅰ, Ⅲ expression, whilecompared with TGF-β1group, negative siRNA group of cell proliferation and collagenexpression has no significant change, and the change of Prx-1siRNA set of further clear,shows the Prx-1siRNA to TGF-β1inducing embryonic lung fibroblast proliferation andcollagen type Ⅰ, Ⅲincrease with facilitating role.5.According to theimmunofluorescence cytochemistry results, compared with control group, TGF-β1stimulate the human embryonic lung fibroblasts8-OHdG levels increased, but comparedwith TGF-β1group, negative transfection group8-OHdG level has no obvious change,while further of Prx-1siRNA group obviously shows that Prx-1siRNA have a promotingeffect to TGF-β1induce the production of ROS in human embryonic lung fibroblasts.6.The total JNK expression in each group has no significant differences; compared withcontrol group, TGF-β1the increase p-JNK level in human embryonic lung fibroblast, butthe level of p-JNK compared with TGF-β1group, negative transfection has no significantchange, while further of Prx-1siRNA group was obviously, indicating that Prx-1siRNAhas a promoting effect of TGF-β1induced phosphorylation of embryo lung fibroblastsJNK.Conclusions TGF-β1can induce human embryonic lung fibroblast cells to produce ROS,and thus promote the activation of JNK, ultimately leading to cell proliferation, collagen Iand III synthesis, and Prx-1gene silencing can be further promoted this TGF-β1rolethrough increased ROS level.