| Objectives: To investigate the effects of Paraquat on type II alveolar epithelial cells of rats in vitro(RLE-6TN);Using the molecular biology techniques,Observe whether paraquat can induce RLE-6TN epithelial-mesenchymal transition in the epithelial cell;Investigate whether the MAPK protein family is involved in PQ-induced EMT in alveolar epithelial cells.;Finally,explain the regulation of JNK/AP-1 signal transduction pathway in paraquat-induced alveolar epithelial mesenchymal transformation.Methods: An in vitro cell model(Rat type II alveolar epithelial cells,RLE-6TN)was established.1.To observe the dose-dependent and time-dependent effect of paraquat on RLE-6TN cells were cultured with different concentration of PQ(0、25、50、100、200、300、400μmol/L)for 24 h and another following 200μmol/L PQ treatment in different time(12、24、36、48、60、72h).The viability of cells was measured by CCK-8.Cells were treated with 200 mol/L PQ in different times(12,24,36h),and apoptosis was detected by flow cytometry Annexin v-fitc assay.Cell cycle was detected by PI single staining.2.To observe the changes of cell morphology,cell biological behavior and epithelial/mesenchymal markers in RLE-6TN cells treated with PQ in different concentrations or in different times.Cells were treated with 200 mol/L PQ in different times(12,24,36h),and cell migration was detected by cell scratch assay.Cell invasion ability was detected by Transwell assay.Immunofluorescence was used to detect the changes in the expression of cell epithelial/interstitial markers.The expression levels of epithelial marker proteins E-cad/ ZO-1 and interstitial marker proteinsα-SMA/Vimentin were detected by western blot with concentrations of 0,25,50 and 100 mol/L PQ at 6,12 and 24 h.3.In order to observe the effect of PQ poisoning on the key proteins of MAPK protein family,RLE-6TN cells were divided into five groups: normal control group,100 mol/L PQ poisoning group,100 mol/L PQ combined with p38 MAPK inhibitor group,100 mol/L PQ combined with JNK inhibitor group,100 mol/L PQ combined with Erk inhibitor group.Western blot was used to detect the expression levels of p-p38 mapk,p-jnk and p-Erk1/2.4.To investigate whether the JNK/AP-1 signaling pathway is involved in the PQ induced EMT of RLE-6TN cells.The concentration of 0,100,200,300 mol/L PQ induced RLE-6TN cells for 24 h,and the expression of JNK,c-jun,c-fos and their phosphorylated proteins in the JNK/AP-1 signaling pathway were detected by western blotting.Transcriptional activity of AP-1 in RLE-6TN cells was induced by PQ using dual luciferase reporter gene assay.5.To investigate effective targets of the JNK/AP-1 signaling pathway,and explore whether the JNK inhibitor SP600125 can be a new target for the treatment of PQ-induced fibrosis.10 mol/L SP600125 was incubated for 2h before adding PQ.The concentration dose without damage to cells was screened by CCK-8,To determine whether the pathway was blocked effectively,detecting expression of p-JNK was by western blot.Flow cytometry Annexin v-fitc assay was used to detect the changes of apoptosis.Cell migration ability was detected by cell scratch test.The expression levels of JNK/AP signaling pathway were detected by western blot,and the expression levels of epithelial marker proteins E-cad,ZO-1 and interstitial marker proteins α-sma and Vimentin were detected.m RNA expression levels of MMP-2,MMP-9,TIMP-1,COL-I and COL-III were detected by RT-PCR.Results:(1)Compared with the control group,the survival rate of RLE-6TN cells decreased significantly with the increase of PQ dose and time(P<0.05),showing a dose-dependent and time-dependent effect.Compared with the control group,the apoptosis rate of RLE-6TN cells in 12,24 and 36 h was decreased(P<0.05)and cell cycle was arrested at S stage(P<0.05).(2)Compared with the control group,200 μmol/L PQ was used to induce RLE-6TN cells for 12,24 and 36 h,and the cell morphology showed that oval tightly connected epithelial cells were transformed into spindle and loosely connected mesenchymal cells.The migration ability of RLE-6TN cells was enhanced(P<0.05).The invasion ability of RLE-6TN cells was enhanced(P<0.05).The fluorescence intensity of epithelial marker E-cad decreased,and interstitial marker FSP-1 is increased.At the concentration of 0,25,50,100 μmol/L PQ infected cells at 6,12 and 24 h,the expression of epithelial marker protein E-cad and ZO-1 was down-regulated,and the expression of interstitial marker protein α-sma and Vimentin was up-regulated(P<0.05).(3)Compared with the control group,protein expressions of p-p38 mapk,p-JNK and p-Erk1/2 in RLE-6TN cells were significantly up-regulated after 24 h induced by 100 μmol/L PQ(P <0.05).Compared with the PQ induction group,the above protein phosphorylation levels in the intervention group(SB203580,JNK SP600125,Erk PD98059)were significantly reduced by 10 μmol/L pretreatment for 1h and 100 μmol/L PQ treatment for 24 h,respectively(P<0.05).(4)Compared with the control group,PQ could significantly up-regulate the expression of p-JNK,p-c-jun and p-c-fos protein(P<0.05).The transcriptional activation of AP1 in the infected group was significantly enhanced compared with that in the control group(P<0.05).(5)1,10μmol/L SP600125 group cell survival rate is not significantly decline(P > 0.05),and 100 μmol/L group SP600125 can obviously inhibit the cell proliferation activity(P < 0.05),10μmol/L SP600125 group in the induction of cell 2 h suck out continue to cultivate,12,24 h after the group cell survival rate is not significantly decline(P > 0.05),10 μmol/L SP600125 group in the induction of cell 2 h after 36 h suck out continues to develop,can significantly inhibit cell proliferation activity(P < 0.05);The 10 μmol/L SP600125 group could significantly inhibit the phosphorylation level of PQ-induced JNK protein.Compared with the infected group,the survival rate of cells in the intervention group was significantly reduced at 12,24 and 36h(P<0.05),and there was no significant change at 48h(P>0.05).Compared with the PQ group,the apoptosis in the intervention group was decreased,but the difference is not significantly significant(P>0.05),and the cell migration ability was significantly reduced in the intervention group(P<0.05).The expression of p-JNK,p-c-jun and p-c-fos protein in the intervention group was effectively inhibited compared with the poisoned group(P<0.05).Compared with the poisoned group,the expression of epithelial markers in the intervention group was significantly increased(P<0.05),while the expression of interstitial markers was significantly decreased(P<0.05).Compared with the infected group,the m RNA expressions of COL-I and COL-III in the intervention group were significantly decreased(P<0.05),and the m RNA expressions of MMP-2/MMP-9 in the other two components were also significantly decreased(P<0.05),and the m RNA expressions of TIMP1 were also significantly increased(P<0.05).Conclusions: MAPK signaling pathway plays an important role in PQ-induced EMT in RLE-6TN cells.JNK mediate the EMT process as the upstream target of the JNK/ AP-1 signaling pathway.Futhermore,SP600125 may be a key target for the reversion of PQ induced EMT... |
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