摘 要: 目的 观察百草枯(PQ)对人肺A549细胞WNT1诱导信号通道蛋白1(WISP1)和基质金属蛋白酶-9(MMP-9)表达的影响,探讨WISP1在PQ诱导肺泡细胞上皮间质转化(EMT)的作用机制。方法 体外培养A549细胞,采用不同浓度PQ染毒,光学显微镜下观察细胞形态学改变,实时荧光定量PCR方法检测A549细胞中E-钙黏蛋白(E-Cadherin)、波形蛋白(Vimentin)、WISP1及MMP-9的表达,Western blot测定E-Cadherin、Vimentin、WISP1和MMP-9蛋白表达。结果 (1)PQ染毒后A549细胞形态发生改变,细胞体积增大,空泡增多,随着PQ染毒浓度增加细胞岀现凋亡。(2)伴随细胞形态改变,E-Cadherin水平随PQ染毒浓度增加而降低,以200 μg/ml PQ时最低(P<0.01);Vimentin浓度增加,以50 μg/ml PQ时最高(P<0.01)。(3)随着PQ染毒浓度增加,WISP1、MMP-9基因和蛋白表达升高,200 μg/ml PQ染毒后第5天WISP1、MMP-9表达达到高峰(P<0.01)。(4)经WISP1 siRNA处理后200 μg/ml PQ诱导细胞的EMT现象减少,E-Cadherin表达较空白siRNA升高(P<0.01),Vimentin浓度下降(P<0.01),WISP1和MMP-9表达降低(P<0.01),两者的降低呈正相关(r=0.986,P<0.01)。结论 WISP1可能参与了PQ诱导的EMT过程,WISP1水平降低抑制了PQ诱导的MMP-9表达。因而推测WISP1与MMP-9可能在PQ所致肺纤维化过程中发挥了作用。 |
关键词: 百草枯(PQ) 细胞上皮间质转化(EMT) WNT1诱导信号通道蛋白1(WISP1) 基质金属蛋白酶-9(MMP-9) E-钙黏蛋白(E-Cadherin) 波形蛋白(Vimentin) |
中图分类号: R994
文献标识码: A
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基金项目: 深圳市龙岗区医疗卫生科技计划项目[LGWJ2021-(046)] |
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Effect of WISP1 on paraquat-induced EMT and MMP-9 expression in alveolar cells |
LIU Jingyan,YU Kuncheng,TAN Changrui
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The Second Affiliated Hospital,School of Medicine,The Chinese University of HongKong;Emergency Department of Longgang District People's Hospital of Shenzhen,Shenzhen,Guangdong 518172,China
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Abstract: Objective To observe the effect of paraquat(PQ)on the expressions of WNT1 induced signal channel protein1(WISP1)and matrix metalloproteinase-9(MMP-9)expression in alveolar epithelial cells(human lung A549 cells),exploring the mechanism of WISP1 in epithelial-mesenchymal transition(EMT)in alveolar cells. Methods A549 cells were cultured in vitro and exposed to different concentrations of PQ,the morphological changes of A549 cells were observed through an optical microscope,the expression of E-Cadherin,Vimentin,WISP1,and MMP-9 in A549 cells was detected using real-time fluorescence quantitative PCR method,and Western blot to detect the mRNA and protein expression levels of E-Cadherin,Vimentin,WISP1 and MMP-9. Results The results showed that(1)After PQ exposure,the morphology of A549 cells changed with increase in cell volume and cytoplasm vacuoles,and apoptosis occurred in cells with the increase of PQ concentration. (2)With the changes in cell morphology,E-Cadherin levels decreased with increasing PQ concentration,until PQ concentration reached 200 μg/ml(P<0.01);while the Vimentin concentration increased with the PQ concentration until it reached 50 μg/ml(P<0.01). (3)With the increase of PQ concentration,the expressions of WISP1 and MMP-9 genes and protein increased and reached a peak on the 5th day after exposure to 200 μg/ml of PQ(P<0.01). (4)After WISP1 siRNA treatment,there were decrease of EMT phenomenon,increase of E-Cadherin expression compared to blank siRNA(P<0.01),Vimentin expression decreased(P<0.01),and inhibition of WISP1 expression with was positively associated with the decreased of MMP-9 expression(r=0.986,P<0.01)in 200 μg/ml of PQ treated cells. Conclusion The results suggested that WISP1 should be involved in the PQ-induced EMT process,the decrease of WISP1 expression could suppress PQ-induced MMP-9 expression,therefore,it is speculated that WISP1 and MMP-9 may play a role in PQ induced pulmonary fibrosis. |
Keywords: paraquat(PQ) epithelial-mesenchymal transition(EMT) WNT1 induced signal channel protein1(WISP1) matrix metalloproteinase-9(MMP-9) E-Cadherin Vimentin |