大肠杆菌诱导奶牛乳腺成纤维细胞MMP-2、MMP-9和uPA系统表达的研究

Escherichia coli induce the expression of MMP-2/MMP-9 and uPA systems in bovine mammary fibroblasts

通过研究大肠杆菌(E.coli)作用奶牛乳腺成纤维细胞(BMFBs)后对基质金属蛋白酶-2(MMP-2)、MMP-9和uPA系统(uPA/uPAR/PAI-1)表达的影响,以进一步阐明MMPs和uPA系统在调控细胞外基质(ECM)代谢中的作用。本研究从患乳腺炎的奶牛乳腺中分离E.coli,制成热灭活的E.coli菌液,采用不同浓度的热灭活E.coli菌液(1×10^5 cfu/mL、1×10^6 cfu/mL、1×10^8 cfu/mL)作用于BMFB,于作用后不同时间点(6 h、12 h、24 h、48 h)通过RTqPCR和western blot检测MMP-2、MMP-9、uPA、uPAR和PAI-1 mRNA的转录水平和蛋白的表达水平,采用明胶酶谱法检测MMP-2和MMP-9的酶活性。结果显示,MMP-2和MMP-9 mRNA的转录水平与对照组相比均有不同程度的升高趋势,且显著变化趋势集中在作用后24 h(p<0.001),其中MMP-2蛋白的表达水平与对照组相比主要呈先下降后升高再下降的趋势,但在作用后12 h极显著下降(p<0.001),在作用后24 h极显著升高(p<0.001);MMP-9蛋白的表达水平与对照组相比主要呈先升高后下降再升高的趋势,在作用后6 h和48 h极显著升高(p<0.01);明胶酶谱法检测MMP-2的酶活性良好且在作用24 h活性最高,MMP-9的酶活性较弱。uPA mRNA的转录水平与对照组相比随作用时间延长均呈不同程度的升高趋势,且高浓度处理组差异显著(p<0.01),uPA的蛋白表达水平与对照组相比主要呈先升高后下降再升高的趋势,高浓度处理组在作用24 h和48 h差异显著(p<0.01);uPAR mRNA的转录水平与对照组相比随作用时间延长主要呈升高趋势,高浓度处理组作用差异显著(p<0.01),uPAR蛋白表达水平与对照组相比主要呈先升高后下降的趋势,在作用48 h极显著降低(p<0.001);PAI-1 mRNA转录水平与对照组相比随作用时间延长主要呈升高趋势,高浓度处理组在作用24 h和48 h PAI-1 mRNA转录水平均差异显著(p<0.01),PAI-1蛋白的表达水平与对照组相比主要呈先降低后升高再降低的趋势,在作用12 h和48 h均极显著降低(p<0.01),在作用24 h极显著升高(p<0.001)。研究表明,热灭活的E.coli菌液诱导BMFB中MMP-2、MMP-9和uPA系统的表达,激活uPA系统参与调控MMP-2和MMP-9的表达,进而参与降解ECM来影响奶牛乳腺纤维化过程,本研究结果为MMP-2、MMP-9和uPA系统成为治疗奶牛乳腺纤维化的靶点提供了一定的依据。

In order to explore the effect of Escherichia coli(E.coli)on the expression of MMP-2,MMP-9 and uPA system in bovine mammary fibroblasts(BMFB),and to illuminate the role of uPA system in regulating extracellular matrix(ECM).In this study,E.coli was isolated from the mammary glands of cow with mastitis to produce heat-inactivated E.coli.BMFBs treated with heat-inactivated E.coli(1×10^5 cfu/mL,1×10^6 cfu/mL,and 1×10^8 cfu/mL)for 6 h,12 h,24 h,and 48 h,respectively.Total RNA and protein were isolated from the treatment and control groups of BMFBs samples.MMP-2/MMP-9/uPA/uPAR/PAI-1 mRNA transcriptional level and protein expression were examined by RT-qPCR and western blot analysis,respectively,and the activities of MMP-2/MMP-9 were determined by gelatin zymography.The results showed that the mRNA transcriptional level of MMP-2/MMP-9 in the experimental groups were upregulated compared with the control groups,showing significant expression elevation peaked at 24 h(p<0.001).And compared with the control groups,the protein expression of MMP-2 mainly were down-up-downregulated,and decreased significantly at 12 h(p<0.001),increased significantly at 24 h(p<0.001).The protein expression of MMP-9 mainly were up-down-up-regulated,increased significantly at 6 h and 48 h(p<0.01).The activity of MMP-2 was good and highest at 24 h,and the activity of MMP-9 was not obvious.The mRNA transcriptional level of uPA in the experimental groups were upregulated as the time increased,compared with the control groups,and the high concentration treatment group was more significant(p<0.01).And compared with the control groups,the protein expression of uPA mainly were up-down-up-regulated,and the high concentration treatment group was more significant(p<0.01).The mRNA transcriptional level of uPAR in the experimental groups mainly were upregulated with increasing time compared with the control groups,and the high concentration treatment group was more significant(p<0.01).The protein expression of uPAR mainly were increased first and then decreased compared with the control groups,and decreased significant at 48 h(p<0.01).The mRNA transcriptional level of PAI-1 in the experimental groups mainly were upregulated as the time increased,compared with the control groups,and the high concentration treatment group was more significant at 24 h and 48 h(p<0.01).The protein expression of PAI-1 mainly were down-up-downregulated compared with the control groups,and decreased significantly at 12 h and 48 h(p<0.01),increased significantly at 24 h(p<0.001).The results showed that the heat-inactivated E.coil induced BMFBs MMP-2/MMP-9/uPA/uPAR/PAI-1 expression,and activated the uPA system involved in regulation of MMPs expression.MMP-2/MMP-9 and uPA system could affect the process of bovine mammary fibrosis by the degradation of ECM,which provided a basis for using uPA system and MMP-2/MMP-9as promising therapeutic targets in bovine mammary fibrosis.