晚期糖基化终产物受体特异性小干扰RNA对MMP-1和TIMP-1表达的影响

Effect of siR NA-mediated down-regulation of receptor for advanced glycation end products on expression of matrix metalloproteinase-1 and tissue inhibitor of metalloproteinase-1 in rat hepatic stellate cells and hepatic fibrosis

目的:探讨晚期糖基化终产物受体(receptor for advanced glycation end products,RAGE)特异性小干扰RNA(small interfering RNA,siRNA)对原代大鼠肝星状细胞(hepatic stellate cells,HSCs)和肝纤维化(hepatic fibrosis,HF)大鼠肝脏基质金属蛋白酶-1(matrix metalloproteinase-1,MMP-1)及金属蛋白酶组织抑制因子-1(tissue inhibitor of metalloproteminase-1,TIMP-1)表达的影响.方法:分离培养原代大鼠HSCs,将RAGE特异性siRNA表达载体pAKD-GR126转染入原代大鼠HSCs,以空白组和转染非特异性siRNA表达载体pAKD-NC组为对照,分别应用实时荧光定量PCR和Western blot检测各组原代大鼠HSCs中RAGE、MMP-1和TIMP-1的mRNA及蛋白的表达;制备CCl_4诱导的HF大鼠模型,将不同治疗剂量的pAKD-GR126经尾静脉导入成模后大鼠,以正常对照组(NC组)、模型组(FM组)和非特异性siRNA表达载体pAKD-NC组(NS组)为对照,分别应用PCR和Western blot检测各组大鼠肝组织中RAGE、MMP-1和TIMP-1的mRNA及蛋白的表达.常规HE及Masson胶原染色,光镜下观察,比较各组肝组织形态学改变.结果:转染pAKD-GR126的原代HSCs中,RAGE和TIMP-1的mRNA和蛋白的表达显著低于空白对照组和pAKD-NC组(均P<0.05),MMP-1的mRNA和蛋白的表达显著高于空白对照组和pAKD-NC组(均P<0.05).动物试验中,与FM组相比,pAKDGR126小剂量治疗组(LT组)、中剂量治疗组(MT组)、高剂量治疗组(HT组)的RAGE和TIMP-1的mRNA和蛋白表达均有不同程度的降低(均P<0.05),MMP-1的mRNA和蛋白表达均有不同程度的增加(均P<0.05),且与pAKD-GR126呈剂量依赖关系.光镜下观察,LT组、MT组和HT组纤维化程度较FM组和NS组相比,均有不同程度的减轻,且以HT组减轻最明显.结论:RAGE特异性siRNA在原代大鼠HSCs和HF大鼠体内均能抑制RAGE和TIMP-1的表达,并促进MMP-1的表达,使大鼠肝脏HF的程度减轻.

AIM：To investigate the effect of small interfering RNA（siRNA）-mediated down-regulation of receptor for advanced glycation end products（RAGE） on the expression of matrix metalloproteinase-1（MMP-1） and tissue inhibitor of metalloproteinase-1（TIMP-1） in primary rat hepatic stellate cells（HSCs） and hepatic fibrosis（HF）.METHODS：In in vitro experiment,primary rat HSCs were cultured and isolated.The pAKDGR126 vector carrying siRNA targeting RAGE was constructed and transfected to primary rat HSCs.Blank cells and cells transfected with unspecific siRNA vector pAKD-NC were used as controls.In in vivo experiment,liver fibrosis was induced in SD rats with CCl_4.pAKD-GR126 was transfected to liver fibrosis rats at different doses via the tail vein.A blank group,a liver fibrosis model group and an unspecific siRNA vector pAKD-NC-transfected group were used as controls.Real-time PCR and Western blot were used to detect the expression of RAGE,MMP-1 and TIMP-1.The histological changes of the liver were observed by HE and Masson staining methods.RESULTS：The mRNA and protein expression of RAGE and TIMP-1 in pAKD-GR126-transfected primary HSCs was significantly lower than that in the blank group and unspecific siRNA vector pAKD-NC-transfected group（P 0.05 for all）.However,the level of MMP-1 in pAKD-GR126-transfected primary HSCs was significantly higher than that in the blank group and pAKD-NC-transfected group（P 0.05 for all）.In vivo,the mRNA and protein expression of RAGE and TIMP-1 was significantly lower and that of MMP-1 was significantly higher in the low-,medium-,and high-dose RAGE siRNA groups than in the liver fibrosis model group（P 0.05 for all）.Compared with the liver fibrosis model group,liver fibrosis was significantly milder in the low-,medium-,and high-dose RAGE siRNA groups,especially the high-dose group.CONCLUSION：RAGE specific siRNA could decrease the expression of RAGE and TIMP-1,increase the expression of MMP-1 in primary rat HSCs and HF rats,and reduce the degree of rat hepatic fibrosis.