目的利用慢病毒干涉下调内源性RNF31表达,研究NF-κB通路的活化及对细胞凋亡的影响。方法将人RNF31的shRNA片段克隆到慢病毒表达载体p Green Puro中,瞬时转染HEK293T细胞,筛选出有效的干涉片段。将重组表达质粒与包装质粒PMD、SPA共转染...目的利用慢病毒干涉下调内源性RNF31表达,研究NF-κB通路的活化及对细胞凋亡的影响。方法将人RNF31的shRNA片段克隆到慢病毒表达载体p Green Puro中,瞬时转染HEK293T细胞,筛选出有效的干涉片段。将重组表达质粒与包装质粒PMD、SPA共转染293T细胞,在24 h、48 h分2次收集慢病毒上清,用流式细胞术检测病毒滴度。将获得的病毒感染HEK293细胞,提取细胞蛋白,Real-time PCR以及Western Blot检测RNF31干涉效果;报告基因实验检测敲低RNF31对NF-κB转录活性的影响;Real-time PCR检测干涉RNF31对TNF-α诱导的NF-κB下游靶基因的影响;Western Blot检测下调RNF31对IκBα活化的影响;Hochest染色检测下调RNF31对细胞凋亡的影响。结果成功构建RNF31干涉慢病毒p Green Puro-RNF31载体并获得慢病毒颗粒,病毒滴度可达3×107pfu/ml。在HEK293细胞中下调RNF31,抑制TNF-α刺激的NF-κB的转录活性,并抑制NF-κB下游靶基因的表达;下调RNF31抑制TNF-α刺激的IκBα的活化;此外,在TNF-α刺激细胞24 h时,RNF31表达下调使细胞凋亡增多。结论 RNF31表达下调抑制TNF-α刺激的NF-κB通路的激活。展开更多
OBJECTIVE Liver fibrosis is a chronic damage process related to the further progression of hepatic cirrhosis and has yet no truly effective treatment is available.This study aimed to investigate the effects of isochlo...OBJECTIVE Liver fibrosis is a chronic damage process related to the further progression of hepatic cirrhosis and has yet no truly effective treatment is available.This study aimed to investigate the effects of isochlorogenic acid A(ICQA)on liver fibrosis induced by carbon tetrachloride(CCl4)and clarify the underlying mechanism.METHODS Rats were treated with CCl4 for eight weeks in order to induce liver fibrosis and simultaneously orally administered with ICQA(10,20 and 40 mg·kg-1).RESULTS ICQA had significant protective effect on liver injury,inflammation,and fibrosis in rats.Meanwhile,ICQA prevented the activation of hepatic stellate cells(HSC)as indicated by inhibiting the overexpres⁃sion of a-smooth muscle actin(a-SMA).In addition,reduced fibrosis was found to be associated with decreased protein expression of high-mobility group box 1(HMGB1)and toll like receptor(TLR)4.Moreover,ICQA supressed the cytoplasmic translocation of HMGB1 in rat liver.Further investigations indicated that ICQA treatment significantly attenuated nuclear translocation of the nuclear factor-κB(NF-κB)p65 and inhibited degradation of IkBa expression in the liver of rats with liver fibrosis.CONCLUSION ICQA has hepatoprotective and anti-fibrotic effects in rats with liver fibrosis through modulating the HMGB1/TLR4/NF-κB signaling pathways.展开更多
文摘OBJECTIVE Liver fibrosis is a chronic damage process related to the further progression of hepatic cirrhosis and has yet no truly effective treatment is available.This study aimed to investigate the effects of isochlorogenic acid A(ICQA)on liver fibrosis induced by carbon tetrachloride(CCl4)and clarify the underlying mechanism.METHODS Rats were treated with CCl4 for eight weeks in order to induce liver fibrosis and simultaneously orally administered with ICQA(10,20 and 40 mg·kg-1).RESULTS ICQA had significant protective effect on liver injury,inflammation,and fibrosis in rats.Meanwhile,ICQA prevented the activation of hepatic stellate cells(HSC)as indicated by inhibiting the overexpres⁃sion of a-smooth muscle actin(a-SMA).In addition,reduced fibrosis was found to be associated with decreased protein expression of high-mobility group box 1(HMGB1)and toll like receptor(TLR)4.Moreover,ICQA supressed the cytoplasmic translocation of HMGB1 in rat liver.Further investigations indicated that ICQA treatment significantly attenuated nuclear translocation of the nuclear factor-κB(NF-κB)p65 and inhibited degradation of IkBa expression in the liver of rats with liver fibrosis.CONCLUSION ICQA has hepatoprotective and anti-fibrotic effects in rats with liver fibrosis through modulating the HMGB1/TLR4/NF-κB signaling pathways.
