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外源性脂联素对内皮素1诱导的肝星状细胞收缩的影响及作用机制
Effect of adiponectin on hepatic stellate cell contraction induced by endothelin-1 and its mechanism of action
文章发布日期:2016年02月05日  来源:  作者:李楠,刘迎娣,郭明洲,等  点击次数:1876次  下载次数:420次

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【摘要】:目的观察外源性脂联素对内皮素(ET)1诱导的肝星状细胞(HSC)-T6收缩的影响,探讨脂联素在此过程中的可能作用机制。方法应用胶原晶格法观察不同浓度(0.25、0.5 μg/ml)脂联素及脂联素(0.5 μg/ml)与左旋硝基精氨酸甲酯(L-NAME)(5 mmol/L)共同作用下对ET-1诱导的HSC-T6细胞收缩的影响;分别采用实时荧光定量PCR、Western Blot检测脂联素(0.5 μg/ml)作用后HSC-T6细胞中ET-1 mRNA及蛋白的表达,采用ELISA检测脂联素(0.5 μg/ml)作用后HSC-T6细胞培养液中ET-1蛋白的表达;采用实时荧光定量PCR及Western Blot检测不同浓度(0.5、1、2 μg/ml)脂联素与ET-1共同作用下HSC-T6细胞中诱导型一氧化氮合酶(iNOS)的mRNA及蛋白的表达,同时检测HSC-T6细胞中AMPK、p-AMPK、Akt、p-Akt蛋白的表达。计量资料多组间比较采用方差分析,进一步两两比较采用Dunnett法。结果(1)胶原晶格实验显示,与空白对照组相比,ET-1组凝胶面积显著收缩[(248±7.3)% vs (71.9±4.1)%, P<0.01];脂联素(0.5 μg/ml)处理后,明显抑制ET-1引起的收缩[(52.7±20.6)% vs (248±7.3)%, P<0.05];L-NAME (5 mmol/L)可以部分抵消脂联素的抑制作用(P>0.05)。(2)脂联素(0.5 μg/ml)可以抑制HSC-T6细胞中ET-1 mRNA及蛋白的表达(P<0.05),同时可以抑制细胞上清液中ET-1的蛋白表达,在脂联素的基础上加入L-NAME后,脂联素的上述抑制作用均得到部分逆转。(3)HSC-T6细胞中有iNOS mRNA表达,加入ET-1后iNOS mRNA表达量明显下降(P<0.01),同时加入 ET-1和脂联素作用后iNOS mRNA表达较ET-1组增加,且随着脂联素浓度升高iNOS mRNA表达量有逐渐升高的趋势,HSC-T6细胞中iNOS蛋白表达与mRNA表达趋势一致;ET-1处理24 h后HSC-T6细胞中p-AMPK表达水平明显减低,在此基础上加入脂联素后, p-AMPK表达水平明显上升,且随脂联素浓度的增加逐渐增加;ET-1处理24 h后HSC-T6细胞中 p-Akt 表达水平明显上升,在此基础上加入脂联素后, p-Akt的表达水平下降,且随脂联素浓度的增加逐渐降低。结论脂联素能够抑制ET-1诱导的HSC收缩,其机制可能是通过激活AMPK,增加NO的合成,减少ET-1的合成分泌,同时阻断Akt信号通路来实现的。脂联素抑制HSC的收缩作用可能是其抗肝纤维化的机制之一。
【Abstract】:ObjectiveTo observe the effect of exogenous adiponectin on hepatic stellate cell (HSC) contraction induced by endothelin-1 (ET-1), and to investigate the possible mechanism of action of adiponectin in this process. MethodsCollagen lattice was applied to observe the effects of adiponectin (0.25, 0.5 μg/ml) alone and adiponectin (0.5 μg/ml) combined with nitro-L-arginine methyl ester (L-NAME) on the contraction of HSC-T6 cells induced by ET-1. Quantitative real-time PCR and Western blot were applied to measure the mRNA and protein expression levels of ET-1 in HSC-T6 cells after the action of adiponectin (0.5 μg/ml), and enzyme-linked immunosorbent assay was applied to measure the protein expression level of ET-1 in HSC-T6 cell culture medium after the action of adiponectin (0.5 μg/ml). Quantitative real-time PCR and Western blot were also applied to measure the mRNA and protein expression levels of inducible nitric oxide synthase (iNOS) in HSC-T6 cells after the combined action of adiponectin (0.25, 0.5 μg/ml) and ET-1, as well as the protein expression levels of AMPK, p-AMPK, Akt, and p-Akt in HSC-T6 cells. Analysis of variance was applied for comparison of continuous data between groups and pairwise comparision between any two groups was made by Dunnett test. ResultsThe collagen lattice experiments showed that compared with the blank control group, the ET-1 group had a significantly reduced gel area [(24.8±7.3)% vs (719±4.1)%, P<0.01]; after the treatment with adiponectin (0.5 μg/ml), the contraction induced by ET-1 was significantly inhibited in the ET-1 group [(52.7±20.6)% vs (24.8±7.3)%, P<0.05]; L-NAME (5 mmol/L) partially counteracted the inhibitory effect of adiponectin (P>0.05). Adiponectin (0.5 μg/ml) inhibited the mRNA and protein expression levels of ET-1 in HSC-T6 cells (P<0.05) and the protein expression level of ET-1 in cell supernatants; after L-NAME was added, the above inhibitory effect of adiponectin was partially reversed. The mRNA expression of iNOS was detected in HSC-T6 cells, and the mRNA expression level of iNOS was significantly reduced after ET-1 was added (P<0.01); after addition of ET-1 and adiponectin, the mRNA expression level of iNOS was significantly increased compared with the ET-1 group, and tended to increase with the increasing concentration of adiponectin; the protein expression level of iNOS had a similar trend. At 24 hours after ET-1 treatment, the expression level of p-AMPK in HSC-T6 cells decreased significantly, and after adiponectin was added, the expression level of p-AMPK increased significantly with the increasing concentration of adiponectin; at 24 hours after ET-1 treatment, the expression level of p-Akt in HSC-T6 cells increased significantly, and after adiponectin was added, the expression level of p-Akt decreased gradually with the reduction in the concentration of adiponectin. ConclusionAdiponectin can inhibit the HSC contraction induced by ET-1, and its mechanism of action is to activate AMPK, increase the synthesis of nitric oxide, reduce the synthesis and secretion of ET-1, and block the Akt signaling pathway. The inhibitory effect of adiponectin on HSC contraction may be one of its anti-fibrosis mechanisms.
【关键字】:肝硬化;肝星状细胞;脂联素;内皮缩血管肽1;一氧化氮合酶
【Key words】:liver cirrhosis; hepatic stellate cells; adiponectin; endothelin-1; nitric oxide synthase
【引证本文】:李楠, 刘迎娣, 郭明洲, 等. 外源性脂联素对内皮素1诱导的肝星状细胞收缩的影响及作用机制[J]. 临床肝胆病杂志, 2016, 32(2): 307-311.

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