补骨脂素和异补骨脂素下调NF-κB活性改善LO2细胞脂代谢紊乱的机制研究
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摘要
探讨补骨脂素和异补骨脂素对正常人肝细胞(LO2)内脂质沉积的改善及作用机制。建立棕榈酸(PA)诱导的人LO2细胞非酒精性脂肪肝模型,给予补骨脂素和异补骨脂素干预,酶法检测细胞内甘油三酯(TG)、总胆固醇(TC)含量,细胞上清液谷丙转氨酶(ALT)、谷草转氨酶(AST)含量; ELISA法检测细胞上清液促炎因子(IL-6,TNF-α)及趋化因子(IL-8,MCP-1)的表达; Western blot法检测细胞内核因子κB(NF-κB) p65磷酸化(p-p65)及非磷酸化蛋白(p65)、转化因子(TGF)β1蛋白表达。结果显示,与模型组比较,补骨脂素组细胞内TG,TC含量降低(P<0. 01,P<0. 05),细胞上清液中ALT和AST含量、促炎因子及趋化因子分泌水平均有降低(P<0. 01,P<0. 05),细胞内p-p65/p65比值及TGF-β1蛋白表达明显下降(P<0. 01);异补骨脂素组细胞内TG含量无明显变化,其余检测指标均有降低(P<0. 01,P<0. 05)。补骨脂素疗效优于异补骨脂素(P<0. 01,P<0. 05)。结果表明,补骨脂素对PA诱导LO2细胞脂肪变性具有改善作用;补骨脂素和异补骨脂素均对PA诱导LO2细胞损伤具有缓解作用,可抑制NF-κB的活性减轻炎症反应并下调TGF-β1表达。
The aim of this paper was to investigate the mechanism and effect of psoralen and isopsoralen in the treatment of lipid accumulation in LO2 cells. Human LO2 cells nonalcoholic fatty liver models were established by using palmitic acid( PA). Then psoralen and isopsoralen were administered for intervention. Intracellular triglyceride( TG) and total cholesterol( TC) content,the cell supernatant alanine aminotransferase( ALT) and aspartate aminotransferase( AST) levels were determined by enzyme method. Cell supernatant proinflammatory cytokines( IL-6,TNF-α) and chemokines( IL-8,MCP-1) were determined by ELISA method. Western blot method was conducted to detect the protein expression of intracellular nuclear factor( NF-κB) p65 phosphorylation( p-p65),nonphosphorylated protein( p65),and transforming factor TGF-β1. Result showed that as compared with the model group,intracellular TG and TC levels,the cell supernatant ALT and AST levels,proinflammatory cytokines and chemokines were decreased( P < 0. 01,P <0. 05); the p-p65/p65 ratio and TGF-β1 protein expression were also significantly decreased( P< 0. 01,P< 0. 05) in psoralen intervention group. As compared with the model cells,intracellular TG content had no significant changes,but all the other indexes were reduced( P<0. 01,P<0. 05) in the cells of isopsoralen intervention group. Psoralen exhibited better effect than isopsoralen( P< 0. 01,P<0. 05). It is concluded that psoralen could improve the adipogenesis of LO2 cells induced by PA; both psoralen and isopsoralen are effective in ameliorating LO2 cells injury induced by PA,reducing inflammation via inhibiting the activation of NF-κB and down-regulating the expression of TGF-β1.
The aim of this paper was to investigate the mechanism and effect of psoralen and isopsoralen in the treatment of lipid accumulation in LO2 cells. Human LO2 cells nonalcoholic fatty liver models were established by using palmitic acid( PA). Then psoralen and isopsoralen were administered for intervention. Intracellular triglyceride( TG) and total cholesterol( TC) content,the cell supernatant alanine aminotransferase( ALT) and aspartate aminotransferase( AST) levels were determined by enzyme method. Cell supernatant proinflammatory cytokines( IL-6,TNF-α) and chemokines( IL-8,MCP-1) were determined by ELISA method. Western blot method was conducted to detect the protein expression of intracellular nuclear factor( NF-κB) p65 phosphorylation( p-p65),nonphosphorylated protein( p65),and transforming factor TGF-β1. Result showed that as compared with the model group,intracellular TG and TC levels,the cell supernatant ALT and AST levels,proinflammatory cytokines and chemokines were decreased( P < 0. 01,P <0. 05); the p-p65/p65 ratio and TGF-β1 protein expression were also significantly decreased( P< 0. 01,P< 0. 05) in psoralen intervention group. As compared with the model cells,intracellular TG content had no significant changes,but all the other indexes were reduced( P<0. 01,P<0. 05) in the cells of isopsoralen intervention group. Psoralen exhibited better effect than isopsoralen( P< 0. 01,P<0. 05). It is concluded that psoralen could improve the adipogenesis of LO2 cells induced by PA; both psoralen and isopsoralen are effective in ameliorating LO2 cells injury induced by PA,reducing inflammation via inhibiting the activation of NF-κB and down-regulating the expression of TGF-β1.
引文
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[20]李劲平,王小静,曾英,等.补骨脂定抗实验性绝经后骨质疏松的效应及作用机制研究[J].中国中药杂志,2013,38(11):1816.
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[23]夏庚,祁飞,杨晋辉,等.核因子-κBp65在肝细胞癌中的表达及意义[J].临床肝胆病杂志,2017,33(12):2342.
[24]袁丹,王玉倩,黄萍,等.药用植物活性成分调控p65核转运总结与展望[J].中国中药杂志,2017,42(17):3286.
[2] Watanabe S,Hashimoto E,Ikejima K,et al. Evidence-based clinical practice guidelines for nonalcoholic fatty liver disease/nonalcoholic steatohepatitis[J]. Hepatol Res,2015,45(4):363.
[3] Mencin A A,Lavine J E. Nonalcoholic fatty liver disease in children[J]. Curr Opin Clin Nutr Metab Care,2011,14(2):151.
[4] Loomba R,Sirlin C B,Schwimmer J B,et al. Advances in pediatric nonalcoholic fatty liver disease[J]. Hepatology,2009,50(4):1282.
[5] Anderson E L,Howe L D,Jones H E,et al. The prevalence of non-alcoholic fatty liver disease in children and adolescents:a systematic review and Meta-analysis[J]. PLoS ONE,2015,10(10):e0140908.
[6] Alkhouri N,Feldstein A E. The TONIC trial:a step forward in treating pediatric nonalcoholic fatty liver disease[J]. Hepatology,2012,55(4):1292.
[7] Fan J G. Epidemiology of alcoholic and nonalcoholic fatty liver disease in China[J]. J Gastroenterol Hepatol,2013,28(Suppl1):11.
[8]朱婵娟,范建高.儿童非酒精性脂肪性肝病[J].实用肝脏病杂志,2013,16(6):483.
[9] Guo X F,Zheng L Q,Li Y,et al. Prevalence and risk factors of being overweight or obese among children and adolescents in northeast China[J]. Pediatr Res,2013,74(4):443.
[10] Fleet S E,Lefkowitch J H,Lavine J E. Current concepts in pediatric nonalcoholic fatty liver disease[J]. Gastroenterol Clin North Am,2017,46(2):217.
[11] Zelber-Sagi S,Godos J,Salomone F. Lifestyle changes for the treatment of nonalcoholic fatty liver disease:a review of observational studies and intervention trials[J]. Therap Adv Gastroenterol,2016,9(3):392.
[12] Fuchs C D,Traussnigg S A,Trauner M. Nuclear receptor modulation for the treatment of nonalcoholic fatty liver disease[J]. Semin Liver Dis,2016,36(1):69.
[13] Wang Y D,Chen W D,Wang M H,et al. Farnesoid X receptor antagonizes nuclear factor kappaB in hepatic inflammatory response[J]. Hepatology,2008,48(5):1632.
[14] Zhou L S,Tang J Q,Xiong X L,et al. Psoralea corylifolia L. attenuates nonalcoholic steatohepatitis in juvenile mouse[J]. Front Pharmacol,2017,8:876.
[15]周俪姗,鄢素琪,熊小丽,等.补骨脂抑制肝组织NF-κB活性治疗幼龄小鼠脂肪肝的机制研究[J].中国中药杂志,2017,42(13):2546.
[16]方珂,陆付耳.胡芦巴丸活性组分改善糖脂代谢紊乱的机理研究[D].武汉:华中科技大学,2017.
[17]李姣,石挺,刘磊,等.人肝细胞系LO2单纯肝脂肪变性细胞模型的建立[J].中兽医医药杂志,2016,35(5):20.
[18]吴疆,魏巍,袁永兵.补骨脂的化学成分和药理作用研究进展[J].药物评价研究,2011,34(3):217.
[19]邱蓉丽,李璘,乐巍.补骨脂的化学成分与药理作用研究进展[J].中药材,2010,33(10):1656.
[20]李劲平,王小静,曾英,等.补骨脂定抗实验性绝经后骨质疏松的效应及作用机制研究[J].中国中药杂志,2013,38(11):1816.
[21] Liu W,Baker R D,Bhatia T,et al.Pathogenesis of nonalcoholic steatohepatitis[J]. Cell Mol Life Sci,2016,73(10):1969.
[22] Magee N,Zou A,Zhang Y. Pathogenesis of nonalcoholic steatohepatitis:interactions between liver parenchymal and nonparenchymal cells[J]. Biomed Res Int,2016,2016:5170402.
[23]夏庚,祁飞,杨晋辉,等.核因子-κBp65在肝细胞癌中的表达及意义[J].临床肝胆病杂志,2017,33(12):2342.
[24]袁丹,王玉倩,黄萍,等.药用植物活性成分调控p65核转运总结与展望[J].中国中药杂志,2017,42(17):3286.