芍药二酮抗肝纤维化及抗炎的活性和机制研究

Activity and mechanism of palbinone against hepatic fibrosis and inflammation

本文旨在研究芍药二酮(palbinone,PB)的抗肝纤维化活性和抗炎活性,并初步探讨内在的调节机制,为其发展成为抗非酒精性脂肪性肝炎(non-alcoholic steatohepatitis,NASH)候选药物奠定活性基础。首先采用磺酰罗丹明B(sulforhodamine B,SRB)方法检测化合物PB对人肝星状细胞LX-2和大鼠肝星状细胞HSC-T6增殖的影响。并在转化生长因子-β1(transforming growth factor beta 1,TGF-β1)诱导的体外肝纤维化细胞模型中,采用实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)和Western blot的方法检测不同浓度芍药二酮对肝纤维化标志基因转录水平及蛋白表达水平的抑制作用,同时分析芍药二酮调控肝纤维化相关基因的作用机制。另外,在脂多糖(lipopolysaccharide,LPS)联合尼日利亚菌素(nigericin)诱导的炎症细胞模型中,采用ELISA的方法检测芍药二酮对于细胞释放白介素-1β(interleukin-1β,IL-1β)水平的影响,同时Western blot检测芍药二酮对炎症信号通路中相关蛋白表达的影响。结果表明,芍药二酮可以显著性抑制肝星状细胞LX-2和HSC-T6的增殖活力,其半数抑制浓度(half maximal inhibitory concentration,IC_(50))值分别为(375.11±55.45)和(260.27±36.81)nmol·L^(-1),并且芍药二酮对TGF-β1诱导的肝纤维化相关基因Ⅰ型胶原α1(collagen typeⅠα1,COL1A1)、TGF-β1、α平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、金属蛋白酶组织抑制因子1(tissue inhibitor of metalloproteinase 1,TIMP1)的表达水平呈现出剂量依赖性的抑制作用。机制研究表明,芍药二酮可能是通过抑制了Yes相关蛋白(Yes-associated protein,YAP)的表达水平,从而减弱了其对于下游纤维化通路的激活作用。此外,芍药二酮还可以通过抑制核因子κB(nuclear factor kappa-B,NF-κB)信号通路的活性,减少炎症因子含半胱氨酸的天冬氨酸蛋白水解酶-1(cysteinyl aspartate specific proteinase-1,caspase-1)和IL-1β的释放,从而发挥抗炎作用。综上所述,芍药二酮不仅能够通过抑制YAP蛋白的表达抑制肝星状细胞的增殖与活化,而且能够同时抑制炎症因子的表达与释放,其对于肝纤维化和炎症的双向调节作用为芍药二酮开发为抗非酒精性脂肪性肝炎药物提供理论支持。

The study aims to investigate the anti-hepatic fibrosis and anti-inflammatory activities of palbinone,and to explore the internal regulatory mechanism,so as to lay an active foundation for its development as an antinon-alcoholic steatohepatitis(NASH)candidate.First,sulforhodamine B(SRB)method was used to detect the effect of palbinone on the proliferation of human hepatic stellate cells LX-2 and rat hepatic stellate cells HSC-T6.Following,in the in vitro hepatic fibrosis cell model that activated by transforming growth factor beta 1(TGF-β1),quantitative real-time PCR(qRT-PCR)and Western blot were used to detect the inhibitory effect of different concentrations of palbinone on the transcription level and protein expression level of hepatic fibrosis markers.And the regulating mechanism of palbinone on fibrosis-related genes was analyzed at the same time.In addition,in the inflammatory cell model that induced by lipopolysaccharide(LPS)and nigericin,ELISA was used to detect the effect of palbinone on the released interleukin-1β(IL-1β)level.At the same time,Western blot was used to detect the effect of palbinone on the related proteins of inflammatory pathway.The results showed that palbinone could significantly inhibit the proliferation activity of LX-2 and HSC-T6,and their half maximal inhibitory concentration(IC_(50))values were(375.11±55.45)and(260.27±36.81)nmol·L^(-1),respectively.In addition,palbinone showed a dose-dependent inhibitory effect on the expression levels of TGF-β1-induced fibrosis-related genes,including collagen typeⅠα1(COL1A1),TGF-β1,α-smooth muscle actin(α-SMA)and tissue inhibitor of metalloproteinase 1(TIMP1).Mechanism study showed that palbinone may decrease the expression level of Yes-associated protein(YAP),thereby weakening its activation effect on the downstream fibrosis pathway.In addition,palbinone also exerted an anti-inflammatory effect by inhibiting the activity of nuclear factor kappa-B(NF-κB)signaling pathway and reducing inflammatory factors cysteinyl aspartate specific proteinase-1(caspase-1)and IL-1β release.In conclusion,palbinone can not only inhibit the proliferation and activation of hepatic stellate cells by inhibiting the expression of YAP,but also inhibit the expression and release of inflammatory factors at the same time.All these studies provide theoretical support for the development of palbinone as an anti-nonalcoholic steatohepatitis drug.