RESUMEN
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 anti-non-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 (IC50) 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 type Ⅰ α 1 (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.
RESUMEN
Liver fibrosis is a tissue repair compensatory response to liver injury caused by various chronic factors, ultimately leading to liver cirrhosis, liver failure and even hepatocellular carcinoma. Abnormal activation of hepatic stellate cells is the cellular basis of liver fibrosis development. Pepstatin Pr, the derivative of pepstatin A, was isolated from Streptomyces sp. CPCC 202950. Our purpose was to investigate the anti-fibrotic activity of pepstatin Pr and explore its molecular mechanism. Hepatic stellate cell LX-2 was stimulated by TGFβ1 and sub- sequently treated with pepstatin Pr. Its cytotoxicity was detected by sulforhodamine B (SRB) assay. The expression of COL1A1, α-SMA and cathepsin D, signaling proteins TGFβ, Smad and YAP/TAZ were detected by Western blot or real-time PCR. The results showed that pepstatin Pr was not cytotoxic to LX-2 cells. And pepstatin Pr significantly reduced the mRNA and protein expression of COL1A1 and α-SMA, which are important liver fibrosis markers. Pepstatin Pr also repressed the protein expression level of cathepsin D, TGFβ1, YAP/TAZ, the phospholation level of Smad2, and YAP nuclear translocation. In conclusion, pepstatin Pr exhibits anti-fibrotic effects in TGFβ1-stimulaed LX-2 cells by mediating YAP-TGFβ-Smad pathway.