| Summary: | 博士 === 國立中興大學 === 生命科學系所 === 100 === Human serum paraoxonase 1 (PON1) is associated with high-density lipoprotein (HDL)and inhibit oxidative modification of low-density lipoprotein (LDL), is thought to a major anti-atherosclerotic component of HDL implication of PON1 in prevention against atherosclerosis. Clinically, statins have been the most widely prescribed drugs for Hypercholesterolemia, some types of statins could increase the expression of PON1. Besides, a numerous of components isolated from plants and their products, such as quercetin, naringenin and resveratrol have anti-oxidative and anti-inflammatory properties, lower cholesterol and also increase the expression of PON1. Therefore, it is necessary to discover continually new compounds that increase expression of PON1. Firstly, we find a folk medicinal plant, Graptopetalum paraguayense, has anti-oxidative, anti-inflammatory, anti-hypertensive, and anti-atherogenic properties. The effects of G. paraguayense on the activity and expression of PON1 were examined using various extracts of the plant; extracts were made in water (GPWE), 50% ethanol (GP50E), and 95% ethanol (GP95E). Of these extracts, GP50E was found to be the most effective at increasing the function and expression of PON1 in a human hepatoma HepG2 cell line. Data from electrophoretic mobility shift assays and promoter-reporter luciferase analysis demonstrated that the DNA binding activity and transactivation ability of NF-kB were enhanced by GP50E. Treatment with NF-kB inhibitors, pyrrolidinedithiocarbamate (PDTC), and BAY 11-7082, significantly attenuated GP50E-induced PON1 production and NF-kB transactivation activity. In addition, GP50E increased the levels of phosphorylated protein kinase B (PKB / AKT). Pharmacological inhibition of AKT by LY294002 effectively suppressed NF-kB activation and PON1 gene expression, suggesting that AKT was an upstream regulator of GP50E-mediated biological events. Overall, the results show that GP50E up-regulated PON1 gene expression via an AKT/NF-kB-dependent signaling pathway in human hepatoma HepG2 cells. Secondly, the effective compounds of medicinal plants were examined for the property of increasing PON1 expression. A botanical alkaloid, berberine (BBR), isolated from a number of medicinal plants (e.g., Berberisaquifolium, Berberisvulgaris, Berberisaristata and Tinosporacordifolia) has been reported to lower the cholesterol level in serum and is thought to display cardioprotective property. However, the effect of BBR on PON1 gene expression remains unclear. To clarify this point, the PON1 arylesterase activity and protein levels were increased by BBR in a dose- and time-dependent manner in human hepatoma HepG2 and Huh7 cells. The result of PON1 upregulation by BBR was confirmed to be concordant with that of upregulation of PON1 mRNA expression. In addition, treatment of HepG2 cells with BBR increased the levels of phosphorylated JNK and its downstream target c-Jun. The PON1 upstream region contained a consensus binding site for AP-1, and the electrophoretic mobility shift assay and chromatin immunoprecipitation analysis indicated the AP-1 factors, especially c-Jun, binding to the upstream sequence of PON1 promoter upon BBR treatment. Moreover, pretreatment with SP600125 (JNK inhibitor) or curcumin (AP-1 inhibitor) markedly attenuated the BBR-induced PON1promoter activity and protein expression. This study addresses that JNK/c-Jun signaling pathway plays a crucial role for the transcriptional regulation of PON1 gene expression by BBR in human hepatoma cells. Thirdly, atherosclerosis is a chronic inflammatory response in the wall of arteries. A number of studies demonstratethat the difference in regulation of PON1 gene in response to various cytokines in inflammation. However, the relationships between PON1 and atherosclerosis-related inflammation remain unclear. In this study, a human hepatoma HepG2 cell line was used as a hepatocyte model to examine the effects of the proinflammatory cytokines on PON1 expression. Our result showed that IL-6 increased the levels of PON1 protein; data from real-time RT-PCR analysis revealed that the IL-6-induced PON1 expression occurred at the transcriptional level. Additionally, increase of IkB kinase activity and IkB phosphorylation, and reduction of IkB protein level were observed in IL-6-treated HepG2 cells compared with untreated culture. This event was accompanied by increase of NF-kBp50 and p65 nuclear translocation. Moreover, treatment with IL-6 augmented the DNA binding activity of NF-kB. Furthermore, pharmacological inhibition of NF-kB activation by PDTC and BAY 11-7082, significantly suppressed the IL-6-induced expression of PON1. In addition, IL-6 increased the levels of phosphorylated protein kinase B (PKB, AKT). Pharmacological inhibition of AKT by LY294002 effectively suppressed NF-kB activation and PON1 gene expression, suggesting that AKT was an upstream regulator in IL-6-mediated biological event. Taken together, these results demonstrate that IL-6 upregulates PON1 gene expression, via an AKT/NF-kB-dependent signaling pathway in human hepatoma HepG2 cells. These observations are the first studies to suggest that 50% ethanol extracts of G. paraguayense , IL-6 and berberine up-regulate PON1 expression via AKT/NF-kB and JNK/c-Jun pathway. These results led to the conclusion that the anti-atherogenic characteristics of G .paraguayense, berberine and IL-6 modulate, at least in part, the upregulation of hepatocyte PON1 gene expression.
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