| Summary: | 博士 === 中山醫學大學 === 營養學研究所 === 101 === Department of Health, Executive Yuan, R.O.C. (TAIWAN) reported that cancer, cardiovascular disease, chronic liver disease and liver cirrhosis are among the top 10 death causes in 2012. In recent years, various phytochemicals from foods and herbs exhibited different biological properties via regulation of gene expression. Andrographis paniculata (A. paniculata) is a traditional medicinal herb which is widely used in many Asia countries. Andrographolide, a diterpenoid, is the major bioactive component of A. paniculata, which has been demonstrated to have various biological actions including anti-diabetes, anti-inflammation, antioxidation, and anti-hepatotoxicity. In this study, we investigated the effect of andrographolide on detoxification and anti-inflammation.
The pi class of glutathione S-transferase (GSTP) is one of the phase II biotransformation enzymes. Our previous study showed that andrographolide upregulates the expression of GSTP. The aim of the first study was to investigate the mechanism by which andrographolide induces GSTP gene expression in rat primary hepatocytes. Andrographolide was dissolved in dimethyl sulfoxide (DMSO) and the final concentration of DMSO in medium is 0.1%. Hepatocytes treated with 40 μM andrographolide had increased Akt phosphorylation at 0.5 h and c-jun phosphorylation at 3 h. However, pretreatment with 20 μM PI3K inhibitors, wortmannin and LY294002 for 1 h, or siPI3K abolished the andrographolide-induced phosphorylation of c-jun at 3 h and GSTP protein expression at 48 h. EMSA showed that pretreatment with wortmannin, LY294002, or siPI3K attenuated andrographolide-induced AP-1-DNA-binding activity. Results of immunoprecipitation indicated that andrographolide increased nuclear c-fos/c-jun heterodimer accumulation. Addition of antibodies against c-jun and c-fos to nuclear extract decreased nuclear protein binding to the AP-1 consensus of DNA sequence. In summary, andrographolide induction of GSTP expression in rat primary hepatocytes is through activation of the PI3K/Akt, phosphorylation of c-jun, nuclear accumulation of AP-1, and subsequent binding to the response element in the target gene.
Oxidative stress is considered to be a major risk factor in aging, inflammation, cancer, atherosclerosis, and diabetes mellitus. NADPH oxidase is one of the major sources of endogenous reactive oxygen species (ROS). In the second study, we used EA.hy926 endothelial-like cells to explore the anti-inflammatory activity of andrographolide. EA.hy926 cells were pretreated with 7.5 μM andrographolide [1:1000 (vol/vol) DMSO/DMEM] for 16 h and then challenged with 1 ng/ml TNFα for an additional 5 min to 6 h. Andrographolide attenuated TNFα-induced ROS generation at 20 min, Src phosphorylation at 5 min, and NADPH oxidase subunits, p47phox and p67phox, membrane translocation at 15 min as well as ICAM-1 gene expression at 6 h. In the small hairpin RNA interference assay, shp47phox abolished TNFα-induced p65 nuclear translocation at 1 h, ICAM-1 gene expression and adhesion of HL-60 cells at 6 h. Moreover, pretreatment with the NADPH oxidase inhibitors, 2 mM apocynin (APO) and 5 μM diphenyleneiodonium (DPI), for 1 h attenuated TNFα-induced p47phox and p67phox membrane translocation and ICAM-1 gene expression. Andrographolide induced heme oxygenase 1 (HO-1) and glutamate cysteine ligase modifier subunit (GCLM) gene expression in a time-dependent manner. Cellular GSH content was increased by andrographolide after 24 h treatment. shGCLM attenuated andrographolide-induced increase in GSH content and reversed the andrographolide inhibition of HL-60 adhesion. shHO-1 showed the similar effect on andrographolide inhibition of HL-60 adhesion to shGCLM. Pretreatment with the 20 μM PI3K inhibitor, LY294002, for 1 h attenuated andrographolide-induced HO-1 and GCLM gene expression and cellular GSH content, but MEK inhibitor, PD98059, had no effect on andrographolide-induced HO-1 and GCLM gene expression. Moreover, pretreatment with LY294002 attenuated andrographolide-induced Nrf2 nuclear translocation and c-jun phosphorylation at 2 h. shNrf2 and shJUN decreased andrographolide-induced HO-1 and GCLM gene expression. The mechanism underlying up-regulation of HO-1 and GCLM by andrographolide was dependent on PI3K/Akt pathway, and both Nrf2 and AP-1 transcriptional factors were involved. Taken together, our results suggest that andrographolide attenuates TNFα-induced inflammation at least partially through suppression of NADPH oxidase activation and induction of HO-1 and GCLM gene expression, which is PI3K/Akt pathway-dependent.
In summary, we suggested that andrographolide has detoxification, antioxidant and anti-inflammatory activities not only through the induction of GSTP in rat primary hepatocytes and HO-1 and GCLM in EA.hy926 cells but also through the suppression of TNFα-induced NADPH oxidase activation. Moreover, the induction of GSTP, HO-1 and GCLM is dependent on PI3K/Akt pathway and Nrf2 as well as AP-1 were demonstrated to be the transcriptional factors involved.
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