Physalin A regulates the Nrf2 pathway through ERK and p38 for induction of detoxifying enzymes

P. alkekengi was purchased from a Kyungdong oriental herbal market, Seoul, Republic of Korea. The voucher specimens (ND4) have been deposited at the Systems Biotechnology Research Center, KIST, Gangneung Institute of Natural Products, Republic of Korea. This plant identified by Dr. Hak Cheol Kwon who responsible for KIST natural products library at KIST Gangneung, institute of natural products. Dried P. alkekengi (2.5 kg) were extracted using 95% ethanol for 4 h by reflux. After filtration, the ethanol were evaporated in a vacuum to obtain the ethanol extract (203 g), which was suspended in distilled water and partitioned using n-hexane, ethyl acetate, and n-butanol. The ethyl acetate fraction (15 g) was chromatographed on a Sephadex LH-20 column, eluted using methanol to obtain five fractions (fractions 1–5). Physalin A was re-chromatographed from fraction 3 using Sephadex LH-20 (methanol) and RP-18 gel [methanol-water (40 → 70%, v/v)] column chromatography to obtain white crystals. The chemical structures of compound 1 was determined by ¹H and ¹³C nuclear magnetic resonance and the results were compared with published data [24].

Dulbecco’s high glucose modified Eagle medium (DMEM) (Hyclone, Logan, UT, USA), fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin were obtained from Thermo Scientific (Waltham, MA, USA). 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and bovine serum albumin were purchased from Sigma-Aldrich (St, Louis, MO, USA).

Hepa-1c1c7 (mouse hepatoma cells) and HepG2 (human hepatocellular carcinoma cells) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were maintained at sub-confluence in the presence of 95% air and 5% CO₂ in a humidified atmosphere at 37 °C. DMEM and α-MEM were used for HepG2 and Hepa-1c1c7 cell cultivation, respectively. The media were supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin.

Specific QR activity was measured using a previously reported QR assay [25, 26], after brief modifications. Hepa-1c1c7 cells (1 × 10⁴ cells/well) were plated on 96-well culture plates and incubated for 24 h before treatment with five compounds isolated from P. alkekengi including physalin A (Fig. 2). The absorbance at 610 nm was determined five times at 50 s intervals using a Synergy HT multi-microplate reader (Bio-Tek Instruments, Winooski, VT, USA).

Cell viability was measured using the WST-1 cell proliferation assay kit (EZ-Cytox; Daeil Lab, Seoul). HepG2 cells (1 × 10⁴ cells/well) were plated in 96-well culture plates and incubated at 37 °C for 24 h, followed by treatment with various concentrations of physalin A. After 24 h of treatment, we added 1/10 diluted EZ-Cytox solution to each well and incubated for 1 h 20 min. Then, absorbance was measured at 450 nm using Synergy HT multi-detection microplate reader.

HepG2 cells (6 × 10⁵ cells/well) were plated in 6-well culture plates and incubated for 24 h prior to physalin A treatment. Nuclear and cytosolic fractions were prepared using a nuclear extraction kit (Cayman, Ann Arbor, MI, USA).

HepG2 cells (6 × 10⁵ cells/well) were plated in 6-well culture plates and incubated for 24 h prior to physalin A treatment. Cell lysate was prepared using ice-cold radioimmunoprecipitation assay (RIPA) buffer (Thermo Scientific) containing phenylmethane sulfonyl fluoride (PMSF) and a protease inhibitor cocktail (Sigma, St. Louis, MO, USA), followed by centrifugation for 25 min at 4 °C. Protein concentrations were determined using protein assay dye reagent concentrates (Bio-Rad, Hercules, CA, USA). Total cell lysates, and nuclear and cytosolic fractions were loaded on 10% sodium dodecyl sulfate polyacrylamide gels, electrophoresed, and then transferred to polyvinylidene difluoride membranes (Bio-Rad). The membranes were blocked using 3% bovine serum albumin (BSA), followed by incubation with primary antibodies against the following proteins: β-actin, NQO-1, HO-1, LaminB (Santacruz Biotechnology, Santa Cruz, CA, USA); Nrf2 (Abcam, Cambridge, MA, USA); ERK, p-ERK, p38, and p-p38 (Cell Signaling Technology, Denvers, MA, USA) in 3% BSA. The western blots were developed using SuperSignal™ West Femto maximum sensitivity substrate reagent (Thermo Scientific).

Total RNA was extracted from HepG2 cells using the RNeasy mini kit (Qiagen, Hilde, Germany) according to the manufacturer’s instruction. cDNA was synthesized using the PrimeScript™ first strand cDNA synthesis kit (Takara, Shiga, Japan) according to the manufacturer’s instruction. qRT-PCR analysis was performed using the LC480 detection system (Roche, Basel, Switzerland).

HepG2 cells (1 × 10⁵ cells/well) were plated in 24-well culture plates and incubated for 24 h, followed by physalin A treatment. The cells in each well were transfected with 0.4 μg ARE-luc reporter construct containing human NQO-1 sequences and 50 ng of pRL-CMV transfection control vector using the TransIT-2020 transfection reagent (Mirus Bio, Madison, WI, USA) according to the manufacturer’s instructions. The reporter assay was performed using a dual luciferase assay kit (Promega, Madison, WI, USA) and Synergy HT multi-detection microplate reader.

HepG2 cells (3 × 10⁶ cells/well) were plated in 100-mm culture dishes and incubated for 24 h, followed by physalin A treatment. The cells were lysed in HKMG buffer (10 mM HEPES (pH 7.9), 100 mM KCl, 5 mM MgCl_2, 1 mM dithiothreitol (DTT), 10% glycerol and 0.5% NP-40), and the cell lysates were incubated overnight with biotinylated oligonucleotides. The sequence of the oligonucleotide is as follows: 5′-AAATCGCAGTCAC- -AGTGACTCAGCAGAATCTGAGCCTAGG-3′. To obtain the nucleotide bound protein, the samples were incubated with streptavidin-agarose resin (Thermo Scientific) for 6 h at 4 °C, and then washed with HKMG buffer. The washed samples were used for western blot analysis.

The cellular distribution of Nrf2 was determined using immunofluorescence assay and confocal microscopy as previously described [27]. In brief, HepG2 cells (2 × 10³ cells/well) were plated on glass coverslips in 24-well plates, and incubated for 24 h prior to treatment. Next, the cells were incubated with primary antibody against Nrf2 (1:150; Cambridge, UK) overnight at 4 °C. Alexa Fluor 488-conjugated anti-rabbit (Invitrogen, Carlsbad, CA, USA) secondary antibody was used at 1:200 dilution for 1 h at room temperature. Antibodies were diluted in phosphate buffered saline-Tween 20 (PBST) containing 5% normal serum and 0.3% Triton X-100. Images were obtained using a Leica TCS SP5 confocal system (Leica, Wetzlar, Germany).

QR activity is a measure of the chemopreventive effect of any compound [7, 28]. We first assessed the specific QR activity of the P. alkekengi extract and five compounds derived from this plant in Hepa-1c1c cells. Results showed that the extract and only physalin A increased specific QR activity in a dose-dependent manner (Fig. 3a-b). Other compounds, such as physalin O, luteolin, methyl chlorogenic acid, and luteolin-7-O-glucoside did not significantly increase QR activity (Fig. 3c-f). The P. alkekengi extract and the isolated compounds did not significantly affect cell viability. Sulforaphane was used as a positive control in these experiments. These results showed that physalin A is an active component responsible for induction of QR activity.

Since physalin A was the active component required for QR activity, we performed cell viability assay using 3.125–100 μM physalin A (Fig. 4a) to determine the non-cytotoxic concentration range that can be used in further experiments involving HepG2 cells. No significant cytotoxicity was observed below 25 μM (Fig. 4a).

To investigate whether physalin A induces QR activity through the Nrf2 pathway, we measured Nrf2 mRNA level and ARE activation. Nrf2 upregulated genes encoding phase II detoxification enzymes, such as NQO-1, by binding to the ARE in the target gene’s promoter regions [8]. qRT-PCR and western blotting showed that physalin A increased NQO-1 mRNA expression (Fig. 4b) and the protein levels of Nrf2 target genes, respectively (Fig. 4e). To determine whether physalin A activated the ARE by promoting Nrf2 binding, we performed an oligonucleotide pull-down assay and a reporter assay using a construct containing the ARE (Fig. 4d). Also, we observed that the binding of Nrf2 to ARE was increased by physalin A in HepG2 cells in a dose-dependent manner (Fig. 4c).

To confirm that physalin A increases Nrf2 expression and nuclear accumulation, we assessed Nrf2 expression at various time points and monitored nuclear translocation of Nrf2 from the cytosol using fluorescent dyes. Nrf2 expression in a time-dependent manner (Fig. 5a) when the cells were treated with 20 μM physalin A. We also observed that physalin A increased Nrf2 nuclear accumulation in dose-dependent manner (Fig. 5b-c) when the cells were treated for 4 h. Thus, these results showed that physalin A induces Nrf2 expression and nuclear accumulation.

A previous study showed that multiple kinases are involved in the translocation of Nrf2 to the nucleus [11‐14]. To identify the kinases required for physalin A-mediated Nrf2 translocation, we assessed Nrf2 expression after pre-treatment with several following kinase inhibitors: LY294002 (inhibitor of PI3K), SP600125 (inhibitor of JNK), U0126 (inhibitor of ERK), or SB202190 (inhibitor of p38). Particularly, treatments with U0126 (inhibitor of ERK) and SB202190 (inhibitor of p38) reduced Nrf2 nuclear accumulation (Fig. 6a). To further confirm if ERK and p38 regulate Nrf2 activation, the phosphorylated-forms of ERK and p38 were investigated in a time-dependent manner. We observed that physalin A increases ERK and p38 phosphorylation. We also determined the expression of HO-1, one of the target genes, in cells treated with physalin A and U0126 or SB202190. Results showed that the expression of Nrf2 and its target genes was considerably reduced after co-treatment with the inhibitor compared to treatment with physalin A alone (Fig. 6c). Thus, these results suggest that physalin A activates the ERK and p38 kinases to induce Nrf2 nuclear translocation.