Protective effects of Cambodian medicinal plants on tert‑butyl hydroperoxide‑induced hepatotoxicity via Nrf2‑mediated heme oxygenase‑1

Introduction

Cambodia is a tropical country featuring plateaus, flat plains, hills and highland, the area of which is covered predominantly in forest. By virtue of its tropical climate, Cambodia has a diverse range of natural resources, amongst which plants are primarily used for complementary medicines by the local population. As many as 515 species amongst 134 families of medicinal plants have been found in Cambodia (1), allowing Cambodians to couple the use of medicinal plants with their traditional treatment of diseases. Cambodian medicinal plants are crucial in folk medicine in communities, with traditional healers offering assurance for their curable effects (2). According to the Ministry of Health in Cambodia, 45% of the Cambodian population use herbal plants for therapeutic purposes, despite a shortage of scientific evidence (3).

Liver disease, predominantly attributed to hepatitis and alcoholism, is considered a primary contributor to morbidity and mortality rates in humans. The accumulation of deleterious substances in hepatocytes caused by infection, injury, exposure to xenobiotics, autoimmunity or genetic disorders may lead to liver damage in the form of inflammation, scarring, cirrhosis or liver dysfunction (4,5). Oxidative stress is critical in liver injury and may be triggered by reactive oxygen species (ROS) generated by various signal transduction pathways (6). Oxidant-induced liver injury is caused by toxins, including tert-Butyl hydroperoxide (t-BHP), which exerts cytotoxic effects through glutathione (GSH) depletion coupled with intracellular over-influx of Ca2+ (4,7). Although there is general recognition that the drugs currently available for treating patients with liver disease are mandatory, natural products originating from plants have attracted considerable interest among hepatologists in terms of their efficacy and safety (8).

Traditional medicinal plants have long been used in folk medicine for ameliorating liver diseases. A number of these plants have shown potential in the treatment of liver disease due to their therapeutic mechanisms. For example, Silybum marianum and Picrorhiza kurroa have been confirmed to be clinically efficacious in the treatment of toxic hepatitis, fatty liver disease, cirrhosis, ischemic injury, radiation toxicity and viral hepatitis, based upon their antioxidative, antilipid peroxidative, antifibrotic, anti-inflammatory, immunomodulatory and liver regenerating effects (9). In addition, phytochemicals, including glycyrrhizin, matrine and silymarin, have been shown to exert therapeutic effects against hepatitis, alcoholic liver disease and liver cirrhosis (10). Phytochemicals protect hepatocytes (11) via the induction of heme oxygenase-1 (HO-1), which is catabolized to produce carbon monoxide, biliverdin and free iron (12). HO-1 is well-known as a valuable therapeutic candidate as it exerts anti-inflammatory, anti-apoptotic and antiproliferative effects (13). In addition, HO-1 is understood to be upregulated in the liver and splanchnic organs during portal hypertension (14) by the activation of nuclear factor-E2-related factor-2 (Nrf2) (15). Nrf2 is known to be important in the regulation of phase II detoxifying enzymes and associated proteins, including HO-1, GSH, catalase, superoxide dismutase, glutathione-S-transferase, γ-glutamyl cysteine ligase, NAD(P)H: quinone oxidoreductase-1, glutathione peroxidase and glutathione reductase (16). Nrf2-regulated phase II detoxifying enzymes can be critical in protecting the liver from oxidative stress imposed by ROS (15,16). Alcoholic liver disease (ALD) is reported to affect the Cambodian population (17) due to high levels of alcohol consumption among men and women (18). Several species of Cambodian medicinal plants have been used for the treatment of ALD via a number of traditional approaches. However, the majority of these plants have received limited scientific investigation, particularly screening assays of their hepatoprotective activities (19).

Thus, the present study was designed to determine the hepatoprotective characteristics of 64 Cambodian plants by examining the cytoprotective effects of the Cambodian plant extracts against t-BHP-induced hepatotoxicity in HepG2 cells. Several laboratories have used human liver-derived HepG2 cells as a model for investigating hepatocyte proliferation and function; these cells perform several of the functions of normal hepatocytes, including the secretion of plasma proteins and extensive degradation of insulin (20). Therefore, HepG2 cells were selected for use in the present study. Selective extracts with hepatoprotective effects were further evaluated to examine their cytoprotective mechanism associated with the activities of ROS, HO-1 and Nrf2.

Materials and methods

Chemicals and reagents

Roswell Park Memorial Institute (RPMI) medium, fetal bovine serum (FBS), trypsin-EDTA solution and antibiotic-antimycotic solution (containing 10,000 units of penicillin, 10,000 µg of streptomycin and 25 µg of amphotericin B/ml) were purchased from Gibco; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Tin protoporphyrin IX (SnPP), an inhibitor of HO activity, was obtained from Porphyrin Products (Logan, UT, USA). All other relevant chemicals and reagents were obtained from Sigma-Aldrich; Merck Millipore (Darmstadt, Germany).

Preparation of Cambodian plants

A total of 64 Cambodian plants were imported from O'reusey Market (Phnom Penh, Cambodia). All plants were identified by Professor Sun Kaing Cheng (Laboratory of Phytochemistry, Faculty of Pharmacy, University of Health Sciences (Phnom Penh, Cambodia). The herbarium specimens (no. WKP-2013-23-WKP-2013-86) were stored at the College of Pharmacy, Wonkwang University (Iksan, Korea). Dry plants (20 g each) were extracted with 70% ethanol (1 liter of each) for 2 h, and the extracts were concentrated in vacuo to obtain 70% ethanol extracts. Plant extraction was supported by the College of Pharmacy, Wonkwang University.

Cell culture and viability assay

Human liver-derived HepG2 cells were obtained from American Type Culture Collection (Manassas, VA, USA). The cells were maintained at 5×105 cells/ml in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, penicillin G (100 U/ml−1), streptomycin (100 mg·ml−1) and L-glutamine (2 mM), and were incubated at 37°C in a humidified atmosphere containing 5% CO2 and 95% air. The cells were pretreated for 3 h with the indicated concentrations of Cambodian plant extract and stimulated for 24 h with t-BHP. The control group was treated with equal volume of DMSO as the extracts. The determination of cell viability was performed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. MTT (2.5 mg·ml−1; 50 µl) was added to each well, containing a cell suspension of 2×105 cells/ml in 96-well plates, at a final concentration of 0.5 mg·ml−1. The mixture was incubated for 3–4 h at 37°C, and the liquid was removed from the wells in turn. Subsequently, DMSO (150 µl) was added to each well, and the absorbance was read at 540 nm on a UV Max microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The relative optical density of formazan in the control group, which contained cells treated with neither glutamate nor the plant extracts, was taken as 100% viability.

Western blot analysis

The human liver-derived HepG2 cells were harvested and pelleted by centrifugation at 200 × g for 3 min at 4°C. The cells were then washed with PBS and lysed with 20 mM Tris-HCl buffer (pH 7.4) containing a protease inhibitor mixture (0.1 mM phenylmethanesulfonyl fluoride, 5 mg/ml aprotinin, 5 mg/ml pepstatin A and 1 mg/ml chymostatin). The protein concentration was determined using a Lowry protein assay kit (P5626; Sigma-Aldrich; Merck Millipore). An equal quantity of protein from each sample (30 µg) was resolved using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then electrophoretically transferred onto a Hybond enhanced chemiluminescence (ECL) nitrocellulose membrane (Bio-Rad Laboratories, Inc.). The membrane was blocked with 5% skimmed milk and sequentially incubated with primary antibodies against HO-1 (1:1,000; cat. no. sc-10789), Nrf2 (1:1,000; cat. no. sc-722), Lamin B (1:1,000; cat. no. sc-6216), actin (1:1,000; cat. no. sc-1616) all obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA), a further anti-HO-1 antibody (1:1,000; Merck Millipore; cat. no. 374090) and horseradish peroxidase-conjugated secondary antibodies (1:1,000; Santa Cruz Biotechnology, Inc.; cat. nos. sc-2741 and sc-2004) followed by ECL detection.

Preparation of nuclear and cytosolic fractions

The cells were homogenized (1:20, w/v) in PER Mammalian Protein Extraction buffer (Pierce; Thermo Fisher Scientific, Inc.) containing freshly added protease inhibitor cocktail I (EMD Millipore, Billerica, MA, USA) and 1 mM phenylmethylsulfonyl fluoride. The cytosolic fraction of the cell was prepared by centrifugation at 15,000 × g for 10 min at 4°C. The nuclear and cytoplasmic extracts of the cells were prepared using NE-PER nuclear and cytoplasmic extraction reagents (Pierce; Thermo Fisher Scientific, Inc.), respectively.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis

Total RNA was isolated from the cells using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.), in accordance with the manufacturer's protocol, and quantified spectrophotometrically at 260 nm. The total RNA (1 µg) was reverse-transcribed using the High Capacity RNA-to-cDNA kit (Applied Biosystems; Thermo Fisher Scientific, Inc.). The cDNA was then amplified using the SYBR Premix Ex Taq kit (Takara Bio, Inc., Shiga, Japan) using a StepOnePlus Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). Briefly, each 20 µl of reaction volume contained 10 µl of SYBR-Green PCR master mix, 0.8 µM of each primer, and diethyl pyrocarbonate-treated water. The primer sequences were designed using PrimerQuest (Integrated DNA Technologies, Cambridge, MA, USA; www.idtdna.com/Primerquest/Home/Index). The primer sequences were as follows: HO-1, forward 5′-CTCTTGGCTGGCTTCCTT-3′ and reverse 5′-GGCTCCTTCCTCCTTTCC-3′ and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), forward 5′-ACTTTGGTATCGTGGAAGGACT-3′ and reverse 5′-GTAGAGGCAGGGATGATGTTCT-3. The optimal conditions for PCR amplification of the cDNA were established according to the manufacturer's protocol. The data were analyzed using StepOne software version 2.3 (Applied Biosystems; Thermo Fisher Scientific, Inc.), and the cycle number at the linear amplification threshold (Cq) values for the endogenous control gene (GAPDH) and the target gene were recorded. Relative gene expression, calculated as the target gene expression normalized to the expression of the endogenous control gene, was calculated using the comparative Cq method (2−ΔΔCq) (21).

Nuclear magnetic resonance (NMR) analysis

The 1H-NMR spectra of the ethanolic extracts of T. crispa and P. weberi were recorded in (CD3) CO2 and D2O using a JEOL JNM ECP-400 spectrometer (400 MHz for 1 h), and chemical shifts were referenced relative to the residual solvent peak. The resultant spectra of the possible functional group present in the plants were determined.

Statistical analysis

The data are expressed as the mean ± standard deviation of at least three independent experiments. To compare three or more groups, one-way analysis of variance, followed by the Newman-Keuls post-hoc test, was used. Statistical analysis was performed using GraphPad Prism software version 3.03 (GraphPad Software, Inc., San Diego, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

Results

Effects of Cambodian plant extracts on t-BHP-induced hepatotoxicity in human liver-derived HepG2 cells

The ethanolic extracts of 64 Cambodian plants on human liver-derived HepG2 cells were assessed in vitro. As shown in Table I, 51 plants exhibited a cytoprotection value of <80%, whereas 19 plants showed a cytoprotection value of >80%. At a final concentration of 100 µg/ml, plant extracts of B. flabellifer (root), C. halicacabum (whole plant), C. trifolia (stem), C. caryophyllus (bark), C. rotundus (rhizome), F. benjamina (stem), Q. indica (whole plant), S. glabra (rhizome) and W. cochinchinensis (stem) resulted in maintained cell viability (Table I). At a final concentration of 300 µg/ml, the plant extracts A. martinii (root), B. bracteata (stem), B. ceiba (bark), D. lomentaceum (stem), M. duperreana (bark), M. citrifolia (fruit), P. humilis (stem), P. weberi (whole plant), P. emblica (fruit) and T. crispa (stem) resulted in maintained cell viability (Table I). The 19 plant extracts with cytoprotective values >80% at a final concentration of 100 µg/ml were used for further analysis (Table II). The results revealed the half maximal effective concentration (EC50) values for B. flabellifer (66.25 µg/ml), C. halicacabum (63.18 µg/ml), C. trifolia (59.23 µg/ml), C. caryophyllus (64.33 µg/ml), C. rotundus (68.75 µg/ml), F. benjamina (62.98 µg/ml), Q. indica (62.9 µg/ml), S. glabra (80.41 µg/ml), W. cochinchinensis (72.95 µg/ml), A. martinii (121.1 µg/ml), B. bracteata (138.9 µg/ml), B. ceiba (148.8 µg/ml), D. lomentaceum (125.7 µg/ml), M. duperreana (105.2 µg/ml), M. citrifolia (157.8 µg/ml), P. humilis (151.3 µg/ml), P. weberi (121.7 µg/ml), P. emblica (144.9 µg/ml) and T. crispa (144.3 µg/ml), as shown in Table II.

Table I. Protective effects of ethanolic extracts (100 and 300 µg/ml) derived from Cambodian plants on t-BHP-induced hepatotoxicity in HepG2 cells.

Table I.

Protective effects of ethanolic extracts (100 and 300 µg/ml) derived from Cambodian plants on t-BHP-induced hepatotoxicity in HepG2 cells.

				Protection (%)
No.	Plant family	Plant species	Plant region	100 µg/ml	300 µg/ml
1	Anacardiaceae	Anacardium occidentale L.	Bark	11.5	45.1
2		Mangifera duperreana pierre	Bark	45.1	85.1
3	Annonaceae	Cananga latifolia finet & gagnep.	Stem	−15.1	−85.1
4		Dasymaschalon lomentaceum finet & gagnep.	Stem	48.8	87.8
5	Apocynaceae	Calotropis procera (aiton) R. Br.	Stem	−33.9	−19.8
6		Streptocaulon juventas (Lour.) merr.	Root	−26.7	−33.1
7		Willughbeia cochinchinensis (Pierre) K. Schum.	Stem	88.7	17.5
8	Araceae	Alocasia macrorrhiza schott.	Stem	−3.3	−17.4
9		Amorphophallus harmandii engl. & gehrm.	Tuber	1.9	19.3
10	Arecaceae	Borassus flabellifer L.	Root	83.1	12.4
11		Borassus flabellifer L.	Male flower	48.4	−30.1
12	Asparagaceae	Dracaena angustifolia (medik.) roxb.	Stem	9.6	51.7
13		Peliosanthes weberi (L. modr.) N. tanaka	Whole plant	42.5	87.6
14	Asteraceae	Blumea balsamifera (L.) DC.	Stem	26.9	47.2
15	Capparaceae	Crateva adansonii DC.	Bark	0.7	1.9
16	Caricaceae	Carica papaya L.	Root	3.9	15.6
17	Combretaceae	Quisqualis indica L.	Whole plant	83.9	20.6
18	Costaceae	Costus speciosus (J. koenig) Sm.	Rhizome	−15.8	−24.1
19	Cyperaceae	Cyperus rotundus L.	Rhizome	89.6	14.4
20	Dilleniaceae	Tetracera indica (christm. & panz) merr.	Stem	1.4	21.2
21	Dipterocarpaceae	Shorea siamensis miq.	Flowers	55.8	69.3
22	Ebenaceae	Diospyros ehretioides wall. ex G. don	Root	46.8	−10.3
23		Diospyros rhodocalyx kurz	Fruit	0.7	3.5
24	Euphorbiaceae	Croton crassifolius geiseler	Rhizome	10.1	−31.2
25	Fabaceae	Bauhinia bracteata (benth.) baker	Stem	36.0	88.1
26		Cassia alata L.	Stem	30.4	30.0
27		Dalbergia hancei benth.	Stem	74.8	48.7
28	Lamiaceae	Gmelina asiatica L.	Stem	4.1	10.8
29		Cinnamomum cambodianum lecomte	Bark	57.0	54.5
30		Cinnamomum caryophyllus (lour.) S. moore	Bark	87.4	5.3
31	Lecythidaceae	Careya arborea roxb.	Bark	−76.1	−16.7
32	Loganiaceae	Strychnos nux-vomica L.	Seed	67.8	75.8
33	Malvaceae	Abutilon indicum (L.) sweet	Stem	10.2	23.0
34		Bombax ceiba L.	Bark	31.8	81.6
35	Meliaceae	Walsura villosa wall. ex wight & arn.	Bark	42.5	−34.3
36	Menispermaceae	Fibraurea tinctoria lour.	Stem	37.9	−80.8
37		Tinospora crispa (L.) hook. f. & thomson	Stem	36.2	88.2
38	Moraceae	Ficus benjamina L.	Stem	87.7	21.9
39		Morus alba L.	Bark	10.4	30.1
40	Pandanaceae	Pandanus humilis lour.	Stem	40.9	85.3
41	Phyllanthaceae	Flueggea virosa (roxb. ex willd.) baill.	Wood	14.2	50.3
42		Phyllanthus emblica L.	Fruit	34.5	85.5
43	Piperaceae	Piper retrofractum vahl	Fruit	−8.5	−12.9
44	Poaceae	Chrysopogon aciculatus (retz.) trin.	Whole plant	50.3	48.6
45		Coix lacryma-jobi L.	Whole plant	65.2	2.0
46		Cynodon dactylon (L.) pers.	Aerial part	70.4	24.0
47		Imperata cylindrica (L.) raeusch.	Rhizome	12.2	27.4
48		Oryza rufipogon griff.	Whole plant	20.3	−15.3
49	Rubiaceae	Anthocephalus chinensis (lam.) rich. ex walp.	Wood	27.1	45.5
50		Gardenia obtusifolia roxb. ex hook. f.	Rhizome	−25.1	−22.8
51		Morinda citrifolia L.	Fruit	30.7	84.3
52	Rutaceae	Aegle marmelos (L.) corrêa	Fruit	0.8	23.6
53		Feroniella lucida swingle	Bark	−52.5	−43.2
54	Sapindaceae	Cardiospermum halicacabum L.	Whole plant	82.5	15.9
55	Simaroubaceae	Brucea javanica (L.) merr.	Whole plant	−65.9	−42.0
56		Smilax glabra roxb.	Rhizome	84.4	15.8
57	Solanaceae	Physalis angulata L.	Whole plant	−4.2	−3.7
58	Urticaceae	Pouzolzia zeylanica (L.) benn. & R. Br.	Whole plant	17.0	3.9
59	Vitaceae	Ampelocissus martinii planch.	Root	45.9	85.9
60		Cayratia trifolia (L.) domin	Stem	88.5	10.9
61		Cissus modeccoides planch.	Stem	59.8	25.4
62		Leea rubra blume ex spreng.	Stem	10.4	30.2
63	Zingiberaceae	Alpinia conchigera griff.	Rhizome	−44.8	−42.0
64		Amomum krervanh pierre ex gagnep.	Fruit	10.7	8.8

[i] Curcumin was used as a reference compound. For pretreatment, samples were incubated for 2 h prior to exposure to t-BHP. t-BHP, tert-butyl hydroperoxide.

Table II. Protective effects of ethanolic extracts from Cambodian plants on t-BHP-induced hepatotoxicity in HepG2 cells.

Table II.

Protective effects of ethanolic extracts from Cambodian plants on t-BHP-induced hepatotoxicity in HepG2 cells.

No.	Plant species	Plant region	EC50 (µg/ml)
1	Ampelocissus martinii planch.	Root	121.1
2	Bauhinia bracteata (benth.) baker	Stem	138.9
3	Bombax ceiba L.	Bark	148.8
4	Borassus flabellifer L.	Root	66.2
5	Cardiospermum halicacabum L.	Whole plant	63.2
6	Cayratia trifolia (L.) domin	Stem	59.2
7	Cinnamomum caryophyllus (Lour.) S. moore	Bark	64.3
8	Cyperus rotundus L.	Rhizome	68.8
9	Dasymaschalon lomentaceum finet & gagnep.	Stem	125.7
10	Ficus benjamina L.	Stem	63.0
11	Mangifera duperreana pierre	Bark	105.2
12	Morinda citrifolia L.	Fruit	157.8
13	Pandanus humilis lour.	Stem	151.3
14	Peliosanthes weberi (L. rodr.) N. tanaka	Whole plant	121.7
15	Phyllanthus emblica L.	Fruit	144.9
16	Quisqualis indica L.	Whole plant	62.9
17	Smilax glabra roxb.	Rhizome	80.4
18	Tinospora crispa (L.) hook. f. & thomson	Stem	144.3
19	Willughbeia cochinchinensis (pierre) K. Schum.	Stem	73.0

[i] Curcumin was used as a reference compound. For pretreatment, samples were incubated for 2 h prior to exposure to t-BHP. EC₅₀, half maximal effective concentration; t-BHP, tert-butyl hydroperoxide.

Effects of Cambodian plant extracts on the mRNA and protein expression levels of HO-1 in human liver-derived HepG2 cells

HO-1 has been reported to be upregulated in hepatocytes (14). Therefore, the present study performed further screening of the 19 plant extracts for the protein expression of HO-1 in the HepG2 cells (Fig. 1). Whether treatment of the HepG2 cells with the selected extracts affected the mRNA expression of HO-1 was subsequently examined. The protein and mRNA expression levels were determined following treatment for 12 h. Amongst the 19 plant extracts, the T. crispa and P. weberi extracts significantly increased the protein (Fig. 1E and J) and mRNA (Fig. 2A and B) levels of HO-1 in a dose-dependent manner, with maximal values observed at 400 µg/ml. The HO-1 inducer, curcumin, was used at a concentration of 20 µM as a positive control, which also increased the expression of HO-1.

Figure 1. Effects of Cambodian plant extracts on the expression of HO-1 in HepG2 cells. (A-J) HepG2 cells were incubated for 12 h with the indicated concentrations of Cambodian plant extracts. Western blot analysis was performed to determine the expression of HO-1. HO-1, heme oxygenase-1.

Figure 2. Involvement of the HO-1 pathway in the cytoprotective effects of T. crispa and P. weberi in HepG2 cells. HepG2 cells were pretreated for 3 h with (A) T. crispa (400 µg/ml) or (B) P. weberi (400 µg/ml) extracts in the presence or absence of SnPP (50 µM), a competitive inhibitor of HO-1 activity, and stimulated for 12 h with t-BHP (60 µM). The data are presented as the mean +standard deviation of three independent experiments. *P<0.05, compared with the control group; **P<0.05, compared with the group treated with t-BHP alone. HO-1, heme oxygenase-1; t-BHP, tert-butyl hydroperoxide; SnPP, Tin protoporphyrin IX.

Effects of the upregulation of HO-1 on t-BHP-induced hepatotoxicity and the inhibition of ROS generation by T. crispa or P. weberi extracts

HO-1 is considered to possess cytoprotective effects against oxidative stress-induced cell damage, notably in HepG2 cells (22). Thus, the present study investigated whether the upregulation of HO-1 triggered by T. crispa or P. weberi extracts mediated these cytoprotective effects. The HepG2 cells were co-treated with 400 µg/ml of T. crispa or P. weberi extracts for 12 h in the absence or presence of SnPP, an inhibitor of HO activity. These inhibitors significantly suppressed T. crispa/P. weberi extract-mediated protection (Fig. 3A and B). The T. crispa/P. weberi extract-induced upregulation of HO-1 was also required for the suppression of t-BHP-induced ROS generation (Fig. 3C and D).

Figure 3. Effects of T. crispa and P. weberi extract-induced upregulation of HO-1 on t-BHP-induced hepatotoxicity and inhibition of ROS. HepG2 cells were treated with 400 µg/ml of (A) T. crispa or (B) P. weberi extracts in the presence or absence of 50 µM of SnPP, and then exposed to t-BHP (60 µM) for 12 h. Exposure of HepG2 cells to t-BHP (60 µM) for 12 h increased the production of ROS. (C) T. crispa or (D) P. weberi extract-induced upregulation of HO-1 effectively inhibited the production of ROS production. The data are presented as the mean + standard deviation of three independent experiments. *P<0.05, compared with the control group; **P<0.05, compared with the group treated with t-BHP alone; #P<0.05, compared with the group treated with T. crispa (400 µg/ml) or P. weberi (400 µg/ml) in addition to t-BHP. HO-1, heme oxygenase-1; t-BHP, tert-butyl hydroperoxide; ROS, reactive oxygen species; SnPP, tin protoporphyrin IX.

Effects of T. crispa and P. weberi extracts on the nuclear translocation of Nrf2

The nuclear translocation of activated Nrf2 is a key upstream regulator for the expression of HO-1 (23). Therefore, the present study examined whether treatment of cells with T. crispa or P. weberi extracts induced the translocation of Nrf2 into the nucleus. The results of the western blot analysis revealed the presence of Nrf2 proteins in the nuclear compartment of HepG2 cells. The cells were incubated with 400 µg/ml T. crispa or P. weberi extracts for 0.5, 1 and 1.5 h. As shown in Fig. 4A and B, the nuclear fractions of the T. crispa or P. weberi extract-treated cells exhibited a gradual increase in the levels of Nrf2, and the levels of Nrf2 simultaneously decreased in the cytoplasmic fractions.

Figure 4. Effects of T. crispa or P. weberi extracts on the nuclear translocation of Nrf2. HepG2 cells were treated with 400 µg/ml of (A) T. crispa or (B) P. weberi extracts for 0.5, 1 and 1.5 h. Nuclei were separated from the cytosol using NE-PER nuclear and cytoplasmic reagents. Western blot analysis was performed, and representative blots of three independent experiments are shown. The data are presented as the mean + standard deviation of three independent experiments. *P<0.05, compared with the control group. Nrf2, nuclear factor E2-related factor 2.

Discussion

Previous studies have shown that several medicinal plants, including Curcuma longa and Glycyrrhiza glabra exert hepatoprotective effects by quenching ROS, which is a primary cause of oxidative damage in hepatocytes (24). HO-1, a heme-degrading enzyme, is involved in anti-inflammatory, anti-apoptotic and antiproliferative effects under various conditions (13). The induction of HO-1 is also likely to be a key therapeutic target in hepato-oxidative damage (25). HepG2 cells have been shown to express HO-1 (26). Several lines of evidence suggest that t-BHP, often used as a model in biological investigations (27), induces apoptotic cell death in HepG2 cells through the depletion of GSH and increase in intracellular Ca2+ concentrations (28). Therefore, t-BHP was used as a cell-death-inducing agent or negative control in the present study. In identifying the hepatoprotective effects of the extracts in hepatocytes, curcumin has been extensively selected as a positive control to identify cytoprotective actions through the upregulation of HO-1 (29,30). Therefore, curcumin was used as a positive control for the screening assay in the present study.

Based upon the results from the preliminary screening for hepatoprotective effects (Table I), 19 active plant extracts were confirmed by performing an additional screen (Table II). HO-1 screening was then performed using western blot analysis (Fig. 1). The induction of cytoprotective enzymes is key to the cytoprotective mechanism. In the present study, it was shown that the ethanolic extracts of T. crispa and P. weberi induced the protein and mRNA expression levels of HO-1 and in HepG2 cells in a concentration-dependent manner (Figs. 1 and 2). In addition, pre-incubation of cells with the T. crispa or P. weberi extracts resulted in enhanced resistance to t-BHP-induced oxidative damage; this effect was attributable to the expression of HO-1 as the inhibition of HO enzyme activity by SnPP significantly reduced T. crispa/P. weberi extract-induced cytoprotection (Fig. 3). The induction of the expression of HO-1 was also required to suppress t-BHP-induced ROS generation. Several reports have indicated that secondary metabolites of plants can activate Nrf2 by binding to Kelch-like ECH-associated protein 1, leading to the upregulation of certain cytoprotective proteins, including HO-1 (15,31). This suggestion is in accordance with the findings of the present study showing that T. crispa or P. weberi extracts significantly increased the levels of Nrf2 and efficiently promoted the translocation of Nrf2 into the nuclei of HepG2 cells (Fig. 4). T. crispa, known as ‘akar seruntum’ or ‘akar patawali’ to the Malays, belongs to the family Menispermaceae and is native to China and Southeast Asia, including Malaysia. Extracts of T. crispa has been used in folk medicine as a therapeutic agent for the treatment of fever, jaundice, hyperglycemia, hypertension, wounds, intestinal worms and skin infections. In addition, several studies have revealed that T. crispa possesses anti-inflammatory, antioxidant, antiallergic, hepatoprotective, antithrombotic, antiviral and anticarcinogenic activities (32,33). In the present study, the ethanolic extract from the aerial parts of T. crispa exerted cytoprotective effects on t-BHP-induced hepatotoxicity in HepG2 cells (EC50 144.3 µg/ml; Table II). The cytoprotective activity may be attributed to the presence of alkaloids, diterpenoid lactones, sesquiterpenoids, phenolics and aliphatic compounds, which are generally found in T. crispa (34), which is partially indicated in Table II. P weberi, widely distributed in Thailand, Lao, Malaysia and Southeast Asia, belongs to the family Liliaceae (35–37), which has antioxidant, antihypertensive, anticholinergic, antiasthmatic and antifungal actions (38,39). In the present study, the leaf extract of P. weberi showed hepatoprotective activity against t-BHP-induced cytotoxicity in HepG2 (EC50 121.7 µg/ml. The cytoprotective activity of P. weberi may be due to the presence of steroidal alkaloids (39). The 1H-NMR analyses of the ethanolic extracts of T. crispa and P. weberi revealed the presence of aliphatic or aromatic compounds (40) (data not shown), which may be responsible for the hepatoprotective properties of these plant extracts. In the 17 other extracts, a different mechanism may occur. Nrf2 induces genes encoding numerous phase II detoxifying enzymes, including HO-1, glutathione, catalase, superoxide dismutase, glutathione-S-transferase, γ-glutamyl cysteine ligase, NADPH quinone oxidoreductase-1, glutathione peroxidase and glutathione reductase (15,16). Further extensive investigations are likely to provide evidence for the other representative mechanisms of hepatoprotection in plant extracts.

In conclusion, among 64 plants extracts from Cambodian medicinal plants, 19 plant extracts showed promising hepatoprotective activities. Of these, the extracts of T. crispa (aerial region) and P. weberi (whole plant) exerted potent cytoprotective effects against t-BHP-induced hepatotoxicity in human derived-HepG2 cells, possibly through the Nrf2-mediated expression of HO-1. Taken together, these results suggested that the ethalolic extracts of T. crispa and P. weberi may possess therapeutic applications in liver disease characterized by oxidative stress.

Acknowledgements

This study was supported by the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (grant nos. 2012R1A1A2042984 and 2008-0062484).

References