Morin, 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), MTT, Hoechst 33342 and STZ were all purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Catalase (sc-271803) and β-actin (sc-8432) primary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Phosphorylated-AMPKα (Thr-172; cat. no. 2531), AMPKα (cat. no. 5831) and FOXO3 (cat. no. 2497) antibodies were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Anti-TATA binding protein (TBP; ab818) antibody was obtained from Abcam (Cambridge, MA, USA). Anti-poly (ADP-ribose) (PAR; cat. no. 4335-MC-100) antibody was purchased from Trevigen (Gaithersburg, MD, USA). All other chemicals and reagents were of analytical grade.

RINm5F rat pancreatic β cells were obtained from the American Type Culture Collection (Manassas, VA, USA). All the cells used in this study were maintained at 37°C in a humidified atmosphere containing 5% CO₂, and were cultured in Roswell Park Memorial Institute-1640 medium supplemented with 10% heat-inactivated fetal bovine serum, streptomycin (100 µg/ml) and penicillin (100 U/ml).

Cells were seeded into 96-well plates at a density of 1×10⁵ cells/ml. After 16 h, cells were incubated for 24 h with various concentrations of STZ dissolved in 0.1 M citrate buffer (pH 4.5); the final concentrations were as follows: 1, 2, 4, 6, 8 and 10 mM. The toxicity of STZ on RINm5F cells was examined using the MTT assay, which is based on the cleavage of a tetrazolium salt by mitochondrial dehydrogenase in viable cells (29).

To evaluate the effects of morin on STZ-induced cell death, cell viability was examined using the MTT assay. Cells were seeded into 96-well plates at a density of 1×10⁵ cells/ml. After 16 h, the cells were treated with various concentrations of morin (10, 25 and 50 µM) for 1 h, followed by exposure to STZ for 24 h at 37°C. Subsequently, 50 µl MTT stock solution (2 mg/ml) was added to each well and incubated for a further 4 h at 37°C. The medium containing MTT was aspirated and the formazan crystals in the viable cells were then dissolved using 150 µl dimethyl sulfoxide. The absorbance of each well was determined using a Multiskan GO Spectrophotometer (Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 490 nm.

Levels of intracellular ROS were determined using the DCFH-DA method as previously described (15). Briefly, RINm5F cells were seeded in 6-well plates at 1×10⁵ cells/ml. After 16 h, the cells were treated with 25 µM morin for 1 h. Following exposure to STZ for 12 h, cells were collected and incubated with 10 µM DCFH-DA at 37°C for 30 min. Subsequently, the cells were rinsed twice and resuspended in PBS, and fluorescence intensity was measured by flow cytometry (FACSCalibur; BD Biosciences, Franklin Lakes, NJ, USA). The data were analyzed using CellQuest (version 7.5.3; BD Biosciences). The relative fluorescence intensity was recorded as the mean value from three repeated experiments. Image analysis for the generation of intracellular ROS was also conducted. Briefly, cells were seeded onto cover slips in 6-well plates at 1×10⁵ cells/ml and were incubated with 5 µM DCFH-DA for 30 min at 37°C as previously described. After washing with PBS, the stained cells were mounted onto a microscope slide in mounting medium (Dako; Agilent Technologies, Inc., Santa Clara, CA, USA). Microscopic images were captured under a laser confocal microscope (Zeiss GmbH, Jena, Germany).

Intracellular NAD⁺ content was measured using the EnzyChrom™ NAD⁺/NADH quantification kit (BioAssay Systems, Hayward, CA, USA), according to the manufacturer's protocol.

The proportion of apoptotic sub-G₁ hypodiploid cells was determined by flow cytometry (30). Cells were initially treated with 25 µM morin for 1 h. Subsequently, STZ was added and cells were incubated for a further 24 h. Harvested cells were washed twice with PBS and fixed in 70% ethanol for 30 min at 4°C. Cells were then rinsed twice with PBS and were incubated for 30 min in the dark at 37°C in 1 ml PBS containing 100 µg propidium iodide (PI) and 100 µg RNase A. Flow cytometric analysis was performed using a flow cytometer (FACSCalibur; BD Biosciences). The proportion of sub-G₁ hypodiploid cells was assessed using histograms generated by the computer programs CellQuest (version 7.5.3; BD Biosciences) and ModFit (version 3.0; Verity Software House, Inc., Topsham, ME, USA).

Cells were plated in 24-well plates at 1×10⁵ cells/ml. After 16 h, cells were treated with 25 µM morin for 1 h and then with STZ at a final concentration of 6 mM for 24 h. Subsequently, a DNA-specific fluorescent dye, Hoechst 33342, at a final concentration of 15 µg/ml, was added to each well and incubated for 10 min at 37°C. The stained cells were then observed using a fluorescence microscope to examine the extent of nuclear condensation.

Total RNA was isolated from cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). cDNA synthesis was performed with 2 µg isolated RNA using SuperScript III Reverse Transcriptase (Invitrogen; Thermo Fisher Scientific, Inc.). qPCR was performed in 96-well optical plates using a CFX-96 Touch™ Real-Time PCR Detection system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The PCR reaction was conducted in a total volume of 20 µl: 10 µl 2X SYBR Premix Ex Taq II kit (Takara Bio, Inc., Otsu, Japan), 1 µl diluted template cDNA (~10 ng) and 10 µM each forward and reverse primers. The PCR protocol was conducted as follows: Pre-incubation at 95°C for 30 sec, followed by 40 cycles of denaturation at 95°C for 5 sec and annealing and extension at 60°C for 34 sec. Primers used in the present study were as follows: β-actin, forward 5′-GAAGATCCTGACCGAGCGTG-3′, reverse 5′-CGAAGTCTAGGGCAACATAGCA-3′; and catalase, forward 5′-AGGTGCTTTTGGATACTTTGAGG-3′ and reverse, 5′-CGACTGTGGAGAATCGGACGG-3′. The quantification cycle (Cq) method was employed to evaluate relative alterations in gene expression and the 2^−ΔΔCq value was calculated after β-actin normalization (31).

Cells were rinsed twice with PBS and lysed in 100 µl lysis buffer [40 mM Tris (pH 8.0), 120 mM NaCl, 0.1% NP-40] on ice for 30 min, after which they were centrifuged at 13,000 × g for 15 min at 4°C. Supernatants were collected and total protein concentrations were determined using the bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology, Haimen, China). Aliquots (40 µg total protein) of the collected supernatants were boiled for 5 min, separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked with 5% non-fat milk for 1 h, and then incubated with primary antibodies (targeting catalase (1:1,000), β-actin (1:5,000), p-AMPKα (1:1,000), AMPKα (1:1,000), FOXO3 (1:1,000), TBP (1:2,000), and PAR (1:1,000) overnight at 4°C), washed with TTBS, and incubated with horseradish peroxidase-conjugated anti-immunoglobulin-G secondary antibodies (cat. nos. 31430 and 31460; 1:5,000 Pierce; Thermo Fisher Scientific, Inc.) for 1 h at room temperature. The blots were visualized using an enhanced chemiluminescence western blotting detection kit (GE Healthcare Life Sciences, Little Chalfont, UK).

Cells were seeded in culture dishes at a density of 1.5×10⁵ cells/ml for 16 h. The cultured cells were then treated with morin for a series of time periods. Cells were harvested and lysed on ice with 1 ml lysis buffer [10 mM Tris-HCl (pH 7.9), 10 mM NaCl, 3 mM MgCl₂ and 1% NP-40] for 4 min. Cell pellets were collected by centrifugation at 3,000 × g for 10 min at 4°C, suspended in 5 µl extraction buffer [20 mM HEPES (pH 7.9), 20% glycerol, 300 mM NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 1 mM DTT and 1 mM PMSF] and incubated on ice for 30 min. The extraction solution was centrifuged at 13,000 × g for 5 min at 4°C, and the supernatants were harvested as nuclear protein extracts for further blotting analysis and stored at −70°C.

Cells were seeded into Petri dishes at a density of 1.5×10⁵ cells/ml. After 16 h, cells were treated with morin for a series of time periods. Catalase activity was determined using a catalase assay kit (S0051; Beyotime Institute of Biotechnology) according to the manufacturer's protocol. In this assay, catalase reacts with hydrogen peroxide (H₂O₂) to produce water and oxygen; unconverted H₂O₂ is then converted to a chromogenic product, which is measured at 520 nm using a spectrophotometer. Catalase activity is presented as U/mg, and 1 unit catalase activity is defined as the amount of enzyme catalyzing the degradation of 1 µmol H₂O₂/min under the conditions of 25°C and pH 7.0. In order to detect the effects of morin on STZ-induced suppression of catalase activity, cells were pretreated with morin for 1 h, and were then incubated with STZ for 24 h. Catalase activity was assessed using the aforementioned catalase assay kit.

Cells were seeded at 1.5×10⁵ cells/ml in 24-well plates and reached ~50% confluence prior to transfection with AMPK siRNA (siAMPK), FOXO3 siRNA (siFOXO3) (both from Invitrogen; Thermo Fisher Scientific, Inc.), catalase siRNA (siCatalase) or control mismatched siRNA (both from Santa Cruz Biotechnology, Inc.). Cells were transfected with 10–50 nM siRNA using Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. A total of 24 h post-transfection, cells were treated with or without morin and/or STZ for 24 h and were subjected to western blot analysis and MTT assay.

All experiments were performed in triplicate and all values are presented as the means ± standard error of the mean. The results were analyzed by one-way analysis of variance in the SigmaStat 3.5 and the post hoc Tukey's test was used to assess statistical significance. P<0.05 was considered to indicate a statistically significant difference.

The effects of various concentrations of STZ on RINm5F cell viability are presented in Fig. 1. A dose-dependent decrease in cell viability was observed when cells were exposed to various concentrations of STZ for 24 h. Following treatment with 6 mM STZ, ~50% inhibition of RINm5F cell viability occurred, and at a concentration of 10 mM, cell viability was markedly reduced to 20%. Based on these results, 6 mM was selected as the appropriate dose of STZ for further experiments.

The protective effects of morin on the viability of STZ-treated cells were measured using the MTT assay. As shown in Fig. 2A, exposure to 6 mM STZ for 24 h markedly reduced the viability of RINm5F cells, whereas pretreatment with morin at 10, 25 and 50 µM for 1 h resulted in amelioration of cell viability. Pretreatment with 25 µM morin exhibited the highest efficiency with regards to the protection of RINm5F cells. In addition, treatment with various concentrations of morin alone did not exhibit any adverse effects on cell viability (Fig. 2B); therefore, morin at 25 µM was chosen as the appropriate dose for subsequent experiments. These results indicated that morin may be capable of protecting RINm5F cells from STZ-induced cell damage.

To confirm the effects of morin on STZ-induced ROS production, ROS fluorescence intensity was detected by flow cytometry. The results of flow cytometric analysis revealed that STZ increased ROS generation; however, pretreatment with morin ameliorated the STZ-stimulated increase in ROS content (Fig. 3A). In addition, intracellular ROS levels were observed under a fluorescence microscope. As presented in Fig. 3B, exposure to STZ for 12 h markedly increased red fluorescence intensity in RINm5F cells, whereas pretreatment with morin reduced red fluorescence intensity upon STZ treatment, thus reflecting a reduction in ROS generation. These results indicated that morin significantly decreased STZ-induced elevated ROS levels, thus suggesting that morin conferred resistance to oxidative stress by suppressing the increase in intracellular ROS levels.

PARP is known to be activated under conditions of oxidative stress and has been well demonstrated in the STZ-induced model of diabetes (32). PARP activity may be examined by western blotting of the PAR protein, as previously described (33). As shown in Fig. 4A, the expression levels of PAR were markedly increased in STZ-treated cells compared with in the control cells, whereas pretreatment with morin inhibited STZ-induced PAR protein expression. As an important substrate for PARP, NAD⁺ is a crucial component in the modulation of cellular metabolism. Therefore, intracellular NAD⁺ levels were measured in the present study. As expected, pretreatment with morin significantly ameliorated the loss of NAD⁺ levels, which was induced by STZ treatment (Fig. 4B). These results indicated that PARP may be activated in response to STZ stimulation, which was accompanied by NAD⁺ depletion; however, morin effectively reversed the increase in PARP activity and the depletion of NAD⁺ levels in STZ-treated cells.

The protective effects of morin against STZ-induced apoptotic cell death were assessed. As shown in Fig. 5A, the combination of morin and STZ markedly increased cell survival rate compared with in STZ-treated cells. A previous study reported that STZ treatment stimulated pancreatic β cells to produce large amount of ROS, which in turn induced cell apoptosis (34). To determine the cytoprotective effects of morin on STZ-induced pancreatic cell apoptosis, cell nuclei were stained with Hoechst 33342 for microscopic examination. The microscopic images presented in Fig. 5B indicated that the nuclei of the control cells were intact, whereas the STZ-treated cells exhibited marked nuclear fragmentation, which is a typical feature of apoptosis. However, when the cells were pretreated with morin, a marked decrease in nuclear fragmentation was observed. In addition, the anti-apoptotic effects of morin on STZ-treated cells were confirmed by flow cytometric analysis with PI staining. As shown in Fig. 5C, apoptotic sub-G₁ DNA content in STZ-treated cells was increased compared with in the control cells. However, prior treatment with morin reduced apoptotic sub-G₁ DNA content in STZ-treated cells. These results indicated that morin reduced apoptotic cell death, thus suggesting that the cytoprotective effects of morin on RINm5F cells may be due to the inhibition of apoptosis.

It is well known that the pancreas is particularly susceptible to STZ-induced free radical damage, due to the low levels of endogenous antioxidant enzymes responsible for scavenging free radicals (35). Since catalase serves an important role in the cellular defense against ROS, and morin has been reported to significantly increase protein expression and enzyme activity of catalase in lung fibroblast cells (15), the effects of morin on catalase expression and activity in RINm5F cells were investigated in the present study. The present results revealed that morin treatment alone induced catalase mRNA transcription in a time-dependent manner (Fig. 6A). In addition, catalase protein expression and enzyme activity were time-dependently increased following treatment with morin (Fig. 6B and C). Treatment with STZ alone for 24 h significantly attenuated the mRNA transcription, protein expression and enzyme activity of catalase in RINm5F cells. Compared with these findings, pretreatment with morin reduced STZ-induced attenuation of catalase mRNA transcription and protein expression (Fig. 6D and E), leading to a significant increase in catalase activity (Fig. 6F).

Since AMPK is an upstream kinase that regulates FOXO3 activation, and the AMPK-FOXO3 signaling pathway has been suggested to be responsible for the induction of antioxidant enzymes (16,36), the effects of morin on AMPK-FOXO3 activation were examined by western blot analysis. As shown in Fig. 7A, 25 µM morin induced phosphorylation of the AMPK catalytic unit AMPKα in a time-dependent manner, thus promoting the nuclear accumulation of FOXO3 (Fig. 7B).

Since catalase is a downstream transcriptional target of FOXO3, the present study aimed to determine whether the AMPK-FOXO3 signaling pathway is involved in the induction of catalase by morin. Following transfection of the pancreatic cells with siAMPK or siFOXO3, cells were treated with morin for 24 h and the protein expression levels of catalase were determined. Silencing of AMPK or FOXO3 expression markedly suppressed the morin-dependent increase in catalase protein expression (Fig. 8A and B). To determine whether morin-enhanced catalase activity confers cytoprotection against oxidative stress, cells were transfected with siCatalase. siCatalase reduced the protective effects of morin against STZ-induced cytotoxicity (Fig. 8C). Taken together, these results revealed that AMPK may be involved in the activation of FOXO3 and the upregulation of catalase.