Rat pancreatic β-cell RINm5F cells were obtained from ATCC (Manassas, VA, USA). Cells were cultured in RPMI-1640 medium (Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) with 10% (v/v) heat-inactivated fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 2 mM glutamine (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), 1% non-essential amino acids (Sigma-Aldrich; Merck KGaA), 100 U/ml streptomycin and 100 U/ml penicillin (Sigma-Aldrich; Merck KGaA) at 37°C in an atmosphere containing 5% CO₂. Icariin was purchased from Sigma-Aldrich (Merck KGaA). Rat IL-1β and IFN-γ proteins were obtained from R&D Systems (Minneapolis, MN, USA).

MTT (Sigma-Aldrich; Merck KGaA) was used to determine cell viability according to the manufacturer's protocols. Briefly, 5 ml MTT solvent (Beyotime Institute of Biotechnology, Haimen, China) was used to dissolve 25 mg MTT to form an MTT solution at 5 mg/ml. A total of 10 µ1 MTT solution was added to each well and incubated for 4 h at 37°C in an incubator. Subsequently, 100 µl formazan solution (Beyotime Institute of Biotechnology) was added for 4 h at 37°C. The optical density of viable cells was measured using a microplate reader (BMG Labtech GmbH, Ortenburg, Germany) at a wavelength of 570 nm.

Biologically synthesized NO is quickly oxidized to form nitrite and nitrate in aqueous solutions (19). Therefore, detecting the nitrite concentration in cell-free culture supernatants using a colorimetric assay may be indicative of NO generation. In brief, RINm5F cells (5×10⁶) or 30 islets were treated with the 5 or 10 µM concentrations of icariin for 3 h, prior to being treated with IL-1β (1 U/ml) and IFN-γ (100 U/ml) for 24 h. Subsequently, 100 µl aliquots of culture supernatant were incubated at room temperature for 5 min with 100 µl modified Griess reagent in a 1:1 mixture of 1% sulfanilamide in 30% acetic acid and 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride in 60% acetic acid (Beyotime Institute of Biotechnology). The absorbance was measured at 540 nm. The NO concentration was calculated from the linear standard curve of serial dilutions of sodium nitrite in a working medium.

Total RNA was extracted from cultured cells using TRIzol reagent (Thermo Fisher Scientific, Inc.). The primer for iNOS was synthesized based on the following previously published sequences (24): Forward, 5′-GAATCTTGGAGCGAGTTGTGG-3′ and reverse, 5′-AGGAAGTAGGTGAGGGCTTGG-3′. First-strand cDNA was obtained using Super M-MLV Reverse transcriptase (BioTeke Corporation, Beijing, China). Reverse transcription was performed at 42°C for 15 min and 72°C for 2 min according to the manufacturer's protocols. PCR was performed using SYBR-Green master mix (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). The following thermocycling conditions were used: Predenaturation at 95°C for 30 sec followed by 40 cycles of amplification at 95°C for 5 sec and annealing and extension at 60°C for 30 sec. GADPH was used to normalize iNOS mRNA expression. GAPDH forward, 5′-GATGACCTTGCCCACAGCCT-3′ and reverse, 5′-ATCTCTGCCCCCTCTGCTGA-3′. The 2^−∆∆Cq method was used to quantify data (24). ABI Prism 7000 software (Applied Biosystems; Thermo Fisher Scientific, Inc.) was used to analyze data.

Following treatment, proteins were extracted from RINm5F cells using a Nuclear and Cytoplasmic Protein Extraction kit (cat. no. P0027; Beyotime Institute of Biotechnology). Protein concentrations were determined using an Enhanced BCA Protein Assay kit (cat. no. P0010S; Beyotime Institute of Biotechnology). A total of 20 µg/lane was separated by 12% SDS-PAGE and transferred to polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked using Blocking Buffer (cat. no. P0023B; Beyotime Institute of Biotechnology) for 2 h at room temperature. Proteins were probed using specific primary antibodies at 4°C overnight, followed by incubation with secondary antibodies at room temperature for 1 h. Specific primary antibodies against pro-caspase-3 (ab44976; 1:500), cleaved caspase-3 (ab13847; 1:500) and cleaved poly ADP-ribose polymerase (PARP; ab32064; 1:2,000) were purchased from Abcam (Cambridge, UK). Secondary antibodies against β-actin (ab8227; 1:2,000) and Larmin A (ab26300; 1:1,000) used in this study were horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG or anti-mouse IgG-HRP (Beyotime Institute of Biotechnology). β-actin and Larmin A were used as internal controls to normalize results. Signals were monitored using a chemiluminescent substrate (KPL, Inc., Gaithersburg, MD, USA). Following electrophoresis, gray values were analyzed using Quantity One v4.4.0.36 software (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

The activity of caspase-3 was conducted using a commercial ELISA kit (cat. no. HC079; Shanghai Gefan Biotechnology Co., Ltd., Shanghai, China) according to the manufacturer's protocols. In brief, cells (1×10⁶) were resuspended in 50 µl lysis buffer (Shanghai Gefan Biotechnology Co., Ltd.) and incubated for 1 h in an ice bath. The supernatant was collected following centrifugation for 10 min at 800 × g at room temperature, following which a colorimetric reagent was added and incubated for 4 h at 37°C. The colorimetric product was monitored using an ELISA reader at a wavelength of 405 nm.

A total of 1×10⁶ cells were washed with PBS and resuspended in binding buffer containing Annexin V-APC and propidium iodide, and incubated at 20–25°C for 10–20 min (Beyotime Institute of Biotechnology). The samples were analyzed using a FACScan flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). The percentage of apoptotic cells in a 10,000-cell cohort was determined using flow cytometry.

Following treatment, nuclear extracts were isolated using the Nuclear Extract kit according to the manufacturer's protocols (Active Motif, Carlsbad, CA, USA; cat. no. 40010). The activity of NF-κB p65 was assessed using an ELISA kit (cat. no. 40596; Active Motif).

Values are presented as the mean ± standard deviation. Statistical comparisons between cell lines were performed using one-way analysis of variance, followed by Dunnett's t-test. GraphPad Prism 7.03 software (GraphPad Software Inc., La Jolla, CA, USA) was used to analyze experimental data and a P<0.05 was considered to indicate a statistically significant difference.

To assess the therapeutic potential of icariin in rat pancreatic β cells, the viability of cultured RINm5F cells was initially examined. As presented in Fig. 1, treatment with icariin up to 10 µM did not result in a significant loss of cell viability. Next, whether icariin protected RINm5F cells from cytokine toxicity was investigated. Treatment with cytokines IL-1β and IFN-γ significantly reduced the cell viability to 49.9±5.2% of the control value (Fig. 1). Pretreatment with icariin significantly abrogated the cytotoxic effects of cytokines on RINm5F cells in a concentration-dependent manner.

NO production was significantly increased following 24 h treatment with cytokines (Fig. 2A). However, the cytokine-induced NO production was effectively inhibited by treatment with 10 µM icariin (Fig. 2A). To investigate the underlying mechanisms responsible for the effects of icariin, RT-qPCR and western blotting were performed to measure the expression of iNOS at the mRNA and protein level, respectively. Treatment with IL-1β and IFN-γ significantly increased the expression of iNOS, while icariin treatment significantly ameliorated this increase at the mRNA and protein level (Fig. 2).

Cytokines are able to promote β-cell death through apoptosis and necrosis (25). Caspase-3 serves a pivotal role in the apoptotic signaling pathway, and so the activation status of caspase-3 was assessed in the present study. Treatment with IL-1β and IFN-γ increased the activity of caspase-3 and cell apoptosis in RINm5F cells, while icariin effectively reversed these effects (Fig. 3). The activation of apoptotic signaling was also confirmed by western blotting (Fig. 3B). Cleaved caspase-3 is the main marker of cell apoptosis (26), and so its expression was assessed. As presented in Fig. 3B, IL-1β and IFN-γ were able to activate caspase-3 and increase the cleavage of PARP in RINm5F cells, while treatment with icariin reduced cleaved caspase-3 and cleaved PARP levels in cytokine-stimulated cells.

NF-κB is a key transcription factor that induces iNOS and regulates subsequent NO production (27). The results of a previous study by our group demonstrated that NF-κB was activated by cytokines or oxidative stress (28). Based on this, it was investigated whether icariin affects the cytokine-induced activation and translocation of NF-κB from the cytosol to the nucleus in RINm5F cells. NF-κB and the nuclear translocation of p65, a key subunit of the NF-κB complex, were significantly promoted by treatment with IL-1β and IFN-γ compared with the control (Fig. 4). In contrast, icariin pretreatment markedly suppressed the cytokine-stimulated activation and nuclear translocation of NF-κB. In summary, these data suggest that icariin may downregulate iNOS expression via inhibiting the cytokine-stimulated activation of NF-κB.