PC12 cells were purchased from the Type Culture Collection of the Chinese Academy of Sciences. PC12 cells were cultured in DMEM (HyClone; GE Healthcare Life Sciences) supplemented with 10% FBS (Zhejiang Tianhang Biotechnology Co., Ltd.) at 37°C in a humidified atmosphere of 5% CO₂/95% air. PC12 cells in the logarithmic phase of growth were plated in 6-well plates at the density of 5×10⁵/ml and divided into three treatment groups: A blank control group, a BSA (300 µg/ml; Beijing ComWin Biotech Co., Ltd.) control group and an AGEs (Biorbyt, Ltd.) group. The AGEs group were treated with different concentrations (100, 200 or 300 µg/ml) of AGEs for 24 h at 37°C, and then subsequent assays were performed. As AGEs were prepared in a non-enzymatic glycosylation reaction using BSA as a substrate, BSA (300 µg/ml) was also used as a control.

Total RNA was extracted from cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) following the aforementioned treatments, according to the manufacturer's protocol. For mRNA analysis, cDNAs were synthesized using a TIANScript First Strand cDNA Synthesis kit (Tiangen Biotech Co., Ltd.), according to the manufacturer's protocol. The temperature protocol for the reverse transcription reaction was 37°C for 60 min. qPCR was performed using a SuperReal PreMix Color (SYBR-Green; Tiangen Biotech Co., Ltd.) in an Mx3000P system (Agilent Technologies, Inc.). The cycling conditions included a pre-denaturation step at 95°C for 15 min, followed by 40 cycles of denaturation at 95°C for 10 sec, annealing at 55°C for 20 sec and extension at 72°C for 32 sec. The primer sequences used in this study are presented in Table I. Quantification cycle (Cq) values for the target genes and the internal control gene, GAPDH, were measured. The expression levels of each mRNA were determined from three independent experiments, and the fold change was calculated using the 2^−ΔΔCq method (23).

Protein levels in PC12 cells were determined after treatment using western blot analysis. Cells were lysed with RIPA lysis buffer (Sigma-Aldrich; Merck KGaA) containing a protease inhibitor cocktail (Sigma-Aldrich; Merck KGaA). Samples were incubated on ice for 30 min and then centrifuged at 16,000 × g for 15 min at 4°C. The supernatant was removed and the protein concentration was determined using the bicinchoninic acid method. Total protein (50 µg) was separated on 12% SDS-PAGE gels and transferred to PVDF membranes. Membranes were blocked with 5% dried skimmed milk/TBS 0.5% Tween-20 (TBST) for 1 h at room temperature. Membranes were incubated overnight at 4°C with a monoclonal rabbit anti-MIF antibody (1:100; cat. no. ab172730; Abcam) or anti-GAPDH antibody (1:1,000; cat. no. CW0101S; Beijing ComWin Biotech Co., Ltd.), and then washed three times with TBST for 10 min each. Subsequently, horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (1:10,000; cat. no. D110058; BBI Life Sciences Corporation) secondary antibody was incubated with the membranes for 1 h at room temperature, which were then washed three times with TBST for 10 min each. The bands were visualized with an ECL reagent (Tanon Science and Technology Co., Ltd.) and detected with the Tanon Imaging System (Tanon Science and Technology Co., Ltd.). The relative intensity of the protein bands was quantified using Image-Pro Plus 6.0 software (Media Cybernetics, Inc.). Experiments were repeated three times.

PC12 cells were plated in 96-well plates at the density of 5×10⁵/ml, and the cells were exposed to various concentrations of AGEs (100–400 µg/ml) at 37°C for 24 h, with 5 replicates/group; equivalent concentrations were applied to the BSA control group. All groups were treated with 10 µl of Cell Counting Kit-8 (CCK-8) reagent (Dojindo Molecular Technologies, Inc.) and incubated for 1–4 h at 37°C before the absorbance was measured at 450 nm using a microplate reader to detect surviving cells.

Aβ_1–40 (Shanghai Aladdin Biochemical Technology Co., Ltd.) was dissolved in sterile PBS and incubated at 37°C for 7 days to induce aggregation prior to treatment. PC12 cells were plated in 96-well plates at the density of 5×10⁵/ml. The blank control group was treated with 100 µl of fresh culture medium and the AD model groups were incubated with 100 µl of medium containing different concentrations (10–40 µg/ml) of Aβ_1–40 at 37°C for 2, 6, 12 or 24 h, with 3 replicates/group (24). All groups were treated with 5 mg/ml MTT solution 10 µl at 37°C for 4 h, the medium was then discarded and 100 µl dimethyl sulfoxide was added to each well to dissolve the formazan crystal product. The absorbance was measured at 570 nm using a microplate reader.

PC12 cells were plated in 12-well plates at the density of 5×10⁵/ml. The cells were exposed to 300 µg/ml AGEs at 37°C for 24 h and then treated with 20 µg/ml Aβ_1–40 at 37°C for 12 h. To assess the effect of AGEs on the expression of inflammatory mediators through MIF and to determine the effect of ISO-1, cells were pre-treated with 7 µM ISO-1 (MedChemExpress LLC) at 37°C for 1 h. The blank control group and the Aβ_1–40 model group were used as control groups, with 3 replicates/group. Following treatment, the number and morphology of the cells were observed under an inverted microscope (magnification ×100). Each sample was analyzed in five randomly selected fields. No significant differences were observed between the BSA control group and the blank control group; therefore, BSA was no longer used as a control.

PC12 cells were plated in 96-well plates at the density of 5×10⁵/ml. The cell groupings and treatments were the same as for the aforementioned microscopy assay, and the activity of cells was determined using the MTT method, according the aforementioned protocol.

PC12 cells were plated in 12-well plates at the density of 5×10⁵/ml. Cells were divided into four groups and exposed to the aforementioned treatments. After treatment, total RNA was extracted and RT-qPCR was conducted using the primers presented in Table I, according the aforementioned protocol.

KEGG (www.kegg.jp) is a database resource that integrates genomic, chemical and systemic functional information (25). By searching for AGEs-associated signaling pathways, map04933 was identified; AGEs-mediated proinflammatory signaling was investigated using this map.

Data are presented as the mean ± SD. Differences among groups were evaluated using one-way ANOVA followed by Tukey's post hoc test using SPSS version 19.0 software (IBM Corp.) and Prism version 5.0 software (GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.

A significant difference in MIF mRNA expression was not observed between the BSA control group and the blank control group; however, compared with the blank control group, the treatment of cells with 200 or 300 µg/ml AGEs produced statistically significant differences, with MIF mRNA expression significantly increased (Fig. 1A). Cells treated with 100 or 300 µg/ml AGEs exhibited significantly increased levels of MIF protein expression compared with the blank control group (Fig. 1B and C). It was hypothesized that a direct intermolecular relationship existed between AGEs and MIF, and that AGEs could induce the expression of MIF mRNA and protein.

To screen for the optimum concentration of AGEs, PC12 cells were treated with different concentrations of AGEs, and the survival rate was determined using a CCK-8 assay. The results revealed that the viability of cells decreased with increasing concentrations of AGEs. The half-maximal effective concentration was calculated to be 335.0 µg/ml using GraphPad Prism 5 software (Fig. 2A). The BSA control group was administered an equivalent concentration of BSA to the optimal concentration of AGEs for PC12 cells (300 µg/ml); no significant difference was observed between the BSA control group and the blank control group (data not shown). The inhibition rate of cells was measured using an MTT assay. As presented in Fig. 2B, the time at which each concentration of Aβ_1–40 inhibited cell survival to the greatest extent was 12–24 h; however, at 24 h, the cells were poorly adherent and a small number of cells were detached. Therefore, cells were treated with Aβ_1–40 for 12 h. Using GraphPad Prism 5 software, it was calculated that the half-maximal inhibitory concentration of PC12 cells induced by Aβ_1–40 was 22.17 µg/ml (Fig. 2C). The optimal concentration of Aβ_1–40 treatment and exposure time to establish an AD model was determined to be 20 µg/ml for 12 h.

Under an inverted microscope (magnification ×100), PC12 cells in the blank control group displayed numerous long dendrites. The blank control cells adhered tightly to the bottom of the culture plate, with dense, clear and visible nucleoli, and exhibited a rapid rate of proliferation (Fig. 3A). Compared with the blank control group, the proliferation of cells in the Aβ_1–40 model group was inhibited, as determined by the decreased number of cells, relaxed intercellular junctions and partial retraction of dendrites (Fig. 3B). Following AGEs treatment, cell proliferation was further inhibited, a large number of cells were detached and more cell fragments were observed; the remaining adherent cells lost their characteristic neuronal features (Fig. 3C). Following pre-treatment with ISO-1, the decrease in cell proliferation was notably attenuated. The number of adherent cells increased, the number of detached cells decreased, cells were strongly adherent, and formed more intercellular junctions with longer and more numerous dendrites (Fig. 3D). Compared with the blank control group, the viability of cells in the Aβ_1–40 model group significantly decreased; the addition of AGEs further decreased cell viability. Following pre-treatment with ISO-1, the survival rate was significantly improved compared with AGEs and Aβ_1–40 alone (Fig. 4). Based on these results, Aβ_1–40 successfully established an AD cell model. AGEs were found to aggravate the cytotoxicity of Aβ_1–40 in the AD cell model and this effect was significantly alleviated by exposure to the MIF inhibitor ISO-1.

Compared with the blank control group, the expression levels of interleukin (IL)-1β, IL-6, tumor necrosis factor-α (TNF-α) and MIF mRNA in cells were significantly increased following treatment with Aβ_1–40. The effects of Aβ_1–40 on MIF expression were markedly smaller compared with the effects on IL expression; it is possible that other factors in the AD cell model may also regulate the expression of MIF. The expression levels of IL-1β and TNF-α were notably further increased by treatment with AGEs, and the expression of MIF and IL-6 was significantly increased. Compared with the group treated with AGEs, pre-treatment of cells with ISO-1 resulted in a significant decrease in the mRNA expression levels of IL-1β, IL-6, TNF-α and MIF (Fig. 5). Based on these results, AGEs aggravated neuroinflammation in the AD model by inducing MIF expression, whereas ISO-1 attenuated this damage.