High glucose increases LPS-induced DC apoptosis through modulation of ERK1/2, AKT and Bax/Bcl-2

Male BALB/c mice, 10 weeks old, were purchased from the Animal Center at Shandong University. All experimental procedures were approved by and performed according to the guidelines of the animal ethics committee of the Shandong University School of Medicine. In brief, mice were injected intraperitoneally with streptozotocin (STZ, 60 μg/g) once a day for 5 days to induce diabetes [14, 15]. Control mice were injected with PBS (Phosphate buffer saline). Seven days after the final injection, blood glucose levels were examined by glucometer (UltraVue, Johnson & Johnson, New Brunswick, NJ, USA). Mice with blood glucose levels greater than 300 mg/dl were considered diabetic. Diabetic mice were given LPS (1 mg in 500 μL PBS) orally once to induce inflammation on the 19th day after the first streptozotocin injection, while control mice were treated with the same volume of PBS. 24 hours later mice were killed and intestines dissected and digested with collagenase. Immune cells were isolated by layering on a Percoll gradient, then DCs were further purified using CD11c magnetic beads (130-052-001, Miltenyi Biotec Inc, Bergisch Gladbach, Germany) [16, 17]. Finally the DCs were collected, stained and analyzed by flow cytometry.

Dendritic cells (DC2.4 cell line) were cultured on collagen coated dishes in complete medium containing RPMI-1640, 10% fetal bovine serum, 1 × 10⁴ units/ml penicillin and 10 mg/ml streptomycin. Cells were passaged using standard cell culture techniques. Culture dishes were placed in an incubator equilibrated with 5% CO₂ at 37°C. The medium was refreshed at intervals of 3 days. To maintain uniform condition, the cells in passages 20 to 30 were used for experiments.

DC2.4 cells were treated with a high dose of glucose (25 mmol/L) for 48 h. Then LPS (100 μg/ml) was added to detect whether the glucose concentration could affect DC apoptosis. After 24 h LPS treatment (cells grown to approximately 80% confluence), the cells were harvested for both western blot analysis and flow cytometry.

Apoptosis of dendritic cells was determined using an Annexin-V and PI apoptosis kit (BU-ap0102, Life Technologies, Carlsbad, CA, USA). After washing twice with PBS, cells were re-suspended in binding buffer and incubated with Annexin-V and PI for 15 min. At least 5000 cells were counted per sample. Apoptotic cells appeared as Annexin-V positive and PI negative.

Hoechst 33258 staining (B33258, Sigma, St Loius, MO, USA) was used to visualize the morphological changes in apoptotic cell nuclei. After being washed with PBS and fixed with 4% paraformaldehyde for 30 min, 0.5 μg/ml Hoechst 33258 was added to the wells and incubated for 5 min. Then cell nuclei were analyzed by fluorescence microscopy. Apoptotic cells were characterized by chromatin condensation and multiple chromatin fragments.

Cells were collected and washed three times with PBS before lysis in lysis buffer (MK163780, ThermoFisher Scientific Inc, Waltham, MA, USA). The protein concentration of the lysates was determined according to the BCA method using the Protein Quantitative Analysis kit (2812 k; CWBIO, CoWin Biotech Co. Ltd., Beijing, China). For electrophoresis, 100 μg of protein was loaded into the wells of a 10% SDS polyacrylamide gel. After separation, proteins were transferred to nitrocellulose membranes, blocked for 3 h at room temperature in blocking buffer (5% nonfat dry milk, Tween-Tris-buffered saline), washed in PBS 3 times and incubated with primary antibody of ABCAM (Rabbit polyclonal to ERK, ab16869, 1: 1:5000; Rabbit polyclonal to AKT, ab66138, 0.15 μg/ml; Rabbit polyclonal to Bax, ab7977, 1:1000; Rabbit polyclonal to Bcl-2, ab18210, 1 μg/ml) and Actin (sc-1616, 1:200, SANTA CRUZ BIOTECHNOLOGY) overnight at 4°C with gentle agitation. Next day after washing three times in PBS, the nitrocellulose membranes were blotted for 1 h at room temperature with secondary antibodies (ab136817, 1:5000, abcam). Finally, immunolabeled protein bands were detected using the ECL(Electro-Chemi-Luminescence) method (Immobilon™ Western, Millipore Corporation, Billerica, MA, USA) and quantified using an Alpha Imager 2200 (ProteinSimple, Santa Clara, CA, USA).

DC2.4 cells were treated as described above. After fixing in 4% paraformaldehyde for 15 min and washing with PBS, cells were permeabilized with 0.1% triton X-100 for 15 min. 3% hydrogen peroxide was used to block endogenous peroxidases. After washing in PBS and blocking in 10% goat serum, cells were incubated with primary antibody of ABCAM (Rabbit polyclonal to ERK, ab16869, 1:1000; Rabbit polyclonal to AKT, ab66138, 1 μg/ml; Rabbit polyclonal to Bax, ab7977, 1 μg/ml; Rabbit polyclonal to Bcl-2, ab18210, 5 μg/ml) and Actin (sc-1616, 1:250, SANTA CRUZ BIOTECHNOLOGY) overnight in a humidified chamber at 4°C. The next day, the cells were washed before being incubated with secondary antibody (SP rabbit HRP kit, CW2035, CWBIO) for 30 min at room temperature. After washing, positive immunostaining was revealed by the DAB(3,3’-diaminobenzidine) method (D8001, Sigma). PBS was used as a negative control for the primary antibodies and Actin was used as a positive control.

Untreated cells had a background level of less than 5% apoptosis both in vivo and in vitro. In cells treated with LPS for 24 h, glucose pre-administration markedly elevated the level of apoptosis (Figure 1 and Figure 2, p < 0.05) while glucose alone had no statistically significant effect (data not shown). However, increased apoptosis was not evident at 15 min and 6 h in LPS treated cells (Figure 2A).To determine the effects of glucose on apoptosis, cells were exposed to glucose for 48 h. Then cell nuclei were stained using Hoechst 33258 and analyzed by fluorescence microscopy. As shown in Figure 3A, cells treated with glucose showed obvious apoptotic features including cytoplasm concentration, chromatin condensation and multiple chromatin fragments. In normal control cells, nuclei were distributed uniformly and were larger in size compared to the other two treated groups. As for these two treated groups, the cells pre-treated with glucose appeared to have a higher degree of apoptosis.

As shown in Figure 4, glucose pre-administration could markedly reduce the levels of phosphorylated (pho)-AKT, pho-ERK and Bcl-2 in cells treated with LPS for 24 h in vitro. However, these changes were not seen in the 15 min and 6 h treated groups. With regards to the expression of Bax, glucose could markedly elevate its expression in cells treated with LPS for 24 h. In addition, similar effects were found in diabetic mouse in vivo that AKT, pho-ERK and Bcl-2 were decreased and Bax was increased Figure 5. These results suggest that the increase in LPS-induced DC apoptosis in high glucose primed cells was mediated through AKT, ERK and Bax/Bcl-2 pathways.

The immunoreactivity for the apoptosis related proteins (AKT, ERK, Bax, Bcl-2) were mainly located in the cytoplasm (Figure 3B). The negative control showed no background staining. Glucose administration decreased the number of positive cells for the antiapoptotic proteins AKT, ERK and Bcl-2 and also reduced the intensity of immunoreactivity of the labeled cells. In comparison, both the number and intensity of the Bax immunoreactive cells was increased.