Endoplasmic reticulum stress regulates cell injury in lipopolysaccharide-induced nerve cells

Cell lines

PC12 cells (ATCC, Manassas, VA, USA) were cultured in high-glucose Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco Cell Culture, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin at 37°C with 5% CO₂. MES23.5 cells (BlueBio, Yantai, China) were cultured in DMEM/F12 medium (Gibco Cell Culture) containing 5% FBS, Sato’s solution, and 1% penicillin–streptomycin at 37°C with 5% CO₂ (Biowest, Riverside, MO, USA). The medium was replaced with serum-free DMEM 2 hours before LPS intervention. Cells were treated with increasing concentrations of LPS (0.1, 1, 10, and 50 µg/mL) for 0, 24, or 72 hours.

CCK-8 assay

Cells were cultured in a 96-well plate. After plasmid transfection for 24 or 72 hours followed by LPS treatment, the medium was replaced with Cell Counting Kit-8 (CCK-8) solution (10 µL per well; Beyotime, Shanghai, China). After incubation for 2 hours at 37°C, the absorbance of each well was detected at a wavelength of 450 nm.

Western blots

The cells were washed twice with pre-cooled phosphate-buffered saline (PBS) and then lyzed for 20 minutes with RIPA Lysis Buffer containing protease inhibitor (Biorbyt, Cambridge, UK) and then centrifuged at 12,000 rpm at 4°C for 20 minutes. The absorbance of each lysate was detected at a wavelength of 562 nm. Total protein was quantified using the bicinchoninic acid protein quantification method with reference standards. The marker (6 µL) or protein sample (20 µL) was added to each lane. The target proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred to a polyvinylidene difluoride membrane. The membranes were blocked with 5% bovine serum albumin for 2 hours and washed three times with PBS containing Tween 20 (TBST) for 10 minutes per wash. The membranes were subsequently incubated with the primary antibody overnight at 4°C (rabbit anti-GRP78, ab108615, 1:5,000; rabbit anti-CHOP, ab179823, 1:1,000; rabbit anti-PERK, ab229912, 1:1,000; rabbit anti-Bax, ab32503, 1:1,000; rabbit anti-Bcl-2, ab59348, 1:5,000; rabbit anti-caspase-3, ab13847, 1:500; rabbit anti-IRE1, ab48187, 1:2,000; rabbit anti-GADPH, ab8245, 1:200; all sourced from Abcam PLC, Cambridge, UK). The membrane was then washed three times with TBST and incubated with a horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit IgG, ab97051, 1:20,000; Abcam) for 2 hours. An electrochemiluminescence solution was prepared and used to visualize the protein bands. Image analysis was performed with Image Lab software (Bio-Rad, Hercules, CA, USA).

Plasmid transfection

Two plasmids with different promoter regions for the overexpression of GRP78 (OverExp GRP78-1 and OverExp GRP78-2) were constructed by Shanghai Hewu Biotechnology (Shanghai, China). The plasmids were transfected into the PC12 and MES23.5 cells with Lipofectamine 2000 (Thermo Fisher Scientific, Rockford, IL, USA) according to manufacturer’s protocol. Briefly, the plasmids were gently pre-mixed with Lipofectamine 2000. The medium in each well of a 96-well culture plate containing PC12 or MES23.5 cells was absorbed and discarded, and 2 mL of serum-containing DMEM was added to each well. The plasmid and Lipofectamine 2000 mixtures were then added to each well. After 48 hours of transfection, real-time PCR (RT-PCR) and western blotting were used to evaluate the transfection efficacy.

RT-PCR

Total RNA was extracted with the TRI Reagent solution (Applied Biosystems, Foster City, CA, USA) and the genomic DNA was removed using DNase I (Qiagen, Inc., Shanghai, China). The RNA was reverse-transcribed into cDNA using the Transcriptor First Strand cDNA Synthesis Kit (Roche, Basel, Switzerland). GRP78 mRNA was amplified with the SYBR Green PCR Master Mix Kit (Applied Biosystems). The PCR reaction system contained 10 µL master mix, 1.6 µL primer mix, 2 µL cDNA template, and 6.4 µL ddH₂O_. The relative expression of GPR78 mRNA was quantified with the 2^−ΔΔCt method. The assay was repeated in triplicate. GADPH was used to normalize the mRNA levels.

TUNEL staining

Cells were analyzed for apoptosis through staining with a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) kit in accordance with the manufacturer’s guidelines (TUNEL Apoptosis Detection Kit; Beyotime). The cells were collected and washed with PBS, then fixed with 4% paraformaldehyde for 30 minutes, followed by incubation with 0.3% Triton X-100 at room temperature for 20 minutes. The biotin-labeled dUTP solution was prepared and incubated with the cells at 37°C for 60 minutes. The reaction termination solution was added, and after incubation for 10 minutes, the cells were washed three times with PBS. The DAB solution (0.5 mL) was then added to develop color, and subsequently, the hematoxylin staining solution was employed to stain the cells.

Statistical analysis

GraphPad PRISM 7 (GraphPad Software, Inc., La Jolla, CA, USA) was used for statistical analysis. The t-test and analysis of variance (ANOVA) were used for comparisons between groups and among groups, respectively. P < 0.05 was considered statistically significant. Data are shown as the mean ± standard deviation (SD). All experiments were repeated in triplicate.

This study was approved by the ethics committee of the Ningbo Hospital of Traditional Chinese Medicine affiliated to Zhejiang Chinese Medical University

LPS significantly reduces neuron viability

PC12 and MES23.5 cells were stimulated with different concentrations of LPS for 0, 24, or 72 hours. Neuron viability in both cell lines was significantly reduced (P < 0.05 to P < 0.001 versus lower LPS concentrations or the control) in a time- and dose-dependent manner (Figure 1a, b). This LPS-induced reduction in cell viability was more obvious in the MES23.5 cells than the PC12 cells. Next, we evaluated the level of ER stress induced in these SAE cell models after 72 hours of treatment with different concentrations of LPS, using GRP78 as a marker of ER stress. We found that the expression levels of GRP78 were significantly increased (P < 0.05 to P < 0.001 versus lower LPS concentrations or the control) in a dose-dependent manner (Figure 1c, d), indicating that UPR was markedly enhanced in both cell lines following LPS treatment. Again, these effects were more evident in the MES23.5 cells.

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Figure 1.

Cell viability and GRP78 expression levels in PC12 and MES23.5 cell models following treatment with lipopolysaccharides (LPS). (a, b) The Cell Counting Kit-8 assay was used to evaluate the cell viability in PC12 and MES23.5 cells, respectively, following treatment with increasing concentrations of LPS for 0, 24, or 72 hours. LPS significantly reduced the cell viability in a time- and dose-dependent manner. (c, d) Western blot analysis of GRP78 expression in PC12 and MES23.5 cells, respectively, following treatment with increasing concentrations of LPS. LPS significantly decreased GRP78 expression in a dose-dependent manner. Data represent the mean ± SD, n = 3. ^$P<0.05 vs. 10 µg/mL LPS. ^△△P<0.01, ^△△△P<0.001 vs. 1 µg/mL LPS. ^#P<0.05, ^###P<0.001 vs. 0.1 µg/mL LPS. *P<0.05 vs. control

UPR regulates neuron viability

The plasmids overexpressing GRP78 (OverExp GRP78-1 and OverExp GRP78-2) were transfected into PC12 and MES23.5 cells. The overexpression of GRP78 was significantly higher (P < 0.01 to P < 0.001) with the OverExp GRP78-1 plasmid than the OverExp GRP78-2 plasmid in both cell lines based on the GRP78 protein (Figure 2a, b) and mRNA (Figure 2c, d) levels. Thus, the OverExp GRP78-1 plasmid was used in all subsequent experiments. The overexpression of GRP78 significantly reduced (P < 0.01 to P < 0.001) the cell viability of the PC12 and MES23.5 cells compared with the model group (LPS treatment alone) in a time-dependent manner (Figure 2e, f), implying that enhanced UPR led to decreased cell viability. An ER stress inhibitor, 4-PBA, ¹⁹ significantly increased (P < 0.05) the cell viability of the two cell lines in a time-dependent manner compared with the model group (Figure 2e, f), suggesting that decreased UPR enhanced cell viability. Thus, UPR induced by LPS may regulate cell viability in a time-dependent manner.

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Figure 2.

Cell viability and GRP78 expression levels in PC12 and MES23.5 cells transfected with GRP78 overexpression plasmids and treated with lipopolysaccharides (LPS). (a, b) Western blot analysis of GRP78 expression. (c, d) Reverse-transcription PCR analysis of GRP78 mRNA expression. Control, no plasmids; Ov-NC, plasmid lacking the GRP78-1 insert; OverExp GRP78-1 and OverExp GRP78-2, plasmids overexpressing GRP78. ***P < 0.001 vs. Ov-NC. ^##P < 0.01 vs. Ov-GRP78-1. (e, f) The overexpression of GRP78 and treatment with the endoplasmic reticulum stress inhibitor, 4-PBA (100 µM), affected the viability of PC12 and MES23.5 cells induced by LPS (1 µg/mL). Control, no LPS treatment or plasmid; Model, LPS treatment only; Model+Ov-NC, LPS treatment plus negative-control plasmid (no GRP78 insert); Model+Ov-GRP78, LPS treatment plus GRP78-1 overexpression plasmid; Model+4-PBA, LPS plus 4-PBA treatment. Data represent the mean ± SD, n = 3. ^##P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. Model+Ov-NC. **P < 0.01, ***P < 0.001 vs. control.

UPR regulates neuron apoptosis

We next used the GRP78-1 overexpression plasmid to evaluate the effects of UPR on cell apoptosis through TUNEL staining. The overexpression of GRP78 significantly enhanced (P < 0.001) cell apoptosis in both the PC12 and MES23.5 cells compared with the Model+Ov-NC group (Figure 3a, b). Moreover, 4-PBA intervention significantly ameliorated (P < 0.001) the effects of LPS on cell apoptosis. These results suggest that LPS induces UPR and further facilitates cell apoptosis.

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Figure 3.

Level of cell apoptosis in PC12 (a) and MES23.5 cells (b) transfected with a GRP78 overexpression plasmid and treated with lipopolysaccharides (LPS) or the endoplasmic reticulum stress inhibitor, 4-PBA. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was used for analysis. Apoptotic cells are stained brown. Control, no LPS treatment or plasmid; Model, LPS treatment only; Model+Ov-NC, LPS treatment plus negative-control plasmid (no GRP78 insert); Model+Ov-GRP78, LPS treatment plus GRP78-1 overexpression plasmid; Model+4-PBA, LPS plus 4-PBA treatment. ***P < 0.001 vs. control. ^###P < 0.001 vs. Model+Ov-NC.

UPR regulates the apoptotic pathway

The expression levels of the ER stress sensor, PERK, and the pro-apoptosis protein, CHOP, were significantly increased (P < 0.05) in both cell lines and MES23.5 only, respectively, by GRP78 overexpression compared with the Model+Ov-NC group (Figure 4a, b). CHOP has been reported to regulate the Bcl-2 protein family, ¹⁴ and the overexpression of GRP78 led to significant reductions (P < 0.01 to P < 0.001) in the expression of Bcl-2. The expression of another pro-apoptosis protein, cleaved caspase-3, was also significantly increased (P < 0.01 to P < 0.001) relative to the Model+OV-NC group (Figure 4c, d). In most cases, treatment with 4-PBA ameliorated the changes in protein expression induced by LPS. Therefore, the results suggested that UPR induces cell apoptosis through the apoptotic pathway.

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Figure 4.

Western blot analysis of the expression of various proteins associated with apoptosis and endoplasmic reticulum (ER) stress in PC12 and MES23.5 cells transfected with a GRP78 overexpression plasmid and treated with lipopolysaccharides (LPS) or the ER stress inhibitor, 4-PBA. (a, b) The expression levels of the ER stress markers, GRP78, CHOP, PERK, and IRE1 in PC12 and MES23.5 cells, respectively. (c, d) The expression levels of the apoptosis-related proteins, Bax, Bcl-2, cleaved caspase-3, and caspase-3, in PC12 and MES23.5 cells, respectively. Control, no LPS treatment or plasmid; Model, LPS treatment only; Model+Ov-NC, LPS treatment plus negative-control plasmid (no GRP78 insert); Model+Ov-GRP78, LPS treatment plus GRP78-1 overexpression plasmid; Model+4-PBA, LPS plus 4-PBA treatment. Data represent the mean ± SD, n = 3. **P < 0.01, ***P < 0.001 vs. control. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. Model+Ov-NC.