Special chemicals and antibodies
Special chemicals include ethyl pyruvate (EP, Sigma-Aldrich Co., Cat# E47808, Saint Louis, MO, USA), CLI-095 (Invivo Gen, Cat# tlrl-cli95, San Diego, CA, USA), C34 (TOCRIS, Cat# 5373, Bristol, UK), recombinant HMGB1 (rHMGB1, ProSpec, Cat# pro-581-b, Rehovot, Israel), BAY 11-7082 (Sigma-Aldrich Co., Cat# B5556), IL-6 (PeproTech Inc., Cat# 400-06-2UG, Rocky Hill, NJ, USA), and neutralizing anti-rat-IL-6 antibody (R&D SYSTEMS, Cat# AF506, Minneapolis, MN, USA).
Primary antibodies include rabbit polyclonal anti-HMGB1 antibody for rat, mouse, and human (Abcam, Cat# ab18256, RRID:AB_444360, Cambridge, UK); rabbit polyclonal anti-AQP4 antibody for rat, mouse, human, and pig (Abcam, Cat# ab46182, RRID: AB_955676); mouse monoclonal anti-TLR4 antibody for rat, mouse, human, pig, baboon, bovine, and Chinese hamster (Novus, Cat# 76B357.1, RRID: AB_839000, Littleton, CO, USA); rabbit polyclonal anti-TLR4 antibody for rat, mouse, human, and rabbit (Boster, Cat# BA1717, RRID:AB_2716293); rabbit polyclonal anti-myeloid differentiation primary response gene 88 (MyD88) antibody for rat and human (Abcam, Cat# ab131071, RRID: AB_11156885); mouse monoclonal anti-IκBα antibody for rat, mouse, human, monkey, bovine, pig, and guinea pig (Cell Signaling Technology, Cat# 4814, RRID: AB_390781, Boston, MA, USA); mouse monoclonal anti-p-IκBα antibody for rat, mouse, human, and monkey (Cell Signaling Technology, Cat# 9246, RRID:AB_2267145); rabbit monoclonal anti-NF-κB antibody for rat, mouse, human, monkey, and bovine (Cell Signaling Technology, Cat# 4764, RRID:AB_823578); mouse monoclonal anti-GAPDH antibody for rat, mouse, and human (Beyotime, Cat# AF0006, RRID: AB_2715590, Shanghai, China); mouse monoclonal anti-Histone H3 antibody for rat, mouse, and human (Beyotime, Cat# AF0009, RRID: AB_2715593); and mouse monoclonal anti-S100β antibody for rat, mouse, human, rabbit, and pig (Boster, Cat# BM0120, RRID:AB_2716291).
Secondary antibodies include horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (absin, Cat# abs20002A, RRID: AB 2716554, Shanghai, China), horseradish peroxidase-conjugated goat anti-mouse secondary antibody (absin, Cat# abs20001A, RRID: AB 2716555), Cy3-conjugated goat anti-rabbit secondary antibody (Boster, Cat# BA1032, RRID: AB_2716305), and FITC-conjugated goat anti-mouse secondary antibody (ZSGB-BIO, Cat# BA1032, Beijing, China, RRID: AB_2716306).
Experimental protocol
Experiment 1. Effects of OGD/R on cellular swelling, HMGB1 and AQP4 expression in spinal cord astrocytes, and HMGB1 and IL-6 levels in the surrounding medium
Oxygen-glucose deprivation and reoxygenation (OGD/R) injury were induced in cultured spinal cord astrocytes. Astrocytic volume as well as HMGB1 and AQP4 expression was subsequently measured at 2, 6, 12, 24, and 48 h during the reoxygenation process after OGD. Enzyme-linked immunosorbent assay (ELISA) was then used to measure HMGB1 and IL-6 levels in the medium at 6, 12, and 24 h during reoxygenation.
Experiment 2. Effects of HMGB1 inhibition on cellular swelling as well as HMGB1, AQP4, and TLR4 expression in spinal cord astrocytes and HMGB1 and IL-6 levels in the surrounding medium after OGD/R
The experimental groups consisted of the following: normal, OGD/R, OGD/R + HMGB1 shRNA, OGD/R + non-targeting shRNA, and OGD/R + EP (HMGB1 inhibitor, 12 μM). With the exception of the normal group, all other group measurements were performed at 6, 12, and 24 h during the reoxygenation process after OGD. Measurements included astrocytic volume and astrocytic morphology and ultrastructure, as well as HMGB1, AQP4, and TLR4 expression. ELISA was used to determine HMGB1 and IL-6 levels in the surrounding medium.
Experiment 3. Role of TLR4/NF-κB signaling pathway in reducing cellular swelling resulting from HMGB1 inhibition in spinal cord astrocytes after OGD/R
To investigate the role of TLR4, spinal cord astrocytes were randomly divided into the following groups: normal, OGD/R, OGD/R + HMGB1 shRNA, OGD/R + non-targeting shRNA, OGD/R + CLI-095 (TLR4 inhibitor, 5 μM), OGD/R + C34 (another TLR4 inhibitor, 15 μM), OGD/R + HMGB1 shRNA + rHMGB1 (10 ng/ml), and OGD/R + EP. The astrocytic volume and the expression levels of TLR4, MyD88, IκBα, p-IκBα, and AQP4 were measured. Nuclear expression levels of NF-κB were also measured, in addition to IL-6 levels in the surrounding medium. Measurements were obtained after undergoing reoxygenation for 24 h after OGD.
To investigate the role of NF-κB, spinal cord astrocytes were randomly divided into the following groups: normal, OGD/R, OGD/R + HMGB1 shRNA, OGD/R + non-targeting shRNA, OGD/R + BAY 11-7082 (NF-κB inhibitor, 5 μM), and OGD/R + EP. The astrocytic volume as well as expression levels of IκBα, p-IκBα, and AQP4 was measured. In addition, expression level of nuclear NF-κB was measured and IL-6 levels in the surrounding medium were measured using ELISA. Measurements were obtained after undergoing reoxygenation for 24 h after OGD.
Experiment 4. Effects of rHMGB1 and IL-6 on regulating AQP4 expression in spinal cord astrocytes
To investigate the role of rHMGB1, spinal cord astrocytes were exposed to rHMGB1 at a series of concentrations (0, 0.1, 1, 10, or 20 ng/ml). After 24 h of exposure, AQP4 expression was measured using Western blot analysis.
We next investigated the role of IL-6 on regulating AQP4 expression in spinal cord astrocytes. First, spinal cord astrocytes were randomly divided into four groups, in which spinal cord astrocytes were exposed to IL-6 (0, 0.1, 1, or 10 ng/ml) for 24 h, and AQP4 expression was measured using Western blot. Second, spinal cord astrocytes were exposed to different astrocyte conditioned media (ACM), which originated from the astrocyte cultures in the OGD/R, OGD/R + HMGB1 shRNA, and OGD/R + non-targeting shRNA groups in Experiment 2. All ACM were harvested after undergoing reoxygenation for 24 h after OGD. The resulting AQP4 expression in spinal cord astrocytes was measured using Western blot. Finally, the neutralizing anti-rat-IL-6 antibody was used to reverse the effect of IL-6 on AQP4 expression in cultured spinal cord astrocytes. Spinal cord astrocytes were randomly divided into the following groups: normal, astrocytes + IL-6 (0.15 ng/ml), astrocytes + IL-6 (0.15 ng/ml) + anti-IL6 antibody (0.1 μg/ml), astrocytes + OGD6h/R24h ACM, and astrocytes + OGD6h/R24h ACM + anti-IL6 antibody (0.1 μg/ml). After 24 h exposure, AQP4 expression was measured using Western blot.
All experiments were repeated at least three times, and the average values were shown. In the normal group of all the experiments, spinal cord astrocytes were cultured in DMEM containing 10% FBS and incubated with 5% CO2 and 95% air at 37 °C. All chemical inhibitors were dissolved in the medium without additional organic solvents. Chemical concentrations and durations used in all experiments were chosen on pilot experiments conducted in our lab.
Western blot
Successive preparations of the plasma membrane and cytoplasmic extracts and nuclear extracts were made using a commercially available protein extraction kit (Beyotime, Cat# P0033) according to the manufacturer’s instructions. Briefly, cultured spinal cord astrocytes were washed with ice-cold PBS, harvested using a cell scraper, and centrifuged at 3000×g for 5 min. Cell pellets were then resuspended in a membrane and cytoplasmic extraction reagent containing phenylmethanesulfonyl fluoride (PMSF), phosphatase inhibitors, and protease inhibitors; vortexed for 5 s; and incubated on ice for 30 min. Lysates were then centrifuged at 12000×g at 4 °C for 10 min to obtain membrane-bound and cytoplasmic protein fractions for later expression analysis, with the exception of NF-κB. For NF-κB analysis, cell pellets were resuspended in a nuclear extraction reagent containing PMSF, phosphatase inhibitors, and protease inhibitors; vortexed for 5 s; and incubated on ice for 30 min. Lysates were centrifuged at 12000×g at 4 °C for 10 min to obtain nuclear protein fraction for NF-κB expression analysis. Prior to Western blot, all protein concentrations were determined using a BCA Protein Assay Kit (Beyotime, Cat# P0012S).
Protein (20 μg per lane) was subjected to electrophoresis using 10% sodium dodecyl sulfate polyacrylamide gels, followed by transfer to a polyvinylidene fluoride membrane (Millipore Corp. Billerica, MA, USA). After transfer, the membrane was blocked in 5% non-fat milk at 37 °C for 2 h. One of the following primary antibodies was then added: anti-HMGB1 (1:1000, Abcam), anti-AQP4 (1:800, Abcam), anti-TLR4 (1:800, Novus), anti-MyD88 (1:800, Abcam), anti-IκBα (1:1000, Cell Signaling Technology), anti-p-IκBα (1:1000, Cell Signaling Technology), anti-NF-κB (1:1000, Cell Signaling Technology), anti-GAPDH (1:1000, Beyotime), or anti-Histone H3 (1:1000, Beyotime). All primary antibodies were incubated at 4 °C overnight. After being washed by PBS containing Tween-20 (PBST), membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:5000, absin) or horseradish peroxidase-conjugated goat anti-mouse secondary antibody (1:5000, absin) at room temperature for 2 h. Bands were visualized using enhanced chemiluminescence (ECL, Beyotime, Cat# P0018) and subsequently analyzed using Quantity One software (Bio-Rad,
http://www.bio-rad.com, RRID: SCR_014280). The expression levels of proteins in the plasma membrane and cytoplasmic extracts were normalized to that of GAPDH. The expression levels of NF-κB in the nuclear extracts were normalized to that of Histone H3. The average band density for the normal group was set at 1.0, and all other band density values were normalized by the average value of the normal group.