Experimental Animals and Reagents
Adult male Sprague-Dawley (SD) rats (weighing 250-280 g) were obtained from the Animal Experimental Center of Chongqing Medical University and used for the in vivo study. Primary astrocytes were extracted from the cerebral cortices of newborn SD rats and cultured.
Glucose-free Dulbecco’s modified Eagle’s medium, Dulbecco’s modified Eagle’s medium (DMEM)/F12 and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, USA). Trypsin and Hank’s solution were obtained from HyClone (Logan, UT, USA). Penicillin/streptomycin (Pen/Strep) and phosphate-buffered saline solution (PBS) were obtained from Beyotime (Shanghai, China). Poly-L-lysine was purchased from Sigma-Aldrich (Milan, Italy). IL-1β, IL-6 and TNF-α enzyme-linked immunosorbent assay (ELISA) kits were obtained from Boster (Wuhan, China), and a CCK-8 kit was obtained from Dojindo (Kumamoto, Japan). TPI-1 was purchased from MedChem Express (New Jersey, USA)
Middle Cerebral Artery Occlusion/Reperfusion (MCAO/R) Model and TPI-1 Dosing
Transient MCAO/R was performed as previously described by our laboratory [24]. Briefly, adult male SD rats were deprived of food and water for 8 h before operation, anesthetized with 3.5% chloral hydrate (350 mg/kg, intraperitoneal injection) and then placed on a heating pad to maintain their body temperature at 37 ± 0.5 °C for the operation. A nylon filament (Beijing Cinontech Co., Ltd., Beijing, China) was inserted into the left middle cerebral artery. Then, the filament was removed after 1 h of ischemia to allow reperfusion. Local cerebral blood flow (CBF) was monitored by a laser Doppler flowmeter (Periflux System 5000, Perimed, Sweden) during the operation. After reperfusion for 24 h, neurological deficit score assays were used to evaluate the success of the MCAO model, and brain tissues were subjected to Western blot analysis, immunoprecipitation and immunohistochemistry. Sham-operated animals underwent the same surgery as the MCAO/R group, except they were not subjected to MCAO.
TPI-1 was obtained from MedChem Express. The SHP-1 inhibitor TPI-1 (HY-100463, MedChem Express) was dissolved in 10% DMSO and diluted to the final concentration in 90% saline (20% SBE-β-CD in saline). We intraperitoneally injected 1 mg/kg TPI-1 into MCAO rats after they awoke. An identical volume of DMSO was injected intraperitoneally as a control.
All analyses conducted in SD rats (N = 377) were divided into two sets of experiments; the first part was performed to study the effect of DJ-1 on inflammation after cerebral I/R, and the second part was performed to study the mechanism by which DJ-1 regulates the NLRX1-TRAF6 interaction. The first part comprised 4 groups, and the second part comprised 8 groups. The groups in the first part were as follows: (1) the sham group (n= 37, 1 died), (2) the MCAO group (n= 39, 3 died), (3) the NC group (MCAO + negative control siRNA) (n= 40, 4 died), and (4) the DJ-1 siRNA group (MCAO + DJ-1 siRNA) (n= 42, 6 died). The groups in the second part were as follows: (1) the sham group (n= 24, 0 died), (2) the MCAO group (n= 26, 2 died), (3) the scramble group (MCAO + scramble) (n= 28, 4 died) (4) the overexpression group (MCAO + DJ-1 overexpression) (n= 28, 4 died), (5) the DMSO group (MCAO + DMSO) (n= 27, 3 died), (6) the TPI-1 group (MCAO +TPI-1) (n= 27, 3 died), (7) the overexpression + DMSO group (MCAO + DMSO + DJ-1 overexpression) (n= 30, 6 died), and (8) the overexpression + TPI-1 group (MCAO +TPI-1 + DJ-1 overexpression) (n= 29, 5 died). In our study, 6 rats were used per experimental group, and each technique was performed in at least triplicate.
Treatment with DJ-1 siRNA or DJ-1 Adeno-Associated Virus (AAV)
DJ-1 siRNA was obtained and used as described in our previous studies [20]. AAV was purchased from Neuron Biotech (Shanghai, China). The following RNA sequences were used to overexpress the rat DJ-1 gene (Gene ID: NM_117287): 5-CCGGACGGCAGTCACTACAGCTACTCAAGAGA and TAGCTGTAGTGACTGCCGTTTTTTTG-3 (overexpression). The scramble AAV did not contain inserts. One month before MCAO, AAV was injected into the left lateral cerebral cortices of the rats. The overexpression efficiency of DJ-1 AAV was confirmed by Western blot analysis.
Evaluation of Neurological Deficits
To assess neurological deficits in the rats on day 1 after MCAO/R, a modified scoring system was applied as described in our previous studies [20]. The scoring system was as follows: grade 0, no neurological damage; grade 1, failure to extend the contralateral forelimb fully; grade 2, circling to right; grade 3, falling to the right; grade 4, no spontaneous autonomic activity or loss of consciousness; grade 5, death.
Quantification of Infarct Volume
After MCAO/R, brains were sliced into 2-mm-thick sections and stained with 2% 2,3,5-triphenyltetrazolium chloride (TTC, Sigma, USA) at 37 °C for 30 min as previously described [20, 25]. Then, the sections were fixed in 4% paraformaldehyde at 4 °C for 24 h. Each section was photographed, and the volume was quantified using ImageJ (version 6.0, NIH, Bethesda, MD, USA). The infarct volume was calculated using the following equation: {[total infarct volume - (ipsilateral hemisphere volume - contralateral hemisphere volume)] / contralateral hemisphere volume × 100%.
HE and Nissl Staining
After neurological examination following MCAO/R, the rats were sacrificed with an overdose of 3.5% chloral hydrate, and their brains were perfused transcardially with 4% paraformaldehyde. The brains were dehydrated and embedded in paraffin after immersion in paraformaldehyde for 24 h. Then, 5-µm coronal sections were stained with hematoxylin and eosin (HE) or 0.1% cresyl violet (Nissl staining) based on standard protocols and mounted for microscopy.
Primary Rat Cortical Astrocyte Culture and Oxygen and Glucose Deprivation/Reoxygenation (OGD/R) Treatment
Astrocytes were cultured as previously described [20]. Briefly, cortical astrocytes were cultured in 75 cm2 flasks at a density of 2.0 × 107/well in DMEM/F12 medium with 10% fetal bovine serum and 1% pen/strep in a humidified incubator in 5% CO2 at 37 °C. After the cells were cultured for 2-3 weeks, the microglia were removed from the flasks by mild shaking, the remaining adherent astrocytes were detached with trypsin, and the recovered cells were plated directly on the bottom of 6-well plates. Astrocytes that spread over the bottom of the plates (7-9 days) were used for further study. Astrocyte purity was determined using the astrocyte-specific marker GFAP.
OGD/R was conducted as previously described. Briefly, astrocytes were established in glucose-free DMEM and were transferred to an incubator with 94% N2, 1% O2, and 5% CO2 at 37 °C for 5 h. The glucose-free DMEM was changed to normal culture medium (DMEM/F12 medium containing 10% fetal bovine serum and 1% pen/strep), and then TPI-1 (10 ng/ml) was added to the culture medium in the 6-well plates. TPI-1 was dissolved in 10% DMSO and diluted to the final concentration in PBS. An identical volume of DMSO and PBS was added to the culture medium as a control. Next, the cells were grown in an incubator with 5% CO2 for 24 h at 37 °C. All the analyses conducted in astrocytes were divided into two sets of experiments; the first part was performed to study the effect of DJ-1 on inflammation after cerebral I/R, and the second part was performed to study the mechanism by which DJ-1 regulates the NLRX1-TRAF6 interaction. The first part comprised 4 groups, and the second part comprised 8 groups. The following groups were used in the first part: (1) the control group, (2) the OGD/R group, (3) the NC group (OGD/R + negative control siRNA), and (4) the DJ-1 siRNA group (OGD/R + DJ-1 siRNA). The following groups were used in the second part: (1) the control group, (2) the OGD/R group, (3) the scramble group (OGD/R + scramble), (4) the overexpression group (OGD/R + DJ-1 overexpression), (5) the DMSO group (OGD/R + DMSO), (6) the TPI-1 group (OGD/R + TPI-1), (7) the overexpression + DMSO group (OGD/R + DMSO + DJ-1 overexpression), and (8) the overexpression + TPI-1 group (OGD/R +TPI-1 + DJ-1 overexpression). Astrocytes were cultivated with 100 nM TPI-1 after OGD. In our study, 6 cultures were performed per experimental group, and each technique was performed in at least triplicate.
DJ-1 Interference and Overexpression in Astrocytes
A lentivirus was obtained and used as described in our previous studies [20]. The following sequence was designed and synthesized to knock down DJ-1: 5-CCGGACGGCAGTCACTACAGCTACTCAAGAGA TAGCTGTAGTGACTGCCGTTTTTTTG-3. The control sequence for DJ-1 knockdown was 5-CCGGTTCTCCGAACGTGTCACGTTTCAAGAGA ACGTGACACGTTCGGAGAATTTTTTG-3. The sequence used to overexpress DJ-1 was 5-ATGGACTACAAGGATGACGATGACAAGGATTACAAAGACGACGATGATAAGGACTATAAGGATGATGACGACAAA-3. The scramble lentivirus did not contain inserts. Three days before OGD/R, lentivirus was added to the culture medium. OGD/R was performed after astrocyte transfection. Western blotting was used to assess the transfection efficiency of the lentivirus.
Cell Viability
To assess astrocyte viability, we used a CCK-8 assay kit (Dojindo, Kumamoto, Japan). Astrocytes were cultured in 96-well plates. Three days before OGD/R, the astrocytes were treated with lentivirus for 72 h. Then, the cells were subjected to OGD/R, and the CCK-8 assay was performed. CCK-8 solution was added to the medium (10 µl/mL). After the astrocytes were cultured with medium at 37 °C for up to 2 h, they were measured with a microplate reader (Bio-Rad, Foster City, CA, USA) at an absorbance of 450 nm.
Immunocytochemistry and Immunohistochemistry
Frozen sections and cells on coverslips were fixed in 4% paraformaldehyde and then washed with PBS. After blocking with 5% FBS/0.01% Triton X-100 in PBS at 37 °C for 1 h, the cells were incubated with the following primary antibodies overnight at 4 °C: NLRX1 (1:100, Abcam, ab105412) and Hsp60 (1:100, Proteintech, 66041-1-1g). The next day, the cells and sections were incubated in a DyLight 488-conjugated goat anti-mouse IgG antibody (1:200, Abbkine, green, A23210) and a DyLight 549-conjugated goat anti-rabbit IgG antibody (1:200, Abbkine, red, A23320) at 37 °C for 30 min. Vectashield containing DAPI was used to mount the coverslips and sections. A laser scanning confocal microscope (LSCM) was used to visualize the sections and glass slides.
Western Blot Analysis
Total protein from cultured astrocytes and ischemic brain tissues was prepared using RIPA buffer with the protease inhibitor PMSF. A total of 50 µg of protein (per lane) was separated by SDS-PAGE (different percentages of SDS-PAGE gels were used to separate and analyze the different proteins) and electrotransferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Boston, MA, USA). The membranes were then blocked with 5% nonfat milk in TBST buffer for 2.0 h at 37 °C and incubated with the following primary antibodies overnight at 4 °C: DJ-1 (1:1000, Abcam, ab76008), anti-β-actin (1:3000, Proteintech, 66009-1-Ig), IL-1β (1:200, Boster, PB0055), IL-6 (1:200, Boster, PB0061), TNF-α (1:200, Boster, PB0082), NLRX1 (1:200, Finetest, FNab05759), TRAF6 (1:200, Santa Cruz Biotechnology, sc-8409), and SHP-1 (1:200, Abcam, ab227503). The next day, the membranes were incubated with secondary antibodies at 37 °C for 2 h. The densities of the bands were detected using an imaging densitometer (Bio-Rad), and the gray values of the bands were quantified using ImageJ. Relative protein expression was normalized to β-actin staining intensity.
Immunoprecipitation and Immunoblot Analyses
In the immunoprecipitation experiments, whole tissue and cell lysates were prepared after I/R and incubated with the indicated antibodies together with protein A/G beads (MedChem Express) overnight. NLRX1 (1:200, Finetest, FNab05759), TRAF6 (1:200, Santa Cruz Biotechnology, sc-8409) and SHP-1 (1:200, Abcam, ab227503) antibodies were used to immunoprecipitate NLRX1, TRAF6 and SHP-1, respectively. The beads were then washed 4 times with lysis buffer, and the immunoprecipitates were separated by SDS-PAGE. The proteins were transferred to PVDF membranes (Millipore, Boston, MA, USA) and then incubated with the indicated antibodies.
ELISA Measurements of TNF-α, IL-1β, and IL-6
After MCAO for 1 h and OGD for 5 h followed by reoxygenation for 24 h, total protein from the ischemic penumbra of the cerebral cortex and cultured astrocytes was prepared using RIPA buffer with the protease inhibitor PMSF. We used TNF-α, IL-1β, and IL-6 ELISA kits (Boster Institute, Wuhan, China) to measure the levels of TNF-α, IL-1β, and IL-6 expression in vivo and in vitro. We assessed the expression of these inflammatory cytokines in collected tissue and cell lysates according to the manufacturer’s instructions. The signal was measured at 450 nm.
Statistical Analysis
All data are shown as the means ± standard errors of the means, and statistical analysis was conducted using GraphPad Prism software (version 6.0). Statistical comparisons were assessed with one-way analysis of variance (ANOVA) followed by Tukey’s test. A value of P < 0.05 indicated statistical significance.