Mice

C57BL/6 wild-type (WT) (male, 8–10 W, 23 ± 3 g, animal approval number: SCXK (Lu) 2019-0001) mice were purchased from Animal Center of Shandong University. OTUD1−/− mice obtained from Professor Chengjiang Gao (Shandong University, Jinan, China) were described previously [18]. All experimental procedures were approved by the Ethics Committee of Shandong University and in accordance with the Institutional Animal Care and Use Committee of Shandong University.

Regents and plasmids

GSK2983559 (TopScience, Cat. T5401) was purchased from TopScience. Poly-d-lysine hydrobromide (Sigma, Cat. P0899) and 2, 3, 5-Triphenyltetrazolium chloride (TTC) (Sigma, Cat. T8877) were obtained from Sigma.

The vectors encoding OTUD1 (WT and C320A) and ubiquitin (WT, K63 and K48), the NF-κB firefly luciferase reporter plasmid and pGL3 promoter-dependent Renilla luciferase reporter plasmid was kindly provided by Professor Chengjiang Gao [18]. His-RIP2 plasmid (Human, GV219) was purchased from GENE.

Primers and siRNAs

The sequences of the primers used in qRT-PCR were listed in Table 1. The 3 siRNAs targeting OTUD1 were designed as below:

Table 1 Sequences of the primers

1st: 5-CCACUUCAGCCCACUCAUUTT-3;

2nd: 5-CCGGAUAUCCCGAAUUGCUTT-3;

3rd: 5-GCUCAGCAAUG GACAUAUTT-3.

Animal model of focal cerebral ischemia

OTUD1−/− and WT mice were subjected to middle cerebral artery occlusion (MCAO) as described previously [7, 19]. Briefly, the mice were initially anesthetized by intraperitoneal injection of 50 mg pentobarbital per kilogram of body weight. The left common carotid artery and left external carotid artery were exposed through a midline neck incision. A MCAO monofilament (CINONTECH, Beijing, Cat. A1-162050) was inserted into the left external carotid artery and advanced into the left internal carotid artery past the MCA origin until the tip reached the proximal anterior cerebral artery, thus occluding the origin of the MCA. A successful occlusion was indicated by a severe reduction in the regional cerebral blood flow to < 20% of the baseline by a laser-Doppler flow meter (Moor, England). Body temperature was kept at 37 °C during operation with a homeothermic blanket. Mice were euthanized after subjected to MCAO without reperfusion at the indicated time. The sham animals were subjected to the same procedure except for the occlusion of the MCA. GSK2983559 (GSK559, RIP2 inhibitor, 3 μl of 200 μM for each mouse) was intraventricularly injected (coordinates: 0.04 mm posterior to the bregma, 0.1 mm lateral to the midline, 0.22 mm ventral to the dura) to mice 30 min before MCAO.

Cell culture and treatment

BV2 cells were purchased from the China center for type culture collection (Wuhan, China), HEK293T cell was a kind gift from Professor Chengjiang Gao. BV2 cells and HEK293T cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Meilunbio, Cat. MA0212) supplemented with 10% FBS (LONSERA, Cat. S711-001S) and incubated with 5% CO2 at 37 °C.

Primary neuron cells were cultured as described in literature [19]. Briefly, cerebral cortex was isolated from E18 mice and neurons were seeded on six-well plates at the density of 7 × 105/well in B27-containing media (Neurobasal medium, Thermo, Cat. A3582901). Neurons were used for experiments after 7 days of culture.

Primary astrocytes cultures were prepared from postnatal 1–2 day mouse pups as previously described [20]. The cerebral cortex was isolated, minced, treated with trypsin at 37 °C for 30 min. After stopping digestion by DMEM/F12 (Meilunbio, Cat. MA0124) medium containing 10% FBS, cells were passed through 100 μm pore-sized membrane (Merck, Darmstadt, Germany) and seeded in T75 flasks. Culture media were refreshed every 2–3 days. Astrocytes were used for experiments after 7 days of culture.

Transfection and treatment

Cells were transfected with plasmid or siRNA using Lipofectamine 2000 (Invitrogen, Cat. 11668019). Cells were deprived of oxygen and glucose (OGD) by incubating in EBSS solution for 90 min with 5% CO2 and 95% N2 at 37 °C. Then, the cells were switched back to reoxygenation (OGD/R) for the indicated time [19].

Immunofluorescence staining

Frozen brain sections (10 μm) or the slides of cells were double labeled with indicated primary antibodies at 4 °C overnight, then gave the secondary antibodies and incubated for 2 h at room temperature. DAPI (Beyotime, Cat.C1005) was incubated for nuclear staining [19].

Antibodies and dilution ratio used in immunofluorescence staining were as follow: anti-OTUD1 (Abcam, Cat. 122481, Rabbit, 1:100), anti-RIP2 (Proteintech, Cat. 15366-1-AP, Rabbit, 1:100; Santa Cruz, Cat. sc-136059, Mouse, 1:100), anti-Flag (Sigma, Cat. F1804, Mouse, 1:200), anti-6*His (Proteintech, Cat. 66005–1-lg, Mouse, 1:200), anti-NeuN (Millipore, Cat. MAB377, Mouse, 1:200), anti-Iba1 (Proteintech, Cat. 10904–1-AP, Rabbit, 1:100; Santa Cruz, Cat. sc-32725, Mouse, 1:100), anti-GFAP (Millipore, Cat. MAB360, 1:100), Alexa Fluor 488 Goat anti-rabbit IgG Ab (Proteintech, Cat. SA00006-2, 1:200), Alexa Fluor 594 Goat anti-Mouse IgG Ab (Proteintech, Cat. SA00006-3, 1:200).

TTC staining

After MCAO, mice brains were cut coronally into 2 mm slices. The slices were stained with 0.2% TTC (dissolved in PBS) at 37 ℃ for 30 min and kept in 4% paraformaldehyde (PFA, Sigma, Cat. P6148). Infarct volumes were calculated as: (contralateral volume − healthy ipsilateral volume)/contralateral volume.

Neurological deficit score in mice

A 4-tiered neurological scoring system were used to determine the outcome by a blinded observer as described previously [21, 22]. Neurological deficit was assessed as follows: 0, normal function; 1, flexion of the torso and contralateral forelimb on lifting the animal by the tail; 2, circling to the contralateral side but normal posture at rest; 3, reclination to the contralateral side at rest; 4, absence of spontaneous motor activity. 5, death.

Histopathology

Mice brains were collected and fixed in 4% PFA, embedded in paraffin, and sectioned. The sections were stained with hematoxylin and eosin (H&E) (Solarbio, Cat. G1120) to observe the neuronal morphology in the cerebral cortex and hippocampus. TUNEL staining was performed by an in situ cell death detection kit (Roche, Cat. 42134700) according to the manufacturer’s instructions as previously described [23].

Western blot and immunoprecipitation(IP)

Western blot analysis of the target proteins in cells and brain tissues was performed as previously described [24].The lysates from cells or brain tissues were extracted using RIPA buffer (Beyotime, Cat. P0013B). Protein concentrations were determined using a BCA protein assay reagent kit (Meilunbio, Cat. MA0082) and 40 μg proteins were electrophoresed on SDS–PAGE, then transferred to polyvinyldifluoridine membranes (Millipore, Cat. 3010040001). The membranes were incubated first with the primary antibodies at 4 °C overnight, and then followed by incubation with HRP-conjugated secondary antibodies at room temperature for 2 h.

For immunoprecipitation, cells were lysed with NP40 buffer and centrifuged, first incubated with the indicated antibody and corresponding IgG controls at 4 °C for 3 h, next, mixed with Protein A+G agarose (Santa Cruz, Cat. Sc-2003), after incubated overnight at 4 °C under rotation, the beads were washed and eluted in SDS sample buffer for further analysis.

Antibodies and dilution ratio used in Western blot and IP assay as follow: anti-OTUD1 (Abcam, Cat. 122481, Rabbit, 1:1000), anti-RIP2 (Proteintech, Cat. 15366-1-AP, Rabbit, 1:100; Santa Cruz, Cat. sc-136059, Mouse, 1:1000), anti-Flag (Sigma, Cat. F1804, Mouse, 1:3000), anti-HA (OriGene, Cat. TA180128, Mouse, 1:3000), anti-6*His (Proteintech, Cat. 66005-1-lg, Mouse, 1:3000), anti-IκBα (CST, Cat. 4814, Mouse, 1:1000), anti-p-NF-κB-p65 (CST, Cat. 3033, Rabbit, 1:1000), anti-β-ACTIN (Affinity, Cat. AF7018, 1:5000), anti-GAPDH (Proteintech, Cat. 60004-1-Ig, Rabbit, 1:5000), HRP-conjugated Affinipure Goat Anti-Mouse IgG (H+L) (Proteintech, Cat.SA00001-1, 1:10,000), HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H+L) (Proteintech, Cat.SA00001-2, 1:10,000).

ELISA assay

The protein levels of IL-6 and TNFα in the supernatants of BV2 and astrocytes cultures were measured by the ELISA kits (eBioscience, E-HSEL-M0003 & E-HSEL-M0009) following the manufacturer’s instructions. Data were quantitatively normalized to the protein concentration of control group.

Reverse transcription and qPCR

Total RNA was extracted using RNA-Quick purification kit (ES Science, Cat. RN001) and 1 μg RNA was reverse-transcripted into cDNA using Hiscript II Q RT SuperMix for qPCR (Vazyme, Cat. R223-01) with the following procedure: 50 °C, 15 min; 80 °C, 15 s. The qPCR was performed with UltraSYBR One Step RT-qPCR Kit (CWBio, Cat. CW2624) as follow protocol: 45 °C, 10 min; 95 °C, 10 s and 65 °C, 45 s for 40 cycles; 95 °C, 15 s; 60 °C, 1 min; 95 °C, 15 s; 60 °C, 15 s. The result was captured and analyzed with BIORAD IQ5 software.

Dual-luciferase reporter assay

Plasmids encoding RIP2, OTUD1 (WT or C320A) were co-transfected with NF-κB firefly luciferase reporter plasmid and pGL3 promoter-dependent Renilla luciferase reporter plasmid into HEK293T cells for 36 h by Lipofectamine 2000. Firefly & Renilla Luciferase Reporter Assay Kit (Meilunbio, Cat. MA0518) was used for Luciferase activity.

Statistical analysis

All results were presented as means ± SD and analyzed with GraphPad Prism 8.0 software. T test, one-way or two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test determined the statistical differences between different groups. P < 0.05 indicates statistically significant.