Animals
All experiments were carried out for the sake of minimizing the number and suffering of animals. All experiments animal operations meet the requirements of the Beijing Administration Rule of Laboratory Animal, and were approved by the Animal Experimental Ethics Review Committee of the Institute of Basic Research for Chinese Medicine, China Academy of Chinese Medical Sciences.
The Sprague–Dawley rats within 12h of birth (Vital River Laboratories, Beijing, China) were used for primary cortical neurons isolation. Male Sprague–Dawley rats (260–280g, Vital River Laboratories, Beijing, China) used for middle cerebral artery occlusion (MCAO) experiment were housed in standard breeding environment without restriction to diet and drinking.
Reagents
Cell culture reagents: Dulbeccoʼs modified Eagle medium (DMEM); Sugar free DMEM; DMEM/F-12 (1:1) and fetal bovine serum (FBS) were obtained from Corning, USA. Neurobasal-A medium; 50 × B27 supplement; 100 × penicillin-streptomycin (PS); 100 × Glutamax-I and 0.25% trypsin were purchased from Gibco, USA.
Click chemistry, pull down and LC-MS/MS reagents: NaVc; CuSO4; TAMRA Azide and Biotin-azide were obtained from Sigma, USA. High capacity neutravidin agarose resin; sequencing grade modified Trypsin; TMT 10 plex reagent set; TEAB and Pierce™ Quantitative Fluorometric Peptide Assay Kit were purchased from Thermo Fisher Scientific, USA. Oasis HLB Extraction Cartridge was obtained from Waters. THPTA was obtained from Click Chemistry Tools.
Other reagents: celastrol (HPLC >98%); edaravone injection (Yangtze River Pharm; China); recombinant human HMGB1 protein (Abcam, United States); Cell Counting Kit-8 (CCK-8; DOJINDO; Japan); Coomassie brilliant blue (CBB; Beyotime; China). For intraperitoneal injection (i.p.), celastrol was dissolved in 1% dimethylsulfoxide (DMSO) and 0.9% physiological saline in order to yield a concentration of 1 mg/3 ml. All other reagents were purchased from Thermo Fisher or Sigma without special instructions.
Rat primary cortical neurons isolation and RAW 264.7 cell culture
The neonatal Sprague–Dawley rats within 12h of birth were used for primary cortical neurons isolation as previously established with minor revise [20]. Briefly, the cortex of newborn rats was sterile separated in pre-cooling (4°C) DMEM/F-12 (1:1). The minced cortex tissue was digested with 0.2mg/ml DNase Ⅰ and 2mg/ml papain, and inactivated by adding 10% volume FBS. The cell suspension was washed twice with DMEM/F-12 (1:1) and re-suspended in DMEM/F-12 (1:1) containing 10% FBS and 1 × PS. The cells suspension passed through 300 mesh sieves were seeded on L-polylysine pre-coated orifice plate or dish and incubated at 37°C incubator with 5% (v/v) CO2. 4-6h later, the DMEM/F-12 (1:1) was replaced with complete Neurobasal-A medium replenished with 1× PS, 1× Glutamax-I and 1× B27. The culture medium was change half every 2-3 days, and all the experiments were carried out on the seventh day unless otherwise stated.
RAW 264.7 cell was cultured in DMEM containing 10% FBS, 1× PS and maintained in a cell incubator. Cells were passage every 2-3 days and TNF-α was testing in cell within 20 passages.
Oxygen glucose deprivation (OGD) insult
The transient OGD model was constructed to simulate cerebral I/R injury in cultured primary neurons as previously described [21]. Briefly, the Neurobasal-A medium was displaced with deoxygenated, sugar free DMEM, and the cells were incubated in a hypoxia chamber (STEMCELL Technologies, Canada ) filled with 95% N2 + 5% CO2 for 4 h in 37°C incubator and returned to normal culture condition according to the experimental requirement. In contrast, control cells were cultured in normal culture conditions. After reaching the established time, cell viability evaluation was determined by CCK8 assay or cells were collected for other experiments. For CCK8 assay, absorbance was measured using a multimode plate reader (PerkinElmer, USA) at 450 nm.
Proteome reactivity profiles of primary neuron treated with cel-p
Fluorescence labeling profiling of cel-p binding proteins was conducted in living primary neuron with or without celastrol competitor and OGD model refer to previous operation [22]. Similarly, increasing concentrations of cel-p (0-1.6 μM) or cel-p (0.8μM) + competitor (celastrol 2 ×, 4 ×, 6 ×, 8 ×) were added into the 6 well plates with or without OGD interfere and incubated for 4 h in cell incubator. Then supernatant of cell lysate were collected and BCA method was used for protein concentration quantification. The click chemistry reaction was conduct with NaVc (100 mM stock solution, final concentration 1 mM), THPTA (100 mM stock solution, final concentration 100 μM), CuSO4 (100 mM stock solution, final concentration 1 mM) and TAMRA Azide (5 mM stock solution, final concentration 50 μM) in equal amounts (100 μg) of extracted protein for 2h at room temperature. The protein was precipitated with 1 ml pre-cooling (-20°C) acetone, and re-dissolved with 30 μl 1× SDS loading buffer. 15 μl of sample was separated with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel, and the labeling profiles were visualized with in-gel fluorescence scanning in laser scanner (Azure Sapphire RGBNIR, USA) and then the gel was stained with CBB.
Cellular imaging of cel-p
Cellular imaging experiment was conduct with fluorescence microscopy as described previously to verify the utility of cel-p for imaging of potential cellular targets [23]. To track the cellular distribution of cel-p, living primary neurons were incubated with 0.8 μM cel-p for 0-6 h. The cells were fixed with 4% paraformaldehyde solution for 10 min and 0.2% Triton-X 100 permeated 15 min. Click chemistry reaction was carry out (regents and concentration referenced 2.3.3) for 2h and washed thrice to remove excessive agents. The nuclei were stained with Hoechst for 10min. The images were obtained with confocal fluorescence microscopy (Leica TCS SP8 SR, Germany).
Pull down, TMT labeling and targets validation
Pull-down, TMT labeling and LC-MS/MS experiments were carried out to identify the interacting cellular targets of celastrol and primary neuron according to previous description with certain modification [24]. Primary neuron cells cultured in 100 mm dishes were divided into the following groups and continue incubated for 4 h : Control (less than 1% DMSO), cel-p (4 μM), and celastrol 8 × (cel-p 4 μM + celastrol 32 μM). Click chemistry reaction was conduct with Biotin-azide (50 mM stock solution, final concentration 50 μM), NaVc, THPTA and CuSO4 (concentration referenced 2.3.3) in equal amounts (1 mg) of extracted protein from each sample for 4 h. The protein was precipitated in pre-cooling (-20°C) acetone, and re-dissolved with 0.1% SDS in PBS. The sample supernatants were poured into the washed agarose resin (50μl of agarose resin in each group) and incubated with gentle rotation for 4 h to make resin capture the biotinylated proteins.
For on agarose resin digestion, the agarose resin was washed thrice with 1% SDS, 0.1 % SDS and 6 M urea in order. The agarose resin was reduced with 6M urea and 100 mM Dithiothreitol (DTT) for 30 minutes. Then the cysteines were blocked with 6M urea and 400 mM IAA and incubated avoid from light for 45 minutes. 2μg trypsin was add into each sample and incubated overnight to digest the proteins captured on the agarose resin. Supernatant containing digestive peptides were separated from the mixture by centrifugation and loaded onto the activated Extraction Cartridge, and then the samples were transferred to a new tube spin dry. The dried samples were re-constituted with 100 mM TEAB, and the digestive peptides were subjected to TMT labeling subsequently on the basis of instructions. TMT 126C and 131N labeled for negative control samples 1 and 2, TMT 127C and 127N labeled for cel-p pull-down samples 1 and 2, TMT 130C and 130N labeled for celastrol 8 × pull-down samples 1 and 2. The labeling reaction was quenched 2 h later with 1M Tris HCl and the labeled samples were converged to a single new tube for LC-MS/MS (Thermo Fisher, Orbitrap Fusion Lumos, USA) identify and quantify.
Cellular Thermal Shift Assay (CETSA)
Target engagement assay of celastrol with HMGB1 was performed the same with previous article with minor modify [25]. The primary neurons in 100 mm dishes were collected with PBS containing protease inhibitor. The cells were subjected to freezing and thawing cycles in liquid nitrogen and repeated mechanical crushing to obtain cell lysate supernatant by centrifugation. Then equivalent supernatant (1 mg) was treated with either DMSO or celastrol (20 μM) 1h at room temperature with gently shaking. The treated supernatant was divided into ten equal parts and heated according to designated temperatures. The cooled samples were centrifugation again to obtain supernatant and conducted Western blot analysis.
Western blot analysis
Supernatants of neuron lysate or rat cerebral cortex tissues lysate were obtained with RIPA lysate and protease inhibitor. For the cytoplasm and nuclear protein extraction, the nuclear-cytosol extraction kit was used on the basis of instructions. Fully reduced and disulfide bond HMGB1 isoforms were detected in rat primary cortex neuron and condensed culture supernatant with non-reducing PAGE gel, and samples were collected avoid from reducing agent (β-mercaptoethanol or DTT). After OGD and celastrol treatments as detailed above, the culture supernatant was collected at 0, 4, 8, 12, 24, 48 h and centrifuged to discard cell debris. Then the supernatant was concentrated 20 folds with Amicon Ultra-4 50kDa and Amicon Ultra-4 10kDa. Protein content was identified using the BCA assay, and the denatured sample was separated with 10%, 12% or 15% SDS-PAGE gel.
Separated protein samples were transferred onto PVDF membranes, blocked in 5% bovine serum albumin (BSA)and incubated overnight at 4°C with anti-HMGB1, anti-HSP70, anti-NF-κB, anti-RAGE, anti-TLR4, PCNA, or β-actin primary antibody and then secondary antibodies (goat anti-rabbit, 1:5000; goat anti-mouse, 1:5000) for 2 h at room temperature. Membranes were washed thrice with TBST after incubation. The bands were visualized with enzyme-linked chemiluminescence in the detection system (Azure C400, USA).
Immunofluorescence staining
For animal Immunofluorescence, after series dewaxing and dehydration, rat cerebral cortex paraffin slice were incubated in antigen retrieval for 10 min in 95°C and permeabilized 15 min in 0.2% Triton X-100. The slices were blocked 1h in 5%BSA, incubated with primary antibodies against HMGB1, HSP70, or NF-κB at 4 °C overnight and 2 h with secondary fluorescence antibodies (goat anti-rabbit, 1:500; goat anti-mouse, 1:500, Abcam) avoiding from light. After 10 min of Hoechst staining, the slices were photographed with laser scanning confocal microscope.
For cell Immunofluorescence, the treated cells were washed with PBS, fixed in 4% paraformaldehyde solution and permeabilized in 0.2% Triton-X 100. The rest procedures were the same with animal Immunofluorescence.
Expression and purification of HMGB1 A box and B box
Recombinant human HMGB1 box A (residues 1–89) and box B (residues 90–175) were cloned in a modified pET-24d vector (Novagen, Madison, WI) expressing a protein with an N-terminal 6-His tag. The E. coli BL21 was transformed with pET24d-HMGB1 A box and pET24d-HMGB1 B box, cultured in LB medium containing 50g/ml kanamycin at 37℃ to an absorbance of 0.8 at 600 nm, and expression was induced with 0.4 mM Isopropyl-D-1-thiogalactopyranoside (IPTG) for 12h at 16℃ before being harvested by centrifugation. Cell pellets were suspended in lysis buffer (20 mM Tris-HCl, pH 8.0, 200mM NaCl, 1mM PMSF) and disrupted by sonication. After centrifugation (12000 g, 30 min, 4℃), the supernatant incorporated His-tagged recombinant A box or B box was applied to a Ni-beads column. The column was washed with 30 ml binding buffer (20 mM Tris-HCl, pH 8.0, 200 mM NaCl and 20 mM or 50 mM Imidazole). A box protein was eluted with elution buffer (20 mM Tris-HCl, pH 8.0, 200 mM NaCl and 200 mM Imidazole). B box protein was eluted with elution buffer (20 mM Tris-HCl, pH 8.0, 200mM NaCl and 100 mM Imidazole). The samples were exchanged by the buffer containing 20 mM Tris-HCl, pH 8.0, 200 mM NaCl and concentrated by using centrifugal filter units according to the protocol provided by manufacturer. The concentration of purified proteins was determined with BCA protein assay reagent kit. The purity and integrity of purified HMGB1 A box and B box was verified by CBB after 15% SDS-PAGE gel separation.
HMGB1 full-length, A box and B box labeling and activity assay
Briefly, recombinant human HMGB1 full-length protein, A box and B box were dissolved or diluted with PBS, and reacted with celastrol, cel-p, IAA, IAA-yne, Glycyrrhetinic acid (GA), Melatonin (Mel) or DTT as needed for a period of time. Subsequently, the click chemistry reaction was conducted as same with title 2.3.3 fluorescence labeling and the protein was separated with 12% (HMGB1 full-length protein) or 15% SDS-PAGE gel (A box and B box). The labeling state was visualized by i fluorescence laser scanner and stained with CBB.
For activity assay of celastrol and HMGB1 protein, recombinant human HMGB1 protein and celastrol combined HMGB1 activity were measured by stimulating TNF-α in RAW 264.7 cells for 24h. For confirming whether celastrol blocked the binding of receptors TLR4 and RAGE to B box, the semi in vivo precipitation of B box complex experiment was conduct as previously with minor revise [26]. 100 μg B box proteins were first reacted with or without equivalent amount (equimolar with B box, 10-2μmol) and five folds amount of celastrol (5 ×10-2μmol) in Ni-beads column for 1 h. Then 500 μg lysate of primary neurons was add into the Ni-beads column and reacted 2 h at 4a, and eluted to precipitate B box complex. The denatured complex sample was subjected to immunoblot analysis with antibodies against TLR4 and RAGE. The same B box with equivalent amount of celastrol elution did not incubate with cell lysate was used for TNF-α analysis in RAW 264.7 cells to research whether celastrol reduced the ability of B box inducd TNF-α secretion.
Induction of MCAO and neurological defect assessment in rats
By inserting an filament to occlude the right middle cerebral artery (MCA) of male Sprague–Dawley rats (250–280g), we established cerebral I/R model in vivo as described previously [27]. Briefly, the rats fasted overnight were anesthetized with continuous supply of 3% isoflurane + 95% oxygen mixture. A 4-0 monofilament was inserting into the internal carotid artery through a tiny incision in the external carotid artery to block the cerebral blood flow of MCA. 90 min later, the filament was withdrawn to resume blood stream providing. The Sham group did the same steps without inserting the filament. Four groups included: (1) Sham group, saline i.p.; (2) Model (M) group, saline i.p.; (3) M + cel 1mg/kg, i.p.; (4) M + eda 6 mg/kg, i.v..
The neurological deficits of rats were assessed at 24, 48, 72 h after reperfusion respectively by an experimenter who was blinded to the group information. Zea-Longa five-point scores was used to assess neurologic deficits according to previous description [27]. The animals without symptoms of neurological impairment or dying after surgery were rejected. 72h after reperfusion, infarct volume was determined by 2, 3, 5-triphenyltetrazolium chloride (TTC) stating and calculated with Image J software as previously [27]. Nissl-stained cells in the rat cerebral cortex were observed at 200 × magnifications with a light microscope to assess Nissl body damage.
Statistical analysis
Data were presented as the mean ± SEM. Raw data were statistically analyzed with Graph Pad Prism 5.0. The density of Westren blot bands was quantified using Image J software. The data were analyzed using one-way ANOVA. Fisher’s least-significant difference post hoc test was used to test the differences between two groups. P value less than 0.05 was considered statistically significant.