Salidroside promotes the repair of spinal cord injury by inhibiting astrocyte polarization, promoting neural stem cell … – Nature.com

Reagents and antibodies

The salidroside was purchased from MCE (HY-N0109, Monmouth Junction, NJ, USA). Primary antibodies used in this study included rabbit anti-STAT3 (ab68153, Abcam, Cambridge, UK), rabbit anti-p-STAT3 (ab76315, Abcam), mouse anti-Nestin (ab6142, Abcam), rabbit anti-Sox2 (ab92494, Abcam), rabbit anti-GFAP (ab7260, Abcam), mouse anti-MAP2 (ab11268, Abcam), rabbit anti-NeuN (ab177487, Abcam), rat anti-C3 (ab11862, Abcam), rabbit anti-GAP43 (ab75810, Abcam), mouse anti-Neurofilament (ab134306, Abcam), rabbit anti--actin (93473, Cell Signaling Technology, Danvers, MA, USA), mouse anti-JNK (66210-1-Ig, Proteintech, Chicago, USA), rabbit anti-p-JNK (ab4821, Abcam), rabbit anti-BrdU (ab152095, Abcam). Alexa Fluor 594 or 488 conjugated goat anti-mouse IgG (H+L) (115-545-062; 115-585-062) or goat anti-rabbit IgG (H+L) (111-545-045; 111-585-045), and Alexa Fluor 647 conjugated goat anti-rat IgG (H+L) (112-605-062) for immunofluorescence staining were purchased from Jackson ImmunoResearch (West Grove, PA, USA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (H+L) and HRP-conjugated goat anti-mouse IgG (H+L) for Western blot were purchased from Invitrogen (31460; 31430, Carlsbad, CA, USA). ELISA kits for IL-1 (88-5019-88) and IL-6 (88-7064-88) were purchased from Invitrogen. The CCK8 kits for cell viability were purchased from DOJINDO (CK04-1000t, Dojindo, Kumamoto, Japan). JSI-124 (HY-N1405) and SP600125 (HY-12041) were purchased from MCE.

The conduction of all animal procedures strictly adhered to the Guidelines for the Care and Use of Laboratory Animals (National Institutes of Health) and approved by the Institutional Animal Care and Use Committee of National Center for Nanoscience and Technology (NCNST21-2101-YC01). Briefly, 45 female C57BL/6J mice (1835g; 8 weeks old) underwent deep anesthesia via intraperitoneal injection of pentobarbital sodium (50mg/kg of body weight). Following this, the dorsal fur of the mice was meticulously removed and disinfected with an iodophor solution. Subsequently, the T10 lamina was excised, revealing the spinal cord. After the dorsal surface of the spinal cord was fully exposed, a rod (1.3mm in diameter; RWD Life Science Corp., C4p01-001, China) was used to compress the spinal cord with a force of 50 kdynes and a dwell time of 10s. After surgery, the muscular and dermal layers were meticulously sutured. The mice received daily abdominal massages to facilitate defecation and urination until the restoration of reflexive micturition control. These SCI models were randomly segregated into three groups: a SCI-only group (n=25/group), a low-dose salidroside treatment group (n=25/group) and a high-dose salidroside treatment group (n=25/group). The number of standardized animal models could meet statistical needs as reported [7]. No mice died before the end of the experiment, and all model mice were included in the final analysis. The SCI-only group functioned as the control. The SCI-only group functioned as the control. Salidroside was administered via intraperitoneal injection at a dosage of 50mg/kg (low-dose) or 100mg/kg (high-dose) once every 2 days until the point of sacrifice. For late-stage drug therapy experiments (n=30/group), intraperitoneal injection of salidroside (100mg/kg) commenced on the 16th day after surgery, along with colivelin (Col) (STAT3 agonist, 1mg/kg, i.p.) or anisomycin (Ani) (JNK agonist, 15mg/kg, s.c.), every two days until euthanasia.

The evaluation of mouse locomotor capacity post-SCI was conducted through Basso Mouse Score (BMS) assessments on days 1, 3, 7,14, 21, and 28 after surgery. Scores range from 0 for complete paraplegia to 9 for normal function.

Gait and motor coordination were assessed at 28th day post surgery. The forelimbs were marked with blue dye, while the hindlimbs were marked with red dye. Mice were placed on a track lined with blotting paper, with an incentive cassette placed at the end to encourage rapid traversal. Footprint patterns were digitized, and representative images were employed to assess coordination.

Mice were positioned on a rotating rod that experienced gradual acceleration. The duration and rotational speed when the mice fell down were recorded. Prior to the official experiment, adaptation trials were conducted for two days, with each mouse undergoing two tests.

In an independent experiment, SCI models were randomly allocated into four groups (n=4/group), each receiving varying concentrations of salidroside. Subsequently, the mice were euthanized, and blood samples were collected for serological analysis. Additionally, major organs were dissected for hematoxylin-eosin (H&E) staining to assess histological changes.

On days 7, 14, 21, and 28 post injury, the mice were humanely euthanized through intraperitoneal administration of pentobarbital sodium (80mg/kg). The hearts of the mice underwent immediate perfusion with 20mL of ice-cold saline followed by 10mL ice-cold paraformaldehyde (4%, w/v). The incision of SCI was opened, and the spinal cord segment encompassing the lesion site was extracted. Spinal cord samples were subsequently fixed in paraformaldehyde (4%, w/v) at 4C for 24h, before dehydrated in a sucrose-PBS gradient (20% and 30%, w/v). Finally, the samples were embedded in OCT and sectioned into continuous longitudinal sections of 10m. All sections were stored at 80C for subsequent immunostaining.

Primary astrocytes were obtained from the brains of 1~3-day-old C57BL/6J mice according to an established protocol [27]. Astrocytes were cultured in T75 flasks pre-coated with poly-L-lysine (Sigma, St.Louis, MO, USA) to obtain primary mixed glial cell cultures using DMEM/F12 (Gibco, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco), 1% penicillin and streptomycin solution (Gibco), 2 mM l-glutamine (Gibco), 100M non-essential amino acids (Gibco), and 2mM sodium pyruvate (Gibco). Subsequent to confluence, the cultures underwent agitation at 180rpm for 30min on an orbital shaker to eliminate microglia. The mixed glial cultures were further treated with 20mL of fresh culture medium and subjected to shaking at 240rpm for 6h, an action that effectively removed oligodendrocyte precursor cells (OPCs). The ensuing astrocytes were employed for subsequent experiments. To induce neurotoxic A1 astrocytes, a mixture termed Activator comprising IL-1 (3ng/mL, I3901, Sigma), TNF (30ng/mL, 8902SF, Cell Signaling Technology), and C1q (400ng/mL, MBS143105, MyBioSource, San Diego, Southern California, USA) was concurrently added to the medium, with cultures maintained for 24h [12]. Following this, the supernatant of neurotoxic A1 astrocytes was replaced with fresh complete medium and continued for an additional 24h. The resultant supernatant (astrocyte conditioned medium, ACM) was collected for subsequent NSC intervention experiments (Supplementary Fig. 1).

Primary NSCs were isolated from the brains of 1~3-day-old C57BL/6J mice using a process resembling the procedure for astrocytes, except that the cell precipitates were resuspended in NSCs growth medium [28]. This growth medium consisted of DMEM/F12 (Gibco) supplemented with 2% N2 (Gibco), 1% B27 (R&D Systems, Minneapolis, MN, USA), bFGF 20ng/ml (R&D Systems), and EGF 20ng/ml (R&D Systems). NSCs were identified by Nestin (red) and Sox2 (green) (Supplemental Fig. 2). Differentiation experiments necessitated the substitution of the medium with neuron complete medium, characterized by Neurobasal medium (Gibco) supplemented with 2% B27 (Gibco), 1% l-glutamine (Gibco), 100 IU/ml penicillin, and 100mg/ml streptomycin. For dispersed cells, neurospheres were digested with Accutase (Gibco) and seeded within well plates pre-coated with poly-L-lysine (Sigma). In cell proliferation experiments, neurospheres or dispersed NSCs were cultivated in NSCs growth medium supplemented with ACM in the presence or absence of Sal (100M), JSI-124 (0.5M), or SP600125 (30M). To assess differentiation, dispersed NSCs were cultivated in neuron complete medium featuring the aforementioned stimulating agents.

The viability of primary astrocytes and NSCs was evaluated with a CCK8 assay (Dojindo). After 24h of incubation with varying concentrations of salidroside, with or without Activator or ACM, wells were rinsed 3 times with PBS, followed by the addition of CCK8 solution (10L; 1:10 dilution) in fresh culture medium (100L) and then incubated for 1h at 37C. The optical absorbance was measured at 450nm using a microplate reader (ELx800; Bio-Tek, Winooski, VT, USA).

Proteins were extracted from cells using a protein extraction kit (Solarbio, Beijing, China). Protein concentration was quantified via a BCA assay kit (Thermo Scientific, Waltham, MA, USA). 20g of protein samples underwent separation by SDS-PAGE, transferring to PVDF membranes (ISEQ00010, Millipore), and then blocking with 5% bovine serum albumin (BSA, Solarbio). Following this, the membranes were incubated with primary antibodies overnight at 4C. Thereafter, HRP-conjugated secondary antibodies were introduced at room temperature for 2h. Reacting bands were visualized using ECL reagent (170-5061, Bio-Rad, Hercules, CA, USA), with protein band densities semi-quantified via Image J (National Institutes of Health, Bethesda, MD, USA).

Spinal cord sections or cultured cells were fixed with pre-cooled 4% paraformaldehyde for 15min at 4C, then permeabilized with 0.3% Triton X-100 for 20min, and blocked with 5% BSA for 1h. The samples were incubated at 4C overnight with primary antibodies, followed by incubation with the corresponding fluorescent secondary antibody for 2h at room temperature. After triple washes with PBS, the nuclei were stained with DAPI (Thermo Fisher Scientific), and fluorescent images were captured using an epifluorescence microscope (AxioVertA1 and ImagerA2), or a confocal fluorescence microscope (LSM710; Carl Zeiss, Germany).

Nissl staining in spinal cord samples was performed on the 14th day post surgery. Subsequent to rinsing the slices with distilled water, they were immersed in a cresol violet solution for 10min. Then slices were rinsed with distilled water, differentiated with 95% ethanol, rinsed with xylene, and finally fixed with neutral balsam. Bright-field images were acquired using an epifluorescence microscope (AxioVertA1 and ImagerA2).

Total RNA was extracted from astrocytes or NSCs post-various treatments using Trizol Reagent (Invitrogen) according to the manufacturers instructions. The concentrations of RNA were gauged by a Biometra Optical Thermocycler (Analytik Jena, Goettingen, Germany). Five hundred nanograms of RNA underwent reverse transcription into cDNA employing the High-capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). The primer sequences used for qPCR amplification were:

C3: 5-ACATGC GCCGAA GCA GA-3 (forward)

5-ACCCGGACCTCAAAA CTGG-3 (reverse)

GAPDH: 5-TGTGATGGGTGTGAACCACG-3 (forward)

5-CAGTGAGCTTCCCGTTCACC-3 (reverse)

NeuN: 5-GACAACCAGCAACTCCACCC-3 (forward)

5-GAGCCCCGCTCGTTAAAAAT-3 (reverse)

GFAP: 5-CATGCCACGCTTCTCCTTGT-3 (forward)

5-ATCATCTCTGCACGCTCGCT-3 (reverse)

iNOS: 5-ACGGACGAGACGGATAG-3 (forward)

5-GGGCTTCAAGATAGGGA-3 (reverse)

Nestin: 5AAGCAGGGTCTACAGAGTCAGATCG-3 (forward)

5-GCTGTCACAGGAGTCTCAAGGGTAT-3 (reverse)

Quantitative real-time PCR was performed using SYBR qRCR premix (Takara, Kyoto, Japan). Cycling conditions encompassed an initial denaturation at 95C for 30s, followed by 40 cycles at 95C for 5s, 60C for 30s, and 72C for 10min. Target gene expression was normalized to GAPDH expression using the CT method.

The concentrations of IL-1 and IL-6 in ACM post-various treatments were quantified using ELISA kits according to the manufacturers protocols. Briefly, the ACM was collected and centrifuged at 2000 rpm for 20min to eliminate debris. 100L of all samples or standards were added to appropriate wells of capture-antibody pre-coated 96 well plates for 1h incubation followed by 100L of detection antibody for another 1h. Then all wells underwent three washes with 1 wash buffer followed by 100L/well of TMB substrate for 10min in darkness. Finally, STOP Solution (0.16M H2SO4) was added and absorbance was read at 450nm wavelengths using a microplate reader (ELx800, Bio-Tek, USA) within 30min.

Results are presented as meanSD of at least three independent experiments. Means were compared by one-way ANOVA followed by Bonferronis post hoc tests for multiple comparisons (SPSS 21; SPSS, Chicago, IL, USA). P values of <0.05 (two tailed) were considered statistically significant. *p<0.05; **p<0.01; ***p<0.001.

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Salidroside promotes the repair of spinal cord injury by inhibiting astrocyte polarization, promoting neural stem cell ... - Nature.com