Contributed equally
Oxysophoridine (OSR) is an alkaloid extracted from
Spinal cord injury (SCI) is a clinically common serious trauma. The occurrence rate has the tendency to rise with increases in annual traffic accidents (
Secondary SCI is further damage that is caused by various factors following primary damage of spinal cord. The mechanism is extremely complicated (
The membrane structure of spinal cord tissues contains abundant lipids. Following damage, the generation and release of oxygen radicals is increased. Oxygen radicals act on the polyunsaturated fatty acids of the cytomembrane to generate lipid peroxidase, change membrane permeability, disintegrate lysosomes and cause necrocytosis, which results in secondary damage of the spinal cord. Free radical scavenging in cells primarily involves two antioxidant systems, including enzymatic defense reactions superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) (
SCI is the continuous destruction of the spinal canal caused by external force, fracture or instant damage of the spinal cord caused by dislocation upon trauma and is the opposite to physical trauma (
The pharmacological action of oxysophoridine (OSR), whose chemical structure is presented in
The experimental protocol was approved by Shandong Provincial Hospital Affiliated to Shandong University Animal Care and Ethics Committee. Female adult Sprague-Dawley rats (weight, 200–230 g, n=50) were purchased from the Experimental Animal Center of Shandong University, and maintained in standard cages (22–24°C and 55–60% humidity) with water and food
Functional recovery was assessed following treatment with OSR using the BBB Locomotor Rating Scale to ensure consistency of the lesion (
Whole blood (500 µl) was centrifuged at 2,000 × g for 10 min at 4°C and serum was collected in every rat to determine the levels of tumor necrosis factor-α (TNF-α; H052), interleukin (IL)-1β (H002), IL-6 (H007), IL-8 (H008), malondialdehyde (MDA; A003-1), SOD (A001-1) and GSH-Px (A005) using commercial ELISA kits from Nanjing Jiancheng Biology Engineering Institute (Nanjing, China) according to the manufacturer's protocol.
Spinal cord tissues were isolated from every rat and homogenized in RIPA assay (Beyotime Institute of Biotechnology, Haimen, China). Protein concentrations were measured using a BCA protein assay kit (Beyotime Institute of Biotechnology, Haimen, China). Proteins (50–80 µg) were fractionated by 12% SDS-PAGE and transferred to a nitrocellulose membrane. The membranes were blocked in 5% skim milk in TBS-Tween-20 (TBS-T; 0.05%) at room temperature for 1 h on a shaker and incubated with the following primary antibodies: Prostaglandin E2 (PGE2; sc-20771; 1:500), intercellular adhesion molecule-1 (ICAM-1; sc-7891; 1:500), cyclooxygenase-2 (COX-2; sc-7951; 1:500), nuclear factor-κB (NF-κB; sc-109; 1:500), Bcl-2-associated X (Bax; sc-6236; 1:500), Bcl-2 (sc-783; 1:500) and GAPDH (sc-367714; 1:500; all from Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at 4°C overnight. The membrane was washed with TBS-T and treated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:3,000), visualized with a BeyoECL Star (Beyotime Institute of Biotechnology) and quantified using Bio-Rad Laboratories Quantity One software version 3.0 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
Data are presented as the mean ± standard deviation using SPSS version 17.0 (SPSS, Inc., Chicago, IL, USA). Statistical differences were determined using one-way ANOVA followed by a Tukey test. P<0.05 was considered to indicate a statistically significant difference.
The BBB scores in the SCI model group were significantly reduced, compared with the sham group (
The present study confirmed the anti-inflammatory effect of OSR on inflammation in SCI rats by measuring the levels of TNF-α, IL-1β, IL-6 and IL-8 in the serum using ELISA kits. The results indicate that TNF-α, IL-1β, IL-6 and IL-8 levels in SCI model rats were significantly higher compared with levels in the sham group (
To confirm the anti-oxidative stress effect of OSR in SCI rats, the levels of MDA, SOD and GSH-Px in the serum were measured using ELISA kits. Compared with the sham group, MDA levels were significantly increased, and SOD and GSH-Px activities were significantly reduced in SCI model rats (
PGE2 protein expression in the spinal cord tissue of SCI model rats was significantly higher compared with the sham group (
The ICAM-1 protein expression in the spinal cord tissue of SCI model rats was significantly increased compared with the sham group (
As demonstrated in
To investigate the involvement of the NF-κB pathway in the anti-inflammatory effect of OSR in SCI rats, NF-κB protein expression in the spinal cord tissue was measured using western blotting. SCI significantly induced NF-κB protein expression compared with the sham group (
To investigate the effect of OSR on apoptosis in the SCI rat model, the Bax/Bcl-2 protein expression ratio in the spinal cord tissue of rats was measured by western blotting. The ratio of Bax/Bcl-2 protein expression in SCI model rats was significantly higher compared with the sham group (
Pathophysiological changes in SCI are divided into primary mechanical damage and secondary damage that occurs subsequent to primary damage (
A previous study demonstrated that inflammatory responses have an important role on the pathogenesis of secondary damage (
SOD has an important role in the oxidation and antioxidant balance, and scavenges free radicals and protect cells from damage (
NF-κB is a transcription factor that has an important regulatory role in inflammatory responses, immune responses, cell growth, differentiation and apoptosis (
The inflammatory response is one of primary mechanisms of aggravating secondary SCI. The process is primarily induced by the toll-like receptor (TLR)-NF-κB signaling pathways (
Bcl-2 is a cytoplasmic protein that has a higher level of expression during development of the central nervous system (
In summary, OSR rescues SCI as treatment with OSR increased BBB scores, reduced spinal cord tissue water content, reduced the production of pro-inflammatory cytokines, increased the levels of anti-oxidant enzymes and reduced the ratio of Bax/Bcl-2 protein expression in SCI model rats, which revealed anti-inflammatory, anti-oxidative stress and anti-apoptosis effects of OSR that may be mediated via NF-κB and the Bax/Bcl-2 pathway. In conclusion, OSR may exert a protective effect on SCI by anti-inflammatory, anti-oxidative stress and anti-apoptosis effects, which indicates that OSR may be a potential therapeutic agent for SCI.
Chemical structure of oxysophoridine.
OSR rescues BBB scores and spinal cord water content in SCI model rats. OSR reduces the effects of SCI on (A) BBB scores and (B) spinal cord water content in SCI model rats. ##P<0.01 vs. sham group and **P<0.01 vs. SCI model group. OSR, oxysophoridine; BBB, Basso, Beatie and Bresnahan; SCI, spinal cord injury; 60 OSR, 60 mg/kg OSR; 120 OSR, 120 mg/kg OSR; 180 OSR, 180 mg/kg OSR.
OSR suppresses inflammation in SCI model rats. OSR suppresses serum levels of (A) TNF-α, (B) IL-1β, (C) IL-6 and (D) IL-8 in SCI model rats. ##P<0.01 vs. sham group and **P<0.01 vs. SCI model group. OSR, oxysophoridine; SCI, spinal cord injury; TNF, tumor necrosis factor; IL, interleukin; 60 OSR, 60 mg/kg OSR; 120 OSR, 120 mg/kg OSR; 180 OSR, 180 mg/kg OSR.
OSR suppresses oxidative stress in SCI model rats. OSR suppresses levels of (A) MDA, and increases levels of (B) SOD and (C) GSH-Px in SCI model rats. ##P<0.01 vs. sham group and **P<0.01 vs. SCI model group. OSR, oxysophoridine; SCI, spinal cord injury; MDA, malondialdehyde; SOD, superoxide dismutase; GSH-Px, glutathione peroxidase; 60 OSR, 60 mg/kg OSR; 120 OSR, 120 mg/kg OSR; 180 OSR, 180 mg/kg OSR.
OSR suppresses the protein expression of PGE2, ICAM-1 and COX-2 in SCI model rats. (A) Representative image of western blot analysis of PGE2, ICAM-1 and COX-2 protein expression. Densitometric analysis indicated that OSR suppressed the protein expression of (B) PGE2, (C) ICAM-1 and (D) COX-2 in SCI model rats. ##P<0.01 vs. sham group and **P<0.01 vs. SCI model group. OSR, oxysophoridine; PGE2, prostaglandin E2; ICAM-1, intercellular adhesion molecule-1; COX-2, cyclooxygenase-2; SCI, spinal cord injury; 60 OSR, 60 mg/kg OSR; 120 OSR, 120 mg/kg OSR; 180 OSR, 180 mg/kg OSR.
OSR suppresses NF-κB protein expression and the ratio of Bax/Bcl-2 in SCI model rats. (A) Representative image of western blot analysis of NF-κB, Bcl-2 and Bax protein expression. Densitometric analysis indicated that OSR reduced (B) NF-κB protein expression and (C) Bax/Bcl-2 ratio in SCI model rats. ##P<0.01 vs. sham group and **P<0.01 vs. SCI model group. OSR, oxysophoridine; NF-κB, nuclear factor-κB; Bax, Bcl-2-associated X; SCI, spinal cord injury; 60 OSR, 60 mg/kg OSR; 120 OSR, 120 mg/kg OSR; 180 OSR, 180 mg/kg OSR.