Contributed equally
Carvacrol (CAR) is a naturally occurring phenolic monoterpene and has been demonstrated to possess a spectrum of pharmacological actions. The present study was designed to assess the neuroprotection of CAR against spinal cord injury (SCI) in rats and to identify the underlying mechanisms. SCI was induced using the modified weight-drop method in Wistar rats. CAR or saline was administered at doses of 25, 50 and 100 mg/kg for 46 days. Neuronal function following SCI was evaluated using the Basso, Beattie and Bresnahan (BBB) locomotor rating scale. Spinal cord edema was assessed by measuring the water content in spinal cord tissues. The oxidative indicators, including malondialdehyde, catalase, superoxide dismutase glutathione peroxidase and 8-isoprotane as well as endothelial nitric oxide synthase (eNOS) activity and caspase-3 were measured using corresponding commercial kits. The protein expression of eNOS and B cell lymphoma-2 (Bcl-2) as well as Bcl-2-associated X protein (Bax) was analyzed by western blot analysis. The SCI-induced rats demonstrated marked reductions in BBB scores. CAR treatment recovered neurological function with decreasing BBB scores. CAR was found to have inhibitory effects on the water content in the spinal cord, oxidative stress, eNOS, nitric oxide production and apoptosis-associated molecules, including Bax and caspase-3 as well as promoting Bcl-2 expression in SCI-induced rats. These results suggested that CAR protects against SCI via mediating oxidative stress and the eNOS signaling pathway.
It is well established that spinal cord injury (SCI) produces primary damage and triggers a prolonged period of secondary lesion. Among the factors associated with the secondary injury, reactive oxygen species have attracted significant attention for their role in the pathogenesis of SCI (
Nitric oxide (NO) is an important endothelium-derived relaxing factor involved in the pathophysiology of SCI. It is well established that NO is produced from the guanidine group of L-arginine by three types of nitric oxide synthase (NOS) enzymes, including endothelial NOS (eNOS), neuronal NOS and inducible NOS (
Carvacrol (CAR) is a natural monoterpenoid phenol compound extracted from the essential oil of the family Lamiaceae, which includes the genera
Wistar rats weighing ~220–260 g were obtained from the Animal Centre of Beijing (Beijing, China). They were kept in a standard environment and allowed free access to water and food. Experimental protocols were performed in accordance with the guidelines of the Care and Use of Laboratory Animals of the Provincial Hospital Affiliated to Shandong University (Jinan, China). The study was approved by the ethics committee of the Provincial Hospital Affiliated to Shandong University (Jinan, China).
CAR (with a purity >98%) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Commercial kits for malondialdehyde (MDA), catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and eNOS were obtained from Nanjing Jiancheng Biotechnology Institute (Nanjing, China). 8-Isoprostane EIA kit (no. 516351) was obtained from Cayman Chemical (Ann Arbor, MI, USA). Other reagents were all of analytical grade.
The rat model of SCI was prepared as described previously with minor modifications (
The motor recovery in SCI rats was evaluated by a locomotor rating scale between 0 (complete paralysis) and 21 (normal locomotion) developed by Basso, Beattie and Bresnahan (BBB) (
Spinal cord edema was assessed by measuring the water content in spinal cord tissues. Following treatment with CAR for 46 days, the impaired spinal cords were dried at 80°C for 48 h in order to determine the dry weight. Water content of the spinal cords was calculated using the following formula: Spinal cord water content (%) = (wet weight − dry weight) / wet weight × 100%.
The oxidative markers, including MDA level and the activities of CAT, SOD and GSH-Px in spinal cord tissues were detected using corresponding commercial kits (Nanjing Jiancheng Bioengineering Institute). Detection of plasma 8-isoprostane levels was performed using the 8-Isoprostane EIA kit.
Following treatment with CAR for 46 consecutive days, the injured spinal cord tissues were homogenized in ice-cold lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 10% glycerol, 1% Nonidet P-40, 5 mM EDTA and 1 mM phenyl-methylsulfonyl fluoride). Supernatant was collected following centrifugation at 12,000 ×g for 20 min and protein quantification was conducted using a BCA kit (Beyotime Institute of Biotechnology, Shanghai, China). Protein (60
The eNOS activity was measured according to the manufacturer's instructions of the Nitric Oxide Synthase Assay kit (no. S0025; Beyotime Institute of Biotechnology). Additionally, NO production in the plasma was analyzed by measuring the supernatant for nitrite using Griess reagent (Promega Corp., Madison, WI, USA).
As a critical molecule in cellular apoptosis, caspase-3 activity was measured by cleavage of chromogenic caspase substrates, Ac-DEVD-pNA. Colorimetric analysis of the quantity of caspase-3 was performed using a spectrophotometer (Alpha-1860S; Shanghai Puyuan Company, Shanghai, China) at a wavelength of 405 nm.
All values are presented as the mean ± standard deviation and were analyzed using SPSS 16.0 software (SPSS, Inc., Chicago, IL, USA). Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Dunnett's test. P<0.05 was considered to indicate a statistically significant difference.
The chemical structure of CAR is shown in
As shown in
At 72 h post-surgery, it was observed that MDA levels were markedly increased and the antioxidant enzymes, including CAT, SOD and GSH-Px were all decreased in spinal cord tissues (P<0.01), compared with the sham control (
The present study further examined whether CAR exerted a protective effect through mediating the eNOS pathway.
In order to determine the effect of CAR on cellular apoptosis following SCI, the protein expression of apoptosis-regulated proteins, including Bcl-2 and Bax was detected by western blot analysis. As shown in
The major findings of the present study illustrated that CAR facilitated the recovery of motor function in SCI-induced rats and that its neuroprotection may be associated with suppressing oxidative stress and the eNOS signaling pathway.
It has been demonstrated that SCI-induced primary damage is irreversible and appears to not be amenable to neuroprotective therapy. However, the secondary impairment occurs in response to the deleterious substances produced following primary trauma (
NO is considered to be one of the major regulators of spinal damage. A previous study revealed that eNOS-derived NO production aggravated the impairment caused by SCI in rats (
The secondary lesion caused by SCI can damage spinal neurons and trigger apoptotic cascades. The pharmacological inhibition of apoptosis may function as a potential therapeutic strategy. Targeted retrograde gene delivery of brain-derived neurotrophic factor was reported to inhibit cellular apoptosis and restore neurological function following SCI (
Taking these results into account, it was concluded that CAR protected the rat spinal cord from injury. The neuroprotective effect of CAR may be associated with suppressing oxidative stress and inhibiting the eNOS signaling pathway following SCI in rats.
Chemical structure of carvacrol.
Effects of CAR on the water content of the spinal cord following SCI (n=10, mean ± standard deviation). **P<0.01, compared with the sham group; ##P<0.01, compared with the SCI group. Sham, sham group; SCI, spinal cord injury group; CAR (25), carvacrol (25 mg/kg)-treated group; CAR (50), carvacrol (50 mg/kg)-treated group and CAR (100), carvacrol (100 mg/kg)-treated group. CAR, carvacrol; SCI, spinal cord injury.
Effects of CAR on the concentration of (A) MDA and on the activities of antioxidant enzymes (B) CAT, (C) SOD and (D) GSH-Px in spinal cord tissues of rats from different groups (n=10, mean ± standard deviation). **P<0.01, compared with the sham group; ##P<0.01, compared with the SCI group. Sham, sham group; SCI, spinal cord injury group; CAR (25), carvacrol (25 mg/kg)-treated group; CAR (50), carvacrol (50 mg/kg)-treated group and CAR (100), carvacrol (100 mg/kg)-treated group. CAR, carvacrol; SCI, spinal cord injury; SOD, superoxide dismutase; GSH-Px, glutathione peroxidase; CAT, catalase; MDA, malondialdehyde.
Effects of CAR on the plasma 8-isoprostane level following SCI (n=10, mean ± standard deviation). **P<0.01, compared with the sham group; ##P<0.01, compared with the SCI group. Sham, sham group; SCI, spinal cord injury group; CAR (25), carvacrol (25 mg/kg)-treated group; CAR (50), carvacrol (50 mg/kg)-treated group and CAR (100), carvacrol (100 mg/kg)-treated group. CAR, carvacrol; SCI, spinal cord injury.
Effects of CAR on the protein level and activity of eNOS as well as plasma NO concentration following SCI (n=10, mean ± standard deviation). (A) Representative images of immunoblots with antibodies against eNOS in injured spinal cords from different groups. eNOS: 133 kDa; GAPDH: 36 kDa. (B) Quantitative analysis of the protein level of eNOS in spinal cords from different groups. The data were normalized to the loading control GAPDH. (C) Measurement of eNOS activity. (D) NO production was detected spectrophotometrically by measuring its metabolite, nitrite. **P<0.01, compared with the sham group; ##P<0.01, compared with the SCI group. Sham, sham group; SCI, spinal cord injury group; CAR (25), carvacrol (25 mg/kg)-treated group; CAR (50), carvacrol (50 mg/kg)-treated group and CAR (100), carvacrol (100 mg/kg)-treated group. CAR, carvacrol; SCI, spinal cord injury; eNOS, endothelial nitric oxide synthase; NO, nitric oxide.
Effects of CAR on the protein expression of Bcl-2 and Bax following SCI (n=10, mean ± standard deviation). (A) Representative images of immunoblots with antibodies against Bcl-2 and Bax in injured spinal cords from different groups. Bcl-2: 26 kDa; Bax: 23 kDa; GAPDH: 36 kDa. (B and C) Quantitative analysis of the protein levels of Bcl-2 and Bax, respectively in spinal cords from different groups. The data were normalized to the loading control GAPDH. **P<0.01, compared with the sham group; ##P<0.01 compared with the SCI group. Sham, sham group; SCI, spinal cord injury group; CAR (25), carvacrol (25 mg/kg)-treated group; CAR (50), carvacrol (50 mg/kg)-treated group and CAR (100), carvacrol (100 mg/kg)-treated group. SCI, spinal cord injury; CAR, carvacrol; Bcl-2, B cell lymphoma-2; Bax, Bcl-2-associated X protein.
Effects of CAR on caspase-3 activity following SCI (n=10, mean ± standard deviation). **P<0.01, compared with the sham group; ##P<0.01, compared with the SCI group. Sham, sham group; SCI, spinal cord injury group; CAR (25), carvacrol (25 mg/kg)-treated group; CAR (50), carvacrol (50 mg/kg)-treated group and CAR (100), carvacrol (100 mg/kg)-treated group. SCI, spinal cord injury; CAR, carvacrol.
Effects of CAR on the motor function of rats 24, 48 and 72 h after SCI.
Group | n | 24 h | 48 h | 72 h |
---|---|---|---|---|
Sham | 10 | 14.21±1.12 | 18.11±1.27 | 19.88±1.47 |
SCI | 10 | 4.98±0.87 |
4.18±0.82 |
4.05±0.63 |
CAR (25 mg/kg) | 10 | 9.78±0.89 |
10.12±0.97 |
11.25±0.76 |
CAR (50 mg/kg) | 10 | 11.63±0.65 |
11.89±0.75 |
12.04±0.95 |
CAR (100 mg/kg) | 10 | 12.85±0.96 |
14.15±0.88 |
16.21±1.06 |
P<0.01, versus sham group;
P<0.01, versus SCI group. CAR, carvacrol; SCI, spinal cord injury.