Delphinidin, a flavonoid polyphenolic compound, is widely found in nature and is used as a food supplement due to its pharmacological activity. The aims of the present study were to examine the anti-inflammatory effect of delphinidin in alleviating spinal cord injury (SCI)-induced inflammation in a rat model and to determine the underlying mechanisms in SCI. The Basso, Beattie, Bresnahan (BBB) scores of rats were assessed to evaluate the effect of delphinidin on the recovery of motor function. ELISA kits were also used to analyze the activities of tumor necrosis factor-α (TNF-α), interleukin (IL)-6, cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2) and caspase-3. In addition, the protein expression levels of nuclear factor (NF)-κB, activator protein 1 (AP-1) and p38-MAPK protein expression were measured using western blot analysis. Treatment with delphinidin significantly increased the BBB scores, as well as inhibited the intramedullary spinal pressure in SCI rats. Delphinidin treatment also significantly suppressed the levels of inflammatory factors and NF-κB protein expression in SCI rats. Finally, treatment with delphinidin significantly inhibited NF-κB stimulation, COX-2 activity, PGE2 production, and AP-1 and p38-MAPK protein expression in SCI rats. These results suggest that the anti-inflammatory effect of delphinidin alleviated inflammation in the SCI rat model via alleviation of the intramedullary spinal pressure through the NF-κB and p38-MAPK signaling pathways.
Spinal cord injury (SCI) is a common trauma that threatens human health seriously and can occur at any age (
SCI involves a series of cytological and molecular biology cascade reactions (
The inflammatory reaction is a major component of secondary SCI and is a key constituent of the pathophysiological mechanism of acute SCI. Inflammation may promote a series of molecular biological events, resulting in the activation of inflammatory cells from the spinal cord tissues and infiltration of the circulatory system, the release of various pro-inflammatory mediators and neurotoxins, and the generation of oxygen radicals and nitroso compounds, and consequently causing cell damage (
Delphinidin (
Delphinidin has been extensively used as a complementary anti-inflammation and trauma treatment in the form of a food supplement (
All experimental procedures were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals published by Liaocheng People's Hospital, Liaocheng Clinical School of Taishan Medical University (Liaocheng, China). The study was approved by the Ethics Committee of Liaocheng People's Hospital. Adult male Sprague-Dawley rats (n=40, weight, 220–250 g, 8–9 weeks-old) were purchased from the Laboratory Animal Center of the Taishan Medical University, and raised in the specific-pathogen-free Laboratory Animal Center at a constant environment with a temperature of 22±1°C, 50–60% humidity, an alternating 12 h light-dark cycle and free access to food/water. The rats were randomly divided into four groups (n=10 rats per group): Sham, SCI model, and two SCI + delphinidin-treated (40 or 200 mg/kg) groups. A total of 24 h after SCI, the delphinidin group rats were treated with 40 or 200 mg/kg delphinidin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 2 weeks.
In order to establish an SCI model, the rats were anesthetized with 35 mg/kg pentobarbital sodium by intraperitoneal injection. The spinal cord was aseptically exposed following laminectomy at T9-10. Next, a 2-mm diameter hammer was implanted at 25 mm from the T9-10 spinal cord and removed after 1 min. Subsequently, manual bladder expression was performed three times daily.
Following delphinidin treatment, the BBB scores of rats were assessed to evaluate the recovery condition of the motor function. The BBB scores ranged between 0 and 21, with the lowest point (score of 0) indicating complete paralysis and the maximum point (score of 21) implying normal function.
Following delphinidin treatment, rats was sacrificed using decapitation under anesthetization (35 mg/kg pentobarbital sodium) and spinal cord (2 mm cephalic and caudally around the injury epicenter) was removed. The tissues were dissolved in RIPA lysis buffer (Beyotime Institute of Biotechnology, Jiangsu, China), and a BCA kit (Beyotime Institute of Biotechnology) was used to quantify the protein concentration. In accordance with the specifications of the ELISA kits, protein samples (10 µg) were used to measure the activities of tumor necrosis factor-α (TNF-α; cat. no. H052), interleukin (IL)-6 (cat. no. H007), cyclooxygenase-2 (COX-2; cat. no. H200), prostaglandin E2 (PGE2; cat. no. H099) and caspase-3 (cat. no. G015; all Jiancheng Bioengineering Institute, Nanjing, China) using a luminometer (MicroLumat Plus LB 96V; Berthold Technologies, Bad Wildbach, Germany).
Following delphinidin treatment, protein samples were obtained from spinal cord tissues. The protein samples (40 µg) were separated by 8–10% SDS-PAGE and transferred into polyvinylidene difluoride membranes (EMD Millipore, Bedford, MA, USA). The membranes were blocked in 5% fat-free milk for 1 h and then incubated at 4°C overnight with primary antibodies, including antibodies against nuclear factor-κB (NF-κB)/p65, activator protein 1 (AP-1), phosphorylated-p38-MAPK and β-actin. Subsequently, the membranes were incubated with secondary antibodies for 2 h at room temperature, and images were captured using ChemiDoc-It TS2 Imager (UVP, LLC, Upland, CA, USA). The relative protein expression was determined using ImageJ2× software (National Institutes of Health, Bethesda, MD, USA).
Data are expressed as the mean ± standard deviation and the Student's t-test was used for single statistical comparisons using SPSS version 13.0 (SPSS, Inc., Chicago, IL, USA) software package. P<0.05 was considered to indicate a statistically significant difference.
The present study investigated whether the anti-inflammatory effect of delphinidin was able to improve the BBB scores in a rat model of SCI. As shown in
The effect of delphinidin treatment on the intramedullary spinal pressure in SCI rats was investigated. There was a significant increase in the intramedullary spinal pressure in SCI rats when compared with the sham group (
To examine the anti-inflammatory effect of delphinidin in SCI rats, the activities of anti-inflammatory factors TNF-α and IL-6 were analyzed by ELISA. The TNF-α and IL-6 activities were found to be markedly increased in SCI rats, compared with the sham group (
In order to investigate the anti-inflammatory effect of delphinidin in SCI rats, the COX-2 and PGE2 levels were investigated by ELISA. The induction of SCI in the rats resulted in enhanced COX-2 activity (
To further determine the effect of delphinidin treatment on apoptosis in the SCI rats, caspase-3 activity was detected in the present study using ELISA. As shown in
To further investigate the anti-inflammatory effect of delphinidin in the rat model of SCI, NF-κB/p65 and Ap-1 protein expression levels were detected using western blot analysis. As shown in
To further study the molecular and cellular effects of delphinidin on SCI, the expression of phosphorylated p38-MAPK in the rat model of SCI was detected. At 2 weeks after SCI, the p-p38-MAPK protein expression in the SCI model group was markedly higher in comparison with that of the sham group (
Acute SCI is a trauma that may result in disability (
The inflammatory reaction is mainly regulated by the expression of several genes, while the NF-κB family is the major regulatory factor of inflammatory gene expression (
At present, TGF-β1 is increasingly investigated due to its association with the majority of fiber hyperplastic diseases (
COX-2 is typically considered to be harmful under pathological conditions, such as inflammation, pain, cellular damage and tumor. However, recent studies have proven that COX-2 also has an effect on the normal physiological function of humans (
It is known that p-p38-MAPK participates in signal transduction in the process of nerve cell apoptosis. Thus, strong or weak expression of p-p38 predicts whether nerve cells are alive (
In conclusion, the anti-inflammatory effect of delphinidin was able to alleviate the SCI-induced inflammation and intramedullary spinal pressure in a rat model of SCI. The present study confirms that the anti-inflammatory effect of delphinidin was achieved in SCI rats through theinhibition of COX-2 and PGE2 production, and the suppression of NF-κB and p38 MAPK signaling pathways.
Chemical structure of delphinidin.
Anti-inflammatory effect of Del alleviates the BBB scores in a rat model of SCI, following treatment with 40 or 200 mg/kg Del. *P<0.01 vs. SCI model group; #P<0.01 vs. sham group. Del, delphinidin; BBB, Basso, Beattie, Bresnahan; SCI, spinal cord injury.
Anti-inflammatory effect of Del treatment (40 or 200 mg/kg) alleviates the intramedullary spinal pressure in a rat model of SCI. *P<0.01 vs. SCI model group; #P<0.01 vs. sham group. Del, delphinidin; SCI, spinal cord injury.
Effect of Del treatment (40 or 200 mg/kg) on inflammatory factors in a rat model of SCI. The treatment increased the (A) TNF-α and (B) IL-6 activities in the spinal cord tissues of SCI rats. *P<0.01 vs. SCI model group; #P<0.01 vs. sham group. Del, delphinidin; SCI, spinal cord injury; TNF, tumor necrosis factor; IL, interleukin.
Del treatment (40 or 200 mg/kg) in a rat model of SCI reduced the COX-2 activity, as determined by ELISA. *P<0.01 vs. SCI model group; #P<0.01 vs. sham group. Del, delphinidin; SCI, spinal cord injury; COX-2, cyclooxygenase-2.
Del treatment (40 or 200 mg/kg) in a rat model of SCI reduced the PGE2 production, as determined by ELISA. *P<0.01 vs. SCI model group; #P<0.01 vs. sham group. Del, delphinidin; SCI, spinal cord injury; PGE2, prostaglandin E2.
Del treatment (40 or 200 mg/kg) in a rat model of SCI reduced the activity of caspase-3, which is an indicator of apoptosis, as determined by ELISA. *P<0.01 vs. SCI model group; #P<0.01 vs. sham group. Del, delphinidin; SCI, spinal cord injury.
Effect of Del treatment (40 or 200 mg/kg Del) on NF-κB expression in a rat model of SCI, as determined by western blot analysis. The anti-inflammatory effect of Del increased the NF-κB/p65 protein expression, shown in the (A) western blots and (B) quantified levels. *P<0.01 vs. SCI model group; #P<0.01 vs. sham group. Del, delphinidin; SCI, spinal cord injury; NF, nuclear factor.
Effect of Del treatment (40 or 200 mg/kg) on Ap-1 in a rat model of SCI as determined by western blot analysis. The anti-inflammatory effect of Del inhibited Ap-1 protein expression, shown in the (A) western blots and (B) quantified levels. *P<0.01 vs. SCI model group; #P<0.01 vs. sham group. Del, delphinidin; SCI, spinal cord injury; AP-1, activator protein 1.
Effect of Del treatment (40 or 200 mg/kg) on p38-MAPK in a rat model of SCI as determined by western blot analysis. The anti-inflammatory effect of Del inhibited p38-MAPK protein expression, shown in the (A) western blots and (B) quantified levels. *P<0.01 vs. SCI model group; #P<0.01 vs. sham group. Del, delphinidin; SCI, spinal cord injury.