Protective effects of orientin against spinal cord injury in rats

Abstract

We aimed to study the reparative effects of orientin against spinal cord injury (SCI) in rats and explore its potential mechanisms. Sprague–Dawley rats were divided into Sham, SCI, Orientin, and SB203580 [an inhibitor of p38 mitogen-activated protein kinase (p38MAPK)] groups. In the SCI group, rats underwent Allen’s beat. SCI animals in Orientin and SB203580 groups were respectively treated with 40 mg kg⁻¹ orientin and 3 mg kg⁻¹ SB203580 once daily. Functional recovery was evaluated based on Basso, Beattie, and Bresnahan scoring. Histopathological analysis was performed using hematoxylin-eosin and Nissl staining. Cell apoptosis was examined by TUNEL staining. The relative quantity of apoptosis-related proteins, glial fibrillary acidic protein (GFAP), neurofilament 200 (NF200), and brain derived neurotrophic factor (BDNF) was detected via western blotting. The indices related to inflammation and oxidation were measured using agent kits. The p38MAPK/inducible nitric oxide synthase (iNOS) signaling activity was detected using real-time quantitative PCR, western blotting, and immunohistochemical staining. Orientin was revealed to effectively mitigate cell apoptosis, neuroinflammation, and oxidative stress in impaired tissues. Meanwhile, orientin exerted great neuroprotective effects by abating GFAP expression, and up-regulating the expression of NF200 and BDNF, and significantly suppressed the p38MAPK/iNOS signaling. Orientin application could promote the repair of secondary SCI through attenuating oxidative stress and inflammatory response, reducing cell apoptosis and suppressing p38MAPK/iNOS signaling.

Introduction

Spinal cord injury (SCI) is a serious and currently incurable trauma imposed on the spinal cords, accompanied by complete or incomplete motor, sensory, and other dysfunctions. A primary mechanical injury and a subsequent secondary injury are involved in SCI. The direct and irreversible trauma that nerve cells, axial membrane, and blood vessels undergo comprise primary SCI, which will inevitably lead to an autodestructive secondary injury, mediated by a change in the biochemical conditions, such as local ischemia, lack of neurotrophic factors, and energy metabolism disorder. The degree of damage from secondary injury is far higher than that of the primary injury.

At the early stage of SCI, the local residual microglia and vascular endothelial cells are stimulated by the local adverse microenvironment, and therefore release several pro-inflammatory cytokines, including tumor necrosis factor alpha (TNF-α) and interleukin-1β [1]. The binding of these pro-inflammatory factors to their death receptors can activate one key death inducible protease caspase-8, which further triggers caspase-9 and caspase-3 activation, leading to the endogenous and exogenous apoptosis of cells in lesion sites. Meanwhile, ischemia and hypoxia are progressively aggravated along with the decrease of blood flow following SCI, and eventually induce masses of reactive oxygen radicals (ROS) to produce. Oxidative stress occurs when the antioxidant system is unable to scavenge excessive ROS. Thereby, a series of destructive reactions are elicited to occur, including lipid peroxidation, protein degradation, and DNA breakage, resulting in cell necrosis and ROS leak in the SCI area [2]. Leaked ROS further exacerbates the inflammatory response and cell apoptosis.

Additionally, traumas were revealed to trigger the phosphorylation of p38 mitogen-activated protein kinase (p38MAPK) by a series of enzyme-linked reactions, leading to the activation of p38MAPK signaling [3,4]. As a consequence, the expression of genes related to cell apoptosis and inflammatory response is upregulated, such as inducible nitric oxide synthase (iNOS). iNOS induces excessive production of endogenous NO, and a high level of NO combines with superoxide anions to form peroxynitrite anions (ONOO−). The ONOO− molecules will be degraded into radicals (hydroxide and nitrogen dioxide), with strong cytotoxicity inflicted upon the host cells. Namely, the activation of p38MAPK signaling contributes to the intense inflammation in the SCI sites by increasing the expression of iNOS [5]. Thus, effectively inhibiting the activity of p38MAPK/iNOS signaling may be a novel avenue for SCI therapy.

In conclusion, cellular, molecular, and biochemical cascades trigger extensive damages from secondary SCI if not controlled effectively. Therefore, screening for drugs that efficiently control the pathological process of secondary injury is significant for SCI repair.

Considering the promising effects of Chinese herbal medicines on neuroprotection and immune function regulation, their active ingredients have been widely used for SCI repair in animal experiments [6,7]. Orientin, a flavonoid monomer compound, is mainly extracted from Passiflora, Stenoloma chusanum, Lonicera japanica, and other medicinal plants. And it has been reported to exert a prominent influence on anti-inflammation, anti-oxidation, anti-apoptosis, and neuroprotection [8,9]. Orientin administration was revealed to effectively improve the anti-oxidative capacity and the neuronal ultrastructure, enhanced the activities of Na⁺/K⁺-adenosine triphosphatase (ATPase) and Ca²⁺/Mg²⁺-ATPase in the kidney, liver, and brain of aging mice induced by d-galactose, and therefore provided a protective effect against aging [10]; and previous studies demonstrated that orientin considerably protected brains from the ischemia/reperfusion injury in rats by relieving oxidative stress [11], as well as through inhibiting the upregulation of Aquaporin-4 and pro-inflammatory cytokines [12].

Prompted by the previous findings, we hypothesized herein that orientin could promote SCI repair based on its beneficial pharmacological action. We also hypothesized that orientin could inhibit the activation of p38MAPK/iNOS signaling and hereby alleviate the damage severity caused by secondary injury. For investigation of these hypotheses, orientin was used to treat SCI model rats, and its protective effects on traumatic spinal cords, including the potential underlying mechanism, were preliminarily analyzed.

Methods

Experimental design

Sprague–Dawley rats (Beijing HFK Bioscience Co. Ltd., Beijing, China) were kept separately in a specific-pathogen-free environment at 25 ± 2°C with 60 ± 10% humidity, a 12 : 12-h light/dark cycle, and free access to food and water. Ten-week-old Sprague–Dawley male rats (weighing 260–290 g) with good physiological conditions were chosen to reduce experimental bias, and randomly divided into SCI, Orientin, Sham, and SB203580 groups. SB203580 (Beyotime Institute of Biotechnology, Shanghai, China) is a p38MAPK signaling inhibitor widely used in animal experiments. Orientin group: Following SCI procedures, rats were immediately administered orientin (Pureone Biotech Co. Ltd., Shanghai, China) through the vena caudalis injection, and orientin (40 mg kg⁻¹ d⁻¹) was used for 10 days [10,12]. SCI group: Rats were subjected to SCI surgery and injected with equal volume of saline daily. Sham group: Rats underwent laminectomy, and were not subjected to Allen’s beat. SB203580 group: After the surgical operation, rats were intraperitoneally injected with 3 mg kg⁻¹ d⁻¹ SB203580 [13].

The timeline of sampling spinal cord tissues was mentioned in Supplementary material I, Supplemental digital content 2, http://links.lww.com/WNR/A758.

Spinal cord injury model

To minimize the suffering of experimental animals, adequate anesthesia with urethane (1.5 mg·kg^-1 body weight) [6] was performed via intraperitoneal injection. The anesthetized animals were placed on their front, and 5 mg penicillin was administrated through an intramuscular injection. After their backs were shaved and sterilized with iodophor, a dorsal 3-cm longitudinal cut was made covering the T₉–T₁₁ vertebrae, and paraspinal muscles were dissected, where the spinal cords were exposed by laminectomy. Spinal traumas were induced by Allen’s beat with a 10-g rod (dropped from a height of 5 cm) at the T₁₀ level. Following the injury, muscle and skin incisions were sewn up. And then animals were placed on a heating pad to maintain the body temperature until they became conscious. Postoperatively, their bladders were manually emptied twice a day until they regained a conditioned response.

The SCI model was established according to the experimental procedures approved by the Animal Ethics Committee of Hebei North University (Hebei, China; reference no 2019-1-9-15). Due to death of 16 rats post-SCI, 76 rats were used in all experimental procedures. After SCI surgery, fentanyl (Yichang Humanwell Pharmaceutical Co. Ltd., Hubei, China) at the dosage of 2.5 μg/kg was applied to ease the pain relief imposed on experimental rats through intramuscular injection according to the drug instructions.

Animal locomotion scale

Following SCI surgery, the hindlimb locomotor function of rats (n = 3) was evaluated once every 3 days until the 30^th day after trauma. The rats were placed in an oval open field with a diameter of 1.5 m × 0.6 m. Three trained evaluators, blinded to the experimental conditions, observed in earnest the hindlimb movements for 2 min, and scored between 0 and 21 for motor function according to the Basso, Beattie, and Bresnahan (BBB) scoring system [14], which includes 21 different criteria.

Tissue sections preparation for light microscopy

After the intraperitoneal anesthesia as aforementioned, intracardiac perfusion was performed to experimental animals, and the spinal cord biopsies (n = 3) were sampled on the 11^th day from trauma for light microscopy. Approximately 0.5 cm tissue segments were selected from each side of the lesion sites. Thereafter, fixation in 4% paraformaldehyde solution for 12 h, dehydration with graded ethanol for 30 min per solution, dimethylbenzene vitrification for 40 min were performed at room temperature (RT), followed by paraffin embedding for 1 h in a 60 °C oven. Finally, 6-μm-thick sections were prepared for hematoxylin-eosin (H&E), Nissl, TUNEL, and immunohistochemical staining.

Hematoxylin-eosin staining

Five tissue sections from every group were collected at random and deparaffinized twice (10 min/time) in dimethylbenzene. Subsequently, these sections were gradually rehydrated in gradient of alcohol solutions (100–70%) for 5 min per solution, and then immersed in deionized water for 2 min. Finally, the sections were stained with H&E reagents (Solarbio Science & Technology Co. Ltd., Beijing, China) for 5 min each in order, respectively. Following air drying for 5 min, the slides were sealed with epoxide resin.

The pathological changes were evaluated using a 6-point scale [6] under an optical microscope (Image M2; Carl Zeiss AG, Oberkochen, Germany). The scoring rules were set as follows: 0, cells are arranged closely, and a normal morphology of neurons was presented; 1, a few eosinophilic cells distributed in the spinal cord tissue; 2, cells shrank slightly and are surrounded by small vacuoles; 3, cells shrank highly, and the enlarged gaps between cells were easily visible; 4, the spinal cord membrane was seriously damaged, and infarction accounted for one third in the gray matter area; 5, large infarction accounted for more than half of the gray matter area, and shrunk cells look necrotic. The histopathological scoring was performed by experienced histopathologists blinded to the experiment grouping.

Nissl staining

Following dewaxing and rehydration according to the above methods, the randomly selected tissue sections were immersed in 1% Methyl Violet at 37 °C for 15 min, and rinsed with double-distilled water twice. They were then treated with Nissl Differentiation agent (Beyotime) for 5 s, followed by dehydration using gradient of alcohol solutions (70–100%) for 5 min each in order. After vitrification in xylene and washes in PBS, the sections were mounted onto a coverslip and studied under the light microscope.

TUNEL assay

Dewaxing and rehydration were conducted as above, and five sections from every group were stained using TUNEL detection kits (Boster Biotech Co. Ltd., Wuhan, China) according to the manufacturer’s instruction. The sections were incubated with 20 μg ml⁻¹ protease K (without DNase) for 30 min at RT, followed by rinsing with Tris-buffered saline (TBS) for 3 min. Endogenous peroxidase was then inactivated by 3% H₂O₂ for 10 min at RT. Next, each sample was covered with 20 μl labeling buffer and 10 μl Biotin labeled Digoxin antibody, followed by incubation in the dark at 37 °C for 1 h. Thereafter, 3,3-diaminobenzidine agent (Beyotime) was then used for coloration and hematoxylin for redyeing. Finally, the tissue sections were washed with TBS, sealed neutral balsam mounting medium, and observed under a light microscope.

Immunohistochemical staining

After dewaxing and rehydration as above, 3% H₂O₂ was used to quench endogenous peroxidase for 30 min, and then incubation with 2% (v/v) normal goat serum was performed to block nonspecific adsorption for 1 h. Thereafter, all sections were incubated with iNOS antibody (Bioss Biological Co. Ltd., Beijing, China; 1 : 500 in PBS, v/v) overnight at 4 °C. Following PBS wash for three times (10 min/time), incubation with secondary antibody (conjugated with fluorescein isothiocyanate) was kept for 2 h. Finally, 0.5 µg/ml Hoechst 33258 (Beyotime) was used to stain nuclei for 5 min at RT, and the superfluous fluorescence dye was washed away using PBS. For quantitative analysis, nine view fields from three independent parallel experiments were used to count the number of iNOS-positive cells and the nuclei stained with Hoechst 33258. And the rates of iNOS-positive cells was calculated.

Sampling for all biological assays

After carbon dioxide euthanasia, the lesion spinal cords (2.0 cm in length) were immediately extracted from experimental rats on the 8^th day from trauma, and washed using precool PBS. One half of each sample was cut into small pieces, weighed and homogenized on ice. Following 4 °C centrifugation at 8000 g for 20 min, the supernatant was collected and frozen in liquid nitrogen for the assay of anti-oxidation, anti-inflammation, and western blotting. The other was ready for real-time quantitative PCR (RT-qPCR).

Anti-oxidation assay

To examine the inhibitory effect of orientin on oxidative stress following SCI, the content of malondialdehyde, and the relative activity of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) were measured by their corresponding methods: thiobarbituric acid microplate method at a wavelength of 530 nm, 5,5-dithiobis-(2-nitrobenzoic acid) colorimetric method at a wavelength of 420 nm, and nitrotetrazolium blue chloride method at a wavelength of 560 nm according to the manufacturer’s instructions. All kits were purchased from Shanghai BestBio Biological Co. Ltd (Shanghai, China).

Anti-inflammation assay

To analyze the anti-inflammatory effects of orientin, ELISA (Mskbio Biological Co. Ltd., Wuhan, China) was performed following the manufacturer’s instructions. The tissue supernatant samples prepared above were used to measure the expression of TNF-α, interleukin-1β, interleukin-4, and interleukin-10. The optical density (OD) values were read in a microplate reader (Multiskan FC; ThermoFisher Corporation, Waltham, USA) at a wavelength of 450 nm.

Western blotting

Western blotting was performed using the tissue supernatants aforementioned. Based on the determination using bicinchoninic acid kit (BestBio Biotech Co. Ltd., Shanghai, China), the concentrations of protein samples were adjusted to 2.5 mg/ml using 2% SDS containing 5× loading buffer. And 10 µl samples containing 25 µg protein were loaded onto the 12% SDS-PAGE, and then the protein bands were electrotransferred onto polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were blocked with 10% dry milk for 1 h, and incubated with the rabbit anti-rat primary antibodies for 2 h. Following washes with PBS-T (PBS containing 0.1% Tween-20) for three times, incubation with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1 : 5000 dilution; ABclonal Co. Ltd., Wuhan, China) was carried out for 1 h. Finally, the protein bands were visualized using chemiluminescence detection reagent (Beyotime). All abovementioned incubations and procedures were carried out at RT. The primary antibodies of proteins detected were used in this paper, involved in cell apoptosis, cleaved caspase-3 (1 : 1000 dilution; Cell signaling Technology Co. Ltd., Shanghai, China), Bax (1 : 1000 dilution; Bioss), and Bcl-2 (1 : 1000 dilution; Bioss); neuroprotection, glial fibrillary acidic protein (GFAP) (1 : 1000 dilution; Bioss), brain derived neurotrophic factor (BDNF) (1 : 1000 dilution; Bioss), and neurofilament 200 (NF200) (1 : 1000 dilution; Bioss); and p38MAPK/iNOS signaling, p38 (1 : 2000 dilution; Bioss), p-p38 (1 : 5000 dilution; ABclonal), and iNOS (1 : 1000 dilution; Bioss). β-actin antibody (1 : 10 000 dilution; ABclonal) was used as the loading control.

Real-time quantitative PCR

To trace the inhibitory effect of orientin on p38MAPK/iNOS signaling at the early stage of SCI, RT-qPCR was carried out. Following euthanization of rats through carbon dioxide intoxication, samples of spinal tissues were taken from rats of four experimental groups on days 2, 4, and 8 from trauma, from which total RNA was extracted using TRNzol reagent (Tiangen Biotech Co. Ltd., Beijing, China). When the OD values were 1.9–2.0 at a wavelength of 260/280 nm, the RNA samples were used for the reverse transcription into cDNA using First-Strand cDNA Synthesis kit (Tiangen). The synthesized cDNA as the RT-qPCR template, RT-qPCR amplifications were conducted using the Premix Taq PCR kit (Tiangen). The thermocycling conductions were as follows: initial denaturation at 94 °C for 5 min, followed by 30 cycles of amplification, including denaturation at 94 °C for 40 s, annealing for 40 s, and extension at 72 °C for 2 min, followed by one cycle at 72 °C for 10 min. β-actin was used as an internal control. Compared with the control, the relative quantity of target gene mRNA was plotted as a fold change using the 2^−ΔΔCq method [15]. The specific primers of related genes and their information are shown in Supplementary Table I, Supplemental digital content 3, http://links.lww.com/WNR/A759.

Statistical analysis

All statistical data were analyzed using SPSS 17.0 software (SPSS, Inc, New York, USA), and are presented as the mean ± SD. The significant differences among multiple groups were determined using one-way analysis of ANOVA followed by Tukey’s post hoc significance test. P < 0.05 was considered to indicate a statistically significant difference.

Results

Locomotor assessment

The hindlimb motor functions of all rats were evaluated using BBB scoring (Fig. 1). Due to the low level of injury imposed, the locomotion behavior of rats in the Sham group quickly recovered (within 6 days). In the SCI group, rats exhibited complete bilateral hindlimb dyskinesia and progressively regained slight motor functions from the 15^th day after trauma; however, their BBB scores were only 5.18 ± 0.98 on day 30 after trauma. Although the locomotion scores in the Orientin group were much lower than those in the Sham group (^**P < 0.01), the experimental animals obtained significant functional recovery compared with the SCI group from day 9 after trauma (^##P < 0.01). In addition, SB203580 was revealed to improve the motor function of SCI rats, however the BBB scores in the SB203580 group were lower than those of the Orientin group (^♦P < 0.05 and ^♦♦P < 0.01) during the functional evaluation.

Fig. 1.

Assessment of neurological function using BBB scoring. Compared to the Sham group, functional recovery was gradually obtained in the other groups from the 3^rd day of trauma. And better functional recovery was obtained in the orientin group than the SCI and SB203580 groups from the 9^th day post-SCI. n = 3. ^**P < 0.01, the other groups vs. Sham group; ^##P < 0.01, Orientin and SB203580 groups vs. SCI group; ^♦P < 0.05 and ^♦♦P < 0.01, Orientin vs. SB203580 group. BBB, Basso, Beattie, and Bresnahan; SCI, spinal cord injury.

Pathomorphological evaluation through hematoxylin-eosin and Nissl staining

On the 11^th day after trauma, H&E staining was performed to evaluate the pathomorphological changes. As presented in Fig. 2a, compared with the normal structure of spinal tissues, severe damages were easily visible in the perilesional tissues: the cell bodies of neurons shrank, the empty vacuoles and a large number of necrotic neurons were clearly seen to surround the shrunken neurons, and the cell nucleus were condensed. A significantly protective effect of orientin against SCI-associated damage was clearly observed: neurons with normal-like morphology had clear boundaries, less edema, and fewer cavities were detected in comparison to the SCI group, and the disordered microstructural structure was ameliorated. The reparative effect of SB203580 on the injured spinal cords was much weaker than that of orientin, where visible vacuoles and shrunken neurons were clearly observed.

Fig. 2.

Histopathological evaluation via H&E and Nissl staining on the 11^th day post-SCI. (a and b) H&E staining and histopathological scoring exhibited orientin exerted a marked protective effect on spinal cords against trauma. (c and d) Nissl staining and quantification of Nissl bodies: compared to the sham group, SCI led Nissl bodies to significantly decrease; But the number and morphology of Nissl bodies were notably preserved by orientin treatment. n = 3. ^**P < 0.01, the other groups vs. Sham group; ^#P < 0.05 and ^##P < 0.01, Orientin and SB203580 groups vs. SCI group; ^♦P < 0.05, Orientin vs. SB203580 group. Magnification, 200×. H&E, hematoxylin-eosin; SCI, spinal cord injury.

Histopathological scoring using a six-point scale (Fig. 2b) showed: rats in the SCI group incurred much higher scores than the other groups; treatment with orientin and SB203580 significantly reduced the histopathological scores associated with SCI, and the scores of the Orientin group were lower than those of the SB203580 group.

Additionally, Nissl staining and quantification analysis (Fig. 2c and d) revealed that: (i) in the Sham group, the gray matter of spinal cords contained normal neurons with abundant Nissl substance hypochromatic chromatin, and conspicuous nucleoli; (ii) neurons in the SCI group were seriously impaired, which was evidenced by a vacuolated cytoplasm without Nissl substance; (iii) SB203580 could ameliorate the damage to a certain extent; (iv) by contrast, orientin treatment better maintained neuron morphology of the gray matter, and abundant Nissl substance could be observed in the cytoplasm of well-maintained neurons.

Cell apoptosis assay

The antiapoptotic effect of orientin was estimated using TUNEL staining (Fig. 3a and b). Compared with a few apoptotic cells detected in the Sham group, a great number of TUNEL-positive cells were observed in the lesion spinal tissues. There was a significant difference in the number of apoptotic cells in one field of vision between the Sham and SCI groups (^**P < 0.01, 16.33 ± 3.78 vs. 86.67 ± 6). The quantification of TUNEL-positive cells demonstrated that although both orientin and SB203580 reduced the rate of apoptotic cells in the damaged tissues compared with the SCI group (^##P < 0.01), the antiapoptotic effect of orientin was more prominent than that of SB203580 (^♦P < 0.05).

Fig. 3.

Analysis of cell apoptosis using TUNEL staining and Western blotting. (a and b) TUNEL staining and its quantitative analysis revealed that in comparison with SCI group, the quantity of TUNEL-positive nuclei was considerably reduced in the orientin group. (c and d) Western blotting of apoptosis-associated proteins and quantification of the protein band densities further verified that orientin notably reduced the relative quantity of c.casp3 and Bax, but up-regulated the Bcl-2. n = 3. ^**P < 0.01, the other groups vs. Sham group; ^##P < 0.01, Orientin and SB203580 groups vs. SCI group; ^♦P < 0.05, Orientin vs. SB203580 group. What were pointed out with arrows means the nuclei of apoptotic cells in all groups. Magnification, 200×. c.casp, cleaved caspase; SCI, spinal cord injury.

The results of western blotting further confirmed the inhibitory effect of orientin on cell apoptosis. As shown in Fig. 3c and d, orientin significantly reduced the relative quantity of pro-apoptosis proteases c. caspase-3 and Bax compared with the SCI group (^##P < 0.01), but notably enhanced the expression level of one anti-apoptosis protein Bcl-2 (^##P < 0.01, orientin vs. SCI group).

Inhibitory effects of orientin on oxidative stress and inflammation

SCI notably increased malondialdehyde content, which was approximately two times more than that of the Sham group (^**P < 0.01) (Fig. 4a). Meanwhile, the relative activity of GSH-Px and SOD in the SCI group only accounted for ~40% of that in the Sham group (^**P < 0.01) (Fig. 4b and c). As compared with SB203580, orientin administration effectively decreased malondialdehyde content and recuperated the relative activity of SOD and GSH-Px to a certain degree (^♦P < 0.05). Meanwhile, the SCI-induced inflammation was significantly suppressed by orientin and SB203580 through reducing the levels of pro-inflammatory cytokines and increasing the levels of anti-inflammatory cytokines (Fig. 4d and e; ^##P < 0.01, Orientin and SB203580 groups vs. SCI group). And SB203580 was revealed to exert more potent inhibitory action on the expression of TNF-α, but had no marked effects on the other three inflammatory factors (^♦P < 0.05, Orientin vs. SB203580 group).

Fig. 4.

Quantification assays of oxidant stress markers and inflammatory cytokines. (a–c) Determining results of MDA, GSH-Px, and SOD revealed the increase in oxidative stress induced by SCI was significantly attenuated by orientin. (d and e) Quantification analysis of pro-inflammatory cytokines (TNF-α and IL-1β), and anti-inflammatory (IL-4 and IL-10) cytokines showed orientin significantly alleviated inflammatory response induced by SCI. n = 3. ^**P < 0.01, the other groups vs. Sham group; ^#P < 0.05 and ^##P < 0.01, Orientin and SB203580 groups vs. SCI group; ^♦P < 0.05, Orientin vs. SB203580 group. GSH-Px, glutathione peroxidase; MDA, malondialdehyde; SOD, superoxide dismutase; SCI, spinal cord injury.

Assay of orientin neuroprotection

The relative quantity of GFAP, NF200, and BDNF was determined by western blotting, to elucidate the neuroprotective effects of orientin on the perilesional tissues. As shown in Fig. 5, the expression of GFAP in the SCI group was increased about four times that of the Sham group (^**P < 0.01), but the expression of NF200 was markedly suppressed by SCI (^**P < 0.01). In addition, SCI led to an increase in BDNF expression, and the difference was statistically significant between the SCI and Sham group (^*P < 0.05). Immediate treatment with orientin and SB203580 significantly abated GFAP expression, and upregulated the expression of NF200 and BDNF (^#P < 0.05 and ^##P < 0.01, orientin vs. SCI group); and the neuroprotection of orientin was notably higher than that of SB203580 (^♦P < 0.05 and ^♦♦P < 0.01, orientin vs. SCI group).

Fig. 5.

Detection of proteins associated to neural repair via western blotting. Western blotting results for (a) GFAP, (b) NF200, and (c) BDNF, and their quantitative analysis (d–f) demonstrated that orientin treatment significantly suppressed GFAP expression, but upregulated NF200 and BDNF. n = 3. ^*P < 0.05 and ^**P < 0.01, the other groups vs. Sham group; ^#P < 0.05 and ^##P < 0.01, Orientin and SB203580 groups vs. SCI group; ^♦P < 0.05 and ^♦♦P < 0.01, Orientin vs. SB203580 group. BDNF, brain derived neurotrophic factor; GFAP, glial fibrillary acidic protein; NF200, neurofilament 200; SCI, spinal cord injury

Suppressive effects of orientin on p38/inducible nitric oxide synthase signaling

The RT-qPCR results showed that SCI surgery incurred a marked increase in the mRNA expression levels of p38 and iNOS (^*P < 0.05 and ^**P < 0.01); and treatment with orientin and SB203580 for 7 days notably suppressed p38 and iNOS expression (^#P < 0.05 and ^##P < 0.01). The quantification analysis (Fig. 6a and b) exhibited that the relative quantity of p38 and iNOS mRNA only accounted for about 70% of those in the SCI group, respectively. The abovementioned results of RT-qPCR were verified via western blotting (Fig. 6c and d). It revealed that orientin treatment could significantly suppress the phosphorylation of p38 (^##P < 0.01), and the relative quantity of iNOS was consequently decreased. Immunohistochemical staining further confirmed that to a certain degree, orientin protection indeed prevented the expression amount of iNOS from increasing induced by SCI (^##P < 0.01).

Fig. 6.

Analysis of p38MAPK/iNOS signaling using RT-qPCR and western blotting. (a and b) On the 2^nd, 4^th, and 8^th day after SCI, RT-qPCR detection showed orientin could obviously suppress the relative quantity of p38 and iNOS mRNA. (c and d) On the 8^th day after SCI, western blotting of p38, p-p38, and iNOS and quantification of the band densities confirmed that orientin significantly suppressed the expression of p38 and iNOS, and the phosphorylation of p38, which was helpful to reduce the activity of p38MAPK/iNOS pathway. (e and f) Immunohistochemical staining and quantitative analysis revealed that compared to SCI group, orientin markedly the rate of iNOS-positive cells. n = 3. ^*P < 0.05 and ^**P < 0.01, the other groups vs. Sham group; ^#P < 0.05 and ^##P < 0.01, Orientin and SB203580 groups vs. SCI group; ^♦P < 0.05 and ^♦♦P < 0.01, Orientin vs. SB203580 group. iNOS, xxx; p38MAPK, p38 mitogen-activated protein kinase; RT-qPCR, real-time quantitative PCR; SCI, spinal cord injury.

Discussion

Although the pathophysiological mechanism of SCI remains obscure, inflammation and oxidative stress have been suggested to be the main inducing factors of secondary SCI [16]. Following SCI, a series of pre-inflammatory factors, including TNF-α, interleukin-1β, and interleukin-6, were released by M1 microglia cells, and therefore astrocytes were activated into reactive astrocytes [2]. In turn, a large number of pre-inflammatory factors are released by the reactive astrocytes, and therefore the inflammatory reaction is further exacerbated. Persistent and excessive inflammatory reaction can deteriorate injury degree. Furthermore, SCI-induced hypoxia and ischemia in the spinal cords leads to hypoxia metabolism in vascular endothelial cells and nerve cells, accompanied by ROS production. Excessive ROS eventually leads to lipid peroxidation of spinal cord tissues, destruction of cell membrane, cell collapse, extracellular Ca²⁺ influx, and arachidonic acid cascade reaction, leading to cell apoptosis. In view of its potent anti-inflammatory, anti-oxidant, and antiapoptotic effects [17], orientin was used for SCI repair in our present study, and its reparative effects and the underlying mechanism were explored. Thankfully, our present study demonstrated that orientin administration markedly ameliorated SCI-induced inflammation by inhibiting the release of pro-inflammatory cytokines (TNF-α and IL-1β) and increasing the content of anti-inflammatory cytokines (IL-4 and IL-10). In addition, treatment with orientin significantly decreased the level of oxidative products (malondialdehyde), and increased the relative activities of antioxidant enzymes (SOD and GSH-Px). These finding proved that based on its efficient anti-oxidative and anti-inflammatory actions, orientin could optimize the microenvironment of SCI area to promote cell survival and SCI repair.

Apoptosis was considered to be an important promoter of neuronal death following SCI and plays a crucial role in paraplegia [16]. Our study demonstrated that, in contrast with the SCI and SB203580 groups, orientin treatment considerably decreased SCI-induced TUNEL-positive cells. And western blotting further confirmed the results of TUNEL staining. Namely, compared with the SCI group, orientin intervention markedly reversed the activation of the executive enzyme (caspase-3 and Bax), and effectively enhanced the expression of Bcl-2, and therefore the Bcl-2 : Bax ratio was increased, which is conductive to suppressing SCI-induced cell apoptosis and the recovery of neurological function of SCI rats.

In addition to the suitable microenvironment, the recovery of motor function in SCI animals was dependent on the differentiation of neural stem cells into neurons and axon regeneration. NF200 and BDNF play an important role in nerve regeneration [18,19]. As one of the unique structural proteins of neurons, NF200 plays a crucial role in the morphology, structure, and physiological function in neurons. BDNF, a broad-spectrum neurotrophic factor, can activate TrkB signaling pathway, and the BDNF/TrkB pathway exerts an important impact on neuroprotection, inflammatory regulation, and synapse plasticity following central nervous system damage [20]. And it has been proved GFAP is upregulated following SCI, and further promotes the formation of glial scars in the SCI region, which is not beneficial for the recovery of nerve function [21]. In the present research, compared to SB203580, orientin was exhibited to exert a significantly therapeutic role in promoting SCI repair by abating the expression of GFAP, and maintaining the expression of NF200 and BDNF at a high level.

An increasing number of studies have shown that the activation of p38MAPK signaling could intensify neuronal apoptosis, neuron degeneration, and neuroinflammation following SCI [20,22]. The phosphorylation level of p38 reflects the degree of tissue damage [5]. p-p38 can upregulate iNOS expression by phosphorylating the activating transcription factor 2. iNOS uses arginine as the substrate to produce excessive NO, resulting in nitrification stress and aggravation of the inflammatory response, which further worsens secondary SCI. SB203580, an inhibitor of p38MAPK signaling, was employed as reference to determine the suppressive effect of orientin against the p38MAPK/iNOS pathway in our study. And it was revealed that orientin application not only downregulated p38 but also inhibited p38 phosphorylation, and further decreased the relative quantity of iNOS in a great part, which benefits to accelerate SCI repair.

In conclusion, the present study suggested that the use of orientin inhibited neuron apoptosis and further promoted the recovery of motor function in the SCI rats, through attenuating inflammation and oxidative stress, enhancing the expression of NF200 and BDNF, and inhibiting p38/iNOS signaling. These findings suggested that elucidating the pathophysiological mechanism of SCI and repairing SCI in clinically relevant model animals are of great significance. The putative therapeutic effects of orientin demonstrated in the present study showed that it will be a potential therapeutic candidate for SCI.

Acknowledgements

This work was supported by Applied Basic Research Project of Zhangjiakou Science and Technology Bureau [grant number 1911020D], General Project of Hebei North University [grant number YB2018034], Morphology Experimental Teaching Center of Hebei Province [grant number XTZX201901], College Students’ innovation and entrepreneurship training program [grant number S202110092005], and projection of Hebei North University (grant number C2023405008).

All authors claim that none of the material in the paper has been published or is under consideration for publication elsewhere.

All animal experiments were approved by the Animal Ethics Committee of Hebei North University (Hebei, China; reference nο. 2019-1-9-15).

Conflicts of interest

There are no conflicts of interest.