Rosmarinic acid exerts a neuroprotective effect on spinal cord injury by suppressing oxidative stress and inflammation via modulating the Nrf2/HO-1 and TLR4/NF-κB pathways CURRENT STATUS: POSTED

Background: Spinal cord injury (SCI) is a severe central nervous system injury for which few efficacious drugs are available. Rosmarinic acid (RA), a water-soluble polyphenolic phytochemical, has antioxidant, anti-inflammatory, and anti-apoptotic properties. However, the effect of RA on SCI is unclear. We investigated the therapeutic effect and underlying mechanism of RA on SCI in vivo and in vitro. Methods: In vivo experiment, The BBB locomotion scale, the inclined plane test, Nissl staining, and spinal cord edema were employed to determine the neuroprotective effects of RA treatment after SCI. Inflammatory and oxidative stress markers were detected by commercial kits and cell apoptosis status was measured by TUNEL staining. A proteomics and bioinformatics approach, together with Western blotting, was used to investigate the effect of RA on the proteome of SCI rats. In vitro experiment, oxidative stress and inflammatory injury were induced by H2O2 and LPS stimulation. Effects of RA on cell viability, apoptosis, inflammatory, and oxidative stress were evaluated. Results: Using a rat model of SCI, we showed that RA improved locomotor recovery after SCI and significantly mitigated neurological deficit, increased neuronal preservation, and reduced apoptosis. Also, RA inhibited activation of microglia and the release of TNF-α, IL-6, and IL-1β and MDA. Moreover, proteomics analyses identified the Nrf2 and NF-κB pathways as targets of RA. Pretreatment with RA increased levels of Nrf2 and HO-1 and reduced those of TLR4 and MyD88 as well as phosphorylation of IkB and subsequent nuclear translocation of NF-κB-p65. Using H2O2- and LPS-induced PC12 cells, we found that RA ameliorated the H2O2-induced decrease in viability and increase in apoptosis and oxidative injury by activating the Nrf2/HO-1 pathway. Also, LPS-induced cytotoxicity and increased apoptosis and inflammatory injury in PC-12 cells were mitigated by CO 2 parts. 1, whether RA protects against H 2 O 2 -induced oxidative injury in PC12 cells. PC12 cells in logarithmic phase allocated into the control, H 2 O 2 and RA (5, 10, and 20 µg/mL) groups. In the control group, cells treated with normal medium, in the H 2 O 2 group

Introduction rats were i.p. injected with 1% pentobarbital (40 mg/kg) before surgery to induce adequate analgesia.
The animals were placed in the prone position. A 2-cm midline incision was made at the level of the T8-T10 vertebra, the paraspinal muscles were separated, and the T8-T10 spinal processes were removed. Laminectomy of ninth vertebra was performed to adequately expose the spinal cord. Next, a vascular clip (30 g force, Oscar, China) was used to compress the spinal cord for 30 s without destroying the dura mater, and the incision sites were sutured. The sham rats were subjected to the same surgery procedure except for compression injury. Bladder massage was performed twice a day to assist urination until micturition. RA (10,20, and 40 mg/kg) was dissolved in normal saline (NS) and administered intraperitoneally (i.p.) daily for 7 or 14 consecutive days. Rats in the sham and SCI groups received an equivalent volume (1 mL) of NS as vehicle.

In vivo experimental design
The animal experiment was divided into two parts. In part 1, we evaluated the effect of RA on SCI rats and determined the mechanism (Fig. 1). Sixty female SD rats were randomly allocated into the sham, SCI, SCI + 10 mg/kg RA, SCI + 20 mg/kg RA, and SCI + 40 mg/kg RA groups (n = 12 each). Six rats per group were euthanized for the neurological-function and histological studies. The remaining rats in each group (n = 6) were used for molecular biological and biochemical experiments. Rats in the sham and SCI groups were injected i.p. with NS, and rats in the three SCI + RA groups received i.p. injection of 10, 20, and 40 mg/kg RA, respectively, once per day for 14 successive days. At 7 days after SCI, the rats were euthanized and the injured spinal cord lesions (6 mm around the center) were collected. The samples were rapidly fixed or frozen for further study. In part 2, we investigated the neuroprotective mechanism of RA on SCI. Sixty female SD rats were randomized into the sham, SCI, SCI + RA (20 mg/kg), SCI + RA (20 mg/kg) + ML385 (500 µg/kg, inhibitor of Nrf2), and SCI + ML385 (500 µg/kg) groups (n = 12 each). Rats in the sham and SCI groups were injected i.p. with NS, rats in the SCI + RA + ML385 group received i.p. injection of 20 mg/kg RA and intrathecal (i.t.) injection of 500 µg/kg ML385 (in DMSO) 1 h before administration of RA, and the rats in the SCI + RA and SCI + ML385 groups were injected i.p. with 20 mg/kg RA and i.t. with 500 µg/kg ML385, once per day, respectively, for 14 consecutive days. The rats were treated in the same manner as in part 1.

2-DE and imaging analyses
The 2-DE analysis was performed as described previously [32,33]. Briefly, 100 mg of spinal cord tissue (SCI group and SCI + 20 mg/kg RA group) were harvested and lysed in 1 mL of lysis buffer.
Whole-cell lysates (80 μg) were added to a 17-cm immobilized pH 3-10 nonlinear gradient strip (Bio-Rad Laboratories) and rehydrated in an Ettan IPG-phor isoelectric focusing system (Bio-Rad) at 20°C and 30 V for 10 h. Sample uptake into the strip was achieved at 20°C using the following settings: 250 V for 30 min, 1,000 V for 1 h, and 500 V for 10 h. Next, the strips were incubated in equilibration buffer containing 1% (w/v) dithiothreitol and 2.5% (w/v) iodoacetamide in 6 M urea, 0.375 M Tris-HCl (pH 8.8), 20% glycerol, and 2% sodium dodecyl sulfate (SDS). The second-dimension separation was performed using 12% SDS-polyacrylamide gel electrophoresis (PAGE) in two steps at 10°C: 70 V/gel for 30 min and 300 V/gel until the bromophenol blue reached the bottom of the gel. The gels were stained using a silver staining kit (CWBIO), spots were detected using a GS-800 calibrated densitometer (Bio-Rad Laboratories), and the images were analyzed using PDQuest™ 2-DE (ver. 8.0.1; Bio-Rad Laboratories). Protein spots were extracted from the 2-DE gels and identified by MALDI-TOF/MS, and protein-protein interaction network and gene ontology (GO) analyses of differential proteins were performed as described previously [32,33].

Cell culture and experimental design in vitro
PC12 cells originate from a rat adrenal pheochromocytoma cell line, have neuron-like characteristics, and have been used for SCI research in vitro [34,35]. PC12 cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Waltham, MA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific) and 1% penicillin/streptomycin (Gibco) at 37°C in a humidified atmosphere with 5% CO 2 . The experiment was divided into two parts. In part 1, we investigated whether RA protects against H 2 O 2 -induced oxidative injury in PC12 cells. PC12 cells in logarithmic phase were allocated into the control, H 2 O 2 , and RA (5, 10, and 20 µg/mL) groups. In the control group, cells were treated with normal medium, and those in the H 2 O 2 group were treated with 200 µM H 2 O 2 . Cells in the RA group were pretreated with RA for 24 h and subsequently with 200 µM H 2 O 2 for 24 h. In part 2, we assessed the protective effect of RA on LPS-induced inflammatory injury in PC12 cells. PC12 cells were allocated into the control, LPS, and RA (5, 10, and 20 µg/mL) groups.
Control cells were treated only with normal medium, and LPS-injury cells were treated with LPS (5 µg/mL) for 12 h. PC12 cells in the RA groups were pretreated with various concentrations of RA for 24 h prior to exposure to LPS (5 µg/mL) for 12 h.

Cell viability assay
The viability of PC12 cells was determined by a Cell Counting Kit-8 (CCK-8) assay. In brief, PC12 cells were seeded onto 96-well plates (5 × 10 3 per well) containing 100 μL of complete medium for 24 h.
Next, 10 μL of CCK-8 (Beyotime; Jiangsu, China) solution was added to each well, and the plate was incubated at 37℃ for 2 h. Finally, absorbance at 450 nm was measured using a microplate reader (Bio-Rad; Hercules, CA).

Lactate dehydrogenase release assay
A lactate dehydrogenase (LDH) release assay was used to evaluate cytotoxicity by detecting the content of LDH in culture supernatant. Medium was harvested and the LDH content was determined using an LDH release assay kit (Beyotime; Jiangsu, China) according to the manufacturer's protocol.
Absorbance at 450 nm was measured using a microplate reader.

Annexin V-fluorescein isothiocyanate/propidium iodide assays of apoptosis
To assess apoptosis, the PC12 cells (1 × 10 5 per well) were seeded onto six-well plates for 24 h. The cells were harvested, washed twice with PBS, resuspended in 195 μL of binding buffer containing annexin V-FITC (5 μL) and propidium iodide (PI; 10 μL) using an apoptosis assay kit (Beyotime; Jiangsu, China), and incubated in the dark for 15 min. The apoptosis rate was analyzed by flow cytometry (BD Biosciences; Piscataway, NJ).

Measurement of reactive oxygen species concentration
The intracellular reactive oxygen species (ROS) concentration was measured using an ROS assay kit with dichloro-dihydro-fluorescein diacetate (DCFH-DA, Beyotime), which is oxidized to fluorescent DCF by hydroperoxides. Cells were collected, resuspended in PBS, and stained with DCFH-DA according to the manufacturer's directions. The level of ROS was determined by flow cytometry.

Detection of cytokine expression and oxidative stress markers
The collected spinal cord tissue was thawed, weighed, and dissolved in 1 mL of 0.9% normal saline at 4°C, and the homogenate was obtained by centrifugation at 10000 g for 15 min. Next, the levels of inflammatory factors (TNF-α, IL-6, IL-1β, and IL-10) in spinal cord supernatant and PC12 cell culture supernatant were assayed using ELISA kits according to the manufacturers' instructions (Yuanye Biotechnology, Shanghai, China). Absorbance at 450 nm was measured using a microplate reader.
Biochemical kits (Beyotime; Jiangsu, China) were used to determine the level of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and malondialdehyde (MDA) in the supernatant of perilesional spinal cord tissue and PC12 cells according to the manufacturer's instructions. Protein contents were measured using a bicinchoninic acid (BCA) assay kit (Beyotime).

Quantitative real time RT-PCR
Total RNA was isolated from spinal cord tissue and PC12 cells using TRIzol™ reagent (Qiagen; Valencia, CA) according to the manufacturer's instructions. Complementary DNA (cDNA) was synthesized from 1 μg of total RNA using a cDNA reverse transcription kit (TaKaRa; Dalian, China) according to the manufacturer's instructions. mRNA levels were assayed by reverse transcription quantitative polymerase chain reaction (RT-qPCR) following a standard protocol with the LightCycler 96 System (Roche; Pleasanton, CA) and detection via SYBR green (SYBR Green Supermix; TaKaRa).
The results were normalized to the GAPDH mRNA level and fold-changes were analyzed using the 2 -ΔΔCt method. The sequences of the primers are listed in Table 1.

Western blotting
Spinal cord tissues and cells were washed twice with cold PBS, lysed in radioimmunoprecipitation assay buffer, and protein levels were quantified using a Bradford Protein Assay Kit (Beyotime; Hangzhou, China). Equal amounts of protein samples were subjected to 10% SDS-PAGE, electrotransferred onto a 0.45-μm polyvinylidene difluoride membrane (Millipore; Billerica, MA), blocked in Tris-buffered saline with Tween 20 containing 5% BSA for 2 h at room temperature, and incubated overnight with the primary antibodies at 4℃. Next, the membranes were incubated with the corresponding secondary antibodies for 1 h and signals were visualized by chemiluminescence (Bio-Rad). Image J software was used to analyze the Western blotting data.

Statistical analysis
Values are means ± standard deviation (SD) of triplicate experiments. Student's t-test and one-way analysis of variance were performed with Statistical Package for the Social Sciences ver. 21.0 software (IBM Corporation, Chicago, IL) to assess the significance of differences; p-values of < 0.05 were considered to indicate statistical significance.

RA protected spinal cord tissue and improved functional recovery from SCI
The chemical structure of RA is presented in Fig. 2A. To explore the therapeutic effect of RA on locomotion recovery after SCI, we performed BBB and inclined plane testing at 2 weeks after injury.
The mean BBB score of the sham group was 21, and those of the SCI and RA-treated groups were below normal (Fig. 2B). At 3 days after injury, there was no significant difference in BBB scores between the SCI and RA treatment groups. At 5 days after injury, BBB scores were higher in the RAtreated group compared to the untreated group. Inclined-plane test scores were consistently higher in the RA-treated group at 5, 7, and 14 days after injury (Fig. 2C). Compared with the sham group, the water content of the SCI group increased significantly at day 7 after injury but was markedly decreased by RA (Fig. 2D). Furthermore, HE staining at day 7 showed large areas of hemorrhage, swelling and destruction of nerve cells, widening of interstitial space, glial cells proliferation, and infiltration of inflammatory cells in the SCI group; these pathologies were significantly improved by RA ( Fig. 2E). Moreover, RA significantly increased the number of Nissl-positive neurons at 7 days after injury in a dose-dependent manner (Fig. 2F-G). Therefore, RA promoted functional recovery and ameliorated tissue damage in SCI rats.

RA increased neurotrophin expression in SCI rats
Immunofluorescence staining showed that levels of GFAP, a marker of astrocytes, increased in the SCI group, suggesting activation of astrocytes; this increase was markedly attenuated by RA (Fig. 3A, C).
Immunofluorescence staining demonstrated that numbers of NeuN-positive cells were significantly lower in the SCI group than in the sham group. Notably, RA significantly increased the number of NeuN-positive cells compared with the SCI group ( Fig. 3B-C). Moreover, the results of Western blotting and immunofluorescence for GFAP and NeuN were consistent. The levels of NF-H and BDNF were increased by RA compared with the SCI group ( Fig. 3D-E). Therefore, RA increased the expression of neurotrophic factors in the spinal cord, possibly promoting positive the neurological outcomes after SCI.

RA suppressed cell apoptosis in the spinal cord of SCI rats
To evaluate the effect of RA on apoptosis, TUNEL staining was performed at day 7 after SCI. TUNELpositive cells were detected at the injury site in the spinal cord of SCI rats, RA decreased the number of apoptotic cells in a dose-dependent manner (Fig. 4A). We next assayed the levels of apoptosisrelated proteins. Immunohistochemistry staining indicated that the number of cleaved caspase-3positive cells in the spinal cord was increased in the SCI rats and was decreased by RA (Fig. 4B).
Additionally, Western blotting showed that levels of Bax, cleaved caspase-9, and cleaved caspase-3 were upregulated in the spinal cord of SCI rats compared to sham rats, while that of Bcl-2 was markedly downregulated. Notably, RA reduced Bax, cleaved caspase-9, and cleaved caspase-3 levels and increased the Bcl-2 level, significantly (Fig. 4C). Therefore, RA inhibited apoptosis in spinal cord tissue after SCI.

RA attenuated oxidative stress in the spinal cord of SCI rats
To investigate the effects of RA on oxidative stress in SCI rats, changes in SOD, CAT, and GSH-Px activities and the MDA content of spinal cord tissue were assayed. The activities of SOD (Fig. 5A), CAT (Fig. 5B), and GSH-Px (Fig. 5C) were significantly decreased and the content of MDA (Fig. 5D) was significantly increased in the SCI group compared to the sham group. RA markedly increased the activities of SOD, CAT, and GSH-Px and decreased the content of MDA, implying attenuation of SCIinduced oxidative damage.

RA inhibited the inflammatory response in the spinal cord of SCI rats
The inflammatory response plays an important role in the pathogenesis of SCI. An increased number of microglia and their activation is critical in the development of inflammation following SCI, so we performed immunofluorescence staining for Iba-1 (microglia marker) on spinal cord tissue at 7 days after SCI with and without treatment with 20 mg/kg RA. A few Iba-1-positive cells were observed in the sham group, while more Iba-1-positive cells were present in the injured spinal cord tissue at 7 days after SCI. The number of Iba-1-positive cells was reduced by 20 mg/kg RA ( Fig. 6A-B).
Therefore, RA significantly inhibited microglial activation in SCI. Next, ELISA showed that the levels of TNF-α, IL-6, and IL-1β in the SCI group were significantly increased compared with the sham group, indicating excessive inflammation. RA significantly reduced the levels of TNF-α, IL-6, and IL-1β and significantly increased that of IL-10 compared to the SCI group ( Fig. 6C-F). Furthermore, Western blotting and RT-PCR showed that the rats in the SCI group had higher TNF-α and IL-10 protein and mRNA levels compared with the sham rats. Also, the TNF-α level was significantly reduced and that of IL-10 was increased by RA ( Fig. 6G-H). Therefore, RA inhibited the inflammatory response in the spinal cord of SCI rats.

Proteomics analysis
We performed proteomics analyses on spinal cord tissue at 7 days after SCI with and without 20 mg/kg RA. SCI rats were treated with 20 mg/kg RA for 7 days, spinal cord tissue was harvested, and proteins were resolved by 2D-PAGE (Fig. 7A). The 16 proteins that showed at least a twofold difference in intensity between the RA-treated SCI rats and SCI rats (p < 0.05) were subjected to further analysis.
The identified proteins-with their theoretical molecular weights, isoelectric points (pI), coverage, MASCOT scores, MS/MS matched sequences, and changes in levels-are listed in Table 2 The online STRING database was used to forecast the interaction networks among the identified proteins (Fig. 7C); HO-1, HSP90, HSP27, and ANXA2 were key nodes and modulators in the network.
GO analysis is a dynamic, controlled vocabulary for the role of genes and proteins in eukaryotic cells.
A GO analysis revealed that 17 functions were annotated as MF, 287 as BP, and 69 as CC (p < 0.05); the top 10 significantly enriched GO terms (threshold false discovery rate p < 0.01) are shown in Fig.   7D-F. A reactome pathway analysis showed that proteins were enriched in the neutrophil degranulation, detoxification of ROS, iron uptake and transport, cellular responses to stress, posttranslational protein phosphorylation, and regulation of insulin-like growth factor (IGF) transport pathways (Fig. 7G). The identified proteins are involved in multiple biological processes and have molecular functions mainly related to energy metabolism, heat shock/stress, oxidative stress, inflammation, and apoptosis. Therefore, the neuroprotective effect of RA on SCI is mediated by improvement of energy metabolism and prevention of oxidative stress and inflammation.

RA modulated the Nrf2/HO-1 and TLR4/NF-κB pathways in the spinal cord of SCI rats
In the STRING analysis, HO-1 was a key node and modulator in the identified network. HO-1 is an antioxidant enzyme downstream of the Nrf2 signaling pathway. Also, HSP27, ANXA2, AKR1B1, and ARG1 are closely related to the NF-κB signaling pathway. Thus, we investigated the effect of RA on the Nrf2 and NF-κB signaling pathways. Compared with the sham and SCI groups, cytoplasmic and nuclear Nrf2 levels were significantly decreased and increased, respectively, by RA (Fig. 8A). We next assayed levels of HO-1, an antioxidant enzyme regulated by Nrf2. RA increased the expression of HO-1 compared to the untreated group (Fig. 8A). Also, RA increased the Nrf2 and HO-1 mRNA levels in injured tissue compared to the untreated group (Fig. 8B). Therefore, RA promoted nuclear translocation of Nrf2 and increased expression of HO-1.

RA attenuated H 2 O 2 -induced oxidative injury and activated the Nrf2/HO-1 pathway in PC12 cells
To confirm the effects of RA on oxidative stress in vitro, PC12 cells were treated with H 2 O 2 followed by RA. Treatment with RA at 5, 10, and 20 μg/mL for 24 h did not cause any apparent cytotoxicity in PC12 cells, while 40 or 80 μg/mL RA decreased PC-12 cell viability (Supp. Fig. 1). Therefore, we used ≤ 20 μg/mL RA in subsequent experiments. A CCK-8 assay showed that H 2 O 2 (200 µM) treatment for 24 h decreased the viability of PC12 cells; this effect was reversed by pretreatment with RA for 24 h (Fig. 9A). The release of LDH, an indicator of cytotoxicity, was used to estimate the protective effect of RA. The LDH level in the culture medium of H 2 O 2 -treated cells was significantly increased compared with that of control cells. RA significantly decreased LDH release from cells in a dosedependent manner (Fig. 9B). The proportion of apoptotic cells was decreased by 5, 10, and 20 μg/mL RA compared with the H 2 O 2 group (Fig. 9C). Likewise, RA reversed H 2 O 2 -induced alterations in the levels of apoptosis-associated proteins (Fig. 9D). Immunofluorescence staining demonstrated that RA pretreatment significantly decreased the number of cleaved caspase-3-positive cells compared with the H 2 O 2 group (Supp. Fig. 2). Also, the increased fluorescence intensity suggested intracellular accumulation of ROS in PC12 cells exposed to H 2 O 2 compared to the control (Fig. 9E). However, pretreatment of PC12 cells with RA prior to H 2 O 2 exposure attenuated the fluorescence intensity, indicating inhibition of ROS generation. Compared to the H 2 O 2 group, cells pretreated with RA exhibited a decreased level of MDA but increased activities of SOD, GSH-Px, and CAT (Fig. 9F).
Furthermore, nuclear Nrf2 and HO-1 levels were increased and cytoplasmic Nrf2 levels were decreased by RA in H 2 O 2 -induced-PC12 cells (Fig. 9G). Immunofluorescence staining demonstrated that RA pretreatment significantly increased the number of HO-1-positive cells compared with the H 2 O 2 group (Fig. 9H-I). Therefore, RA increased cell viability, reduced oxidative stress, and suppressed apoptosis in the presence of H 2 O 2 -induced oxidative injury by activating the Nrf2/HO-1 signaling pathway.

RA attenuated LPS-induced inflammatory injury and inhibited the NF-κB pathway in PC12 cells
To verify the involvement of the NF-κB pathway in RA-mediated inhibition of inflammation in vitro, neuronal PC12 cells were treated with LPS followed by RA. A CCK-8 assay indicated that pretreatment of PC12 cells with 5, 10, and 20 μg/mL RA significantly ameliorated the LPS-induced reduction in their viability (Fig. 10A). Also, RA significantly suppressed LPS-induced cytotoxicity and apoptosis, as evidenced by decreased LDH release (Fig. 10B), upregulation of Bcl-2, and downregulation of Bax, cleaved caspase-3, and cleaved caspase-9 (Fig. 10C). Also, immunofluorescence staining showed that RA pretreatment significantly reduced the number of cleaved caspase-3-positive cells compared with the LPS group (Supp. Fig. 3). The levels of TNF-α, IL-6, and IL-1β were significantly decreased and that of IL-10 was increased by RA pretreatment (Fig. 10D). The levels of TNF-α and IL-10 mirrored these results (Fig. 10E). The marked increase in the TLR4, MyD88 and phosphorylated IκBα and IKKα/β levels indicated activation of the NF-κB pathway in LPS-induced PC12 cells. However, RA decreased the expression of TLR4, MyD88, p-IκBα, and p-IKKα/β (Fig. 10F). Compared to the LPS group, pretreatment with RA reduced the nuclear translocation of p65 (Fig. 10G). Also, immunofluorescence staining showed that RA significantly reduced the number of NF-κB p65-positive cells compared with the LPS group (Fig. 10H-I). Therefore, RA exerted anti-apoptotic and antiinflammatory effects on LPS-injured PC12 cells by suppressing the TLR4/NF-κB signaling pathway.

ML385 reversed the effects of RA on SCI
To verify involvement of the Nrf2 signaling pathway in the neuroprotective effect of RA on SCI, an Nrf2 inhibitor (ML385) was used. The BBB scores and inclined-plane test revealed that the effects of RA were significantly inhibited by ML385 at days 7 and 14 ( Fig. 11A-D). ML385 significantly increased the water content of spinal cord tissue at day 7 post-injury under the SCI plus RA condition (Fig. 11E). Moreover, HE staining revealed that ML385 significantly increased injury of single RA treatment (Fig. 11F). The effects of combined treatment with RA and ML385 on neuronal survival were assayed by Nissl staining; the combination decreased the number of surviving neurons in the anterior horn (Fig. 11G-H). Furthermore, TUNEL staining showed that the number of apoptotic cells was decreased significantly by RA; this effect was reversed by ML385 (Fig. 11I). Also, SCI-mediated apoptosis was diminished by RA (as determined by assaying the levels of Bcl-2, Bax, cleaved caspase-9, and cleaved caspase-3); this effect was reversed by ML385 (Fig. 11J). Therefore, RA activated the Nrf2 signaling pathway to promote functional recovery and inhibit apoptosis in SCI.

RA exerted a neuroprotective effect by modulating the Nrf2 and NF-κB pathways
Next, we explored the effect of RA on SCI-induced oxidative stress and inflammation after inhibiting the Nrf2 signaling pathway. The SCI-mediated decrease in SOD, CAT, and GSH-Px activity and increase in the MDA content were attenuated by RA; this effect was significantly diminished by ML385 (Fig. 12A). The levels of proinflammatory cytokines (TNF-α, IL-6, and IL-1β) were significantly decreased and that of the anti-inflammatory cytokine IL-10 was increased by RA pretreatment, and the anti-inflammatory activity of RA was significantly reduced by ML385 (Fig. 12B). Similar results were observed for the protein levels of TNF-α and IL-10 (Fig. 12C). Western blotting showed that nuclear Nrf2 and HO-1 levels were significantly decreased and cytoplasmic Nrf2 levels were increased by ML385 under the SCI plus RA condition (Fig. 12D). Moreover, Western blotting indicated that administration of RA alone significantly reduced expression of p-IκBα and p-IKKα/β compared to the SCI group; this was abrogated by ML385 (Fig. 12E). Also, expression of p65 (nucleus) was increased and that of p65 (cytoplasmic) was significantly increased by ML385 under the SCI plus RA condition (Fig. 12F). Therefore, RA inhibited the NF-κB signaling pathway indirectly by activating the Nrf2 signaling pathway. The above results indicate that RA protected SCI rats against oxidant stress and inflammation by activating the Nrf2 signaling pathway and inhibiting the NF-κB signaling pathway.

Discussion
Traumatic SCI is a major cause of death and long-term paralysis worldwide [36]. SCI patients frequently have severe physical activity disorders, placing a heavy burden on families and society.
However, currently available drugs, surgery, and other therapies have limited efficacy and various adverse effects. Most traumatic SCI does not involve physical transection of the spinal cord, but damage to the spinal cord due to contusion, compression, or traction injury. Such incomplete SCI can cause persistent severe secondary damage, which can be treated [37]. Therefore, preservation of living cells during secondary injury is a major therapeutic target for SCI. Plant-derived drugs for SCI are safe, effective, and have few side effects [7]. RA is a polyphenolic phytochemical extracted from a range of Labiatae herbs with powerful antioxidant, anti-inflammatory, and neuroprotective effects in vitro and in vivo [13][14][15][16][17]. We evaluated the ability of RA to promote functional recovery from SCI. The results demonstrate that RA activates the Nrf2/HO-1 pathway and inhibits the TLR4/NF-κB pathway to reduce oxidant stress, inflammation, and apoptosis, thereby ameliorating the reduced locomotor function and tissue damage of acute SCI (Fig. 13)..
Evaluation of neurologic function is necessary for assessment of recovery from SCI. We performed BBB and inclined-plane tests to assess the locomotor function of SCI rats. RA remarkably improved locomotor function recovery, ameliorated tissue damage, and reduced the water content ratio in the SCI rats. Neuronal injury plays a key role in the progression and prognosis of acute SCI. The number of Nissl bodies was significantly reduced in the SCI rats. Nissl bodies comprise rough endoplasmic reticulum of synthetic proteins that reflect the neural structures of the brain and spinal cord and are related to the nutritional status of neurons; they may dissolve or even disappear under pathological conditions. Similarly, decreased density of NeuN-positive cells indicates loss of neurons in SCI. BDNF is a neurotrophic factor that regulates the growth and differentiation of neurons during development and the plasticity and survival of adult neurons and glial cells [38]. NF is a neuron-specific intermediate filament protein and a major neuron-specific protein and cytoskeleton that plays an important role in preserving the spatial configuration of nerve cells and signal transduction and is closely related to axon regeneration [39]. Astrocytes are the most abundant type of glial cell in the CNS and actively participate in neuropathological responses. Astrocytes typically proliferate in response to SCI. Activated astrocytes produce a series of proinflammatory cytokines, which regulate neuroinflammatory responses. In addition, astrocytes produce chemokines that attract inflammatory cells through the blood-brain barrier [40]. Inflammatory activation of astrocytes leads to the formation of dense scars that affect the regeneration of axons [41]. RA prevented neuron loss and promoted neuronal regeneration, as indicated by an increase in the number of Nissl bodies and the expression of NeuN, BDNF, and NF-H, and abolition of astrocyte activation.
Neuronal death, including necrosis and apoptosis, is a hallmark of CNS diseases. Apoptosis leads to further neurological damage and loss of function during secondary SCI. Inhibition of neuronal apoptosis plays a crucial role in functional and pathological recovery following SCI [42]. Apoptosis is a type of programmed cell death regulated by signal transduction pathways. Bcl-2-family proteins are important regulators of apoptosis and include anti-apoptotic proteins (e.g.,, Bcl-2) and proapoptotic proteins (e.g.,, Bax). A higher Bax/Bcl-2 ratio can form ion channels causing the release of cytochrome c from mitochondria into the cytosol, leading to activation of caspases-9 and -3, inducing apoptosis [43]. Bax, cleaved caspase-9, and cleaved caspase-3 were upregulated, and Bcl-2 was downregulated, in the SCI group, implying that SCI induces apoptosis. However, RA inhibited activation of cleaved caspase-9 and cleaved caspase-3 and reduced the Bax/Bcl-2 ratio, indicating inhibition of apoptosis. Furthermore, the number of TUNEL-positive cells was decreased by RA, also implying inhibition of apoptosis. SCI-induced apoptosis and mitochondrial dysfunction lead to ROS and lipid peroxide accumulation, which is implicated in the deterioration of SCI [44]. The abundance of lipids in spinal cord tissue means that it is easily injured by lipid peroxidation and ROS. SOD catalyzes the disproportionation of superoxide anion and is the main antioxidant enzyme that scavenges ROS, protecting against ROSinduced damage [24].

CAT decomposes H 2 O 2 into H 2 O and O 2 and protects cells from H 2 O 2 -induced
damage [45]. GSH-Px removes H 2 O 2 and lipid peroxides, protecting the cell membrane [23]. MDA is the final product of polyunsaturated fatty acids, and its content is an important indicator of lipid peroxidation [24]. Therefore, SOD, CAT, and GSH-Px activity and the MDA content can be measured as indicators of oxidative stress damage secondary to SCI. Our results indicate that RA attenuated the increased MDA content and SOD, CAT, and GSH-Px activities, protecting SCI rats from oxidative injury and overproduction of ROS.
Excessive inflammation is also implicated in the pathogenesis of SCI by contributing to neuronal apoptosis and multiple secondary injuries [6]. Microglia are unique to the CNS. After trauma or infection, expression of CD11b is increased in microglia, triggering secretion of IL-10, TNF-α, IL-6, IL-1β, and ROS [46]. In SCI, activated microglia secrete proinflammatory cytokines, induce astrocyte activation, promote glial scar formation, destroy the oligodendrocytes that are responsible for myelination of multiple axons, and induce axonal death [47]. Thus, the microglia-mediated inflammatory response plays an important role in secondary injury after SCI, and blocking the activation of microglia and the production of proinflammatory mediators may have therapeutic potential for SCI. RA attenuated microglia activation and TNF-α, IL-6, and IL-1β secretion, and promoted release of IL-10 in the injured spinal cord, so the amelioration by RA of SCI may be related to microglia inhibition and its anti-inflammatory activity.
The levels of HO-1, HSP90, HSP27, ANXA2, AKR1B1, and ARG1, which are associated with the Nrf2 and NF-κB signaling pathways, were markedly changed. HO-1 is an important antioxidant enzyme of the downstream Nrf2 signaling pathway [23,24]. Inhibition of HSP90 prevents atherosclerosis in experimental diabetes by inducing Nrf2-dependent cytoprotective mechanisms [48]. Salari et al. reported that treatment of human macrophages with recombinant HSP27 induced degradation of IκBα, nuclear translocation of the NF-κB p65 subunit, and activation of downstream NF-κB transcriptional activity [49]. In addition, ANXA2 induced activation of NF-κB through TLR4, resulting in proinflammatory cytokine production [50]. AKR1B1 is upregulated in SCI rats, interacts with the NF-κB pathway and promotes astrocyte proliferation and an inflammatory response [51]. ARG1, as a marker of anti-inflammatory M2 microglia, is associated with induction of NF-κB via TLR4 [52].
Next, we performed a bioinformatics analysis (protein network, GO, and pathway analyses) of the 16 proteins. STRING analyses revealed that the proteins were closely clustered in an interaction network centered on HO-1, HSP90, HSP27, and ANXA2. Also, 17 GO functions were annotated as MF, 287 as BP, and 69 as CC. In addition, a reactome pathway analysis showed that the proteins were enriched in the neutrophil degranulation, detoxification of ROS, iron uptake and transport, cellular responses to stress, post-translational protein phosphorylation, and regulation of the IGF transport pathway. Therefore, the neuroprotective effect of RA on SCI may be mediated by improved energy metabolism and prevention of oxidative stress and inflammation.
Nrf2 interacts with antioxidant response elements, followed by activation of downstream antioxidant proteins [19]. Also, Nrf2 regulates the expression of a panel of antioxidants and detoxification enzymes and plays an important role in regulating oxidative stress and inflammation. Especially in CNS diseases, Nrf2 plays a key role in defending against potential oxidative stress or insults [53]. Therefore, modulating Nrf2 may prevent oxidative-stress-induced injury in CNS diseases. Nrf2 activators exert a neuroprotective effect in animal models of SCI. Zhang et al. found that sinomenine attenuated traumatic SCI by inhibiting oxidative stress and inflammation by activating the Nrf2 pathway [24]. Normally, the binding of Nrf2 to Keap1 in the cytoplasm prevents its nuclear translocation. Stimulation by physicochemical factors disrupts this binding to Keap1, leading to nuclear translocation of Nrf2, whereby it binds to antioxidant response elements and activates transcription of genes encoding detoxification enzymes and cytoprotective proteins, such as NQO1, SOD, and HO-1 [21,22]. HO-1 prevents oxidative stress, inflammation and metabolic disorders [23,24]. RA promotes nuclear translocation of Nrf2 and increases HO-1 levels. Similarly, the antioxidant and anti-apoptotic activities of RA reportedly protect against ischemic stroke by upregulating Nrf2 and HO-1 expression [54].
NF-κB is a central transcription factor in the inflammatory response and regulates cell proliferation and apoptosis [29]. Inactive NF-κB exists in the cytoplasm in the form of a trimer that aggregates with IκB. Upon stimulation by bacterial or viral infection, inflammatory factors, or LPS, activation of inhibitor of NF-κB kinase (IKK) triggers dissociation of the cytoplasmic NF-B/IB complex. The IKKmediated phosphorylation and proteasomal degradation of IB facilitates the nuclear translocation of active NF-B transcription factor subunit (p65), causing release of IL-6, IL-1β, and TNF-α [55,56]. In addition, the Toll-like receptor 4 (TLR4)-mediated myeloid differentiation factor-88 (MyD88) signaling pathway plays an important role in regulating the inflammatory response [57]. TLR4 is activated by LPS, resulting in the recruitment and activation of the signal adaptor MyD88 by the interaction between the intracellular TH domain of TLR4 and the carboxy-terminal TH domain of MyD88. The activation of MyD88 triggers activation of the NF-κB signaling pathway and the production of proinflammatory cytokines [58]. We found that the anti-inflammatory effect of RA on SCI was due to downregulation of TLR4, MyD88, p-IKKα/β, p-IBα, and p65 (nucleus), and upregulation of p65 (cytoplasm). Similarly, RA reportedly mitigated the LPS-induced neuroinflammatory response by inhibiting TLR4/NF-κB-mediated inflammasome activation [59].
In support of the above, RA prevented H 2 O 2 -induced cytotoxicity, oxidative damage, and apoptosis, and activation of the Nrf2/HO-1 pathway in PC12 cells. Also, RA protected PC12 cells from LPSinduced cytotoxicity, inflammatory damage, and apoptosis, and inactivated the TLR4//NF-κB pathway.
To investigate whether the effect of RA on SCI was dependent on the presence of Nrf2, we used a Nrf2 inhibitor (ML385). The Nrf2 level in spinal cord tissue was significantly decreased by ML385. Also, ML385 reversed RA-induced functional recovery and inhibition of apoptosis in SCI rats. This was confirmed by the contents of SOD, CAT, GSH-Px, and MDA and the levels of TNF-α, IL-6, IL-1β, and IL-10. Also, the inhibition of Nrf2 reversed the RA-mediated antioxidant and anti-inflammatory effects.
Nrf2 has been reported to exert an anti-inflammatory effect and to suppress activation of NF-κB [27].

Conclusion
In conclusion, RA exerted a neuroprotective effect in vivo and in vitro by inhibiting oxidative stress, inflammation, and apoptosis via the Nrf2, NF-κB, and caspase-3 pathways, respectively. Also, RA activated the Nrf2/HO-1 signaling pathway and inhibited the TLR4/NF-κB signaling pathway. Finally, RA improved the locomotor function of SCI rats, implying its potential as a target for the development of therapeutics for SCI.   Figure 1 The diagrammatic of study design in vivo.  The results are expressed as means ± SD from three independent experiments (n = 6 per group). #p < 0.05 or ##p < 0.01 vs. Sham group; *p < 0.05 or **p < 0.01 vs. SCI group.          Proposed mechanisms of RA in protecting SCI and promoting nerve repair. RA exerts neuroprotective effect against oxidative stress and inflammation through activating the Nrf2/HO-1 signaling pathway and inhibiting the NF-κB signaling pathway in SCI rats.

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