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Salvianolic acid B attenuates spinal cord ischemia-reperfusion–induced neuronal injury and oxidative stress by activating the extracellular signal–regulated kinase pathway in rats

  • Author Footnotes
    1 These authors contributed equally to this work.
    Jun Fu
    Footnotes
    1 These authors contributed equally to this work.
    Affiliations
    Department of Orthopaedic Oncology, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi, China
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  • Author Footnotes
    1 These authors contributed equally to this work.
    Hong-bin Fan
    Footnotes
    1 These authors contributed equally to this work.
    Affiliations
    Department of Orthopaedic Oncology, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi, China
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  • Zheng Guo
    Correspondence
    Corresponding author. Department of Orthopaedic Oncology, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi 710032, China. Tel.: +86 029 84775567; fax +86 029 84775568.
    Affiliations
    Department of Orthopaedic Oncology, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi, China
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  • Zhen Wang
    Correspondence
    Corresponding author. Department of Orthopaedic Oncology, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi 710032, China. Tel.: +86 029 84775577; fax +86 029 84775578.
    Affiliations
    Department of Orthopaedic Oncology, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi, China
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  • Xiang-dong Li
    Affiliations
    Department of Orthopaedic Oncology, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi, China
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  • Jing Li
    Affiliations
    Department of Orthopaedic Oncology, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi, China
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  • Guo-xian Pei
    Affiliations
    Department of Orthopaedic Oncology, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi, China
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  • Author Footnotes
    1 These authors contributed equally to this work.
Published:December 09, 2013DOI:https://doi.org/10.1016/j.jss.2013.11.1118

      Abstract

      Background

      Salvianolic acid B (SalB), the main bioactive compound isolated from the traditional Chinese medicinal herb broad Radix Salviae Miltiorrhizae exerts a spectrum of pharmacologic activities. We investigated the effects of SalB treatment in a rat model of spinal cord ischemia and reperfusion (I/R) injury and the underlying mechanism.

      Materials and methods

      SalB was administered at 1, 10, or 50 mg/kg after spinal cord ischemia. The potential protective effects on spinal cord injury were determined by spinal cord edema, infarct volume, and motor function assessment of the hind limbs.

      Results

      SalB treatment significantly decreased spinal cord edema and infarct volume and preserved motor function of the hind limbs in a dose-dependent manner. SalB administration ameliorated the generation of oxidative products and preserved antioxidant defense activities in the injured spinal cord at both 4 and 24 h after I/R injury. Moreover, SalB prolonged the I/R injury–induced activation of extracellular signal–regulated kinase (ERK), and blocking ERK activation with PD98059 partially prevented the neuroprotective effects of SalB.

      Conclusions

      These findings demonstrate the neuroprotective effects of SalB in a spinal cord I/R injury model and suggest that SalB-induced neuroprotection was mediated by ERK activation.

      Keywords

      1. Introduction

      Paraplegia after open surgery repair is a devastating complication encountered in the clinical practice, and the incidence of paraplegia is approximately 5%–8% as reported in thoraco-abdominal aneurysm repair surgery [
      • Svensson L.G.
      • Crawford E.S.
      • Hess K.R.
      • et al.
      Experience with 1509 patients undergoing thoracoabdominal aortic operations.
      ]. In spite of the progress of surgical adjuncts and pharmacologic interventions, spinal cord ischemia and reperfusion (I/R) injury induced by temporary interruption of blood supply is still one of the most prevalent causes [
      • Saxena P.
      • Abu Hasan F.
      • Merry C.
      Spinal cord ischemia following coronary artery bypass surgery.
      ]. The cellular and molecular mechanisms of spinal cord I/R injury are not fully understood; therefore, there are no scientifically established methods for the treatment or prevention of spinal cord injury to date [
      • de Haan P.
      • Kalkman C.J.
      • Jacobs M.J.
      Pharmacologic neuroprotection in experimental spinal cord ischemia: a systematic review.
      ]. Thus, it is imperative to find preventative agents for spinal cord I/R injury.
      In recent years, increasing evidence supports the beneficial effects of natural products on neuronal cell death and functional deficits. Natural compounds with neuroprotective potential could be a critical strategy for the treatment of I/R injury [
      • Kam A.
      • Li K.M.
      • Razmovski-Naumovski V.
      • et al.
      The protective effects of natural products on blood-brain barrier breakdown.
      ,
      • Chen T.
      • Liu W.
      • Chao X.
      • et al.
      Neuroprotective effect of osthole against oxygen and glucose deprivation in rat cortical neurons: involvement of mitogen-activated protein kinase pathway.
      ]. Radix Salviae Miltiorrhizae, also known as Danshen, is a popular traditional Chinese herb widely used in treating a number of diseases including coronary artery disease and other cardiovascular disorders [
      • Ji X.Y.
      • Tan B.K.
      • Zhu Y.Z.
      Salvia miltiorrhiza and ischemic diseases.
      ,
      • Wang Z.S.
      • Luo P.
      • Dai S.H.
      • et al.
      Salvianolic acid B induces apoptosis in human glioma U87 cells through p38-mediated ROS generation.
      ]. Salvianolic acid B (SalB), the most abundant and bioactive water-soluble compound extracted from Salviae Miltiorrhizae, has been well recognized as a neuroprotective agent with antioxidative and anti-inflammatory activities [
      • Cao W.
      • Guo X.W.
      • Zheng H.Z.
      • et al.
      Current progress of research on pharmacologic actions of salvianolic acid B.
      ,
      • Chen T.
      • Liu W.
      • Chao X.
      • et al.
      Salvianolic acid B attenuates brain damage and inflammation after traumatic brain injury in mice.
      ]. Pretreatment with SalB exerts protective effects on cerebral and myocardial I/R injury [
      • Fan H.
      • Yang L.
      • Fu F.
      • et al.
      Cardioprotective effects of salvianolic acid A on myocardial ischemia-reperfusion injury in vivo and in vitro.
      ,
      • Tang M.
      • Feng W.
      • Zhang Y.
      • et al.
      Salvianolic acid B improves motor function after cerebral ischemia in rats.
      ]. However, there have been no studies investigating the effects of SalB on spinal cord I/R injury. In the present study, we examined the neuroprotective efficacy of SalB in a rat model of spinal cord I/R injury as well as the potential molecular mechanisms.

      2. Materials and methods

      2.1 Subjects

      Male Sprague Dawley rats weighting 350–450 g were housed at 20°C–22°C with a 12-h light/dark cycle. Female rats were not studied because their hormones (estrogen/progesterone) fluctuation during the menstrual cycle might influence the results. Standard animal chow and water were freely available. All experimental protocols were performed in accordance with NIH Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23, revised 1996). All efforts were made to minimize animal numbers and suffering. SalB (purity > 98%) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China) and dissolved in saline.

      2.2 Spinal cord I/R injury

      Spinal cord I/R injury was performed as has been previously described [
      • Lu K.
      • Liang C.L.
      • Liliang P.C.
      • et al.
      Inhibition of extracellular signal-regulated kinases 1/2 provides neuroprotection in spinal cord ischemia/reperfusion injury in rats: relationship with the nuclear factor-kappaB-regulated anti-apoptotic mechanisms.
      ]. Briefly, rats were anesthetized with chloral hydrate (40 mg/kg, intraperitoneal injection) and placed in the supine position. The thoracic aorta was exposed via a 3- to 4-cm midline incision, and spinal cord ischemia was induced by occluding the descending thoracic aorta for 20 min with a bulldog clamp. Blood flow was then restored by removing the clamp, and Ampicillin was injected intramuscularly once a day for 3 d postoperatively to prevent infection. Blood pressure and heart rate were continuously monitored by a calibrated pressure transducer connected to an invasive pressure monitor (Spacelabs Medical, Inc, Redmond, WA). Arterial blood was sampled for the determination of blood gases and plasma glucose. The hemodynamic parameters were recorded 10 min before ischemia, 10 min after ischemia, and 10 min after reperfusion. SalB (1, 10, or 50 mg/kg) was injected via the tail vein at the initiation of spinal cord ischemia, and PD98059 (20 μM) was given intrathecally 30 min before injury.

      2.3 Evaluation of spinal cord edema

      A wet-dry method was used to measure the water content of the spinal cord tissue, an index of spinal cord edema. Briefly, rats were sacrificed by cervical dislocation at 72 h after reperfusion, and the spinal cord tissue samples were weighed immediately (wet weight). The dry weight was determined after heating the tissues for 48 h at 100°C, and the spinal cord water content was obtained by the following calculations: % H2O = (1 − dry weight/wet weight) × 100%.

      2.4 Spinal infarction assay

      The spinal cord infarct volume was determined by 2,3,5,-triphenyltetrazolium chloride staining as described previously [
      • Zhu J.W.
      • Chen T.
      • Guan J.
      • et al.
      Neuroprotective effects of allicin on spinal cord ischemia-reperfusion injury via improvement of mitochondrial function in rabbits.
      ]. The animals were sacrificed by cervical dislocation at 14 d after injury, and the lumbar enlargements of the spinal cord (L1–L5) were sectioned into 2.0-mm thick coronal slices for staining. The histologic analyses were performed in a blinded manner, and the areas of spinal cord infarction were determined by loss of 2,3,5,-triphenyltetrazolium chloride staining. The infarction volume was measured in each slice and summed up using computerized planimetry (PC-Based Image Tool software, Scion Corporation, CA, USA).

      2.5 Motor function measurement

      The assessment of locomotor behavior is an important measure of long-term functional recovery after spinal cord injury. In the present study, hind limb locomotor function was evaluated at 2, 4, 6, 8, 10, 12, and 14 d after injury using the modified Basso, Beattie, and Bresnahan Locomotor Rating Score (BBB score), a 21-point open field locomotion score. The BBB score assesses locomotor functions on a scale from 0 (complete hind limb paralysis) to 21 (normal locomotion). Each point represents a specific set of characteristics displayed by the animals during open field locomotion. Each evaluation was completed by two people who were blinded to the group treatments.

      2.6 Measurement of oxidative products

      The ischemic spinal cord tissues were dissected at 4 or 24 h after reperfusion, and the tissue protein concentration was determined using a standard commercial BCA kit (Rockford, IL, USA). The protein expression level of 8-iso-PGF2α was detected by using an enzyme-linked immunesorbent assay kits and a plate reader. Malonaldehyde (MDA) content was also measured using a commercial kit according to the manufacturer's instructions (Cayman Chemical Company, Ann Arbor, MI).

      2.7 Detection of antioxidant enzymes activities

      Using the same spinal cord samples as in Section 2.6, we also measured the antioxidant enzymatic activity within the lumbar enlargement. The enzyme activities of catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPX), and glutathione S-transferase (GST) in the tissue homogenate were measured according to the technical manual of the detection kits (Cayman Chemical Company). The enzymatic activity of CAT and SOD were expressed as Units per gram and Units per milligram of protein, respectively. The enzymatic activity of GPX and GST were expressed as the percentage of sham-treated control rats.

      2.8 Western blot analysis

      The homogenates obtained in Section 2.7 were also used for Western blot analyses. Forty micrograms of protein was resolved on a 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis gel and transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% nonfat milk and incubated with the primary antibodies for phosphorylated extracellular signal–regulated kinase (p-ERK), extracellular signal–regulated kinase (ERK), and β-actin. The membranes were then washed and incubated for 1 h at room temperature with the appropriate secondary antibodies. The optical density of each band was quantified using Image J (Scion Corporation, CA, USA).

      2.9 Statistical analysis

      Statistical analyses were performed using SPSS 16.0 (Scion Corporation, CA, USA), a statistical software package. Statistical evaluation of the data was performed by one-way analysis of variance. A value of P < 0.05 was considered statistically significant.

      3. Results

      3.1 Physiological and hemodynamic parameters

      No significant differences in rectal temperature, arterial Pao2, Paco2, pH, or blood glucose was observed between any of the groups as measured 10 min before ischemia, 10 min after ischemia, and 10 min after reperfusion. Laser Doppler flowmetry signal showed that the mean arterial pressure was equivalently reduced to approximately 20% of the baseline after abdominal aorta occluding and recovered equivalently after reperfusion in all groups.

      3.2 Effect of SalB on neuronal injury after spinal cord I/R injury

      I/R injury–induced spinal cord edema was assessed using a wet-dry evaluation of spinal cord water content. As shown in Figure 1A, I/R injury significantly increased spinal cord water content at 72 h after reperfusion, which was partially reversed by SalB treatment in a dose-dependent manner. Moreover, the spinal cord infarct volumes in the 10 mg/kg SalB and 50 mg/kg SalB treatment groups were lower than the sham-treated group, whereas the spinal cord infarct volume in the 1 mg/kg SalB treatment group was similar to that in the sham-treated group.
      Figure thumbnail gr1
      Fig. 1Effect of SalB on neuronal injury after spinal cord I/R injury. The animals were treated with normal saline or SalB at different concentrations (1, 10, or 50 mg/kg) after injury. Spinal cord water content at 72 h (A, n = 8/group) and infarct volume at 14 d (B, n = 8/group) were assayed. The data are represented as the mean ± standard deviation. #P < 0.05 versus Sham group and P < 0.05 versus Vehicle group.

      3.3 Effect of SalB on locomotor behavior after spinal cord I/R injury

      Locomotor behavioral outcomes between the sham- and SalB-treated rats were evident using the BBB scale over the time course of 2, 4, 6, 8, 10, 12, and 14 d after reperfusion (Fig. 2). All animals exhibited normal locomotor behavior (BBB = 21) before surgery and survived until the final locomotor behavioral assessment. The rats in the sham group exhibited flaccid paraplegia after recovery from anesthesia and then developed spasms over the next 2 d. Treatment with either 10 mg/kg SalB or 50 mg/kg SalB significantly improved the locomotor behavior of animals with spinal cord I/R injury from 2 to 14 d after reperfusion. However, no statistically significant difference in BBB scores was found between the 1 mg/kg SalB group and sham group.
      Figure thumbnail gr2
      Fig. 2Effect of SalB on motor function after spinal cord I/R injury. The animals were treated with normal saline or SalB at different concentrations (1, 10, or 50 mg/kg) after injury, and the locomotor function was evaluated by using the BBB scores at 2, 4, 6, 8, 10, 12, and 14 d after reperfusion (n = 8/group). The data are represented as the mean ± standard deviation. *P < 0.05 versus Vehicle group.

      3.4 Effect of SalB on oxidative products

      Oxidative stress in the spinal cord of I/R-injured rats was suggested by a rise in 8-iso-PGF2α and MDA levels at both 4 and 24 h after reperfusion (Fig. 3). Treatment with SalB significantly decreased the protein expression of 8-iso-PGF2α and MDA measured at 4 and 24 h after reperfusion in a dose-dependent manner. However, 1 mg/kg SalB was not effective at inhibiting MDA production measured at 4 h after reperfusion (P > 0.05).
      Figure thumbnail gr3
      Fig. 3Effect of SalB on oxidative products. The rats were treated with normal saline or SalB at different concentrations (1, 10, or 50 mg/kg) after injury, and the expression levels of MDA (A, n = 8/group) and 8-iso-PGF-2α (B, n = 8/group) were detected at 4 and 24 h after reperfusion. The data are represented as the mean ± standard deviation. *P < 0.05 versus Vehicle group.

      3.5 Effect of SalB on endogenous antioxidant enzymes

      Furthermore, we detected the activity of endogenous antioxidant enzymes in the spinal cord tissues at 4 and 24 h after reperfusion, which was shown in Figure 4. Spinal cord I/R injury–induced a slight increase in the activities of CAT and SOD at 4 h after injury, and the enzymatic activities of CAT, SOD, GPX, and GST were significantly decreased at 24 h after reperfusion compared with the sham-treated group, indicating the damaged endogenous antioxidant system after spinal cord I/R injury. SalB treatment markedly preserved the activities of CAT, SOD, GPX, and GST at 24 h after reperfusion compared with the vehicle group, with no effects on endogenous antioxidant enzyme activity at 4 h after reperfusion (P > 0.05).
      Figure thumbnail gr4
      Fig. 4Effect of SalB on endogenous antioxidant enzymes. The rats were treated with normal saline or SalB at different concentrations (1, 10, or 50 mg/kg) after injury, and the expression levels of CAT (A), SOD (B), GPX (C), and GST (D) were detected at 4 and 24 h after reperfusion (n = 8/group). The data are represented as the mean ± standard deviation. #P < 0.05 versus Sham group and *P < 0.05 versus Vehicle group.

      3.6 Effect of SalB on the activation of ERK

      Based on the aforementioned results, we chose the 50 mg/kg SalB treatment to investigate the potential molecular mechanism for the protective effects of SalB during spinal cord I/R injury. As shown in Figure 5, the expression of ERK and p-ERK were determined by Western blot up to 24 h after reperfusion. The protein expression of p-ERK started to increase notably 1 h after injury, decreased at 6 h, and returned to baseline at 12 h after reperfusion. p-ERK levels in the SalB-treated group tended to be higher than those observed in the sham group at 1 h after injury, but they were not statistically significantly different (P > 0.05). In contrast, at 6, 12, and 24 h after reperfusion, SalB significantly increased ERK1/2 phosphorylation. In addition, neither I/R injury nor SalB had any effect on the total protein levels of ERK.
      Figure thumbnail gr5
      Fig. 5Effect of SalB on the activation of ERK. The rats were treated with normal saline or 50 mg/kg SalB after injury. The expression of ERK and p-ERK were determined by Western blot up to 24 h after reperfusion (A, n = 6/group), and the relative activity of ERK was calculated (B). The data are represented as the mean ± standard deviation. P < 0.05 versus Vehicle group.

      3.7 Effect of ERK inhibition on SalB-induced protection

      To mechanistically investigate the relationship between the neuroprotective effect of SalB and its action on ERK regulation, the activation of ERK was blocked by the specific inhibitor PD98059 (20 μM). As shown in Figure 6A and B, the results of spinal cord water content and infarct volume indicated that the SalB-induced protective effect against spinal cord I/R neuronal injury was partially prevented by PD98059. Similarly, treatment with PD98059 significantly reduced the SalB-induced preservation of motor function compared with SalB-treated rats at both 7 and 14 d after reperfusion (Fig. 6C). In addition, we also examined the effects of PD98059 on the expression of endogenous antioxidant enzymes after spinal cord I/R injury and SalB treatment. As shown in Figure 7, PD98059 treatment significantly decreased the activities of CAT, SOD, GPX, and GST both at 4 and 24 h after reperfusion, indicating an ERK-dependent mechanism on the activities of endogenous antioxidant enzymes in our spinal cord I/R injury model.
      Figure thumbnail gr6
      Fig. 6Effect of ERK inhibition on SalB-induced neuroprotection. The rats were treated with normal saline or 50 mg/kg SalB in the presence or absence of pretreatment with the ERK inhibitor PD98059 (PD, 20 μM). The spinal cord water content at 72 h (A, n = 8/group) and infarct volume at 14 d (B, n = 8/group) were assayed. The locomotor function was evaluated using the BBB scores at 7 and 14 d after reperfusion (C, n = 8/group). The data are represented as the mean ± standard deviation. *P < 0.05.
      Figure thumbnail gr7
      Fig. 7Effect of ERK inhibition on SalB-induced antioxidative activity. The rats were treated with normal saline or 50 mg/kg SalB in the presence or absence of pretreatment with the ERK inhibitor PD98059 (PD, 20 μM). The expression levels of CAT (A), SOD (B), GPX (C), and GST (D) were detected at 4 and 24 h after reperfusion (n = 8/group). The data are represented as the mean ± standard deviation. #P < 0.05 versus Vehicle group and *P < 0.05 versus SalB group.

      4. Discussion

      In the present study, we demonstrated the neuroprotective effect of SalB in a rat model of spinal cord I/R injury. This protection was sustained for at least 2 wk after injury and was associated with improved locomotor function. Moreover, SalB prolonged the activation of ERK after ischemia and recirculation, and pretreatment with the ERK inhibitor PD98059 partially prevented the SalB-induced neuroprotective effects.
      Although many neuroprotective compounds derived from natural products are in various stages of clinical trials, SalB represents unique advantages. Radix Salviae Miltiorrhizae and its related species have been widely used for thousands of years in traditional Chinese medicine with little reported toxicity [
      • Ho J.H.
      • Hong C.Y.
      Salvianolic acids: small compounds with multiple mechanisms for cardiovascular protection.
      ]. Salvianolic acid is the main water-soluble compound in Radix Salviae Miltiorrhizae, and SalB is the most abundant component with commercial value for both food and medicinal purposes [
      • Wang Z.S.
      • Luo P.
      • Dai S.H.
      • et al.
      Salvianolic acid B induces apoptosis in human glioma U87 cells through p38-mediated ROS generation.
      ]. SalB exhibits higher scavenging activities than vitamin C against HO·, O2·−, 1,1-diphenyl-2-picryl-hydrazyl radicals and 2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) radicals [
      • Zhao G.R.
      • Zhang H.M.
      • Ye T.X.
      • et al.
      Characterization of the radical scavenging and antioxidant activities of danshensu and salvianolic acid B.
      ]. With its polyphenolic structure, SalB can freely permeate through the phospholipids bilayers and immediately interact with various target molecules, even in an energy-deficient environment [
      • Ho J.H.
      • Hong C.Y.
      Salvianolic acids: small compounds with multiple mechanisms for cardiovascular protection.
      ,
      • Liu S.
      • Sun Y.
      • Chen H.
      • et al.
      Magnetic screening of the potential targeted protein of salvianolic acid B using T7 phage display library.
      ]. Due to the first-order absorption, SalB reaches its maximum plasma concentration within 0.5–1 h and can be detected up to 180 min after oral administration [
      • Zhang J.
      • Yu H.
      • Sheng Y.
      • et al.
      HPLC determination and pharmacokinetic studies of salvianolic acid B in rat plasma after oral administration of Radix Salviae Miltiorrhizae extract.
      ,
      • Wu Y.T.
      • Chen Y.F.
      • Hsieh Y.J.
      • et al.
      Bioavailability of salvianolic acid B in conscious and freely moving rats.
      ]. In our present study, we observed a significant decrease in spinal cord edema when the concentration of SalB was decreased to 1 mg/kg, which could be easily obtained by oral administration or intravenous injection. More clinical trials are needed to confirm these results from both basic science and clinical data.
      Oxidative stress–induced cell damage has long been implicated in I/R injury [
      • Kleikers P.W.
      • Wingler K.
      • Hermans J.J.
      • et al.
      NADPH oxidases as a source of oxidative stress and molecular target in ischemia/reperfusion injury.
      ,
      • Chen T.
      • Fei F.
      • Jiang X.F.
      • et al.
      Down-regulation of Homer1b/c attenuates glutamate-mediated excitotoxicity through endoplasmic reticulum and mitochondria pathways in rat cortical neurons.
      ]. Overwhelming evidence has accumulated implying that decreased endogenous antioxidant enzyme activities play a predominant role inI/R conditions by promoting oxidative stress [
      • Tan D.X.
      • Manchester L.C.
      • Sainz R.M.
      • et al.
      Physiological ischemia/reperfusion phenomena and their relation to endogenous melatonin production: a hypothesis.
      ]. In the present study, spinal cord I/R injury induced high levels of oxidative stress at 4 h after injury, as indicated by increased expression of MDA and 8-iso-PGF2α, whereas the activities of endogenous antioxidant enzymes (CAT and SOD) were slightly increased. At 24 h after spinal cord I/R injury, the endogenous antioxidant enzyme activities were all decreased, and the oxidative stress was augmented, which agrees with previous studies [
      • Zhu J.W.
      • Chen T.
      • Guan J.
      • et al.
      Neuroprotective effects of allicin on spinal cord ischemia-reperfusion injury via improvement of mitochondrial function in rabbits.
      ]. Due to its polyphenolic structure, SalB is thought to be a free radical scavenger, which has been demonstrated by neutralizing free radical assays such as the 1,1-diphenyl-2-picryl-hydrazyl radical scavenging test or the 2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) assay [
      • Zhao G.R.
      • Zhang H.M.
      • Ye T.X.
      • et al.
      Characterization of the radical scavenging and antioxidant activities of danshensu and salvianolic acid B.
      ,
      • Sun Y.
      • Zhu H.
      • Wang J.
      • et al.
      Isolation and purification of salvianolic acid A and salvianolic acid B from Salvia miltiorrhiza by high-speed counter-current chromatography and comparison of their antioxidant activity.
      ]. Additionally, SalB can inhibit lipid peroxidation of rat liver microsomes induced by iron/cysteine and vitamin C/NADPH and the hemolysis of rat erythrocytes induced by H2O2 in vitro [
      • Liu G.T.
      • Zhang T.M.
      • Wang B.E.
      • et al.
      Protective action of seven natural phenolic compounds against peroxidative damage to biomembranes.
      ]. SalB is also effective in lowering plasma cholesterol levels, reducing endothelial damage and inhibiting atherosclerosis in rabbits [
      • Wu Y.J.
      • Hong C.Y.
      • Lin S.J.
      • et al.
      Increase of vitamin E content in LDL and reduction of atherosclerosis in cholesterol-fed rabbits by a water-soluble antioxidant-rich fraction of Salvia miltiorrhiza.
      ]. Here, we showed that SalB treatment ameliorated the generation of oxidative products and preserved the antioxidant enzyme activities in the injured spinal cord at both acute and late phases (4 and 24 h) after reperfusion. These data indicate that the antioxidant properties of SalB may be due not only to its direct scavenging of free radicals but also the recruitment of the endogenous antioxidative system.
      ERK, one of the best characterized members of the mitogen-activated protein kinase family, is a widely expressed protein kinase intracellular signaling molecule, which mediates a range of activity from metabolism, motility, and inflammation to cell death and survival [
      • Shioda N.
      • Han F.
      • Fukunaga K.
      Role of Akt and ERK signaling in the neurogenesis following brain ischemia.
      ,
      • Ma W.
      • Quirion R.
      The ERK/MAPK pathway, as a target for the treatment of neuropathic pain.
      ,
      • Chen T.
      • Cao L.
      • Dong W.
      • et al.
      Protective effects of mGluR5 positive modulators against traumatic neuronal injury through PKC-dependent activation of MEK/ERK pathway.
      ,
      • Chen T.
      • Zhang L.
      • Qu Y.
      • et al.
      The selective mGluR5 agonist CHPG protects against traumatic brain injury in vitro and in vivo via ERK and Akt pathway.
      ]. The activation of ERK is usually increased under I/R conditions [
      • Kitagawa H.
      • Warita H.
      • Sasaki C.
      • et al.
      Immunoreactive Akt, PI3-K and ERK protein kinase expression in ischemic rat brain.
      ], which was also confirmed in our present study as evidenced by a temporal increase of phosphorylated ERK at 1 h after reperfusion. However, whether the increased activation of ERK is protective or detrimental is highly debatable. Despite the volume of evidence supporting the detrimental effects of ERK activation after ischemic injury (through the promotion of inflammation and oxidative stress), several previous studies have demonstrated that increased ERK phosphorylation is involved in the neuroprotective effects of growth factors, estrogen, and preconditioning, as well as some natural products [
      • Chen T.
      • Liu W.
      • Chao X.
      • et al.
      Neuroprotective effect of osthole against oxygen and glucose deprivation in rat cortical neurons: involvement of mitogen-activated protein kinase pathway.
      ,
      • Kaminska B.
      MAPK signalling pathways as molecular targets for anti-inflammatory therapy–from molecular mechanisms to therapeutic benefits.
      ]. The dual roles of ERK1/2 activation are likely not only related to its responses to a diverse array of stimuli and cell surface receptors but also dependent on the duration and extent of phosphorylation [
      • Sawe N.
      • Steinberg G.
      • Zhao H.
      Dual roles of the MAPK/ERK1/2 cell signaling pathway after stroke.
      ]. A previous study showed that the inhibitory effect of salvianolic acid on homocysteine-induced A10 cell proliferation was via the ERK-dependent pathway [
      • Hung Y.C.
      • Wang P.W.
      • Pan T.L.
      • et al.
      Proteomic screening of antioxidant effects exhibited by radix Salvia miltiorrhiza aqueous extract in cultured rat aortic smooth muscle cells under homocysteine treatment.
      ]. In our present study, we found that SalB treatment prolonged the spinal cord I/R injury–induced activation of ERK, and PD98059 partially prevented the neuroprotective effects of SalB, which indicates a beneficial effect of SalB through an increase of ERK phosphorylation.

      5. Conclusion

      In summary, our present study showed that SalB, one of the bioactive compounds extracted from the root of Danshen, protects rats against spinal cord I/R–induced neuronal injury. These protective effects were associated with an improvement in motor function and the inhibition of oxidative stress. Moreover, SalB treatment prolonged the activation of ERK induced by spinal cord I/R injury, and its neuroprotection was partially dependent on ERK activation. The effects of SalB in treating experimental spinal cord I/R injury may lead to interesting therapeutic perspectives in future clinical trials.

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