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Journal of Ethnopharmacology 139 (2012) 504– 512

Contents lists available at SciVerse ScienceDirect

Journal of Ethnopharmacology

jo ur nal homep age : www.elsev ier .com/ locate / je thpharm

anax notoginsenoside produces neuroprotective effects in rat model of acutepinal cord ischemia–reperfusion injury

ing Ning, Xiaoqian Dang, Chuanyi Bai, Chen Zhang, Kunzheng Wang ∗

epartment of Orthopedics, The Second Affiliated Hospital of Medical School, Xi’an Jiaotong University, No.157, Xiwu Road, Xi’an, Shaanxi 710004, China

r t i c l e i n f o

rticle history:eceived 3 July 2011eceived in revised form5 November 2011ccepted 17 November 2011vailable online 4 December 2011

eywords:anax notoginsenosideeuroprotectivecute spinal cord ischemia–reperfusion

njurycute inflammatory reactionerebral edemapoptosiseuronal morphology

a b s t r a c t

Ethnopharmacological relevance: Acute spinal cord ischemia–reperfusion injury (SCII) is associated withpathological changes, including inflammation, edema, and neuronal apoptosis. Panax notoginsenoside(PNS), an important traditional Chinese medicine, has shown a variety of beneficial effects, includinghomeostasis maintenance, anti-myocardial ischemia activities, and neuroprotective functions. However,whether it can produce neuroprotective effects in SCII and the underlying mechanisms remain largelyelusive.Aim of the study: In the present study, we investigated the effects of PNS on neurological and histopatho-logical changes after SCII as well as the underlying mechanisms.Materials and methods: Sixty-four adult rats were randomly assigned into one of the four groups: the shamgroup, the ischemic group, the PNS group, and the Methylprednisolone group. A rat model of SCII wasadopted from a commonly used protocol that was initially proposed by Zivin. Neurological function wasevaluated with the Basso, Beattie and Bresnahan (BBB) locomotor rating scale. Histopathological changeswere examined with hematoxylin and eosin staining as well as Nissl staining. Immunohistochemistryand Western blot were conducted to compare the changes in tumor necrosis factor-�, interleukin-1�,interleukin-10, aquaporin-4 (AQP-4), member 6 of the TNF receptor superfamily (Fas), and Fas ligand(FasL) in the spinal cord. Finally, neuronal apoptosis was measured by electron microscopy.Results: The BBB scores of the PNS-treated injured animals were significantly increased. The grosshistopathological examination showed restored neuronal morphology and increased number of neuronsafter the PNS treatment. The PNS treatment decreased SCII-induced up-regulation of cytokine levels.

In addition, PNS suppressed the increased expression of AQP-4 after SCII, suggesting an anti-edemaeffect. Finally, PNS treatment inhibited injury-induced apoptosis and reduced the expression levels ofapoptosis-related proteins, Fas and FasL, confirming its anti-apoptosis effects against SCII.Conclusion: The current findings suggest that PNS produces robust neuroprotective effects in spinal cordischemia–reperfusion injury, and this role may be mediated by its anti-inflammation, anti-edema, and anti-apoptosis actions.

. Introduction

Acute spinal cord ischemia–reperfusion injury (SCII), a seri-us and debilitating central nervous system injury, can induce anmmediate or delayed paraplegia (Kuniyoshi et al., 2003). It is a

ajor complication of surgeries in the thoracic and thoracoab-ominal aneurysm with the incidence of 3–18% (MacArthur et al.,005). Although many strategies, including temporary shunts orartial bypass, drainage of the cerebrospinal fluid, pharmacologi-

al measures, and hypothermia (McCullough et al., 1988; Svenssont al., 1993; Tabayashi et al., 1993), have been developed to increaseschemic tolerance in the spinal cord, the incidence of paraplegia

∗ Corresponding author. Tel.: +86 13809195901.E-mail address: lancetning@126.com (K. Wang).

378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2011.11.040

© 2011 Elsevier Ireland Ltd. All rights reserved.

remains high and consequently, this poses a persistent and devas-tating threat to patients (Etz et al., 2008).

Although the exact mechanism of SCII remains elusive, it is gen-erally believed that inflammatory cytokines play a pivotal role intriggering a cascade of events which leads to cell apoptosis. Theearly accumulation of inflammatory cytokines in and around themicrovessels at the ischemic zones has been widely reported (Clarket al., 1994; Jean et al., 1998; Fleming et al., 2006), which can be thecause of spinal cord edema and neuronal apoptosis (Samantarayet al., 2008).

Recent studies have suggested that traditional Chinesemedicine, including tetramethylpyrazine (Fan et al., 2006) and

resveratrol (Liu et al., 2011), can be very helpful in the treatmentof SCII. Another effective medicine is Panax notoginseng (Burk)F.H. Chen (PNG), which belongs to the family of Araliaceae and hasbeen used as a traditional Chinese herbal medicine for thousands

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f years. Panax notoginsenoside (PNS), a compound isolated fromNG, is the main effective ingredient of PNG. The main componentsf PNS are ginsenoside Rb1 (29.86%), Rg1 (20.46%), Rd (7.96%), Re6.83%), and notoginsenoside R1 (2.74%) (Chen et al., 2008). Thehemical structure of each component has also been determinedecently (Yang et al., 2010). Extensive studies have demonstratedts broad physiological and pharmacological functions, including

aintaining homeostasis, protecting against thrombotic events,nd treating hyperlipidemia, atherosclerosis, coronary atheroscle-otic heart disease, and cancer (Yang and Qin, 2003; He et al.,007; Li et al., 2007). PNS also has pleiotropic benefits, such asnti-inflammation, anti-edema, anti-oxidation, and anti-apoptosisNg, 2006; Zheng et al., 2008). Particularly, PNS can protect neu-ons in animal models of cerebral ischemia–reperfusion injury (Lit al., 2009). However, to our knowledge, there is no study onts neuroprotective effects against SCII and more importantly, thenderlying mechanism of its beneficial effects remains unknown.

In this study, we used a rat model of SCII and provided fur-her evidence that PNS could produce neuroprotective effects afterCII through its roles in anti-inflammation, anti-edema, and anti-poptosis.

. Materials and methods

.1. Animals and experimental groups

This study was approved by the ethic committee of College ofedicine, Xi’an Jiaotong University, and performed in accordanceith the policies of Chinese animal research committees and guide-

ines from U.S. National Institute of Health (NIH Publication No.6-23, revised 1996).

Sprague-Dawley rats were purchased from the Experimentalnimal Facilities of Xi’an Jiaotong University. Sixty-four adult rats

260–320 g) were randomly assigned into one of the four groupsn = 16 per group): Group A (the sham group), Group B (the ischemicroup), Group C (the PNS group), and Group D (the Methylpred-isolone group). Methylprednisolone, a standard clinical drug inhe treatment of acute spinal cord injury, was used as a posi-ive control in this study. Rats in Group A were exposed to theperational area without injury; rats in Group B had spinal cordschemia–reperfusion injury and 0.9% saline intraperitoneal injec-ion 30 min before aortic clamping and reperfusion; and rats inroups C and D received intraperitoneal injection of PNS (30 mg/kg,unming Pharmaceutical Group Corporation Ltd., China; approvalumber: GYZZ Z53020662; catalog number: 08FL03) and Methyl-rednisolone (30 mg/kg, Pfizer, Belgium, dissolved in 0.9% saline),espectively, 30 min before aortic clamping and reperfusion. All ani-als were housed in separated cages with free access to food andater. Room temperature was set at 25 ± 3 ◦C with standard 12 h

ight/dark cycle.

.2. Rat model of acute SCII

The rat model of SCII was adopted from a commonly used pro-ocol (Zivin and Degirolami, 1980). The SCII model proposed byivin et al. has been developed into a mature and reliable model.lthough initially developed in rabbits, many following studiesave proved its effectiveness in rats (Usul et al., 2004; Wang and

iang, 2009; Tian et al., 2011). All animals were prohibited fromrinking during the morning of the surgery. Animals were anes-hetized with chloral hydrate (40 mg/kg, intraperitoneal injection,

aini Chemical, China) and placed in the supine position. After a 3-o 4-cm medial incision, the abdominal aorta was exposed at theevel of the left renal artery. Four hundred units of heparin weredministered 5 min before the aortic occlusion, and spinal cord

acology 139 (2012) 504– 512 505

ischemia was induced with the aorta clamped by a bulldog clampjust below the left renal artery. After the occlusion, the pulsation ofthe femoral artery disappeared. The blood flow was obstructed for30 min. Then the bulldog clamp was removed, and the abdominalwall was closed with a sterile 6-0 silk suture. Ampicillin (ShuangyePharmaceutics, China) was injected to the lower limb muscles oncea day for 3 days postoperatively to prevent infection. Body tempera-tures were closely monitored. All the rats were housed individuallywith free access to food. Bedding in each cage was changed everyday to keep it dry. After the injury, bladder massage was performedtwice a day to stimulate autonomic urinary reflex. Rats were sacri-ficed 3 days after the surgery.

2.3. Evaluation of neurological function

Locomotor recovery after SCII was scored in an open field testaccording to the Basso, Beattie and Bresnahan (BBB) locomotorrating scale from 0 (complete paralysis) to 21 (normal locomo-tion) (Basso et al., 1996). BBB scores measured a combinationof rat hind limb movement, joint movement, weight support,fore/hind limb coordination, trunk position and stability, stepping,paw placement, toe clearance, and tail position, which representsthe sequential recovery stages that rats usually attained after SCII.Rats were allowed to move freely for 4 min. Locomotion activityof the hind limb was evaluated at 24, 48 and 72 h postoperatively.Scoring standard was detailed as follows: the first part evaluatedthe activity of the hind limb joints, the second part evaluated thepace and coordination of the hind limbs, and the third part eval-uated the delicate activity of the paws during locomotion. Thelocomotion was scored by two independent observers blind to thedesign of the experiment.

2.4. Hematoxylin and eosin (HE) and Nissl staining

Three days after the surgery, the rats (n = 6 for each group) weretranscardially perfused with 0.1 mol/L phosphate buffered saline(PBS) and then with 4% paraformaldehyde (PFA) in PBS for 30 min.Spinal cords were dissected and kept in 4% PFA for post-fixationovernight. After dehydration, the spinal cords were embeddedwith paraffin and serial coronal sections with the thickness of5 �m were collected. To assess the histopathological changes, thesections were further subjected to HE and Nissl staining using well-established protocols.

2.5. Immunohistochemistry

Sections obtained in 2.4 were used for immunohistochemistry.Horseradish peroxidase (HRP)-labeled Streptavidin kit (Bosen,China) was used for immunohistochemical studies for tumor necro-sis factor-� (TNF-�), interleukin-1� (IL-1�), interleukin-10 (IL-10),aquaporin-4 (AQP-4), membrane 6 of the TNF receptor superfam-ily (Fas), and Fas ligand (FasL). The following primary antibodieswere used: rabbit anti-TNF-� (1:200), rabbit anti-IL-1� (1:200),rabbit anti-IL-10 (1:200), rabbit anti-AQP-4 (1:200), rabbit anti-Fas (1:200), and rabbit anti-FasL (1:200) (all from Bosen, China).HRP-conjugated goat anti-rabbit secondary antibody (Santa Cruz,USA) was used for 3,3′-diaminobenzidine staining using well-established protocols. Images were acquired with a biologicalimaging microscope (Olympus, Japan), and analyzed with Image-ProPlus (MediaCybernetics, USA).

2.6. Western blot

For Western blot, the rats (n = 6 for each group) were immedi-ately sacrificed and the spinal cord was taken out. Proteins wereextracted with RIPA lysis buffer kit (Bio-Tek, USA) and the protein

506 N. Ning et al. / Journal of Ethnopharm

Fig. 1. Neurological function measured by BBB locomotion scores from 0 to 72 hafter SCII in rats. (A) the sham group; (B) the ischemic group; (C) the PNS group;and (D) the Methylprednisolone group. Significant differences were observed fora(

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ll three-time points. *p < 0.01 versus Group A (sham); **p < 0.01 versus Group Bischemic). Data were presented as mean ± SD (n = 16 each group).

evels were determined by a Brefeldin A kit (Bio-Rad, USA). Theamples were subjected to SDS-PAGE gel electrophoresis (12%, 10%,2%, 10%, 8%, and 8% gels for TNF-�, IL-1�, IL-10, AQP-4, Fas, andasL, respectively). The following primary antibodies were used forestern blot: rabbit anti-TNF-� (1:500), rabbit anti-IL-1� (1:500),

abbit anti-IL-10 (1:500), rabbit anti-AQP-4 (1:500), rabbit anti-Fas1:500), and rabbit anti-FasL (1:500). HRP-conjugated goat anti-abbit secondary antibody was used. �-Actin was used as a loadingontrol. Protein bands were visualized with enhanced chemilu-inescence and images were acquired and analyzed with FR-200

ystem (Furi, China). Protein expression levels were normalized tohe levels of �-actin.

.7. Electron microscopy (EM)

For EM, the rats (n = 4 for each group) were immediately sacri-ced and the spinal cord was taken out. Spinal cord tissues wereut into chops of 1 mm3 and fixed in 2.5% glutaraldehyde at 4 ◦Cor 5 h. After washing with PBS (pH 7.4), the tissues were fixedith 1% osmic acid, gradually dehydrated with ethanol and ace-

one, embedded with epoxy resin, and then stored at 70 ◦C forolymerization for 48 h. Sections (60 nm) were collected with aeichert-Jung Ultracut microtome (Leica, Germany). Sections wereouble stained with uranyl acetate and lead citrate trihydrate,ried, and observed under transmission EM (Zeiss, Germany).

.8. Statistical analysis

Data were analyzed by SPSS 16.0 (SPSS, USA) and representeds mean ± standard deviation (SD). Group difference was analyzedy one-way analysis of variance followed by Fisher’s post hoc tests.

p value less than 0.05 was considered statistically significant.

. Results

.1. Neurological function evaluation

The evaluation of neurological function in the open field tests summarized in Fig. 1. The results showed that BBB locomotioncores in the sham group (Group A) were similar to normal valuesBasso et al., 1996), indicating that no neural dysfunction occurredn the sham group. SCII markedly decreased BBB scores at all time

oints post-injury (p < 0.01) in the ischemic group (Group B), indi-ating severe injury in neural function. In contrast to the ischemicroup, the PNS group (Group C) and Methylprednisolone groupGroup D) showed a significant increase in BBB scores (p < 0.01 at all

acology 139 (2012) 504– 512

time points), suggesting that PNS treatment improved the recoveryof neural function after SCII (Fig. 1).

In addition to behavioral changes, we also compared histopatho-logical alterations in the spinal cord after injury by HE staining(Fig. 2A). The results showed that the spinal cord in the shamgroup (Group A) had integrative infrastructures and clear bound-ary between the gray and white matters (Fig. 2a1). Blood vesselsand central canal also displayed normal morphology (Fig. 2a1).These observations indicated no neuronal apoptosis and glial pro-liferation in the sham group. In stark contrast, boundaries in theischemic group (Group B) became obscure and broad hemorrhagewas spread out in both gray and white matters (Fig. 2b1). Patches ofnecrosis were seen in the gray matter and liquefaction was foundsurrounding the damaged tissues. In addition, gaps between cellsand blood vessels became remarkably larger. A large portion ofneurons demonstrated condensed nucleus, darkly red stained cyto-plasm, and apoptotic bodies. Reactive glial cells were also foundsurrounding neurons like “satellites.” The extent of neuronal dam-age in Groups C and D was between that of Groups A and B (Fig. 2c1and d1). Under PNS or Methylprednisolone treatment, the spinalcord lacked clear infrastructures and cellular boundaries, but hem-orrhage, necrosis, and peripheral tissue edema were relatively mildas compared with in Group B. Some neurons remained, however,dimly stained, indicating blurring structures. The number of glialcells was also increased, suggesting glial proliferation.

Nissl staining demonstrated that neurons in the sham group(Group A) displayed integrative and granular-like morphology(Fig. 2a2). The plasma was densely stained with toluidine blue, indi-cating active supply of neuronal nutrients and adequate energysynthesis. However, in the ischemic group (Group B), neuronsdecreased in number and showed irregular morphologies (Fig. 2b2).Intracellular toluidine blue staining was also significantly reduced,dimly spread out, and located irregularly, indicating that SCII-induced neuronal necrosis and apoptosis led to neuronal lossand the remaining neurons suffered from poor-energy-synthesis-induced neuronal dysfunction. Under PNS or Methylprednisolonetreatment (Groups C and D), we found retained neurons, althoughthe number was reduced (Fig. 2c2 and d2). Tissue morphologywas generally maintained with lighter staining in the cytoplasmand granular-like neurons, showing that with PNS or Methylpred-nisolone intervention, neurons partially restored their function,especially in cellular nutrient supply and energy synthesis.

3.2. Anti-inflammation effects of PNS

The anti-inflammatory effects of PNS were examined bythe expression of several inflammatory factors using immuno-histochemical (Fig. 3A) and Western blot (Fig. 3C) methods.Immunohistochemical staining of IL-1�, IL-10, and TNF-� was allnegative in gray matters and peripheral white matters in the shamgroup (Group A), suggesting no obvious neuronal inflammationin this area (Fig. 3Aa). In the ischemic group (Group B), strongstaining of IL-1�, IL-10, and TNF-� was observed, suggesting a dra-matic infiltration of inflammatory cells in both gray matters andperipheral white matters (p < 0.01 for all three factors, Fig. 3Ab). Incontrast, after PNS or Methylprednisolone treatment, we observedreduced immunostaining of IL-1�, IL-10, and TNF-�, indicating thatthe infiltration of inflammatory cells was greatly relieved (p < 0.01for all three factors, Fig. 3Ac and Ad). The expression levels ofinflammatory factors are presented as percentages in Fig. 3B.

The representative Western blot gels for TNF-� (17 kD), IL-1�(30 kD), IL-10 (18 kD) and �-actin (42 kD) are showed in Fig. 3C.

The expression levels of these proteins were quantified and nor-malized to the levels of �-actin and presented as percentages ofchanges from the levels in the sham group (Group A) (Fig. 3D). Allthree proteins were expressed at relatively low levels in the sham

N. Ning et al. / Journal of Ethnopharmacology 139 (2012) 504– 512 507

Fig. 2. (A) HE staining of the spinal cord (400×). HE staining showed normal neuronal and glial morphology in Group A (sham). In Group B (ischemic), the injured spinal cordexhibited typical necrosis, including broad hemorrhage, edema, reactive gliosis, and neuronal apoptosis with condensed nuclei. In Groups C (PNS) and D (Methylprednisolone),neurons displayed normal morphology with clear boundary. Compared with the situation in Group B, only mild glial proliferation, hemorrhage, and edema occurred in Group C.(B) Nissl staining of the spinal cord (400×). Nissl staining showed that in Group A (sham), neurons exhibited a large amount of granule-like, dense toluidine blue staining in thec in numt displ

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he number of Nissl bodies restored as compared with that in Group B and neurons

roup (Group A) and were significantly increased after SCII (Group, p < 0.01). Expression levels of these proteins in Groups C and Dhowed a dramatic drop compared with those in Group B (p < 0.01).hese results indicated that similar to Methylprednisolone, PNSould effectively decrease inflammation in damaged spinal cordissues, although it could not completely ameliorate the symptoms.

.3. Anti-edema effects of PNS

The anti-edema effects of PNS were examined by the expres-ion of AQP-4, a member of the aquaporin family of integralembrane proteins, using immunohistochemical (Fig. 4A) and

estern blot (Fig. 4C) methods. The sham group (Group A) demon-

trated little positive AQP-4 staining in the central ependymalells and the gliocytes around blood vessels, suggesting low AQP-4xpression in these areas (Fig. 4Aa). In the ischemic group (Group

ber or even disappeared in neurons. In Groups C (PNS) and D (Methylprednisolone),ayed relatively normal morphology.

B), strong positive staining of AQP-4 was observed, suggestingmarked edema (p < 0.01, Fig. 4Ab). After PNS or Methylprednisolonetreatment, AQP-4 staining was significantly recued (p < 0.01), indi-cating that SCII-induced edema was relieved (Fig. 4Ac and Ad).The expression levels of AQP-4 are presented as percentages inFig. 4B.

The representative Western blot gels for AQP-4 (30 kDa) and �-actin (42 kDa) are shown in Fig. 4C. The expression level of AQP-4was quantified and normalized to �-actin and presented as per-centages of changes from that in Group A in Fig. 4D. AQP-4 wasexpressed at low levels in the sham group (Group A) and signifi-cantly increased after SCII (Group B, p < 0.01). Its expression level

in Groups C and D showed a dramatic drop as compared with thatin Group B (p < 0.01). These results indicated that like Methylpred-nisolone, PNS could effectively treat SCII-induced edema althoughit could not fully ameliorate the symptoms.

508 N. Ning et al. / Journal of Ethnopharmacology 139 (2012) 504– 512

Fig. 3. (A and B) Immunohistochemistry of inflammatory factors in the spinal cord. (A) Representative immunohistochemistry images (400×) of TNF-�, IL-1�, and IL-10 at72 h post-lesion. There was no obvious expression of inflammatory factors in Group A (sham). In Group B (ischemic), dramatic increases in TNF-�, IL-1�, and IL-10 wereobserved in the impacted location. In Groups C (PNS) and D (Methylprednisolone), the infiltration of inflammatory cells was reduced, revealed by decreased expression ofTNF-�, IL-1�, and IL-10. (B) Quantification of immunohistochemistry results (*p < 0.01 versus Group A; **p < 0.01 versus Group B). (C and D) Western blot of inflammatoryf �, ando in Gr( roup

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actors in the spinal cord. (C) Representative Western blot images of TNF-�, IL-1f these inflammatory factors was significantly higher in Group B (ischemic) thanMethylprednisolone) than in Group B (*p < 0.01 versus Group A; **p < 0.01 versus G

.4. Anti-apoptosis effects of PNS

The anti-apoptosis effects of PNS were analyzed by examin-ng the ultrastructures of neurons, using EM (Fig. 5A), and thexpression of Fas and FasL, two apoptosis-related proteins, usingmmunohistochemical (Fig. 5B) and Western blot (Fig. 5D) meth-ds.

EM examination showed no apoptotic cells in the sham groupGroup A), in which the nuclear membranes were integrative andranules in the chromatin were delicate and evenly distributedFig. 5Aa). Neither introcession of the mitochondrial membranesor enlargement of the granular endoplasmic reticulum (ER) was

ound. Nucleus was round with no formation of apoptotic bod-es (Fig. 5Aa). In comparison, a large amount of apoptotic cells

ere found in the ischemic group (Group B, Fig. 5Ab). Neuronshrank and exhibited abnormal morphology with vacuolated cyto-lasm, broken or missing mitochondrial ridges, and dramaticallynlarged ER. Nuclear membranes were introverted and the chro-atin was attached to them. Finally, the ultrastructures of the

eurons in Groups C and D demonstrated a much clearer morphol-gy, including an evenly distributed chromatin, integrative nuclearembranes with fewer introcession, and intact granular ER anditochondria (Fig. 5Ac and Ad). These results suggested that similar

o Methylprednisolone, PNS could effectively reduce SCII-inducedpoptosis of spinal cord neurons.

The sham group (Group A) showed no positive Fas and FasLtaining in neurons or glial cells (Fig. 5Ba), whereas in the ischemic

roup (Group B), the positive staining was widely distributed inoth white and gray matters (Fig. 5Bb). Under PNS or Methylpred-isolone treatment (Groups C and D), the levels of positive cellsere dramatically decreased (Fig. 5Bc and Bd), consistent with

IL-10 at 72 h post-lesion. (D) Quantification of Western blot results. Expressionoup A (sham). Their expression was significantly lower in Groups C (PNS) and DB).

reduced structure damage in the spine cord. Quantification of theexpression levels is shown in Fig. 5C.

The Western blot results of Fas and FasL were consistent withimmunohistochemical results. The levels of Fas and FasL were lowin the sham group (Group A) and significantly increased after SCIIin Group B. Both PNS and Methylprednisolone treatments partiallyreversed SCII-induced increase in expression levels (Fig. 5D and E).These results indicated that PNS could exert the neuroprotectiveeffects by preventing the formation of apoptotic body and reducingthe expression of apoptosis-related proteins.

4. Discussion

SCII not only results in direct damage by ischemia and anoxiabut also inflicts ensuing pathological changes, including acuteinflammatory reaction, edema, lipid peroxidation, calcium over-load, excitatory amino acid poisoning (Simpson et al., 1990), andapoptosis (Sakurai et al., 1998). It is noteworthy that in most cases,the secondary damage, rather than the primary damage, becomesthe critical obstacle to successful treatment. The primary damageinitiated by an ischemic injury is usually irreversible; however, thesecondary damage is an active process occurring at the molecularand cellular levels and thus is reversible and modifiable. This makesthe treatment and recovery of SCII doable (Ueno et al., 1994). Sofar many strategies, including temporary shunts or partial bypassand drainage of cerebrospinal fluid have been developed, but theireffects are controversial and paraplegia remains a persistent com-

plication.

In this study, we employed a rat model of SCII to examine theprotective effects of PNS against SCII injury. To assess the neuro-logical function recovery of the spine cord, we used the BBB rating

N. Ning et al. / Journal of Ethnopharmacology 139 (2012) 504– 512 509

Fig. 4. (A and B) Immunohistochemistry of AQP-4 in the spinal cord. (A) Representative immunohistochemical images (400×) of AQP-4 at 72 h post-lesion. AQP-4 expressionwas low in Group A (sham). In Group B (ischemic), dramatic increases in AQP-4 expression were observed in the impacted location. In Groups C (PNS) and D (Methylpred-nisolone), the AQP-4 expression was lower than in Group B. (B) Quantification of immunohistochemical results (*p < 0.01 versus Group A; **p < 0.01 versus Group B). (C andD) Western blot of AQP-4 in the spinal cord. (C) Representative Western blot band images of AQP-4 at 72 h post-lesion. (D) Quantification of Western blot results. Expressionsof AQP-4 was significantly increased in Group B (ischemic) compared with that in Group A (sham), whereas its expression was significantly lower in Groups C (PNS) and D( sus Gr

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o analyze the locomotive function 1–3 days after SCII and showedignificant improvement in neurological function in PNS-treatednimals. This confirms that PNS can ameliorate the motor deficitsnduced by SCII.

The spinal cord is located in the vertebral canal and surroundedy meninges. A transverse section of the spinal cord shows autterfly-shaped core (the gray matter), which contains an enor-ous number of neurons of varying sizes and shapes, and the

urrounding white matter, which contains nerve fibers, neuroglias,nd blood vessels (Craven, 2004). In the present study, we observedevere hyperemia and edema as well as dramatically less neuronsnd more reactive gliosis in the SCII-treated rats by HE stain-ng. However, after the PNS treatment, we observed significantmprovement in all these conditions, suggesting that PNS may pro-ect the tissue structure in the spinal cord. We then used Nissltaining to identity the pathological changes of the spinal cord afterCII and confirmed our hypothesis on the effects of PNS. Nissl bod-es, which contain rough ER and free ribosomes, are the sites toynthesize protein and supply nutrition and energy to neurons.hus the number and morphology of the Nissl body indicate theitality and function of protein synthesis in neurons. The nissl bodyay reduce in number, dissolve, or even disappear under various

athological conditions (Pullen, 1990). In this study, we observedhat the number of neurons and Nissl bodies were both dramati-ally decreased in SCII-treated rats, suggesting neuronal responseso the injury. In contrast, in the PNS-treated rats, both the number

nd morphology of the neurons and Nissl bodies were improved.hese histological data suggest that PNS can preserve the structurend function of the spinal cord after acute ischemia–reperfusionnjury.

oup B).

Accumulating evidence has indicated that leukocytes play a piv-otal role in acute ischemia–reperfusion injury, and inflammation isan essential event in the secondary damage after SCII. Shortly afterSCII, leukocytes begin to accumulate in and around the microves-sels of the ischemic zones (Jean et al., 1998; Fleming et al., 2006),infiltrate into the spinal cord, stimulate the neuronal, glial andendothelial cells, and produce inflammatory factors, including TNF-�, IL-1�, and IL-10 (Barone et al., 1991). These cytokines triggerthe inflammatory cascade to produce even more inflammatory fac-tors and affect the gene expression of glial cells. Ultimately, theseries of events in inflammation break down the blood spinal cordbarrier and further deteriorate the ischemic and anoxic injury.In the present study, we used immunohistochemistry and West-ern blot to investigate the inflammatory factors. Both methodsconsistently showed that TNF-�, IL-1�, and IL-10 were signifi-cantly up-regulated in the spinal cord after SCII, which was inaccordance with previous studies (Seekamp et al., 1998; Fleminget al., 2006) and indicated that inflammatory factors could be pro-duced after SCII and were involved in its secondary damage. Wealso observed that PNS could reduce the activity of leukocytes,decrease expression of these inflammatory factors, and relieve thesecondary inflammation-induced injury, suggesting that PNS hasanti-inflammation function against the secondary damage of SCII.

Edema is another evident pathophysiological change in thespinal cord after SCII. During the secondary damage of SCII, manyvasoactive substances are produced. They can break down the

blood spinal cord barrier, increase the permeability of blood capil-lary, and finally lead to edema in the spinal cord (Olsson et al., 1992).AQP plays an important role in water transport in many vital sys-tems, including respiratory, urinary, and nervous systems. AQP-4

510 N. Ning et al. / Journal of Ethnopharmacology 139 (2012) 504– 512

Fig. 5. A Electron microscopy showing neuronal apoptosis in the spinal cord (10,000×). In Group A (sham), normal neurons displayed intact nuclear membranes, evenlydistributed chromatin granules, and no apoptotic bodies. In Group B (ischemic), neurons shrank and exhibited abnormal morphology with vacuolated cytoplasm, condensedchromatin, introverted nuclear membrane, and increased apoptotic bodies. Neurons in Groups C (PNS) and D (Methylprednisolone), however, showed less condensedchromatin and clearer nuclear membrane. (B and C) Immunohistochemistry of Fas/FasL in the spinal cord. (B) Representative immunohistochemistry images (400×) ofFas/FasL at 72 h post-lesion. No positive staining was observed in Group A (sham). In Group B (ischemic), dramatic increases in Fas and FasL were observed in the impactedlocation. In Groups C (PNS) and D (Methylprednisolone), Fas and FasL staining was reduced. (C) The quantification of immunohistochemistry results (*p < 0.01 versus GroupA; **p < 0.01 versus Group B). (D and E) Western blot of Fas/FasL in the spinal cord. (D) Representative Western blot band images of Fas and FasL at 72 h post-lesion. (E)Q igheri versu

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uantification of Western blot results. Expression of Fas and FasL was significantly hn Groups C (PNS) and D (Methylprednisolone) (*p < 0.01 versus Group A; **p < 0.01

s a member of the AQP family and widely expressed in the ner-ous system. In particular, a positive correlation between AQP-4nd spinal cord edema has been recently reported (Oshio et al.,004). In the current study, after SCII, both immunohistochemistrynd Western blot methods confirmed that AQP-4 expression wasignificantly increased in the spinal cord. The PNS treatment couldemarkably decrease its expression in the spinal cord, indicatinghat PNS could exert anti-edema effects against SCII.

Apoptosis is programmed cell death characterized by DNA early

egradation. On the morphological level, apoptosis involves manypecific ultrastructural changes, including membrane shrinkage,hromatin condensation and margination, and formation of apop-otic bodies (Elmore, 2007). Previous studies have shown that

in Group B (ischemic) than in Group A (sham). This increase was partially reverseds Group B).

apoptosis is common in spinal cord neurons after SCII (Hayashiet al., 1998). In the present study, EM examination showed thatafter SCII, neurons shrank in size and exhibited abnormal morphol-ogy with vacuolated cytoplasm, condensed chromatin, introvertednuclear membrane, and increased apoptotic bodies. In the PNS-treated rats, such ultrastructural abnormalities were significantlyameliorated. A variety of genes participate in apoptosis, suchas Fas and FasL, bcl-2 family, and caspase family. For example,Fas/FasL can dramatically enhance immune reactions after spinal

cord injury (Zurita et al., 2001). After injury, FasL drives the inter-action among three Fas monomers, and then activates the deathdomain of Fas to transmit signals to Fas-associated protein withdeath domain, death-associated protein 6, and receptor-interacting

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N. Ning et al. / Journal of Ethno

rotein, which activate the caspase family and thus initiate apo-tosis. In our study, both immunohistochemistry and Western blotesults showed that PNS inhibited Fas and FasL expression afterCII, which could reduce and/or delay neuronal apoptosis. Combin-ng the EM, immunohistochemistry, and Western blot results, weonclude that PNS can produce an anti-apoptosis effect to protecthe spinal cord after SCII by regulating the expression of Fas/FasL.

In the current study, we demonstrated that PNS exerts sig-ificant protective effects in anti-inflammation, anti-edema, andnti-apoptosis during acute spinal cord ischemia–reperfusionnjury. Our discovery is consistent with previous studies on theherapeutic effects of pseudo-ginseng: anti-inflammation (Li andhu, 1999; Wang et al., 2006; Chang et al., 2007), anti-cerebraldema (Zhou et al., 2008), and anti-neuronal apoptosis (Yang et al.,008; Li et al., 2009). Based on our research and previous studies, weelieve that the anti-inflammation, anti-edema, and anti-apoptosisunctions of PNS are related to the pharmacological mechanismsnderlying the therapeutic role of pseudo-ginseng in the treatmentf spinal cord ischemia–reperfusion injury.

In summary, PNS can inhibit the injury-induced up-regulationf inflammatory factors, which are produced in the damage zones,nd relieve inflammatory cascade. PNS also significantly decreasesQP-4 expression in the spinal cord after injury. In addition, PNS

nhibits Fas and FasL expression after SCII. Finally, PNS can pre-erve the structure of the injured spinal cord and retain neuronalunction. In conclusion, we believe that PNS produces robust neu-oprotective effects in the rat model of acute SCII, which may be dueo its anti-inflammation, anti-edema and anti-apoptosis actions.

cknowledgments

We would like to thank the Graduate student experiment centerf the School of Medicine, Xi’an Jiaotong University, for their exper-mental apparatus. We also thank Prof. Shui-Ping Han (Departmentf Pathology, School of Medicine, Xi’an Jiaotong University) and Dr.hen Zhang (Department of Orthopedics, the Second Affiliated Hos-ital of Medical School, Xi’an Jiaotong University) for their technicalssistance and helpful discussion.

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