Protective effect of GCSB-5, an herbal preparation, against peripheral nerve injury in rats

Journal of Ethnopharmacology 136 (2011) 297–304 Contents lists available at ScienceDirect Journal of Ethnopharmacology journa l homepage: www.e lsev ier .com/ Protective effect of GCSB-5, an herbal preparation injury i Tae-Hoo i Kim Sun-Mee a School of Pha b Jaseng Hospit a r t i c l Article history: Received 11 Ja Received in revised form 12 April 2011 Accepted 14 April 2011 Available online 4 May 2011 Keywords: Achyranthis bidentata Blume Eucommia ulm GCSB-5 Motor function Oxidative stre Peripheral ner nam in, A Cibotium baromets J. Smith, Glycine max Meriill, and Eucommia ulmoides Oliver), has been widely used in Asia for treatment of neuropathic and inflammatory diseases. This study investigated the protective effect of GCSB-5 against peripheral nerve injury in vitro and in vivo. Materials and methods: After left sciatic nerve transection, rats received oral administration of GCSB-5 (30, 100, 300, and 600mg/kg), or saline (vehicle), respectively, once daily for 8 weeks. Motor functional 1. Introdu Peripher practice du and can cau can subsequ nerve injur lular enviro neurons, re (ROS) (Stoll plays a role involved in Antioxidant ∗ Correspon Cheoncheon-d Tel.: +82 31 29 E-mail add 0378-8741/$ – doi:10.1016/j. oides Oliver al recovery ss ve injury recovery and axonal nerve regeneration were evaluated by measurement of sciatic functional index (SFI), sensory regeneration distance, and gastrocnemiusmusclemass ratio. Themyelinated axon number was counted by morphometric analysis. In the in vitro study, the effects of GCSB-5 on H2O2-induced oxidative damage in SH-SY5Y cells were investigated by measurement of cell viability, production of reactive oxygen species (ROS), lipid peroxidation, release of lactate dehydrogenease (LDH), and cellular glutathione contents. Neurite outgrowth was also determined. Results: After 8 weeks of nerve transection, SFI, regeneration distance, and gastrocnemius muscle mass ratio and myelinated axon number showed a significant decrease and these decreases were attenuated by GCSB-5. GCSB-5 significantly inhibited H2O2-induced cell death and oxidative stress, as evidenced by decreases in production of ROS and lipid peroxidation and release of LDH, and by increase in total GSH content. Conclusions: The neuroprotective effect afforded by GCSB-5 is due in part to reduced oxidative stress. © 2011 Elsevier Ireland Ltd. All rights reserved. ction al nerve injuries are commonly encountered in clinical e to accidental trauma, acute compression, or surgery, se temporary or life-long neuronal dysfunctions that ently lead to economic or social disability. Peripheral y sets in motion a dramatic change in the intracel- nment, including molecular composition in lesioned sulting in overproduction of reactive oxygen species and Muller, 1999). Release of ROS at the site of injury in modulation of the inflammatory response and is necrotic and apoptotic cell death after nerve injury. s counteract the detrimental effects of ROS and pro- ding author. School of Pharmacy, Sungkyunkwan University, 300 ong, Jangan-gu, Suwon-si, Gyeonggi-do 440-746, South Korea. 0 7712; fax: +82 31 292 8800. ress: [email protected] (S.-M. Lee). tect against oxidative injury, which is considered to be an essential role in themechanisms of neuroprotection and functional recovery (Maher, 2006). Melatonin, a neurosecretory product of the pineal gland,which functions as an antioxidant and free radical scavenger, showed a beneficial effect on nerve recovery after sciatic nerve dissection (Atik et al., 2011). Traditionally, nerve transection injuries have been remedied by a number of surgical methods, including nerve transfer, nerve grafts, and end-to-side neurorrhaphy (Matsumoto et al., 2000). Despite early diagnosis and improved modern surgical tech- niques, recovery of function can never reach the pre-injury level. Administration of nerve growth factor (NGF) and brain-derived neurotrophic factor has attracted the attention of researchers as a possible therapy for treatment of peripheral nerve injuries in order to accelerate axonal regeneration and target muscle reinner- vation.However, systemically delivered recombinant neurotrophic factor has shownunexpected side effects in clinical trials (Wei et al., 2009). Therefore, it is important to find agents that can promote functional recoverywithout toxicity problems. Lu et al. (2010) have see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. jep.2011.04.037 n rats n Kima, Seong-Jin Yoona, Woo-Cheol Leea, Joon-K Leea,∗ rmacy, Sungkyunkwan University, Suwon 440-746, South Korea al, 635 Sinsa-dong, Gangnam-gu, Seoul, South Korea e i n f o nuary 2011 a b s t r a c t Aim of the study: GCSB-5 (traditional herbs (Saposhnikovia divaricata Schisk locate / je thpharm , against peripheral nerve a, Joonshik Shinb, Sangho Leeb, e: Chungpa-Juhn), an herbal medicine composed of 6 crude chyranthis bidentata Blume, Acanthopanax sessiliflorum Seem, 298 T.-H. Kim et al. / Journal of Ethnopharmacology 136 (2011) 297–304 suggested Astragalus membranaceus extract as a potential nerve growth-promoting factor, being salutary in aiding growth of axons in the peripheral nerve. Recently, Ginsenoside Rg1, which showed ROS scavenging activity, was reported to promote nerve regenera- tion after n GCSB-5 and ankylos ture of six root of Achy rum Seem, maxMeriill has been w Asia. Rooto such asONO of root of A regeneratio ron apopto (Ding et al. ROS scaven properties 1995) and have been r in vitro and been report Therefor peripheral using a rat human SH- 2. Materia 2.1. Materi Dulbecc fetal bovine 100�g/mL BRL (Grand (PBS) was Austria). 2,7 obtained fr Nerve grow son,WI, USA from Sigma 2.2. Prepara Six crud (Umbellifer ranthaceae (Araliaceae soniaceae) and bark o was boiled free-dried a with molec extract of as a powde purchased ticated by Seoul, Kore study were Orential M high-perfor dient extrac for root of S (0.311–0.312mg/g) for root of Achyranthis bidentata Blume, acan- thosideD (0.577–0.578mg/g) for bark ofAcanthopanax sessiliflorum Seem, onitin-4-O-�-d-glucopyranoside for rhizome of Cibotium baromets J. Smith, genistin (0.0426–0.0427mg/g) for seed ofGlycine eriill oid B-5 latio ima le Sp hanB ppro and al In ere g/kg d thr y tra y sci ylon The ls we esthe min B-5 rally (Chu follow b)GC (con nerv rame am g , and nctio scia 2, 4, et a w pr olor 0 cm (PL) h the ed fo 38. 13. his fo of −1 stro we dose s we sep rate erve injuries (Ma et al., 2010). has been used clinically for treatment of arthritis, disc, ing spondylitis (Chung et al., 2010). It consists of amix- crude drugs (root of Saposhnikovia divaricata Schiskin, ranthis bidentata Blume, bark of Acanthopanax sessiliflo- rhizome of Cibotium baromets J. Smith, seed of Glycine , andbarkofEucommiaulmoidesOliver); eachcrudedrug idely used for treatment of inflammatory diseases in fAchyranthis bidentataBlumecanscavenge free radicals, O−, HOCl, andOH− (Ida et al., 1998).Moreover, extracts chyranthis bidentata Blume accelerate peripheral nerve n via enhancement of nerve growth, preventing neu- sis, and inducing neuronal differentiation of PC12 cells , 2008). Bark of Eucommia ulmoides Oliver also shows ging activity (Yen and Hsieh, 2000). Anti-inflammatory of bark of Acanthopanax sessiliflorum Seem (Kim et al., bark of Eucommia ulmoides Oliver (Hong et al., 1998) eported. Anti-inflammatory activity of GCSB-5 in both in vivo acute and chronic animal models has recently ed (Chung et al., 2010). e, this study investigated the effect of GCSB-5 on nerve injury and molecular mechanisms of protection, in vivo sciatic nerve transection and repair model and SY5Y neuroblastoma cell line in vitro. l and methods als o’s modified Eagle’s medium (DMEM), heat-inactivated serum (FBS), and Pen/Strep (100U/mL penicillin, and streptomycin, respectively) were obtained from Gibco Island, NY, USA). Dulbecco’s phosphate buffer saline purchased from PAA Laboratories GmbH (Pasching, -Dichlorodihydrofluorescein diacetate (H2DCFDA)was om Molecular Probes (Invitrogen, Carlsbad, CA, USA). th factor (NGF) was purchased from Promega (Madi- ). Unless stated otherwise, all chemicalswere obtained Chemical Co. (St. Louis, MO, USA). tion and composition of GCSB-5 e herbs [root of Saposhnikovia divaricata Schiskin ae) 4.44g, root of Achyranthis bidentata Blume (Ama- ) 4.44g, bark of Acanthopanax sessiliflorum Seem ) 4.44g, rhizome of Cibotium baromets J. Smith (Dick- 2.78g, seed of Glycine max Meriill (Fabaceae) 2.78g, f Eucommia ulmoides Oliver (Eucommiaceae) 1.39g] in tap water (1 L) for 3h and then the extract was nd then subjected to ultrafiltration and components ular weight over 10,000 were excluded to obtain the GCSB-5 (3.47g, 17.1%). The filtrate was lyophilized r and kept at 4 ◦C until use. The crude drugs were from an herbal market, Seoul, Korea, and authen- Dr. S.H. Lee, Jaseng Hospital of Oriental Medicine, a. The voucher specimens of the plants used in this deposited in the herbarium of Jaseng Hospital of edicine. The validation of GSCB-5 was performed by mance liquid chromatography analysis of each ingre- t using six indicator biological components: cimifugin aposhnikovia divaricata Schiskin, 20-hydroxyecdysone max M mia ulm GCS to regu 2.3. An Ma byDae were a mittee Nation mals w (100m expose sharpl surger 10/0 n 1996). anima the an 2.4. Ad GCS tered o study to the sham( section sciatic the pa the sh pooled 2.5. Fu The and 1, of Bain SFI, pa water c (10×8 length on bot and us SFI = − + In t an SFI 2.6. Ga At 8 lethal scissor used to un-ope and geniposide (0.431–0.432mg/g) for bark of Eucom- es Oliver (Lee and Cha, 2008; Cha and Lee, 2009). was further standardized for quality control according ns imposedby theKorea Food andDrugAdministration. l treatments and surgical procedure rague-Dawley rats, weighing 200–220g, were supplied iolinkCo., Ltd., Eumseong, Korea. All animal procedures vedby the SungkyunkwanUniversityAnimal CareCom- wereperformed inaccordancewith theguidelinesof the stitutes of Health. In preparation for surgery, the ani- anesthetized by intraperitoneal injection of ketamine ) and xylazine (10mg/kg). The right sciatic nerve was ough a gluteal muscle-splitting incision. The nerve was nsected 10mm distal to the sciatic notch using micro- ssors and immediately repaired with four epineurial sutures under an operating microscope (Kanaya et al., wound was closed in layers using silk sutures and the re returned to their cages and allowed to recover from sia. istration of GCSB-5 was dissolved in normal saline (vehicle) and adminis- once a day. Dose selection was based on a previous ng et al., 2010). The animals were assigned randomly ing groups (10 animals per group); (a) vehicle-treated SB5-treated sham(c) vehicle-treated sciatic nerve tran- trol), (d–g)GCSB-530, 100, 300, and600mg/kg-treated e transection. No differences were observed in any of ters between the vehicle- and GCSB-5-treated rats in roup; therefore, the results of groups (a) and (b) were were referred to as sham. nal assessment tic functional index (SFI)was determinedbefore surgery 6, and 8 weeks after surgery according to the method l. (1989) for transected nerve injury. For calculation of ints were obtained by moistening the hind paws with swhile ratswalkedalongawhitepaper coveredcorridor ). Black paw prints were left on the paper. Paw print , total toe spread (TS), and intermediary toe spread (IT) normal (N) and experimental (E) sides were measured r calculation of the SFI: 3([EPL − NPL]/NPL) + 109.5([ETS − NTS]/NTS) 3([EIT − NIT]/NIT) − 8.8 rmula, an SFI of 0 indicates normal nerve function, and 00 indicates total impairment. cnemius muscle weight eks after surgery, the animals were euthanized with a of ether. Skins were removed from the hindlimb, and re inserted under the Achilles’s tendon; the blade was arate the gastrocnemius muscle. Both the operated and d sides of the gastrocnemius muscles of each animal T.-H. Kim et al. / Journal of Ethnopharmacology 136 (2011) 297–304 299 were weighed immediately while still wet at the time of euthana- sia. Values were expressed as a ratio by dividing the wet weight of the gastrocnemius muscle of the operated side by that of the un-operated side. 2.7. Regene Regener using the pi surgery, an nerve was microsurgic intervals. W pinched, th movement The distanc first respon 2.8. Neurom After co surgery, a 1 distal and p in a solution Following fi tetroxide, a ded in Spur using a mic dine blue fo Co., Tokyo, the unbiase examiners w 2.9. Cell cul The SH-S the America SH-SY5Y ce 10% FBS, 20 37 ◦C in a h carried out 2.10. Neuri SH-SY5Y 6×104 cells and reversi proliferatio days, cellsw ence or abs the total nu rites (length blind. After phase-cont tion, Olymp photograph bearing cell Triplicate w aged for eac twice. 2.11. Cell vi Cellswe (1×10−4, 1 phate buffe incubation, 500�M of H2O2 was added to each well and the plates were further incubated for 24h before determination of cell via- bility. Cell viability of SH-SY5Y cells was assessed using a modified MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-tetrazolium bro- assay at is n (d trati r 4h s add tical late acta ivity tome b Co OS p SY5Y and t aton ith hlor at 3 d twi addi cenc vale, ngth alon d pe s acc trati activ otal ls we (1× (500 with in × g hosp SH le of 41 gluta DPH tatis ults a resu n th riate ration distance ation distance of the sensory nerve fiberswas evaluated nch-reflex test (Dahlin and Kanje, 1992). At 7 days after imals of each group were anesthetized and the sciatic re-exposed. The sciatic nerve was then pinched with al forceps in a distal-to-proximal direction at 0.5mm hen the tips of the fastest growing sensory axons were ey elicited a reflex response,which could be observed as of the leg and contractions of the muscles on the back. e between the repair site and the place where the rat ded was taken as the regeneration distance. orphometry mpletion of functional assessments at 8 weeks after cm segment of sciatic nerve, including the segment roximal to the coaptation site was excised and fixed of phosphate-buffered 2.5% glutaraldehyde overnight. xation, the nerve tissue was post-fixed in 0.5% osmium nd then dehydrated with gradient alcohol, and embed- r’s resin. The tissue was then cut to a thickness of 2�m rotome with a dry glass knife, and stained with tolui- r a lightmicroscope (Olympus CKX 41, OlympusOptical Japan). Myelinated axons were quantified according to d counting rule (Kaplan et al., 2005), and conducted by ho were blind to the experimental design. ture and treatments Y5Y human neuroblastoma cell linewas obtained from n Type Tissue Culture Collection (Rockville, MD, USA). llswere cultured inDMEMmediumsupplementedwith mM glutamine, and 1% Pen/Strep and maintained at umidified atmosphere with 5% CO2. Experiments were 24h after the cells were seeded. te outgrowth cells were plated in 6-well plates at a density of /well and treated with 0.4�M aphidicolin, a specific ble inhibitor of DNA polymerase ˛ and ı, which ceases n and extends long neurites (LoPresti et al., 1992). At 5 erewashed and treatedwithNGF10ng/mL in the pres- ence of GCSB-5 or melatonin. At 96h after incubation, mberof cells and thenumberof cells providedwithneu- at least double that of cell diameter) were counted on the incubation period, cultures were observed under rast, using an inverted microscope (200× magnifica- us BX51/Olympus DP71, Olympus, Japan) and then ed by using a digital camera. The percentage of neurite- s was determined after at least 100 cells were counted. ells were run in experiments and the data were aver- h treatment group. The entire experimentwas repeated ability re pre-incubatedwith various concentrations ofGCSB-5 ×10−3, 1×10−2 and 1×10−1 mg/mL), a vehicle, phos- red saline (PBS), or melatonin (500�M) for 2h. After mide] salt th solutio concen anothe ide wa The op microp 2.12. L Act tropho (IVDLa 2.13. R SH- plates, or mel batedw 2,7-dic 30min washe for an fluores Sunny wavele 2.14. M Lipi tration concen acid re 2.15. T Cel GCSB-5 tonin twice pended 10,000 sium p Total G length yeast and NA 2.16. S Res of the betwee approp (Mosmann, 1983).MTT is a yellow-colored tetrazolium reduced to a purple formazan. After incubation, MTT issolved in PBS) was added to each well with a final on of 5mg/mL, and the incubation was continued for at 37 ◦C. Finally, an equal volume of dimethyl sulfox- ed to each well to solubilize any deposited formazan. density of each well was measured at 560nm using a reader (Emax, Molecular Devices, Sunnyvale, CA). te dehydrogenase (LDH) activity of LDH in the culture media was analyzed by spec- try procedures using the ChemiLab LDH assay kit ., Ltd., Suwon, Korea). roduction cells were cultured to 70–80% confluence in 96-well reatedwithvarious concentrationsofGCSB-5, a vehicle, in (500�M) for 2h; the plates were then further incu- 25�MH2DCFDA,which becomes a fluorescent product, ofluorescein, in thepresence of awide variety of ROS, for 7 ◦C. After removal of excess H2DCFDA, the cells were cewithDMEMandwere incubatedwithH2O2 (500�M) tional 30min. Cellular fluorescence was measured in a emicroplate reader (SpecraMaxM5,MolecularDevices, CA, USA) at excitationwavelength 485nmand emission 530nm. dialdehyde (MDA) level roxidation was evaluated by measuring MDA concen- ording to the method of Buege and Aust (1978). MDA onswere calculated by the absorbance of thiobarbituric e substances at 532nm. glutathione (GSH) contents re incubated with H2O2 in the presence or absence of 10−4, 1×10−3, 1×10−2 and 1×10−1 mg/mL) or mela- �M) for 30min. After incubation, cells were washed ice-cold PBS, collected by tripsin-EDTA, and resus- 1% picric acid. The homogenate was centrifuged at for 10min, and an equal volume of 100mM potas- hate buffer (pH 7.4) was added to the supernatant. vel was determined spectrophotometrically at a wave- 2nm using the method reported by Tietze (1969) with thione reductase, 5,5′-dithio-bis(2-nitrobenzoic acid) . tical analysis re reported as a mean± S.E.M. The overall significance lts was examined using two-way ANOVA. Differences e groupswere considered significant at p 300 T.-H. Kim et al. / Journal of Ethnopharmacology 136 (2011) 297–304 Fig. 1. Effects surgery. Rats w saline once a d rats per group 3. Results 3.1. Motor SFIs for not shown) in Fig. 1, at 1 to approxim SFI values in period of o tional recov the GCSB-5 6, and 8 we greater imp 3.2. Gastroc As show operated an surgery wa trocnemius This decrea 600mg/kg ( 3.3. Sciatic In the c was 6.84± the control 600mg/kg G 3.4. Neurom At 8 wee transverse s in the contr cant decrea GCSB-5 at t 3.5. Neurot SH-SY5Y nous NGF, w (data not sh trol group the number ffects tion d tered orally with GCSB-5 30, 100, 300 and 600mg/kg or saline once a day gery. The values are represented asmeans± S.E.M for 10 rats per group. Sig- y different (*p T.-H. Kim et al. / Journal of Ethnopharmacology 136 (2011) 297–304 301 Fig. 3. Effects (A) Sham grou GCSB-5 30, 10 from sham. Sig 3.9. MDA le Addition MDA level increase wa 1×10−1 mg 3.10. Cellul As show decrease of This decrea 1×10−2, an 4. Discussi Peripher quent injur strategies h areoftendis regeneratio cal manipu regeneratio ies have sho effective in regeneratio Phoenix ha shown pote ofGCSB-5on themyelinatedaxoncounts8weeksafter surgery. (A-F), histologicalmicrogra p, (B) control group, (C–F) GCSB-30, 100, 300 and 600mg/kg-treated group, respectively, 0, 300 and 600mg/kg or saline once a day after surgery. The values are represented as m nificantly different (+p 302 T.-H. Kim et al. / Journal of Ethnopharmacology 136 (2011) 297–304 Fig. 4. Effects counted. The v (**p T.-H. Kim et al. / Journal of Ethnopharmacology 136 (2011) 297–304 303 Fig. 5. Effects are represente from control. S neuronal ph as retinoic a in neurite o is defined a axons and d and nerve r within a ne incubation ber of neur group, indic bility. These nerve regen tion. 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Protective effect of GCSB-5, an herbal preparation, against peripheral nerve injury in rats 1 Introduction 2 Material and methods 2.1 Materials 2.2 Preparation and composition of GCSB-5 2.3 Animal treatments and surgical procedure 2.4 Administration of GCSB-5 2.5 Functional assessment 2.6 Gastrocnemius muscle weight 2.7 Regeneration distance 2.8 Neuromorphometry 2.9 Cell culture and treatments 2.10 Neurite outgrowth 2.11 Cell viability 2.12 Lactate dehydrogenase (LDH) activity 2.13 ROS production 2.14 Malondialdehyde (MDA) level 2.15 Total glutathione (GSH) contents 2.16 Statistical analysis 3 Results 3.1 Motor functional recovery 3.2 Gastrocnemius muscle mass ratio 3.3 Sciatic nerve regeneration distance 3.4 Neuromorphometry 3.5 Neurotrophic activity 3.6 Cell viability 3.7 LDH release 3.8 ROS production 3.9 MDA level 3.10 Cellular total GSH contents 4 Discussion References