Hindawi BioMed Researh International Volume 217, Artile ID 8234878, 1 page https://doi.org/1.1155/217/8234878 Retration Retrated: Study of Effet of Salvianoli Aid B on Motor Funtion Reovery in Rats with Spinal Cord Injury BioMed Researh International Reeived 2 August 217; Aepted 2 August 217; Published 24 September 217 Copyright 217 BioMed Researh International. This is an open aess artile distributed under the Creative Commons Attribution Liense, whih permits unrestrited use, distribution, and reprodution in any medium, provided the original work is properly ited. BioMed Researh International has retrated the artile titled Study of Effet of Salvianoli Aid B on Motor Funtion Reovery in Rats with Spinal Cord Injury [1]. The artile was found to ontain a substantial amount of material from the following published artile: Xiao-bin Bi, Yu-bin Deng, Dan-hui Gan and Ya-zhu Wang, (28), Salvianoli aid B promotes survival of transplanted mesenhymal stem ells in spinal ord-injured rats. Ata Pharmaologia Sinia, 29: 169 176. doi: 1.1111/j.1745 7254.28.71.x. Additionally, Figure 1 inthisartile[1]isthesameasfigure1 in the 28 artile, but it shows signs of manipulation. Referenes [1] C. Xun, Y. Hu, M. Lu, S. Wang, and D. Lv, Study of effet of salvianoli aid B on motor funtion reovery in rats with spinal ord injury, BioMed Researh International,vol.214,ArtileID 93185,6pages,214.
Hindawi Publishing Corporation BioMed Researh International Volume 214, Artile ID 93185, 6 pages http://dx.doi.org/1.1155/214/93185 Researh Artile Study of Effet of Salvianoli Aid B on Motor Funtion Reovery in Rats with Spinal Cord Injury Chong Xun, Yang Hu, Ming Lu, Shouyu Wang, and Deheng Lv Department of Orthopedi Surgery, First Affiliated Hospital of Dalian Medial University, Zhongshan Road, No. 222, Dalian, Liaoning 116, China Correspondene should be addressed to Deheng Lv; deheng lv@hotmail.om Reeived 2 Deember 213; Revised 6 February 214; Aepted 11 February 214; Published 18 Marh 214 AademiEditor:KotaV.Ramana Copyright 214 Chong Xun et al. This is an open aess artile distributed under the Creative Commons Attribution Liense, whih permits unrestrited use, distribution, and reprodution in any medium, provided the original work is properly ited. In this study effet of salvianoli aid B was observed on motor funtion reovery of rats with spinal ord injury. 5 rats were seleted and after induing SCI their reovery under ontrolled onditions was studied using and (as ontrol). Both ompounds were introdued intraperitoneally in respetive groups of traumati rats at the same time intervals for 28 days. It was observed that introdued at 5 mg/kg/day resulted in better motor funtion reovery. BBB sore was reorded whih inreased signifiantly along with the redution in avity area observed by bright field mirosopy of tissues, that is, from 1 to 1 and from.2 ±.5 mm 2 to.1 ±.3 mm 2, in treated group, respetively, ompared to group. Statistial analysis was arried out using SPSS software (SPSS, Chiago, IL, USA), values were expressed as mean ± SEM, and P value <.1 was onsidered signifiant. Effet of on expression of NF-kB p65 and IkBα was studied and OD values of densitometry of western blots were taken. MPO ativity was also studied. It was observed that treatment of signifiantly redued the expression of both ompounds in treated group as ompared to ontrol group after 28 days of treatment. 1. Introdution Traditionally used in Chinese mediine the bioative ompound extrated from Salvia miltiorrhiza has been responsible for exhibiting harateristis like anti-inflammatory and neuroprotetive both in vivo and in vitro [1, 2]. The antiinflammatory and antioxidant ativities of may be attributed to the presene of phenoli hydroxyl group in the struture of the ompound [3]. Liu et al., 26 [4], showed that an penetrate the blood brain barrier and an trigger differentiation and proliferation of stem ells of nervous system and an also protet neurons from apoptosis [5]. Other possible therapeuti uses of identified so far inlude the protetion against amyloid β protein (Aβ) indued ytotoxiity [6]andprotetionagainstTNF-αinjury in human aorti vasular endothelial ells [7]. That is why this ompound is now used for treating ardiovasular diseases as well as for stroke [8]. In the treatment of spinal ordinjury(sci)ithasbeenshowntoplayanimportant role by reduing inflammatory responses and affeting the proesses that ould lead to seondary regeneration; in experimental models of erebral ishemia and brain injury the neuroprotetive effet of has been demonstrated [9 11]. In traumati injury of spinal ord a number of ellular and moleular events our that an be inluded in primary and seondary injury pathways. The pathology of SCI an be inreased signifiantly by seondary injury in assoiation with the primary injury [12, 13]. A major omponent ontributing to the pathogenesis in ase of seondary injury in SCI is the inflammatory responses [14]. These responses are mediated by indued or enhaned gene expression. In this ase the major omponent is nulear fator-kb (NF-kB) with the family of transription fators like (Rel, RelA/p65, RelB, p5, and p52) [15, 16]. The inhibition of ativation of NF-kB has been demonstrated as a possible strategy for the attenuation of seondary damage in SCI [17, 18]. The diret inhibition of IkB kinase (IKK) has been demonstrated in different studies to regulate the inhibition of NF-kB gene produts [19, 2]. Therefore targeting of IKK/NF-kB pathway an result in improving the reovery of loomotor funtion by the redution of infiltration of inflammatory ells and
2 BioMed Researh International apoptosis after SCI in rats and has been reported in different studies [21, 22]. Inthisstudywehavemadeanefforttoexplorethe use of as a potential inhibitor of IKK/NF-kB pathway and evaluation of expression of IkBα and NF-kB p65 was arried out by seleting two experimental groups of rats with traumati SCI where and (ontrol) groups were tested. The study of funtional reovery of loomotor funtion in rats using Basso-Beattie-Bresnahan (BBB) sale was also performed and MPO ativity was studied in both groups to establish the effet that treatment of might have on reduing inflammation after injury. 2. Materials and Methods (moleular formula: C 36 H 3 O 16,moleularweight:746, purity: 98.5%, Green-Valley, Shanghai, China) was used in the experiment while phosphate buffered saline () was used as a ontrol. A total of 4 adult SD female rats (weight: 2 23 g) were obtained and divided into two main groups of and (2 rats per group). In eah group there were four subgroups having 5 rats eah (n = 5). Four subgroups were monitored till 28 days where in eah week one subgroup was used. was injeted intraperitoneally in all the subgroups of group from, 1 to 28 days andwasinjetedinalltheorrespondingsubgroups of group like in the same amount after injury at similar time interval for the same duration of, 1, 2 to 28 days. In every assigned group the treatment was stopped at the end of the week subsequently while in the other subgroups the treatment ontinued and potential reovery of SCI was studied. At T9-T1 laminetomy was performed after administering 1% hloral hydrate anesthesia. A 1 g NYU impat rod entered above T9 was dropped from 12.5 mm height and a onsistent partial, inomplete SCI was indued and then the postinjury are was taken out aording to the previous reports and bladder was emptied manually twie aday.forsalbandgroupstheratsweregivensalb 5 mg/kg per day intraperitoneally and in group was given only as a ontrol. Then transardial perfusion was made using 4% paraformaldehyde after animals were anaesthetized with 1% hloral hydrate. T9-T1 portion of spinal ord was removed andthenimmersedinthesamefixativefor24hrs.tissue was taken and setioned sagitally after embedding in paraffin for 24 hours. For all the subgroups of and groups setions were olleted on mirosopi slides and after the removal of paraffin graded ethanol was used to rehydrate the slidesandthenallsetionswerestainedwithhematoxylin eosin(he)forgeneralpurposehistology.behaviortesting of rats was done using Basso-Beattie-Bresnahan (BBB) sale at different points before and after injury (1, 7, 14, 21, 28 d) in both and ontrol groups. Here a sore of represents absene of loomotion while 21 shows normal loomotor funtion and subsequent points show the improvement in funtion up till 21 [23]. Statistial analysis was arried out using SPSS software (SPSS, Chiago, IL, USA) and the values were expressed as the mean ± SEM, one way ANOVA followed by Bonferroni post ho test was used for multiple omparison, and P value of <.1 was onsidered signifiant. NF-kB p65 and phosphorylated IkBα were tested aording to the previously desribed methods [24] with slight modifiation. Total proteins were extrated from the 1 mm spinal ord segment that ontained the injury epienter using Total Protein Extration kit (Applygen Tehnologies In., Beijing, China). The onentration of proteins was determined using BCA Protein Assay Kit (Applygen Tehnologies In., Beijing, China), following the manufaturer s protool. Samples were diluted in sample buffer and boiled for 5 min and then 5 mirogram of protein from eah sample was loaded on 4 2% polyarylamide gel, then separated by eletrophoresis, and transferred to polyvinylidene difluoride membrane. After bloking, the membrane was inubated with speifi primary antibodies: mouse anti-rat NF-kB p65 monolonal antibody (1 : 1; Santa Cruz Biotehnology Santa Cruz, CA, USA) and monolonal rabbit anti-rat phosphorylated IkBα (Ser32) antibody (1 : 5; Cell Signaling Tehnology, Danvers, MA, USA). The reative protein bands were visualized using horseradish peroxidase-onjugated anti-rabbit or anti-mouse IgG antibodies (1 : 2; Jakson, West Grove, PA, USA) and an ECL western blotting kit (Applygen Tehnologies In, Beijing, China), whih was performed aording the manufaturer s instrutions. The membranes were exposed to X-ray film for 1 s to 1 min. A polylonal rabbit anti-atin antibody (1 : 5; Santa Cruz Co., Santa Cruz, CA, USA) was used to detet atin in the samples as a loading ontrol. The protein bands were sanned and digitized, and the optial density (OD) of eah band was determined using the Gel-Pro analyzer 4. software. Studies have shown that in ase of spinal ord injury inflammatory responses are triggered beause inflammatory ells at the site of injury release neurotoxins and other inflammatory mediators that an result in the generation of reative oxygen and nitrogen speies resulting in ellular damage [25]. In order to investigate the anti-inflammatory effet of we studied the infiltration of leukoytes within the injured spinal ord. As myeloperoxidase ativity an be used as an indiator of polymorphonulear leukoyte aumulation so we studied the MPO ativity in both groups. 3. Results After arrying out the treatment with by administering at the rate of 5 mg/kg/day in group and 5 mg/kg/day in the group intraperitoneally from to 28 days the spinal ord setions were seleted to monitor the effets of treatment on SCI using bright field mirosopy (Figures 1(a) and 1(b)). HE stained sagittalsetionswere used to measure the avities in spinal ord and from 15 setions in every rat in all subgroups of and group. The avity was measured by average area in all subgroups of both groups and it was observed that signifiantly redued the avity area from.2 ±.5 mm 2 to.1 ±.3 mm 2 ompared with the group (P <.1, Figure 2) inthe fourth subgroup of both and groups (i.e., from 21
BioMed Researh International 3 (a) Figure 1: Bright field mirosopy showing redution in avity area. (a) Bright field mirosopy showing the avity area in group in rats of fourth subgroup after 28 days of injury. (b) Bright field mirosopy showing redution in the avity area in the orresponding fourth group after 28 days of treatment with. Lesion area (mm 2 ) Comparison of and groups bars showing redution in.25 avity area.2.15.1.5 Figure 2: Graphs showing statistially signifiant (P <.1) redution in the avity area in group treated with P <.1 ompared with ontrol. to 28 days of treatment). A differene in the performane in loomotor funtion was observed in treated animals as ompared to group where group rats demonstrated partial weight supported ambulation and after 14 days of Sal B injetions a signifiant statistial differene in BBB sore was observed while after 28 days a signifiant differene of 8 points on BBB sale between and groups was ahieved (Figure 3). The graph shows signifiant differene in BBB sore between and groups where P <.1 was signifiant. In order to observe the effet of on IKK/NF-kB pathway,theexpressionofnf-kbp65inspinalordtissues was observed using western blotting where after 24 hours of SCI the inrease in NF-kB p65 protein was observed in both groups and then a signifiant attenuation of NF-kB p65 was observed among the treated group as ompared to group after 28 days of the treatment (P <.1) (Figures 4 and 5). The expression of phosphorylated IkBα was also observed using western blotting where again after 24 hours an inrease in phosphorylated IkBα was found in and groups; however, a signifiant attenuation in the expression of BBB sore 12 1 8 6 4 2 (b) b b 1 7 14 21 28 Time in days after injury Figure 3: Time ourse of loomotor reovery in rats in treated animals ompared with ontrol. A statistial differene was observed after 2 weeks of the treatment and signifiant differene at four weeks of the treatment. Data is presented here as mean ± SEM, b P <.5, P <.1, in group as ompared to ontrol group. NF-κB p65 β-atin 65 kda 43 kda Figure 4: Western blotting result showing expression of NF-kB p65 in both and groups after 28 days of treatment with. IkBα was observed in group after postinjury treatment (P <.1)after28daysoftreatment(Figures6 and 7). It was observed that infiltration of leukoytes is inreased in SCI in spinal ord tissues but it an be ontrolled and reduedbythehelpofsalb.theevaluationofmpoativity in subgroups of both and ontrol groups was performed and it was observed that the treatment of
4 BioMed Researh International OD values showing NF-κB amount in both groups and ontrol groups showing the amount of NF-κB 3.5 3 2.5 2 1.5 1.5 Figure 5: Graphs showing a differene in OD values of ontrol and treated animal groups. The OD values are from the subgroup 4 after 28 days of the treatment beause a remarkable improvement in loomotor reovery and redution in the avity area was observed in this group. Signifiant derease in the expression of NF-kBisobservedasomparedtotheontrol, P <.1. IκBα β-atin 4 kda 43 kda Figure 6: Western blotting showing expression of IkBα in both Sal B and groups after 28 days of and administration in respetive groups. andownregulatetheinfiltrationofspinalordtissuesby neutrophils.aomparisonofresultofmpoativityinthe fourth subgroup of both and is shown in Figure 8 where P <.1 isfoundsignifiantasomparedtothe ontrol group. The use of therefore an redue tissue damage by inhibition of neutrophil infiltration. 4. Disussion InthepresentstudyitanbeonludedthattheuseofSal B signifiantly improved the loomotor funtion reovery in rats with indued spinal ord injury as ompared to the group where was not administered. A dose of 5 mg/kg/day improved BBB sore in rats as evident in the results as ompared to the ontrol group and moreover the avity area was also redued in this group. The most obvious improvement was observed in the group that reeived for 28 days after injury. There an be many reasonsattributedtothisreoveryinludingtheprotetive effet on neural ells that were injured by SCI and reovery of these neurons. A number of partiular genes that are alled neuroprotetive genes an be indued in a variety of onditions like eletrial stimulation, erebral ishemia, and brain injury [26]; therefore, the indution of suh genes diretly by an result in the treatment of SCI. OD values showing IκBα expression in and groups 1.9.8.7.6.5.4.3.2.1 and groups showing the amount of IκBα Figure 7: OD values showing the redution in expression of IkBα in treated group with a statistial differene, P <.1, as ompared to the ontrol group after 28 days of treatment. MPO ativity values.9.8.7.6.5.4.3.2.1 and ontrol groups bars showing MPO ativity omparison Figure 8: Graphi representation of MPO ativity in treated group and ontrol group. The inrease of MPO ativity in group was signifiantly redued in group ( P <.1). It has been reported that seondary inflammation is regulated by IKK/NF-kB pathway [27] and if this pathway is suessfully targeted the pathogenesis in SCI models an be redued [17]. The seletive inhibition of IKKβ, themain atalyti subunit of IKK, an result in redued inflammatory ells and neuronal apoptosis after SCI in rats [28]. Studies have shown that inflammatory responses are signifiant ontributors to the seondary ompliations after SCI [29]. NF-kB being the major transriptional regulator is very important fator and its ativation an be done by various pathways, one of whih involves IkB kinase omplex [3], and IKKβ is the main atalyti subunit of IKK that an ativate NF-kB by phosphorylation of inhibitory protein of IkB [31]. It is this reason that NF-kB expression and IkBα level were monitored in both groups where group showed a signifiant derease in the expression of NF-kB p65 and IkBα. NF-kB p65 is one of the different Rel family proteins like RelB, -Rel, p5, and p52. The results of Sal
BioMed Researh International 5 B group showed downregulation of infiltration of spinal ord tissues by neutrophils providing additional evidene of therapeuti potential of. Different ompounds have been used from Chinese herbal mediine for this purpose and has shown suessful results in the treatment of SCI in rats as, for example, Lua et al. [32] reently reported butein a ompound obtained from Caragana jubata and Rhus verniiflua to exhibit inhibition of NF-kB pathway and help regulate the expression of these genes induing seondary inflammation in SCI. However, the use of alone for treating SCI in rats is the first report of this potential use of the ompound to the best of our knowledge. Also the parameters studied are physiologial as well as ellular expressions, both providing signifiant evidene of the possible uses of the ; therefore, further studies in this regard will be helpful in exploring this new possible therapeuti agent. The seletive inhibition of stimulatory pathways along with the physiologial reovery like avity redution, improvement in motor funtions provides a possible inite for future investigations that should be explored further. Conflit of Interests The authors delare that there is no onflit of interests regarding the publiation of this paper. Referenes [1] L. Zhou, Z. Zuo, and M. S. Chow, Danshen: an overview of its hemistry, pharmaology, pharmaokinetis, and linial use, JournalofClinialPharmaology,vol.45,no.12,pp.1345 1359, 25. [2] S.X.Wang,L.M.Hu,X.M.Gao,H.Guo,andG.W.Fan, Antiinflammatory ativity of salvianoli aid B in miroglia ontributes to its neuroprotetive effet, Neurohemial Researh, vol.35,no.7,pp.129 137,21. [3] J.H.HoandC.Y.Hong, Salvianoliaids:smallompounds with multiple mehanisms for ardiovasular protetion, Journal of Biomedial Siene,vol.18,no.1,artile3,211. 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