Combined Rosiglitazone and Forskolin Have...

Research ArticleCombined Rosiglitazone and Forskolin Have NeuroprotectiveEffects in SD Rats after Spinal Cord Injury

Qing-qi Meng 12 Wei Lei 2 Hao Chen3 Zhen-cheng Feng1 Li-qiong Hu1

Xing-liang Zhang2 and Siming Li 1

1Department of Orthopedics Guangzhou Red Cross Hospital Jinan University 396 Tongfu Road Guangzhou 510120 China2Laboratory Research Center Guangdong Medical University Zhanjiang 524001 China3Department of Gastroenterology Guangdong General Hospital and Guangdong Academy of Medical SciencesGuangzhou 51000 China

Correspondence should be addressed to Siming Li drsmlihotmailcom

Received 15 January 2018 Revised 22 April 2018 Accepted 8 May 2018 Published 21 June 2018

Academic Editor Elisabetta Mueller

Copyright copy 2018 Qing-qi Meng et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The peroxisome proliferator-activated receptor gamma (PPAR-120574) agonist rosiglitazone inhibits NF-120581B expression and endogenousneural stem cell differentiation into neurons and reduces the inflammatory cascade after spinal cord injury (SCI) The aim ofthis study was to explore the mechanisms underlying rosiglitazone-mediated neuroprotective effects and regulation of the balancebetween the inflammatory cascade and generation of endogenous spinal cord neurons by using a spinal cord-derived neural stemcell culture system as well as SD rat SCI model Activation of PPAR-120574 could promote neural stem cell proliferation and inhibit PKAexpression and neuronal formation in vitro In the SD rat SCI model the rosiglitazone + forskolin group showed better locomotorrecovery compared to the rosiglitazone and forskolin groups MAP2 expression was higher in the rosiglitazone + forskolin groupthan in the rosiglitazone group NF-120581B expression was lower in the rosiglitazone + forskolin group than in the forskolin group andNeuN expression was higher in the rosiglitazone + forskolin group than in the forskolin group PPAR-120574 activation likely inhibitsNF-120581B thereby reducing the inflammatory cascade and PKA activation likely promotes neuronal cell regeneration

1 Introduction

Acute spinal cord injuries (SCI) are severe and often perma-nent and canprove expensive for patients and the communitySCI is the highest cause of death in young people (15ndash29years of age) [1] There are currently no treatments whichhave a proven positive effect on neurological outcome [2 3]following acute SCI Spinal cord injury results in primaryinitial damage and subsequent secondary damage [4] Thesecondary damage due to early inflammatory responses afterspinal cord injury is the main cause for neurological defectsand exacerbates the initial degree of injury [5] and thesecondary damage usually caused by ischemia cellular andtissue edema and oxidative damage [6]

In our previous research we found that activation ofperoxisome proliferator-activated receptor-gamma (PPAR-120574)could promote locomotor recovery after spinal cord injury

[7] PPAR-120574 is a PPAR nuclear receptor subtype Thereare two types of PPAR-120574 ligands natural ligands such asfatty acids and eicosanoid derivatives [8] and syntheticligands such as thiazolidinediones ketones (thiazolidine-dione TZD) of which rosiglitazone (rosiglitazone) andpioglitazone (pioglitazone) are used in clinical medicineas a treatment for type 2 diabetes [9] Rosiglitazone ispart of the thiazolidinediones nuclear hormone receptorsuperfamily and is known for its anti-inflammatory actionsvia the activation of PPAR-120574 Rosiglitazone attenuates theexpression of proinflammatory genes and cytokine produc-tion by regulating ligand activation of transcription factors[10 11] And rosiglitazone has neuroprotective effects inneurodegenerative diseases and spinal cord injury [12 13]Wepreviously reported that application of the PPAR-120574 agonistrosiglitazone could significantly inhibit activation of thekey inflammatory cascade target nuclear transcription factor

HindawiPPAR ResearchVolume 2018 Article ID 3897478 11 pageshttpsdoiorg10115520183897478

2 PPAR Research

kappa B (nuclear factor kappa B NF-120581B) after spinal cordinjury [7]

NF-120581B is extracted from B lymphocytes and is namedbecause of its binding to the immunoglobulin chain geneenhancer sequence-specific 120581B [14] The NF-120581B family oftranscription factors has an essential role in inflammationand innate immunity The active form of NF-120581B is a dimertypically a P50 P65 heterodimer and is themain componentof the NF-120581B family involved in gene transcription [15]Members of the NF-120581B family participate in many physiolog-ical processes including regulation of the immune responsestimulation of immune cell maturation and the productionand survival of essential transcription factors for B cells [15]

In our preliminary research we found that PPAR-120574could inhibit NF-120581B activation and promote proliferationand inhibit neural differentiation of endogenous neural stemcells [7] The NF-120581B and PPAR-120574 signaling pathways areclosely related PPAR-120574 activation along with inhibitionof the NF-120581B-cytokine cascade could protect against dex-tran sulfate sodium-induced colitis in mice [16] PPAR-120574could also attenuate inflammation via NF-120581B inhibition inlipopolysaccharide-induced peritonitis [17] In addition tofunctions in glucose and lipid metabolism and cell prolifer-ation and differentiation PPAR-120574 can also alleviate the acuteand chronic inflammatory response after nerve injury andameliorate neuronal apoptosis and ischemic brain injury bysuppressing NF-120581B-driven p22phox transcription [18]

Imielski et al [19] reported distinct loss of hippocampaldentate gyrus neuronal tissue in NF-120581B gene-deficient micereactivation of NF-120581B could promote the formation of den-tate gyrus neurons and neural network and improve neuronalorganization and animal behavior The underlying mecha-nism is likely related to the activation of cAMPPKA afteractivation of NF-120581B cAMP (Cyclic Adenosine Monophos-phate) was discovered in 1958 by Earl [20] increased levelsof cAMP lead to a transient enhancement of transmitterrelease [21] and regulation of cAMP and cGMP togetherensures coordinated development of one axon and multipledendrites PKA could enhance transmitter release at thesynaptic connection between the sensory and motor neuron[22] PKA is implicated in the growth of axons and survivalof newborn neurons [19 23] Forskolin is a unique diterpeneactivator of cyclic AMP-generating systems [24] Forskolin-mediated activation of adenylate cyclase stimulates nerveregeneration in vivo [25] Sulaiman et al [26] found thattreatmentwith forskolin alone and in combinationwith TGF-120573 resulted in an increase in the number of regenerated axonscompared with saline-only control

These findings were consistent with our preliminaryfindings that activation of PPAR-120574 could inhibit the expres-sion of NF-120581B in a SD rat spinal cord injury model whileinhibiting neuronal survival We therefore hypothesized thatPPAR-120574 activation in SD rats inhibits neurons via PKAwhich is downstream of NF-120581B In in vitro experimentswe intend to verify that PPAR-120574 activation inhibits NF-120581B expression which in turn could inhibit the cAMPPKApathway After confirming the inhibition of cAMPPKA byPPAR-120574 animal experiments will be initiated to determinewhether simultaneous activation of PPAR-120574 and PKA could

inhibit inflammation and promote neuronal formation Inthis study we explored the role of PPAR-120574 and PKA infunctional recovery after spinal cord injury in SD ratsWe aimed to determine the mechanisms underlying thisrecovery as well as the balance between the inflammatorycascade and generation of endogenous spinal cord neurons

2 Materials and Methods

21 In Vitro Experiments

211 Extraction and Culture of Spinal Cord-Derived NeuralStem Cells One-day-old SD rats were killed by cervicaldislocation and immersed in 75 alcohol Three sets ofeye scissors and tweezers were prepared in sequence andplaced in beakers with 75 alcohol numbered 1 2 and 3respectively The skin was cut using the first scissors andboth sides of the spine were cut from the rib fossa fromthe bottom up to remove the modified fascia and fat tissueattached to the spine by using the second scissors With 1mL of serum-free DMEMF12 medium drawn with a 1 mLsyringe syringe needle was flushed from one end of the spineand washed 3 times repeatedly to flush out the spinal cordThe spinal cord was then cut into 2ndash3 pieces by using thethird pair of scissors and the pieces of spinal cord tissue wereremoved into a Petri dish filled with culture medium andsubjected to repeated aspiration using a syringe 5 to forma uniform suspension The spinal cord pieces were sievedthrough 200-mesh sieve in order to separate them into singlecells After suspension in serum-free DMEMF12 sampleswere centrifuged for 5 min (1000 rpm)The cells were stainedwith trypan blue counted and seeded at a density of 1 times 106cells ml in culture flasks and incubated at 37 ∘C in a 5 CO

2

humidified atmosphere Culture mediumwas changed after 3days and a half Peculiar ldquonerve ballrdquo formation was observedusing an inverted microscope

After 5 to 7 days the spinal cord-derived stem cells wereproliferated and expanded in vitro for 1 passage The cellswere separated mechanically by using pipettes and 200-meshsieve and centrifuged for 5 min (1000rmin)The supernatantwas discarded and 2times105ml spinal cord-derived stem cellswere inoculated onto the surface of a sterile-10 Petri dish andincubated in medium containing DMEMF12 + 2 B27 +20 ngml b FGF + 20 ngml EGF This free-serum culturesystem was used in order to screen NSCs Each time mediumis changed and every passage flasks were changed to discardthe adherent cells Thus NSCs are naturally purified duringthe cultivation process The purity and quality of NSC werecalculated by staining with NestinDAPI

Neural stem cells derived from the spinal cord weredivided into three groups namely the rosiglitazone grouptreated with rosiglitazone (1 120583molL in DMSO [Abcamab142179]) the G3335 group treated with G3335 (PPAR-120574-antagonist 01 120583molL) and DMSO and the vehicle grouptreated with DMSOAfter 3 days continuous culture prolifer-ation of NSCs was evaluated by cell number using CellometerAuto 1000 (USA Nexcelom)

Neural stem cells were grown in medium supplementedwith 10 fetal bovine serum (the medium was used to

PPAR Research 3

(a) (b)

(c) (d)

Figure 1 Representative images showing the surgical process in SD rats (a-b) Dorsal skin preparation and disinfection and exposed T9-T10spinous lamina with sterile surgical instruments and spinal cord (c-d) Rats were fixed on an operating table and subjected to instant impactagainst the spinal apparatus The animalsrsquo bodies shook with the lower limbs rapidly retracting and bouncing and tail cocking and quicklyfalling The impacted local spinal cord surface was dark purple and hind limbs appeared completely paralyzed postoperatively

induce differentiation)The early neuronalmarkerMAP2wasdetected by QRT-PCR and western blot

212 QRT-PCRDetection ofMAP2 PKA andPPAR-120574Expres-sion Total RNAwas isolated using the TrizolRNA isolationreagent (Invitrogen) and quantitative data was recordedcDNAs were reverse-transcribed using the RevertAid FirstStrand cDNA Synthesis Kit (Fermentas) The target genefragments were amplified using real time-PCR (RT-PCR)Maxima SYBR Green (Thermo Scientific Waltham MAUSA) was used for staining The target gene expression wasnormalized to glyceraldehyde-3-phosphate dehydrogenase(GAPDH) primers MAP2 (forward 51015840- AACATACCA-CCAGCCCTTTG -3 1015840 reverse 51015840 -GCCTTTCCTTCGTCT-TTCCT-31015840) PKA (forward 51015840-AGCCAAAGCCAAGGA-AGATT-31015840 reverse 51015840-AGCATCACTCGCCCAAAG-31015840 )PPAR-120574 (forward 51015840-CGAGAAGGAGAAGCTGTTGG-31015840 reverse 51015840- TCAGCGGGAAGGACTTTATG-31015840 )

213 Western Blot Detection of MAP2 PKA and PPAR-120574Expression Total cellular protein was extracted and 25 120583gwas used for SDS-PAGE After blocking the membrane for30min rinsing with TBST buffer three times and incubationwith anti-MAP2 (11000 Abcam ab5392) anti-PKA (1400Abcam ab211265) and anti-PPAR-120574 (1500 Abcam ab45036)for 12 h at 4 ∘C the membranes were washed three times

in wash buffer (02 gelatin 005 Tween 20 in TBS) Themembranes were then incubated for 1 h with horseradishperoxidase-labeled secondary antibody washed again andexposed to ECL color development reagents Themembraneswere developed using the ChemiDoc-It TM TS2 ImagingSystem (Bio-Rad) and relative optical density was analyzedusing the ImageJ2x software (National Institute of HealthBethesda MD USA)

214 Detection of cAMP by ELISA The production of cAMPwas determined by ELISA after rosiglitazone treatmentThe supernatants from the neural stem cell cultures wereharvested and cAMP level was determined using an ELISAkit (GBD Co) according to the manufacturerrsquos instructions

22 In Vivo Experiments

221 Animals SD rats were housed in a temperature-controlled room at 27∘C Injured rats underwent manualbladder expression twice a day Under halothane anesthesia[induction 4 maintenance 2 in an oxygen and nitrousoxide (5050) mixture] SCI was induced in adult femaleSprague-Dawley rats by dropping an impactor (10 g weightrod 25 mm in diameter) from a height of 25 mm as previ-ously reported [7] and T9ndashT10 laminectomy was performed[Figure 1] SCI rats were randomly divided into 4 groupsvehicle group (n = 3) rosiglitazone group (n = 3) forskolin

4 PPAR Research

group (n = 3) and rosiglitazone + forskolin group (n = 3)Rosiglitazone (3 mgkg Abcam) or vehicle (001 M PBS) wasinjected ip at 5 min 6 h and 24 h after SCI Forskolin (01mgkgd Abcam) was injected ip continuously for 7 days

222 BBB Score Motor function recovery after SCI wasstudied using the BassondashBeattiendashBresnahan locomotor ratingscale (BBB scale) [27] If there was no spontaneous hind limbmovement the scorewas 0 and a score of 21 indicated normallocomotion To ensure that all animals began with a score of21 all rats were tested prior to injuryThe rats were then testedat 8 h and on days 1 3 7 14 21 and 28 after injury (or up tothe day the animal was euthanized) Each rat was scored for4 min by two observers blinded to the study groups

223 Spinal Cord Harvest The three rats from each groupwere anesthetized by chloral hydrate (10 033 mLkg)and the damaged spinal cord (1 cm from the center of theinjury point) was immediately removed 28 days after surgerySpinal cord tissues (T8ndashT10) were harvested and dissociatedin trypsin (05 mgml) and collagenase (05 mgml) Weseparated the debris from cells as described in previousstudies [28]

224 QRT-PCR Analysis of MAP2 NF-120581B and NeuN Expres-sion Total RNA from 10 mm long spinal cord segmentscontaining the injury epicenter obtained from the 4 groupswere extracted using Trizol (Invitrogen USA) at 28 dafter injury RNA was detected by electrophoresis beforereverse transcription The results showed no degradationOD260280 was between 18 and 19The cDNAwere reverse-transcribed using Revert Aid-First Strand cDNA Synthesiskit (Thermo Scientific) cDNA was used as a template forreverse transcription polymerase chain reaction analysis (RT-PCR) amplification The 2-ΔΔCt method was used for dataanalysis The target gene expression was normalized to thatof glyceraldehyde-3-phosphate dehydrogenase (GAPDH)primers MAP2 (forward 51015840- AACATACCACCAGCCCTT-TG -3 1015840 reverse 51015840 -GCCTTTCCTTCGTCTTTCCT-31015840) NF-120581B (forward 51015840AACACTGCCGAGCTCAAGAT-31015840 reverse51015840CATCGGCTTGAGAAAAGGAG-31015840 ) NeuN (forward 51015840-CGTGGAAAGTGTGGCTGAA-31015840 reverse 51015840-ACCATA-GTCTTCAAAGTCCCG-31015840)

225 Western Blot Analysis of MAP2 NF-120581B and NeuNExpression Spinal cord tissues (1 cm) were isolated using thelesion site as the epicenter Spinal cord tissues were homog-enized in RIPA lysis buffer with phosphatase and proteaseinhibitor (benzamidine 1120583M leupeptin 5120583gml Na

3VO4

200120583M sodium pyrophosphate 200120583M and phenylmethyl-sulfonyl fluoride 200120583M) Tissue homogenates were incu-bated for 20 min at 4∘C and centrifuged at 25000g for 30minat 4∘C Proteins were quantified with the bicinchoninic acidprotein assay kit (Thermo Scientific) and 10 120583g of protein wasloaded in each lane Samples were electrophoresed onto a 12sodium dodecyl sulfatepolyacrylamide gel (SDSPAGE) andtransferred to PVDF membranes (Millipore MassachusettsUSA) After blocking with 10 BSA in TBST (25 mM Tris-HCl 015 M saline and 1 Tween 20) at room temperaturefor 2 h the membranes were incubated with the following

primary antibodies at 4∘C overnight anti-GAPDH (11000Abcam) anti-MAP2 (1800 Abcam) anti-NF-120581B (1400Abcam) and anti-NeuN (1500 Abcam) The membraneswere then incubated with horseradish peroxidase-conjugatedsecondary antibody (15000 Abcam) for 2 h at 4∘C Duringthe experiment the low-temperature environment is strictlyguaranteed and protease inhibitors are added to ensureprotein quality

The membranes were developed using the ChemiDoc-It TM TS2 Imaging System and relative optical density wasanalyzed using Quantity one v44 software (Bio-Rad)

226 PKADetermination Theproduction of PKAwas deter-mined by ELISA The supernatants from spinal cord tissuewere harvested and PKA expression was determined usingan ELISA kit (GBD Co) according to the manufacturerrsquosinstructions

23 Statistical Analysis All statistical analyses were con-ducted using SPSS 140 software The BBB scores for eachanimal were averaged and used to determine the groupmeaneach day The BBB score data were expressed as mean plusmnSD Comparisons among groups were performed by one-wayanalysis of variance (ANOVA) followed by a TukeyndashKramermultiple comparisons post hoc test

3 Results

31 Neural Stem Cell Properties of NSCs Derived from RatSpinal Cord Under the light microscope the primary neuralcells appeared round with small cell bodies and singlescattered shiny halos Most of the cells grew in suspensionand formed neural stem cell spheres while some cells grewadherently At 3-5 days dozens of cloned spheres wereformed by the suspension of cell clusters and the clonedspheres showed spherical shape with no obvious protrusionsThree days after inoculation the NSCs derived from ratspinal cord rapidly proliferated to form cellular spheres orneural spheres (Figure 2(a)-(a1)-(a2)) Immunofluorescencestaining showed Nestin expression in the neurospheres andcytoplasm coloring was seen Immunofluorescence stainingwas performed to identify neural stem cells Nestin DAPIgt90 (Figure 2(b1)-(b2)-(b1))

32 In Vitro Activation of PPAR-120574Could Promote Neural StemCell Proliferation Table 1 shows the number of ENSCs afterPPAR-120574 activation Second generation of NSCs were selectedfrom each group and cultured after adding rosiglitazone (1120583molL) or G3335 (01 120583molL) Cells were cultured for 3days NSCs of all three groups proliferated and the NSCproliferation in the rosiglitazone group was significantlygreater than that in the negative control group (P lt 005 n=3)NSC proliferation in the G3335 group was clearly neutralizedThere was no significant difference in NSC number betweenthe negative control and rosiglitazone + G3335 groups

33 In Vitro Activation of PPAR-120574 Could Inhibit PKA Expres-sion and Neuronal Formation in Spinal Cord-Derived NeuralStem Cells We examined the mRNA expression of PKA

PPAR Research 5

Table 1 Quantity of NSCs (n = 3 mean plusmn SE)

Cell number (times105) Ratio (compared with vehicle group)Vehicle group 365 plusmn 013 1Rosiglitazone group 458 plusmn 021lowast 125Rosiglitazone + G3335 group 357 plusmn 017 098Comparison of the NSC number in the three groups Each experiment was repeated at least three times lowast P lt 005 (n = 3) There were significant differencesbetween the rosiglitazone group and negative control group Statistical methods one-way ANOVA followed by pairwise comparison by the Holm-Sidakmethod

(a)

(a1)

(a2)

(b1)

(b2) (b)

Figure 2 Neural stem cells from rat spinal cord (a)-(a1)-(a2) Cells gather into neurospheres (b1) DAPI immunofluorescence staining (b2)Nestin-CY3 immunofluorescence staining (b) Nestin-CY3-DAPI-immunofluorescence staining (merge) Immunofluorescence staining wasperformed to identify neural stem cells Nestin DAPI gt90 Each experiment was repeated three times

(Figure 3(a)) MAP2 (Figure 3(b)) and NF-120581B (Figure 3(c))in the three groups PKA mRNA expression in the rosiglita-zone group (055 plusmn 004) was lower than that in the vehicle(100 plusmn 008) and when the activation of PPAR-120574 is blockedthe expression of PKA is restored in rosiglitazone + G3335(093plusmn 008) groups (Plt 001 n = 3)The expression ofMAP2mRNA in the rosiglitazone group (040 plusmn 004) was lowerthan that in the vehicle (082 plusmn 006) and when the activationof PPAR-120574 is blocked this inhibition could be partly reversed

in rosiglitazone + G3335 (100plusmn 008) groups (Plt 001 n = 3)Compared with the vehicle (100 plusmn 005) and rosiglitazone +G3335 (064 plusmn 003) groups the expression of NF-120581B mRNAsignificantly decreased with the activation of PPAR-120574 in therosiglitazone group (031 plusmn 0002 P lt 001 n = 3)

In the WB experiment we observed similar trends ofindicators in each group

The activation of PPAR-120574 could inhibit PKA expression(Figures 3(d) and 3(j)) expression in the rosiglitazone group

6 PPAR Research

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Figure 3ThemRNA and protein expression of PKAMAP2 andNF-120581B (a) Relative PKAmRNA expression level (b)MAP2 relativemRNAexpression level (c) Relative NF-120581BmRNA expression level (d) Relative PKA protein expression level (e) Relative MAP2 protein expressionlevel (f) Relative NF-120581B protein expression level (g) Representative western blots showing the effects of rosiglitazone and G3335 on MAP2NF-120581B and PKA (h) cAMP expression level Data were expressed as meansplusmnSD (n = 3) Each experiment was repeated at least three timeslowast P lt 005 compared with vehicle group and P lt 005 compared with rosiglitazone group (one-way ANOVA followed by a TukeyndashKramermultiple comparisons posttest)

(028 plusmn 004) which was lower than that in the vehicle (068 plusmn010) and rosiglitazone + G3335 (051 plusmn 005) groups (P lt 001n = 3)

The expression of neuronal formation marker MAP2protein (Figures 3(e) and 3(j)) was significantly decreased

after adding rosiglitazone (011 plusmn 002) and was lower in therosiglitazone group than in the rosiglitazone + G3335 (038 plusmn006) and vehicle (052 plusmn 004) groups (P lt001 n = 3)

Compared with the vehicle (040 plusmn 006) and rosiglita-zone + G3335 (030 plusmn 005) groups the expression of NF-120581B

PPAR Research 7

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Figure 4 BBB scores after SCI Data were expressed as means plusmnSD (n = 3) Each experiment was repeated at least three times lowast Plt 005 compared with the vehicle group P lt 005 compared withrosiglitazone + forskolin group

protein (Figures 3(f) and 3(j)) was significantly decreasedin the rosiglitazone group (011 plusmn 002 P lt 001 n = 3respectively)

In ELISA experiment cAMP levels in cells treated withrosiglitazone (rosiglitazone group) (261477plusmn18229 ) arelower than vehicle group (538088plusmn36774) After G3335 wasused to block the activation of PPAR-120574 the cAMP levels inrosiglitazone + G3335 group (415345plusmn18906) were higherthan rosiglitazone group (Figure 3(h))

34 Simultaneous Activation of PPAR-120574 and PKA Was MostEffective in Promoting Locomotor Recovery All rats had aBBB score of 21 before SCI and lost locomotor function 8h after SCI At the 24 h point the BBB scores of all thegroups were still not gt2 On day 3 compared with the vehicleand rosiglitazone + forskolin groups the rosiglitazone groupshowed significantly higher BBB scores (P lt 005 n = 8)On days 21 to 28 compared with the vehicle group therosiglitazone + forskolin group showed significantly higherBBB scores (P lt 005 n = 8) the rosiglitazone + forskolingroup also showed higher BBB scores than those of theforskolin or rosiglitazone groups respectively (P lt 005 n= 8) On day 28 all the groups showed the highest BBBscores since SCI (BBB score 988 plusmn 146 1188 plusmn 136 1538plusmn 185 and 1313 plusmn 181 respectively for the vehicle forskolinrosiglitazone + forskolin and rosiglitazone groups P lt 005n = 8) (Figure 4)

35 mRNA Expression of MAP2 NeuN and NF-120581B followingSCI On day 28 after spinal cord injury when PKA wereactivated by forskolin the expression of MAP2 was higherin the forskolin group (185 plusmn 014) (Figure 5(a)) than in theother three groups (P lt 001 n = 3) While PPAR-120574 agonistrosiglitazone negatively regulates the neuronal formation therosiglitazone group (063 plusmn 008 P lt 001 n = 3) showed thelowest expression of MAP2

Rosiglitazone could inhibit the inflammation and pro-mote the mature neuron survival and when combined withPKA agonist forskolin the expressions of mature neuronmarker NeuN (Figure 5(b)) in the forskolin + rosiglitazonegroup (161plusmn013) and the rosiglitazone group (158 plusmn 007)were higher than other groups Using forskolin alone showedno protective effect on mature neuron survive the lowestexpression of NeuN was found in the forskolin group (092plusmn 041)

Rosiglitazone is most critical in inhibiting the inflamma-tory response the expression of inflammation marker NF-120581B(Figure 5(c)) was lowest in rosiglitazone group (043 plusmn 005)When PPAR-120574 and PKA are simultaneously activated theexpression of NF-120581B in forskolin+ rosiglitazone group (051plusmn 006) was still lower than forskolin group (094 plusmn 004) andthe vehicle group (100 plusmn 007)

36 Relative Protein Expression of MAP2 NeuN and NF-120581Bfollowing SCI Indicators of each group in the WB experi-ment were the same as those in RT-PCR MAP2 expression(Figures 5(d) and 5(j)) was higher in the forskolin group (021plusmn 003) than in the vehicle (013plusmn 002) and rosiglitazone (008plusmn 002) groups MAP2 expression was higher in the forskolin+ rosiglitazone group (017 plusmn 003) than in the rosiglitazonegroup

When the PPAR-120574was activated by rosiglitazone alone inrosiglitazone group (017 plusmn 003) and or PPAR-120574 and PKA aresimultaneously activated in forskolin + rosiglitazone groups(019plusmn 004) theNF-120581B expression (Figures 5(f) and 5(j)) waslower than that in the other two groups (vehicle group 047 plusmn010 forskolin group 044plusmn 008) NF-120581B expression betweenthe forskolin + rosiglitazone and rosiglitazone groups has nosignificant differences

Rosiglitazone combined with forskolin has the best pro-tective effect on mature neuron survival and the expressionof NeuN (Figures 5(e) and 5(j)) in forskolin + rosiglitazonegroup (045 plusmn 007) compared with vehicle group (023 plusmn007) and forskolin (017 plusmn 004) groups When PPAR-120574 wasactivated by rosiglitazone alone the expression of NeuN inrosiglitazone group (042 plusmn 007) was higher than forskolinand vehicle group

37 Detection of PKA Expression by ELISA following SCIPKA expression in forskolin group (063plusmn002) was higherthan other groups This effect decreased when PPAR-120574 andPKA are simultaneously activated (forskolin + rosiglita-zone group 049plusmn002) But the forskolin + rosiglitazonegroup still showed higher PKA expression than that ofthe rosiglitazone (017plusmn001) and vehicle groups (027plusmn002)(Figure 5(h)) Considering the effect of rosiglitazone on NF-120581B these data suggest that rosiglitazone likely inhibits PKAthrough NF-120581B

38 Relative Protein Expression of PPAR-120574 following SCIAfter rosiglitazone was used alone the expression of PPAR-120574 in rosiglitazone group (052plusmn007) was higher than invehicle (028plusmn006) and forskolin groups (025plusmn004) Andno difference was found between rosiglitazone group androsiglitazone + forskolin (R+F) group (047plusmn004) (Figures6(a)-6(b))

8 PPAR Research

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2

1

00

6

7

5

4

3

2

1

00

(a)

(b)

(c)

(h)

(d)

(e)

(f)

(g)

Figure 5 The mRNA and protein expression of MAP2 NeuN and NF-120581BThe ELISA results of PKA (a) Relative MAP2 mRNA expressionlevels (b) Relative NeuN mRNA expression level (c) Relative NF-120581B mRNA expression level (d) Relative MAP2 protein expression level(e) Relative NeuN protein expression level (f) Relative NF-120581B protein expression level (g) Representative western blots showing the effectsof rosiglitazone forskolin and forskolin + rosiglitazone on MAP2 NF-120581B and NeuN (h) PKA expression levels detected by ELISA Datawere expressed as meansplusmnSD (n = 3) Each experiment was repeated at least three times lowast P lt 005 compared with vehicle group P lt 005compared with the forskolin + rosiglitazone group

4 Discussion

This study was undertaken to evaluate the effect of simultane-ous activation of PPAR-120574 and PKA on promoting locomotorrecovery in SD rats the expression of the inflammatory factor

NF-120581B survival of mature neurons and the generation ofnewbornneurons after spinal cord injuryOur results indicatethat activating PPAR-120574 and PKA simultaneously could pro-mote locomotor recovery and the neuronal formation afterspinal cord injury Compared to activating PPAR-120574 or PKA

PPAR Research 9

Vehicle RosigForskolin R+F

PPA

R-

rela

tive p

rote

in le

vel

6

5

4

3

2

1

00

lowast

lowast

(a)

PPAR-

vehicle rosig forskolin rosig+forskolin

-Actin

(b)

Figure 6 (a-b)Theprotein expression of PPAR-120574and representative western blots showing the effects of rosiglitazone forskolin and forskolin+ rosiglitazone on PPAR-120574 Each experiment was repeated at least three times

individually activating PPAR-120574 and PKA simultaneouslycould also inhibit the expression of inflammatory factor NF-120581B and protect mature neurons

There are few prolific sources of neural stem cells includ-ing the telencephalon [29] Lu et al [30] reported the presenceof neural stem cells around the ependymal membrane ofthe spinal cord Our results verified the presence of neuralstem cells in the spinal canal Morales-Garcia [31] foundthat activation of PPAR-120574 could promote neural stem cellproliferation in vitro we could verify that the activationof PPAR-120574 promotes proliferation of spinal cord-derivedneural stem cells in vitro which was reversed when PPAR-120574activation was inhibited

The interaction of PPAR-120574 and NF-120581B has been impli-cated in certain central neural diseases [32] PPAR-120574 caninhibit the activity of NF-120581B by binding to the subunits P65and P50 of NF-120581B and competing for common transcriptioncoactivators such as SRC-1 and p300CBP (CREB-bindingprotein) or upregulate inhibitor kappa B (I120581B) protein whichprevents NF-120581B nuclear translocation which is a prerequisitefor NF-120581B activation PPAR-120574 could also indirectly inhibitNF-120581B by activating transcription factor Nrf2 which reducesgeneration of prooxidative molecules that are required forNF-120581B activation PPAR-120574 therefore mediates the inhibitionof NF-120581B thereby reducing the central nervous systeminflammatory cascade and effecting neuroprotection [33]Under conditions of induced differentiation we found thatafter PPAR-120574 activation the expression of NF-120581B is inhibitedPKA expression was also inhibited in this process and wasrestored when PPAR-120574 was inhibited This is likely related toPPAR120574-mediated inhibition of NF-120581B since PKA is founddownstream of NF-120581B however the feedback mechanismwas not clear LKB1 a substrate of PKA plays an importantrole in axogenesis [34] Koichiro et al [35] suggested that theactivation of PPAR-120574by rosiglitazone stimulated NSC growthand inhibited the differentiation of NSCs into neurons Wefound that PPAR-120574 activation in a neural stem cell differ-entiation induction system inhibits the MAP2 expression

which is consistent with the results of Koichiro These resultsconfirmed that the inhibitory effect of PPAR-120574 on neuronaldifferentiation of spinal cord-derived neural stem cells couldoccur via NF-120581B and PKA in vitro In order to show theinhibition of the cAMP pathway by rosiglitazone cAMPlevels in cells were measured by ELISA And we see thatchanges in CAMP levels are consistent with changes in PKAlevels

In order to confirm PPAR-120574 and PKA activation after SCIthe protein expression of PPAR-120574 and PKA was observedin vivo And we found a corresponding increase in theexpression of PPAR-120574 and PKA after applying rosiglitazoneand forskolin respectively We used NeuN as an in vivomarker of mature neuronal survival following SCI NeuN isan antigen used widely in research and diagnostics to identifypostmitotic neurons In the present study we observed thatthe NeuN expression in the forskolin group was lower thanthat in the rosiglitazone and forskolin + rosiglitazone groupsThis suggests that rosiglitazone inhibits inflammation andcombined with forskolin promotes neuronal formation andhas the best protective effect on mature neural stem cellsurvival Forskolin alone had no effect on the survival ofmature neurons in the inflammatory cascade following spinalcord injury

MAP2 is a marker for dendritic lesions in CNS injuryMAP-2 is extremely sensitive to many factors and recentinvestigations have revealed dynamic functions for MAP2 inthe growth differentiation and plasticity of neurons Thesediscoveries indicate that modification and rearrangement ofMAP2 are an early obligatory step in many processes thatmodify neuronal function [36] In the present experimentstudy we observed that the expression of MAP2 in theforskolin group was higher than that in the rosiglitazoneand forskolin + rosiglitazone groups which is likely relatedto the activation of PKA PKA is implicated in the growthof axons and survival of newborn neurons [20 21] Thusforskolin likely plays an important role in promoting neuronformation while rosiglitazone negatively regulates neuron

10 PPAR Research

formation We also examined the expression of cAMP in vitroand PKA in vivo which was consistent with the expressionof MAP2 suggesting that forskolin affects neuron formationthrough cAMPPKA

Compared with the rest of the control group there washigher expression of NeuN in the spinal cord injury segmentNeuroprotection by genistein against oxidative stress injuryin cortical neurons occurs via inhibition of NF-120581B JNK andERK signaling pathway [37] In the adult mammalian brainNF-120581B activity seems to be crucial in regulating structuralplasticity and replenishment of granule cells within thehippocampus throughout life We suggest that inhibiting theexpression of NF-120581B is a key strategy to inhibit the secondaryinflammatory cascade and promote the locomotor recoveryin SD rats after spinal cord injury Further the activation ofPPAR-120574 inhibits NF-120581B expression in a SD rat spinal cordinjury model

Genetic ablation of NF-120581B resulted in severe defects inthe neurogenic region (dentate gyrus) of the hippocam-pus [19] Reactivation of NF-120581B resulted in integration ofnewborn neurons and eventual regeneration of the dentategyrus accompanied by a complete recovery of structural andbehavioral defects These effects were attributed to NF-120581B-mediated control of PKA transcription and regulation ofaxogenesis [19 23] Sulaiman et al [38] found that forskolincould attenuate the adverse effects of long-term Schwanncell denervation on peripheral nerve regeneration in vivoFurther forskolin had protective effects on behavioral deficitsand neuropathological changes in a mouse model of cerebralamyloidosis [39] and could contribute to more efficientproduction of DAergic neurons from human-derived NPCsfor therapy of neurodegenerative diseases [40] In the presentstudy we found that forskolin could improve the numberof nerve dendrites (MAP2 marker) in a SD rat spinal cordinjury model which was consistent with the aforementionedfindings During this process we observed that forskolinincreased the expression of PKA which also confirmed thatthe effect of forskolin on neuronal formation is being throughPKA

In the present study we found that simultaneous activa-tion of PPAR-120574 and PKA elicited the best results in termsof BBB score However simultaneous activation of PPAR-120574and PKA was not as efficacious as activating PKA alone inpromoting the generation of newborn neurons and activatingPPAR-120574 alone in inhibiting inflammation The recovery oflocomotor function after spinal cord injury in rats is likelyrelated to the secondary damage caused by the inflammatorycascade as well as neuronal cell regeneration after injuryThe balance between the two is therefore important toachieve

5 Conclusion

By combining the inhibition of NF-120581B and the survivaland generation of neurons activation of PPAR-120574 and PKAsimultaneously could produce the best results in termsof promoting motor function after spinal cord injury inSD rats The underlying mechanism likely involves thePPAR-120574-mediated inhibition of NF-120581B and the consequent

amelioration of the inflammatory cascade andPKA-mediatedpromotion of neuronal cell formation

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

The authors declare that they have no conflicts of interest

Authorsrsquo Contributions

Qing-qi MengWei Lei and HaoChen contributed equally tothis work and should be considered as co-first authors

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China [Grant nos 81301038 and 81741067] theGuangzhou Science Foundation [Grant no 201607010010]and the Guangzhou Health Science and Technology Project[Grant no 20151A011014]

References

[1] S Kabu YGao B K Kwon andV Labhasetwar ldquoDrug deliverycell-based therapies and tissue engineering approaches forspinal cord injuryrdquo Journal of Controlled Release vol 219 pp141ndash154 2015

[2] M V S Varma J Lin Y-A Bi et al ldquoQuantitative pre-diction of repaglinide-rifampicin complex drug interactionsusing dynamic and static mechanistic models Delineatingdifferential CYP3A4 induction and oatp1b1 inhibition potentialof rifampicinrdquo Drug Metabolism and Disposition vol 41 no 5pp 966ndash974 2013

[3] J R Wilson N Forgione and M G Fehlings ldquoEmergingtherapies for acute traumatic spinal cord injuryrdquo CanadianMedical Association Journal vol 185 no 6 pp 485ndash492 2013

[4] M Ahmadian J M Suh N Hah et al ldquoPPAR gamma signalingand metabolism the good the bad and the futurerdquo NatureMedicine vol 99 no 5 pp 557ndash566 2013

[5] C A Oyinbo ldquoSecondary injury mechanisms in traumaticspinal cord injury a nugget of this multiply cascaderdquo ActaNeurobiologiae Experimentalis vol 71 no 2 pp 281ndash299 2011

[6] C Raposo and M Schwartz ldquoGlial scar and immune cellinvolvement in tissue remodeling and repair following acuteCNS injuriesrdquo Glia vol 62 no 11 pp 1895ndash1904 2014

[7] Q Meng X Liang P Wang et al ldquoRosiglitazone enhances theproliferation of neural progenitor cells and inhibits inflamma-tion response after spinal cord injuryrdquoNeuroscience Letters vol503 no 3 pp 191ndash195 2011

[8] A J Kroker and J B Bruning ldquoReview of the structuraland dynamic mechanisms of PPAR120574 partial agonismrdquo PPARResearch vol 2015 Article ID 816856 15 pages 2015

[9] R J de Groot S C Baker R S Baric et al ldquoMiddle east respi-ratory syndrome coronavirus (MERS-CoV) Announcement ofthe coronavirus study grouprdquo Journal of Virology vol 87 no 14pp 7790ndash7792 2013

PPAR Research 11

[10] AHyong V Jadhav S Lee et al ldquoRosiglitazone a PPARgammaagonist attenuates inflammation after surgical brain injury inrodentsrdquo Brain Research vol 1215 pp 218ndash224 2008

[11] H Liu M E Rose S Culver X Ma C E Dixon andS H Graham ldquoRosiglitazone attenuates inflammation andCA3 neuronal loss following traumatic brain injury in ratsrdquoBiochemical and Biophysical ResearchCommunications vol 472no 4 pp 648ndash655 2016

[12] J M Madeira S M Schindler and A Klegeris ldquoA new look atauranofin dextromethorphan and rosiglitazone for reductionof glia-mediated inflammation in neurodegenerative diseasesrdquoNeural Regeneration Research vol 10 no 3 pp 391ndash393 2015

[13] X-G Li X-J Lin J-H Du S-Z Xu X-F Lou and ZChen ldquoCombination of methylprednisolone and rosiglitazonepromotes recovery of neurological function after spinal cordinjuryrdquo Neural Regeneration Research vol 11 no 10 pp 1678ndash1684 2016

[14] J Napetschnig and H Wu ldquoMolecular basis of NF-120581B signal-ingrdquo Annual Review of Biophysics vol 42 no 1 pp 443ndash4682013

[15] G Bonizzi and M Karin ldquoThe two NF-120581B activation pathwaysand their role in innate and adaptive immunityrdquo Trends inImmunology vol 25 no 6 pp 280ndash288 2004

[16] T Takagi Y Naito N Tomatsuri et al ldquoPioglitazone a PPAR-120574 ligand provides protection from dextran sulfate sodium-induced colitis in mice in association with inhibition of the NF-120581B-cytokine cascaderdquo Redox Report vol 7 no 5 pp 283ndash2892002

[17] Y-F Zhang X-L Zou J WU X-Q Yu and X Yang ldquoRosigli-tazone a Peroxisome Proliferator-Activated Receptor (PPAR)-120574 Agonist Attenuates Inflammation Via NF-120581B Inhibition inLipopolysaccharide-Induced Peritonitisrdquo Inflammation vol 38no 6 pp 2105ndash2115 2015

[18] J-S Wu H-D Tsai W-M Cheung C Y Hsu and T-N LinldquoPPAR-120574 Ameliorates Neuronal Apoptosis and Ischemic BrainInjury via Suppressing NF-120581B-Driven p22phox TranscriptionrdquoMolecular Neurobiology vol 53 no 6 pp 3626ndash3645 2016

[19] Y Imielski J C Schwamborn P Luningschror et al ldquoRegrow-ing the adult brain NF-120581Bcontrols functional circuit formationand tissue homeostasis in the dentate gyrusrdquo PLoS ONE vol 7no 2 Article ID e30838 2012

[20] E W Sutherland ldquoStudies on the mechanism of hormoneactionrdquo Science vol 177 no 4047 pp 401ndash408 1972

[21] M Brunelli V Castellucci and E R Kandel ldquoSynaptic facili-tation and behavioral sensitization in Aplysia possible role ofserotonin and cyclic AMPrdquo Science vol 194 no 4270 pp 1178ndash1181 1976

[22] M Shelly B K Lim L Cancedda S C Heilshorn H Gaoand M-M Poo ldquoLocal and long-range reciprocal regulation ofcAMP and cGMP in axondendrite formationrdquo Science vol 327no 5965 pp 547ndash552 2010

[23] B Kaltschmidt and C Kaltschmidt ldquoNF-KappaB in long-termmemory and structural plasticity in the adult mammalianbrainrdquo Frontiers in Molecular Neuroscience vol 8 no Novem-ber pp 1ndash11 2015

[24] K B Seamon and J W Daly ldquoForskolin A unique diterpeneactivator of cyclic AMP-generating systemsrdquo Journal of CyclicNucleotide Research vol 7 no 4 pp 201ndash204 1981

[25] S L Kilmer and R C Carlsen ldquoForskolin activation of adeny-late cyclase in vivo stimulates nerve regenerationrdquo Nature vol307 no 5950 pp 455ndash457 1984

[26] W Sulaiman and T D Dreesen ldquoEffect of local applicationof transforming growth factor-120573 at the nerve repair site fol-lowing chronic axotomy and denervation on the expressionof regeneration-associated genesrdquo Journal of Neurosurgery vol121 no 4 pp 859ndash874 2014

[27] D M Basso M S Beattie and J C Bresnahan ldquoA sensitiveand reliable locomotor rating scale for open field testing in ratsrdquoJournal of Neurotrauma vol 12 no 1 pp 1ndash21 1995

[28] H XNguyenMD Galvan andA J Anderson ldquoCharacteriza-tion of early and terminal complement proteins associated withpolymorphonuclear leukocytes in vitro and in vivo after spinalcord injuryrdquo Journal of Neuroinflammation vol 5 article no 262008

[29] S Temple ldquoThe development of neural stem cellsrdquo Nature vol414 no 6859 pp 112ndash117 2001

[30] P Lu R Ahmad and M H Tuszynski ldquoNeural stem cells forspinal cord injuryrdquo in Translational Neuroscience pp 297ndash315Springer 2016

[31] J AMorales-Garcia R Luna-Medina C Alfaro-Cervello et alldquoPeroxisome proliferator-activated receptor 120574 ligands regulateneural stem cell proliferation and differentiation in vitro and invivordquo Glia vol 59 no 2 pp 293ndash307 2011

[32] J M Zolezzi M J Santos S Bastıas-Candia C Pinto J AGodoy and N C Inestrosa ldquoPPARs in the central nervoussystem roles in neurodegeneration and neuroinflammationrdquoBiological Reviews vol 92 no 4 pp 2046ndash2069 2017

[33] X R Zhao N Gonzales and J Aronowski ldquoPleiotropic role ofPPAR120574 in intracerebral hemorrhage an intricate system involv-ing Nrf2 RXR and NF-120581Brdquo CNS Neuroscience amp Therapeuticsvol 21 no 4 pp 357ndash366 2015

[34] M Shelly L Cancedda S Heilshorn G Sumbre and M-M Poo ldquoLKB1STRAD Promotes Axon Initiation DuringNeuronal Polarizationrdquo Cell vol 129 no 3 pp 565ndash577 2007

[35] K Wada A Nakajima K Katayama et al ldquoPeroxisomeproliferator-activated receptor 120574-mediated regulation of neuralstem cell proliferation and differentiationrdquo The Journal ofBiological Chemistry vol 281 no 18 pp 12673ndash12681 2006

[36] G Johnson and R Jope ldquoThe role of microtubule-associatedprotein 2 (MAP-2) in neuronal growth plasticity and degener-ationrdquo Journal of Neuroscience Research vol 33 no 4 pp 505ndash512 1992

[37] Y Qian L Cao T Guan et al ldquoProtection by genistein oncortical neurons against oxidative stress injury via inhibition ofNF-kappaB JNK and ERK signaling pathwayrdquo PharmaceuticalBiology vol 53 no 8 pp 1124ndash1132 2015

[38] OA R Sulaiman andTGordon ldquoTransforming growth factor-120573 and forskolin attenuate the adverse effects of long-termSchwann cell denervation on peripheral nerve regeneration invivordquo Glia vol 37 no 3 pp 206ndash218 2002

[39] B A Owona C Zug H J Schluesener and Z-Y ZhangldquoProtective Effects of Forskolin on Behavioral Deficits andNeuropathological Changes in a Mouse Model of CerebralAmyloidosisrdquo Journal of Neuropathology amp Experimental Neu-rology vol 75 no 7 pp 618ndash627 2016

[40] X Wang X Li K Wang et al ldquoForskolin cooperating withgrowth factor on generation of dopaminergic neurons fromhuman fetal mesencephalic neural progenitor cellsrdquo Neuro-science Letters vol 362 no 2 pp 117ndash121 2004

Stem Cells International

Hindawiwwwhindawicom Volume 2018


MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine


Behavioural Neurology

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

2 PPAR Research

kappa B (nuclear factor kappa B NF-120581B) after spinal cordinjury [7]

NF-120581B is extracted from B lymphocytes and is namedbecause of its binding to the immunoglobulin chain geneenhancer sequence-specific 120581B [14] The NF-120581B family oftranscription factors has an essential role in inflammationand innate immunity The active form of NF-120581B is a dimertypically a P50 P65 heterodimer and is themain componentof the NF-120581B family involved in gene transcription [15]Members of the NF-120581B family participate in many physiolog-ical processes including regulation of the immune responsestimulation of immune cell maturation and the productionand survival of essential transcription factors for B cells [15]

In our preliminary research we found that PPAR-120574could inhibit NF-120581B activation and promote proliferationand inhibit neural differentiation of endogenous neural stemcells [7] The NF-120581B and PPAR-120574 signaling pathways areclosely related PPAR-120574 activation along with inhibitionof the NF-120581B-cytokine cascade could protect against dex-tran sulfate sodium-induced colitis in mice [16] PPAR-120574could also attenuate inflammation via NF-120581B inhibition inlipopolysaccharide-induced peritonitis [17] In addition tofunctions in glucose and lipid metabolism and cell prolifer-ation and differentiation PPAR-120574 can also alleviate the acuteand chronic inflammatory response after nerve injury andameliorate neuronal apoptosis and ischemic brain injury bysuppressing NF-120581B-driven p22phox transcription [18]

Imielski et al [19] reported distinct loss of hippocampaldentate gyrus neuronal tissue in NF-120581B gene-deficient micereactivation of NF-120581B could promote the formation of den-tate gyrus neurons and neural network and improve neuronalorganization and animal behavior The underlying mecha-nism is likely related to the activation of cAMPPKA afteractivation of NF-120581B cAMP (Cyclic Adenosine Monophos-phate) was discovered in 1958 by Earl [20] increased levelsof cAMP lead to a transient enhancement of transmitterrelease [21] and regulation of cAMP and cGMP togetherensures coordinated development of one axon and multipledendrites PKA could enhance transmitter release at thesynaptic connection between the sensory and motor neuron[22] PKA is implicated in the growth of axons and survivalof newborn neurons [19 23] Forskolin is a unique diterpeneactivator of cyclic AMP-generating systems [24] Forskolin-mediated activation of adenylate cyclase stimulates nerveregeneration in vivo [25] Sulaiman et al [26] found thattreatmentwith forskolin alone and in combinationwith TGF-120573 resulted in an increase in the number of regenerated axonscompared with saline-only control

These findings were consistent with our preliminaryfindings that activation of PPAR-120574 could inhibit the expres-sion of NF-120581B in a SD rat spinal cord injury model whileinhibiting neuronal survival We therefore hypothesized thatPPAR-120574 activation in SD rats inhibits neurons via PKAwhich is downstream of NF-120581B In in vitro experimentswe intend to verify that PPAR-120574 activation inhibits NF-120581B expression which in turn could inhibit the cAMPPKApathway After confirming the inhibition of cAMPPKA byPPAR-120574 animal experiments will be initiated to determinewhether simultaneous activation of PPAR-120574 and PKA could

inhibit inflammation and promote neuronal formation Inthis study we explored the role of PPAR-120574 and PKA infunctional recovery after spinal cord injury in SD ratsWe aimed to determine the mechanisms underlying thisrecovery as well as the balance between the inflammatorycascade and generation of endogenous spinal cord neurons

2 Materials and Methods

21 In Vitro Experiments

211 Extraction and Culture of Spinal Cord-Derived NeuralStem Cells One-day-old SD rats were killed by cervicaldislocation and immersed in 75 alcohol Three sets ofeye scissors and tweezers were prepared in sequence andplaced in beakers with 75 alcohol numbered 1 2 and 3respectively The skin was cut using the first scissors andboth sides of the spine were cut from the rib fossa fromthe bottom up to remove the modified fascia and fat tissueattached to the spine by using the second scissors With 1mL of serum-free DMEMF12 medium drawn with a 1 mLsyringe syringe needle was flushed from one end of the spineand washed 3 times repeatedly to flush out the spinal cordThe spinal cord was then cut into 2ndash3 pieces by using thethird pair of scissors and the pieces of spinal cord tissue wereremoved into a Petri dish filled with culture medium andsubjected to repeated aspiration using a syringe 5 to forma uniform suspension The spinal cord pieces were sievedthrough 200-mesh sieve in order to separate them into singlecells After suspension in serum-free DMEMF12 sampleswere centrifuged for 5 min (1000 rpm)The cells were stainedwith trypan blue counted and seeded at a density of 1 times 106cells ml in culture flasks and incubated at 37 ∘C in a 5 CO

2

humidified atmosphere Culture mediumwas changed after 3days and a half Peculiar ldquonerve ballrdquo formation was observedusing an inverted microscope

After 5 to 7 days the spinal cord-derived stem cells wereproliferated and expanded in vitro for 1 passage The cellswere separated mechanically by using pipettes and 200-meshsieve and centrifuged for 5 min (1000rmin)The supernatantwas discarded and 2times105ml spinal cord-derived stem cellswere inoculated onto the surface of a sterile-10 Petri dish andincubated in medium containing DMEMF12 + 2 B27 +20 ngml b FGF + 20 ngml EGF This free-serum culturesystem was used in order to screen NSCs Each time mediumis changed and every passage flasks were changed to discardthe adherent cells Thus NSCs are naturally purified duringthe cultivation process The purity and quality of NSC werecalculated by staining with NestinDAPI

Neural stem cells derived from the spinal cord weredivided into three groups namely the rosiglitazone grouptreated with rosiglitazone (1 120583molL in DMSO [Abcamab142179]) the G3335 group treated with G3335 (PPAR-120574-antagonist 01 120583molL) and DMSO and the vehicle grouptreated with DMSOAfter 3 days continuous culture prolifer-ation of NSCs was evaluated by cell number using CellometerAuto 1000 (USA Nexcelom)

Neural stem cells were grown in medium supplementedwith 10 fetal bovine serum (the medium was used to

PPAR Research 3

(a) (b)

(c) (d)









4 PPAR Research






3VO4






3 Results




PPAR Research 5



(a)

(a1)

(a2)

(b1)

(b2) (b)






6 PPAR Research

MAP2

NF-B

PKA

-Actin


12

10

8

6

4

2

00

12

10

8

6

4

2

00

7000

6000

5000

4000

3000

2000

1000

0

12

10

8

6

4

2

00

10

8

6

4

2

00

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

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lowast

lowast

lowastlowast

PKA

rela

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RNA

leve

l








PKA

rela

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rote

in le

vel

MA

P2re

lativ

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A le

vel

MA

P2re

lativ

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tein

leve

l 6

5

4

3

2

1

00

5

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3

2

1

00NF-

B re

lativ

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A le

vel

NF-

B re

lativ

e pro

tein

leve

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cAM

P pm

olm

l pro

tein

(a)

(b)

(c)

(h)

(d)

(e)

(f)

(g)






PPAR Research 7

8h 1d 3d 7d 14d 21d 28d

lowast

lowast

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lowast

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lowastlowast



0

2

4

6

8

10

12

14

16

18

BBB

scor

e













8 PPAR Research

MAP2

NeuN


NF-B

-Actin

lowast

lowast

lowast lowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast

lowast

lowast

Neu

N re

lativ

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A le

vel

Neu

N re

lativ

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tein

leve

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P2 re

lativ

e mRN

A le

vel

MA

P2 re

lativ

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tein

leve

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NF-

B re

lativ

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A le

vel

NF-

B re

lativ

e pro

tein

leve

l

PKA

pm

olg

prot

ein

min






12

10

8

6

4

2

00

1214161820

108642

00

25

20

15

10

5

00 000

05

10

15

20

25

30

6

5

4

3

2

1

00

6

5

4

3

2

1

00

6

7

5

4

3

2

1

00

(a)

(b)

(c)

(h)

(d)

(e)

(f)

(g)


4 Discussion



PPAR Research 9


PPA

R-

rela

tive p

rote

in le

vel

6

5

4

3

2

1

00

lowast

lowast

(a)

PPAR-


-Actin

(b)








10 PPAR Research





5 Conclusion



Data Availability






Acknowledgments


References










PPAR Research 11




































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















PPAR Research 3

(a) (b)

(c) (d)









4 PPAR Research






3VO4






3 Results




PPAR Research 5



(a)

(a1)

(a2)

(b1)

(b2) (b)






6 PPAR Research

MAP2

NF-B

PKA

-Actin


12

10

8

6

4

2

00

12

10

8

6

4

2

00

7000

6000

5000

4000

3000

2000

1000

0

12

10

8

6

4

2

00

10

8

6

4

2

00

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast

PKA

rela

tive m

RNA

leve

l








PKA

rela

tive p

rote

in le

vel

MA

P2re

lativ

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A le

vel

MA

P2re

lativ

e pro

tein

leve

l 6

5

4

3

2

1

00

5

4

3

2

1

00NF-

B re

lativ

e mRN

A le

vel

NF-

B re

lativ

e pro

tein

leve

l

cAM

P pm

olm

l pro

tein

(a)

(b)

(c)

(h)

(d)

(e)

(f)

(g)






PPAR Research 7

8h 1d 3d 7d 14d 21d 28d

lowast

lowast

lowast

lowastlowast

lowast

lowastlowastlowast

lowast

lowast

lowast

lowast

lowastlowast



0

2

4

6

8

10

12

14

16

18

BBB

scor

e













8 PPAR Research

MAP2

NeuN


NF-B

-Actin

lowast

lowast

lowast lowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast

lowast

lowast

Neu

N re

lativ

e mRN

A le

vel

Neu

N re

lativ

e pro

tein

leve

l

MA

P2 re

lativ

e mRN

A le

vel

MA

P2 re

lativ

e pro

tein

leve

l

NF-

B re

lativ

e mRN

A le

vel

NF-

B re

lativ

e pro

tein

leve

l

PKA

pm

olg

prot

ein

min






12

10

8

6

4

2

00

1214161820

108642

00

25

20

15

10

5

00 000

05

10

15

20

25

30

6

5

4

3

2

1

00

6

5

4

3

2

1

00

6

7

5

4

3

2

1

00

(a)

(b)

(c)

(h)

(d)

(e)

(f)

(g)


4 Discussion



PPAR Research 9


PPA

R-

rela

tive p

rote

in le

vel

6

5

4

3

2

1

00

lowast

lowast

(a)

PPAR-


-Actin

(b)








10 PPAR Research





5 Conclusion



Data Availability






Acknowledgments


References










PPAR Research 11




































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















4 PPAR Research






3VO4






3 Results




PPAR Research 5



(a)

(a1)

(a2)

(b1)

(b2) (b)






6 PPAR Research

MAP2

NF-B

PKA

-Actin


12

10

8

6

4

2

00

12

10

8

6

4

2

00

7000

6000

5000

4000

3000

2000

1000

0

12

10

8

6

4

2

00

10

8

6

4

2

00

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast

PKA

rela

tive m

RNA

leve

l








PKA

rela

tive p

rote

in le

vel

MA

P2re

lativ

e mRN

A le

vel

MA

P2re

lativ

e pro

tein

leve

l 6

5

4

3

2

1

00

5

4

3

2

1

00NF-

B re

lativ

e mRN

A le

vel

NF-

B re

lativ

e pro

tein

leve

l

cAM

P pm

olm

l pro

tein

(a)

(b)

(c)

(h)

(d)

(e)

(f)

(g)






PPAR Research 7

8h 1d 3d 7d 14d 21d 28d

lowast

lowast

lowast

lowastlowast

lowast

lowastlowastlowast

lowast

lowast

lowast

lowast

lowastlowast



0

2

4

6

8

10

12

14

16

18

BBB

scor

e













8 PPAR Research

MAP2

NeuN


NF-B

-Actin

lowast

lowast

lowast lowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast

lowast

lowast

Neu

N re

lativ

e mRN

A le

vel

Neu

N re

lativ

e pro

tein

leve

l

MA

P2 re

lativ

e mRN

A le

vel

MA

P2 re

lativ

e pro

tein

leve

l

NF-

B re

lativ

e mRN

A le

vel

NF-

B re

lativ

e pro

tein

leve

l

PKA

pm

olg

prot

ein

min






12

10

8

6

4

2

00

1214161820

108642

00

25

20

15

10

5

00 000

05

10

15

20

25

30

6

5

4

3

2

1

00

6

5

4

3

2

1

00

6

7

5

4

3

2

1

00

(a)

(b)

(c)

(h)

(d)

(e)

(f)

(g)


4 Discussion



PPAR Research 9


PPA

R-

rela

tive p

rote

in le

vel

6

5

4

3

2

1

00

lowast

lowast

(a)

PPAR-


-Actin

(b)








10 PPAR Research





5 Conclusion



Data Availability






Acknowledgments


References










PPAR Research 11




































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















PPAR Research 5



(a)

(a1)

(a2)

(b1)

(b2) (b)






6 PPAR Research

MAP2

NF-B

PKA

-Actin


12

10

8

6

4

2

00

12

10

8

6

4

2

00

7000

6000

5000

4000

3000

2000

1000

0

12

10

8

6

4

2

00

10

8

6

4

2

00

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast

PKA

rela

tive m

RNA

leve

l








PKA

rela

tive p

rote

in le

vel

MA

P2re

lativ

e mRN

A le

vel

MA

P2re

lativ

e pro

tein

leve

l 6

5

4

3

2

1

00

5

4

3

2

1

00NF-

B re

lativ

e mRN

A le

vel

NF-

B re

lativ

e pro

tein

leve

l

cAM

P pm

olm

l pro

tein

(a)

(b)

(c)

(h)

(d)

(e)

(f)

(g)






PPAR Research 7

8h 1d 3d 7d 14d 21d 28d

lowast

lowast

lowast

lowastlowast

lowast

lowastlowastlowast

lowast

lowast

lowast

lowast

lowastlowast



0

2

4

6

8

10

12

14

16

18

BBB

scor

e













8 PPAR Research

MAP2

NeuN


NF-B

-Actin

lowast

lowast

lowast lowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast

lowast

lowast

Neu

N re

lativ

e mRN

A le

vel

Neu

N re

lativ

e pro

tein

leve

l

MA

P2 re

lativ

e mRN

A le

vel

MA

P2 re

lativ

e pro

tein

leve

l

NF-

B re

lativ

e mRN

A le

vel

NF-

B re

lativ

e pro

tein

leve

l

PKA

pm

olg

prot

ein

min






12

10

8

6

4

2

00

1214161820

108642

00

25

20

15

10

5

00 000

05

10

15

20

25

30

6

5

4

3

2

1

00

6

5

4

3

2

1

00

6

7

5

4

3

2

1

00

(a)

(b)

(c)

(h)

(d)

(e)

(f)

(g)


4 Discussion



PPAR Research 9


PPA

R-

rela

tive p

rote

in le

vel

6

5

4

3

2

1

00

lowast

lowast

(a)

PPAR-


-Actin

(b)








10 PPAR Research





5 Conclusion



Data Availability






Acknowledgments


References










PPAR Research 11




































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















6 PPAR Research

MAP2

NF-B

PKA

-Actin


12

10

8

6

4

2

00

12

10

8

6

4

2

00

7000

6000

5000

4000

3000

2000

1000

0

12

10

8

6

4

2

00

10

8

6

4

2

00

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast

PKA

rela

tive m

RNA

leve

l








PKA

rela

tive p

rote

in le

vel

MA

P2re

lativ

e mRN

A le

vel

MA

P2re

lativ

e pro

tein

leve

l 6

5

4

3

2

1

00

5

4

3

2

1

00NF-

B re

lativ

e mRN

A le

vel

NF-

B re

lativ

e pro

tein

leve

l

cAM

P pm

olm

l pro

tein

(a)

(b)

(c)

(h)

(d)

(e)

(f)

(g)






PPAR Research 7

8h 1d 3d 7d 14d 21d 28d

lowast

lowast

lowast

lowastlowast

lowast

lowastlowastlowast

lowast

lowast

lowast

lowast

lowastlowast



0

2

4

6

8

10

12

14

16

18

BBB

scor

e













8 PPAR Research

MAP2

NeuN


NF-B

-Actin

lowast

lowast

lowast lowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast

lowast

lowast

Neu

N re

lativ

e mRN

A le

vel

Neu

N re

lativ

e pro

tein

leve

l

MA

P2 re

lativ

e mRN

A le

vel

MA

P2 re

lativ

e pro

tein

leve

l

NF-

B re

lativ

e mRN

A le

vel

NF-

B re

lativ

e pro

tein

leve

l

PKA

pm

olg

prot

ein

min






12

10

8

6

4

2

00

1214161820

108642

00

25

20

15

10

5

00 000

05

10

15

20

25

30

6

5

4

3

2

1

00

6

5

4

3

2

1

00

6

7

5

4

3

2

1

00

(a)

(b)

(c)

(h)

(d)

(e)

(f)

(g)


4 Discussion



PPAR Research 9


PPA

R-

rela

tive p

rote

in le

vel

6

5

4

3

2

1

00

lowast

lowast

(a)

PPAR-


-Actin

(b)








10 PPAR Research





5 Conclusion



Data Availability






Acknowledgments


References










PPAR Research 11




































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















PPAR Research 7

8h 1d 3d 7d 14d 21d 28d

lowast

lowast

lowast

lowastlowast

lowast

lowastlowastlowast

lowast

lowast

lowast

lowast

lowastlowast



0

2

4

6

8

10

12

14

16

18

BBB

scor

e













8 PPAR Research

MAP2

NeuN


NF-B

-Actin

lowast

lowast

lowast lowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast

lowast

lowast

Neu

N re

lativ

e mRN

A le

vel

Neu

N re

lativ

e pro

tein

leve

l

MA

P2 re

lativ

e mRN

A le

vel

MA

P2 re

lativ

e pro

tein

leve

l

NF-

B re

lativ

e mRN

A le

vel

NF-

B re

lativ

e pro

tein

leve

l

PKA

pm

olg

prot

ein

min






12

10

8

6

4

2

00

1214161820

108642

00

25

20

15

10

5

00 000

05

10

15

20

25

30

6

5

4

3

2

1

00

6

5

4

3

2

1

00

6

7

5

4

3

2

1

00

(a)

(b)

(c)

(h)

(d)

(e)

(f)

(g)


4 Discussion



PPAR Research 9


PPA

R-

rela

tive p

rote

in le

vel

6

5

4

3

2

1

00

lowast

lowast

(a)

PPAR-


-Actin

(b)








10 PPAR Research





5 Conclusion



Data Availability






Acknowledgments


References










PPAR Research 11




































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















8 PPAR Research

MAP2

NeuN


NF-B

-Actin

lowast

lowast

lowast lowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast

lowast

lowast

Neu

N re

lativ

e mRN

A le

vel

Neu

N re

lativ

e pro

tein

leve

l

MA

P2 re

lativ

e mRN

A le

vel

MA

P2 re

lativ

e pro

tein

leve

l

NF-

B re

lativ

e mRN

A le

vel

NF-

B re

lativ

e pro

tein

leve

l

PKA

pm

olg

prot

ein

min






12

10

8

6

4

2

00

1214161820

108642

00

25

20

15

10

5

00 000

05

10

15

20

25

30

6

5

4

3

2

1

00

6

5

4

3

2

1

00

6

7

5

4

3

2

1

00

(a)

(b)

(c)

(h)

(d)

(e)

(f)

(g)


4 Discussion



PPAR Research 9


PPA

R-

rela

tive p

rote

in le

vel

6

5

4

3

2

1

00

lowast

lowast

(a)

PPAR-


-Actin

(b)








10 PPAR Research





5 Conclusion



Data Availability






Acknowledgments


References










PPAR Research 11




































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















PPAR Research 9


PPA

R-

rela

tive p

rote

in le

vel

6

5

4

3

2

1

00

lowast

lowast

(a)

PPAR-


-Actin

(b)








10 PPAR Research





5 Conclusion



Data Availability






Acknowledgments


References










PPAR Research 11




































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















10 PPAR Research





5 Conclusion



Data Availability






Acknowledgments


References










PPAR Research 11




































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















PPAR Research 11




































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of























of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















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