ResearchArticle Lycium barbarum Polysaccharide Promotes

Research ArticleLycium barbarum Polysaccharide PromotesMaturation of Dendritic Cell via Notch Signaling andStrengthens Dendritic Cell Mediated T LymphocyteCytotoxicity on Colon Cancer Cell CT26-WT

WeiWang1 Mingxing Liu1 YangWang1 Tao Yang1 Dongsheng Li1 Feng Ding1

Hongzhi Sun1 Guang Bai 1 and Qing Li 2

1Department of General Surgery The First Affiliated Hospital of Jinzhou Medical University Jinzhou Liaoning 121001 China2Department of Internal Medicine TheThird Affiliated Hospital of Jinzhou Medical University Jinzhou Liaoning 121001 China

Correspondence should be addressed to Guang Bai Baiguang69255163com and Qing Li liqing610557163com

Received 6 April 2017 Revised 13 September 2017 Accepted 25 September 2017 Published 28 January 2018

Academic Editor Chang G Son

Copyright copy 2018 Wei Wang 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

Lycium barbarum polysaccharide (LBP) is the major function component of Lycium barbarum L and has been previously reportedto induce the phenotypic and functionalmaturation of dendritic cells (DCs) aswell as activatingT lymphocytes In the current studythe immunologic cytotoxicity promoting effect of LBP was assessed and the underlying mechanism was explored The impact ofLBP on the phenotype maturation and immunogenicity of DCs was assessed The activity of Notch pathway which is involvedin the regulation of LBP on DCs was detected Afterwards the influence of LBP on cytotoxicity of DC-mediated cytotoxicity Tlymphocytes (CTLs) to CT26-WT colon cancer cells was further assessed Administration of LBP induced the phenotypic andfunctional maturation of DCs After being subjected to LBP the expression of Notch and Jagged and Notch targets Hes1 and Hes5was all upregulated The cytotoxicity of DC-mediated CTLs was strengthened by administration of LBP Additionally cytotoxicityof DC-mediated CTLs on CT26-WT colon cancer cells also increased with effector-target ratio In conclusion LBP could inducethe phenotypic and functional maturation of DCs via Notch signaling and promote the cytotoxicity of DC-mediated CTLs whichcould be employed as a promising adjuvant for cancer immunotherapy

1 Introduction

The concept of immune contexture is mainly emerged basedon studies that are mostly performed with human colorectalcancer (CRC) [1] Study of Pages et al showed that highdensity of intratumoralmemory T cells was closely associatedwith the disease free time and overall survival rate of CRCpatients [2] In addition to the findings in CRC Becht and hiscolleagues also proved the beneficial effect of high densitiesof T cells with a Th1 orientation and of cytotoxic CD8+ Tcells in non-small cell lung cancer (NSCLC) [3 4]Thereforeharnessing the immune system of cancer patients (immuno-therapy in oncology) has been proposed as promising thera-peutic approach for treatment of types of cancers

Generally the immune system can prevent the develop-ment and progression of cancer through a mechanism calledimmune surveillance However once the process of immunesurveillance fails due to variable causable factors [5] it willlead to the immune selection of tumor cell variants with re-duced immunogenicity and allow progression of tumors [67] Currently it is commonly recognized that functioning ofan effective immunotherapy majorly depends on the roles ofimmune cells playing in the development of tumors espe-cially those with antigen presentation capability that is den-dritic cells (DCs) [8] and those with cytolytic activity that iscytotoxicity T lymphocytes (CTLs) [9] Thus efficient induc-tion of antitumor immunity via DC-basedmethods is emerg-ing in recent decades [10ndash12] However induced maturation

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2018 Article ID 2305683 10 pageshttpsdoiorg10115520182305683

2 Evidence-Based Complementary and Alternative Medicine

of DC by microbial products (such as LPS) or inflammatorycytokines (such as TNF) [13ndash15] is toxic and applicationlimited Therefore exploration of nontoxic vehicles that arecapable of inducing DC maturation and immunogenicity isimperative

Lycium barbarum L is a well-recognized East Asia herbalmedicine used for treatment and prevention of disease suchas insomnia liver dysfunction diabetes visual degenerationand cancer [16] The bioactivity of Lycium barbarum L ismainly attributed to its polysaccharide-protein complex [17ndash19] The major component of Lycium barbarum polysaccha-ride (LBP) consists of six monosaccharides (galactose glu-cose rhamnose arabinose mannose and xylose) and 18amino acids [16] Recent studies infer that LBP can also en-hance the immune function of patients [18 19] Verified byseveral studies administration of LBP can induce the pheno-typic and functional maturation and immunogenicity of DCsas well as activating T lymphocytes [16 20 21] Neverthelesseven with the confirmation of the potency of LBP in promot-ing efficiency of immunotherapy the underlying mechanismthroughwhich LBPmodulates the function of DCs and CTLsremains unrevealed

Thus in the current study the impact of LBP on thephenotype maturation and immunogenicity of DCs isolatedfrom mice was investigated To reveal the pathways involvedin the regulating effect of LBP on DCs the expression ofNotch pathway members was quantified The Notch pathwayis reported to be a key signaling transduction in the differ-entiation of DCs [22 23] Thereafter the influence of LBP oncytotoxicity of DC-mediated CTLs to CT26-WT colon cellswas further assessed Findings outlined in the current studyshowed that administration of LBP promoted the maturationand immunogenicity of DCs via Notch signaling whichstrengthened the cytotoxicity ofDC-mediatedCTLs to tumorcells

2 Materials and Methods

21 Chemicals Cell Culture and Animals LBPs (purity8432) for clinical application with an endotoxin contentlt 01 Eumg were purchased from Pharmagenesis Inc (New-town PAUSA) Antibody againstNotchwas purchased fromAbcam (Catl number ab52627 Cambridge MA) Antibodyagainst Jagged was purchased from Bioss (Catl number bs-1448R Beijing China) Murine colon cancer cell line CT26-WT was obtained from Chi Scientific (Shanghai China) andcultured in DMEM supplemented with 10 FBS and peni-cillinstreptomycin at 37∘C in an atmosphere of 5 CO

2and

95 air until a density that allowed cell division throughoutthe course of the experiment BALBc mice were purchasedfrom Changsheng Biotechnology (Liaoning China) andmaintained in cages at room temperature (20ndash25∘C) with aconstant humidity (55plusmn5) with access to food and water adlibitum in a 12 12-h light-dark cycle All animal experimentswere conducted in accordance with the Guide for the Careand Use of Laboratory Animals published by the NationalResearch Council and under IACUC approval The use ofanimals was reviewed and approved by the Animal CareReview Committee ofThe First Affiliated Hospital of JinzhouMedical University

22 Collection of Bone Marrow Cells (BMCs) and Inductionof DCs Themice were anesthetized with overdose pentobar-bital sodium and sacrificed by neck removing method andsterilized with 75 alcohol for 10min Thereafter humerusand tibia were removed and administrated with 75 alcoholfor 2min Then the marrow cavities of tissues were washedwith 1640 medium and BMCs in the suspensions were sep-arated with 70 120583m Cell Strainers and centrifuged with Tris-NH4Cl Lysis Buffer to collect BMCs for induction ofDCsThe

collected BMCs incubated with 1640 medium supplementedwith 10 fetal bovine serum (FBS) and with 20 ngml mGM-CSF and 20 ngml rmIL-4 at 37∘C in an atmosphere of5 CO

2and 95 air for 3 days Afterwards the suspension

was replaced with fresh medium and suspended cells werediscarded After another 5 days the DCs were collectedcentrifuged and resuspended in 1640medium for subsequentassays

23 LBP Administration For assessing the effect of LBP onthe phenotype maturation and immunogenicity of DCs5ml healthy DCs (5 times 105ml) were cultured in 60mm platesadministrated with different doses of LBP (0120583gml 1120583gml10 120583gml and 100 120583gml) for different time courses (24 hand 48 h) The treatment combination with the strongestDCmaturation inducing effect was employed for subsequentassays

24 Isolation of T Lymphocytes from Mice and Induction ofCTLs by DCs T lymphocytes were isolated from spleen ofmice spleen tissues were grinded and cells were suspendedby PBS The mixture was filed with 74-120583m filter gauze andthen subjected to centrifugation for at 1000 rpm for 10minto discard supernate Then the precipitation was added with2ml RBC Lysis Buffer and incubated at room temperaturefor 5min After being subjected to centrifugation at 1000 rpmfor 10min the precipitation was resuspended using 1640medium Afterwards the suspension was mixed with equalvolume of lymphocyte separation medium and then cen-trifuged at 2500 rpm for 25minThe interlayer of the suspen-sionwas collected resuspended using PBS and centrifuged at1000 rpm for 5minThe precipitation was resuspended using1640 medium and incubated with DCs or LBP treated DCs ata ratio of 5 1 (2 times 106 DCs and 1 times 107 T lymphocytes) in solu-tion containing 500 IUml IL-2 and 50 ngml CD3 for 4 days[9] Then the CD8+ CTLs were purified by positive selectionusing Cell Isolation Kit (Catl number 130-049-401 MiltenyiBiotec China) according to the manufacturersrsquo instructionThe phenotypic characteristics of CD8+ CTLs were assessedby detecting expression status of CD3 and CD8 by flowcytometry as described below

25 Cytotoxicity Assessment of CTLs on CT26-WT CellsCT26-WT cells were employed as target cells for assessmentof cytotoxicity of different CTLs (effector cells) For ELISAdetection of IFN-120574 two groups were classified (A) T +E group CT26-WT cells treated with CTLs (effector-targetratio 100 1) for 20 h and (B) T + E + LBP group CT26-WT cells treated with CTLs (effector-target ratio 100 1) and100 ngml LBP for 20 h For flow cytometry detection of cell

Evidence-Based Complementary and Alternative Medicine 3

apoptosis six groups (two groups for effector-target ratio)were set up (A) T + E group CT26-WT cells treated withCTLs (effector-target ratio 10 1 20 1 40 1 respectively) for20 h and (B) T + E + LBP group CT26-WT cells treatedwith CTLs (effector-target ratio 10 1 20 1 and 40 1 resp)and 100 120583gml LBP for 20 h For NK activity determinationby MTT assay 18 groups (six groups for effector-target ratio)were classified (A) T group health CT26-WT cells (B) Egroup CTLs (effector-target ratio 10 1 20 1 and 40 1resp) (C) T + E group CT26-WT cells treated with CTLs(effector-target ratio 10 1 20 1 and 40 1 resp) for 20 h (D)T + LBP group health CT26-WT cells treated with 100 120583gmlLBP for 20 h (E) E + LBP group CTLs (effector-target ratio10 1 20 1 and 40 1 resp) treated with 100 ngml LBP for20 h and (F) T + E + LBP group CT26-WT cells treated withCTLs (effector-target ratio 10 1 20 1 and 40 1 resp) and100 120583gml LBP for 20 h

26 Phenotypic and Maturation Assessment of DCs Uponcompletion of the 48 h culture cells in different groupswere collected The morphology of DCs was observed underinverted phase contrast microscope (AE31 Motic XiamenChina) at 400x magnification Then the expressions of CD80and CD86 on DCs were detected with flow cytometry

27 Enzyme-Linked Immunosorbent Assay (ELISA) Produc-tion of TGF-120573 IL-10 and IL-12 in supernatant of DC cultures(Catl number EK0515 Catl number EK0417 and Catl num-ber EK0422 Boster China) and production of IFN-120574 (Catlnumber 69-90110 MSK Wuhan China) in supernatant weremeasured using ELISA kits according to the manufacturersrsquoinstructions

28 Reverse Transcription Real Time PCR (RT2-PCR) TotalRNA in cells from different groups was extracted usingRNA Purified Total RNA Extraction Kit according to themanufacturerrsquos instruction (Calt number RP1201 BioTekeBeijing China) 120573-Actinwas selected as the internal referencegene RNA was reversely transcribed to cDNA templatesusing super M-MLV reverse transcriptase (Calt numberRP6502 BioTeke Beijing China) and final qPCR reactionmixture (20120583L) consisted of 10 120583L of SYBR GREEN master-mix (Catl number SY1020 Solarbio China) 05120583L of each ofthe primers (Notch forward 51015840-CAAGGCACGGAGGAA-GAAG-31015840 and reverse 51015840-CCAGGTGAGTGTCAGGCATA-31015840 Jagged forward 51015840-CAACGACCGTAATCGCATC-31015840and reverse 51015840-GAAGTGGGCAATCCCTGTG-31015840 Hes1forward 51015840-TGACTGTGAAGCACCTCCG-31015840 and reverse51015840-AAGCGGGTCACCTCGTTCA-31015840 Hes5 forward 51015840-CGCTCGCTAATCGCCTCCA-31015840 and reverse 51015840-CGG-TCCCGACGCATCTTCT-31015840120573-actin forward 51015840-CTGTGC-CCATCTACGAGGGCTAT-31015840 and reverse 51015840-TTTGAT-GTCACGCACGATTTCC-31015840) 1 120583L of the cDNA templateand 8 120583L of Rnase-free H

2O Amplification parameters were

as follows denaturation at 95∘C for 10min followed by 40cycles at 95∘C for 10 s 60∘C for 20 s and 72∘C for 30 sand the reaction was stopped at 4∘C for 5min Relativeexpression levels of the targeted molecules were calculated

with Exicycler 96 (BIONEER) according to the expressionof 2minusΔΔct

29 Western Blotting Assay Total protein product wasextracted by incubating cells with 1 PMSF-RIPA (Catlnumber ST506 Catl number P0013B Beyotime Biotechnol-ogy China) and then centrifuged at 12000 rpm for 10min120573-Actin was selected as the internal reference protein Pro-tein concentrations of different samples were determinedusing the BCA method (Catl number P0012 BeyotimeBiotechnology China) 40120583g protein (20120583L volume) wassubject to a 10 sodium dodecylsulfate polyacrylamide gelelectrophoresis (SDS-PAGE) and transferred onto polyvinyli-dene difluoride (PVDF) sheets Then the membranes werewashed with TTBS and incubated in skim milk powdersolution for 1 h Primary antibodies against Notch (1 1000)Jagged (1 500) and 120573-actin (1 1000) were incubated withmembranes at 4∘C overnight Secondary HRP goat anti-rabbitgoat anti-murine IgG antibodies (1 5000) were addedto the mixture and incubated with the membranes for 45minat 37∘C The blots were developed using Beyo ECL Plusreagent The results were detected in the Gel Imaging Systemand the relative expression levels of targeted proteins werecalculatedwithGel-Pro-Analyzer (Media Cybernetics USA)

210 MTT Assay MTT assay was performed to determinethe viability of CT26-WT cells in different groups Briefly50 120583L exponentially growing cells (2 times 105 cellsml) wereseeded into a 96-well plate in triplicate Then 5mgml MTTwas added to each well and incubated for 4 h at 37∘C TheOD values at 490 nm in different wells were recorded using aMicroplate Reader The NK activity was calculated based onthe expression 1 minus ((ODT+E minusODE)ODT)

211 Flow Cytometry Cells in different groups were collectedwith centrifugation at 1500 rpm for 5minThe apoptotic rateswere measured using an Apoptosis Detection Kit (Catl num-ber KGA106 KeyGEN BioTECH Nanjing China) accordingto the instructions formanufacturers briefly 5 120583LAnnexin Vwas added to different wells After incubation with AnnexinV for 10min at room temperature the cells were resuspendedwith 1x binding buffer and added with 5120583L Propidium Iodide(PI) After a 15-min incubation in the dark the apoptotic rateswere analyzed using a FACScan flow cytometry (Accuri C6BD USA)

212 Statistical Analysis All the data were expressed in theform of mean plusmn SD Studentrsquos 119905-test ANOVA and posthoc tests with Duncan method were performed with asignificance level of 005 using GraphPad Prism 6 (GraphPadSoftware San Diego CA)

3 Results

31 Morphology and Surface Antibody Characteristics ofDCs Morphology of DCs was shown in Figure 1(a) Threedays after culture DCs were grown from confluence intosuspension Thereafter cell colonies were formed by cells


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Figure 1 Morphology and Maturation Detection of BMCs Derived DCs (a) Seven days after induction for BMCs 5 times 105ml DCs wereincubated with LBP at 100120583gml for 48 h and cells morphology was detected using an inverted phase contrast microscope The cells werecharacterized by long dendrites which are typical features of DCs (b) 5 times 105ml DCs were incubated with LBP at different doses (0120583gml1 120583gml 10120583gml and 100 120583gml) for 48 hThe expression of CD80 and CD86 on DCs was detected using flow cytometry methodThe meanfluorescent intensity of CD80 and CD86 was strongest under treatment of 100120583gml LBP (c) 5 times 105ml DCs were incubated with 100120583gmlLBP for 24 and 48 h respectivelyThe expression of CD80 andCD86 onDCswas detected using flow cytometrymethodThemean fluorescentintensity of CD80 andCD86was strongest after 48 h incubation Each assay was represented by three independent replicates lowast119875 lt 005 versusthe other three groups 119875 lt 005 versus 24 h group

with processes on the surface Seven days after culturecells were characterized by long dendrites which are typicalfeatures of DCs By employing flow cytometry the antibodiespresented on the surface of the cells were also detected Theadministration of LBP further increased the expression ofthe two antibodies on the cell surface and the effect wasboth dose- and time-dependent with 48 h administration of100 120583g LBP had the strongest inducing effect (Figures 1(b) and1(c) Figure S1 in Supplementary Material available onlineat httpsdoiorg10115520182305683) Therefore the sub-sequent assays were performed by incubating DCs with

100 120583g LBP for 48 h The results showed that the DCs weresuccessfully induced from BMCs and that LBP could furtherpromote the maturation process

32 Administration of LBP Induced the Production of IL-12and Suppressed the Production of IL-10 as well as TGF-120573The levels of IL-10 and TGF-120573 in the supernatants of DCswere decreased by administration of LBP and the differencesbetween DC and LBP groups were statistically significant(119875 lt 005) (Figures 2(a) and 2(b)) In contrast the levels of


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Figure 2Quantitative Analysis Results of IL-12 IL-10 andTGF-120573Levels ((a) (b) (c)) 5times 105mlDCswere incubatedwith LBP at 100 120583gml for48 h and production of IL-12 IL-10 and TGF-120573 in supernatant of DC cultures wasmeasured using ELISA kits according to themanufacturersrsquoinstructions Administration of LBP induced the expression of IL-12 while suppressing the expression of IL-10 and TGF-120573 Each assay wasrepresented by three independent replicates lowastlowast119875 lt 001 versus DC group

IL-12 in the supernatants of DCs were significantly increased(119875 lt 005) (Figure 2(c))

33 Administration of LBP Initiated Notch Signaling in DCsTo uncover the mechanism which drives the function of LBPon DCs the expression of Notch and Jagged was detectedboth at mRNA and at protein levelsThe expression of targetsof Notch including Hes1 and Hes5 was detected with RT2-PCR It was found that incubation with LBP enhanced theexpression of all indicators (Figures 3(a) and 3(b)) Given thefact that Notch signaling is crucial to the differentiation ofDCs the results of molecular detection confirmed the con-clusion that LBPwas capable of inducing the differentiation ofDCs as illustrated by morphology and antibodies expressiondetection which depended on Notch signaling activation

34 Administration of LBP Strengthened the Cytotoxicity ofDC-Mediated CTLs on Colon Cancer Cell CT26-WT CTLswere successfully induced by DC incubation and the cyto-toxicity characteristics of DC-mediated CTLs were evenstrengthened by administration of LBP the proportion ofCD3+CD8+ cells significantly increased after being subjectedto LBP for 4 days (545plusmn426 versus 809plusmn793) (Figure 4(a))Moreover the production of IFN-120574 by CTLs was also aug-mented by administration of LBP representing the promis-ing potential of LBP to promote immunologic cytotoxicity(Figure 4(b)) As a result the cytotoxicity of CTLs on CT26-WT cells was increased the total apoptotic rates of CT26-WTcell in LBP treated group and the NK activity of LBP treatedCTLs were significantly increased compared with normalCTLs (119875 lt 005) (Figure 5) Additionally effector-target ratioalso served as a factor that influenced the cytotoxicityof CTLs although no significant difference was detectedbetween effector-target ratios 10 1 and 20 1 the apoptoticrate and NK activity in 40 1 group were dramatically higherthan the other two groups (119875 lt 005)

4 Discussion

The application of natural compounds as potential improve-ments for human health keeps increasing in popularity inrecent years Since ancient years oriental herbalmedicine has

been used for treatment of malignancy in Eastern countriesLBP which is the biologically active compounds of Lyciumbarbarum L is proved to have biological activities such asanticancer antioxidant hypoglycemic and immunologicalactivities [24ndash26] Previous studies have shown that admin-istration of LBP induces cell cycle arrest in colon cancer [27]and promotes the maturation of DCs as well as increasingcytotoxicity of CTLs [16 20 21] The findings of the previousstudies took a step further in the current study after con-firming the inducing effect of LBP on DCs the mechanisminvolved in the function of LBP on DCs and its effect on DC-mediated CTL cytotoxicity on tumor cells were also exploredOur results showed that LBP treatment activated the Notchsignaling in DCs representing the pathway through whichLBP promoted DCs maturation Moreover the effect of LBPon the production of IL-10 IL-12 and TGF-120573 might explainthe mechanism driving the LBP function on activation andfunction differentiation of CTLs

It was demonstrated in the current study that LBP treat-ment upregulated the expression of CD80 andCD86 on BMCderived DCs suggesting that LBP induced the phenotypicmaturation of DCs Moreover LBP also promoted functionmaturation of DCs which was validated by the augmentedproduction of IL-12 and suppressed production of IL-10 andTGF-120573 IL-12 is a functional DC maturation marker with amolecular mass of 70 kDa which is a potent IFN-120574 inducerfor CTLs [28 29] To the contrary IL-10 and TGF-120573mediateactive suppression of specific T lymphocytes responses as anessential mechanism in immune response [30]The increasedIL-12 and decreased IL-10 and TGF-120573 along with the en-hanced DC activation by LBP treatment would promote theIFN-120574-secreting T cell differentiation following T cell activa-tion [9] Indeed in ELSIA assay the production of IFN-120574was significantly upregulated Previous studies have demon-strated that IFN-120574+ CTLs are critical for antitumor immuno-therapies [9]Therefore the enhancedDCactivationandhigh-er frequency of IFN-120574+ CTLs evidently indicated that LBPwas a promising adjuvant therapeutic modality along withimmunotherapies

The pathway through which LBP exerted its function onDC maturation was also preliminarily investigated in thecurrent study It was found that administration of LBP would


DC DC + LBP DC DC + LBP


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Western blotting Western blotting

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GAPDH

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GAPDH

(b)

Figure 3Detection of Notch Signaling (a) 5 times 105ml DCs were incubated with LBP at 100120583gml for 48 h and then subjected to RNA PurifiedTotal RNA Extraction Kit to collect total RNAThe expression levels of Notch Jagged Hes1 and Hes5 were detected with RT2-PCR methodAdministration of LBP up-regulated expression of Notch Jagged Hes1 andHes5 at mRNA level (b) 5 times 105ml DCs were incubated with LBPat 100 120583gml for 48 hThen cells were incubated with 1 PMSF-RIPA and the centrifuged at 12000 rpm for 10min to collect total proteinTheexpression levels of Notch and Jagged were detected with western blotting assay Administration of LBP up-regulated expression of Notchand Jagged at protein Each assay was represented by three independent replicates lowastlowast119875 lt 001 versus DC group


T + ECD

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Figure 4 Detection of DC-Mediated CTLsrsquo Dierentiation (a) T cells were incubated with DCs or LBP treated DCs at a ratio of 5 1 (2 times 106DCs and 1 times 107 T lymphocytes) for 4 days Then the CD8+ CTLs were purified by positive selection using Cell Isolation Kit The phenotypiccharacteristics of CD8+ CTLs were assessed by detecting expression status of CD3 and CD8 by flow cytometryThe proportion of CD3+CD8+T lymphocytes was increased in T + E + LBP group (b) CT26-WT cells were cells treated with CTLs (effector-target ratio 100 1) for 20 h orCTLs (effector-target ratio 100 1) and 100 ngml LBP for 20 h Production of IFN-120574 in T cells was detected with ELISA assay Production ofIFN-120574 was increased in T + E + LBP group Each assay was represented by three independent replicates lowastlowast119875 lt 001 versus T + E group

induce the activation of Notch signaling The differentiationof DC is modulated via a network of soluble and cell-boundfactors in stroma [22] Notch signaling is one of the mostmajor components of the process Generally Notch signalingis initiated upon the binding of the extracellular domain ofNotch receptors to Notch ligands Jagged and Delta Withregard to DC differentiation function of Notch signalingis exerted through its cooperation with Wnt pathways [22]Then the activated Wnt pathway will result in sustainedupregulation of IL-12 [31] However the inhibition of IL-10 and TGF-120573 associated with the activated Notch signalingis confusing in that the activation of Notch signaling inDCs always induces the production of IL-10 by TH1 cellsAnd activated Wnt signaling due to Notch activation is alsoreported to upregulate TGF-120573 signaling duringmyofibroblast

differentiation [32ndash34] Subjecting DCs to LBP in the currentstudy seemed to lead to a contrary conclusion to previousstudies which made it hard to evidently elaborate themechanism through which LBP affected DC maturation andcytokines secretion Given that the activated Notch signalingwas associated with decreased IL-10 and TGF-120573 it was likelythat the modulating effect of LBP on DCs comprised morecomplicated pathways as we had expected in the first place

5 Conclusions

Being widely used as an injection in clinical patients in Chinato improve immune functions LBP has showed its promisingpotential for further exploration as an effective adjuvant forcancer immunotherapy In the current study administration


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Q1-UL01

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Figure 5Detection of Cytotoxicity of CTLs (a) CT26-WT cells treated with CTLs (effector-target ratio 10 1 20 1 and 40 1 resp) for 20 h orwith CTLs (effector-target ratio 10 1 20 1 and 40 1 resp) and 100 120583gml LBP for 20 hThe apoptosis rate was detected with flow cytometryThe cell apoptosis rate was increased by LBP administration and effector-target ratio (b) CT26-WT cells CTLs and LBP were incubated indifferent combinations for 20 h [CT26-WT cells CTLs and CT26-WT cells + CTLs (effector-target ratio 10 1 20 1 and 40 1) CT26-WTcells + 100 120583gml LBP CTLs + 100120583gml LBP and CT26-WT cells + CTLs (effector-target ratio 10 1 20 1 and 40 1) + 100120583gml LBP] Theviability of CT26-WT cells was detected with MTT assay and represented by OD values at 490 nm The NK activity was calculated based onthe expression 1 minus ((ODT+E minus ODE)ODT) NK activity of CTLs was increased by LBP administration and effector-target ratio Each assaywas represented by six independent replicates lowast119875 lt 005 versus T + E group 119875 lt 005 119875 lt 005 versus 10 1 and 20 1 effector-target ratio


of LBP not only induced the phenotypic and functionalmaturation of DCs but also promoted the cytotoxicity of DC-mediated CTLs We also attempted to preliminarily study thepathways involved in the effect of LBP on DC but the acti-vated Notch signaling due to LBP treatment introducedmorequestions to the mechanism related to LBP function on DCsTo comprehensively understand the effect of LBP on immuneresponses more work will be needed in the future

Conflicts of Interest

The authors declare that they have no conflicts of interest

Authorsrsquo Contributions

WeiWangandMingxing Liu contributed equally to this study

Acknowledgments

This study was supported by the Special Clinical Founda-tion of Wujieping Medical Foundation Society (Grant no32067501281) and the Foundation for President of LiaoningMedical University (Grant no xzjj20130233)

References

[1] E Becht N A Giraldo M-C Dieu-Nosjean C Sautes-Frid-man and W H Fridman ldquoCancer immune contexture andimmunotherapyrdquo Current Opinion in Immunology vol 39 pp7ndash13 2016

[2] L M Coussens and Z Werb ldquoInflammation and cancerrdquo Na-ture vol 420 no 6917 pp 860ndash867 2002

[3] M-C Dieu-Nosjean M Antoine C Danel et al ldquoLong-termsurvival for patients with non-small-cell lung cancer withintratumoral lymphoid structuresrdquo Journal of Clinical Oncologyvol 26 no 27 pp 4410ndash4417 2008

[4] J Goc C Germain T K D Vo-Bourgais et al ldquoDendritic cellsin tumor-associated tertiary lymphoid structures signal a Th1cytotoxic immune contexture and license the positive prognos-tic value of infiltrating CD8+ T cellsrdquo Cancer Research vol 74no 3 pp 705ndash715 2014

[5] J B Swann and M J Smyth ldquoImmune surveillance of tumorsrdquoThe Journal of Clinical Investigation vol 117 no 5 pp 1137ndash11462007

[6] G P Dunn L J Old and R D Schreiber ldquoThe immunobiologyof cancer immunosurveillance and immunoeditingrdquo Immunityand Immunoediting vol 21 no 2 pp 137ndash148 2004

[7] S Pernot M Terme T Voron et al ldquoColorectal cancer andimmunity what we know and perspectivesrdquo World Journal ofGastroenterology vol 20 no 14 pp 3738ndash3750 2014

[8] J Kumar V Kale and L Limaye ldquoUmbilical cord blood-derivedCD11c+ dendritic cells could serve as an alternative allogeneicsource of dendritic cells for cancer immunotherapyrdquo Stem CellResearch ampTherapy vol 6 no 1 2015

[9] Y Liu X Tian S Jiang et al ldquoUmbilical cord blood-deriveddendritic cells infected by adenovirus for SP17 expressioninduce antigen-specific cytotoxic T cells against NSCLC cellsrdquoCellular Immunology vol 298 no 1-2 pp 18ndash24 2015

[10] L Fong and E G Engleman ldquoDendritic cells in cancer immun-otherapyrdquo Annual Review of Immunology vol 18 pp 245ndash2732000

[11] P Brossart S Wirths W Brugger and L Kanz ldquoDendritic cellsin cancer vaccinesrdquo Experimental Hematology vol 29 no 11 pp1247ndash1255 2001

[12] J Banchereau B Schuler-Thurner A K Palucka and GSchuler ldquoDendritic cells as vectors for therapyrdquo Cell vol 106no 3 pp 271ndash274 2001

[13] F Sallusto and A Lanzavecchia ldquoEfficient presentation of solu-ble antigen by cultured human dendritic cells is maintained bygranulocytemacrophage colony-stimulating factor plus inter-leukin 4 and downregulated by tumor necrosis factor alphardquoThe Journal of Experimental Medicine vol 179 no 4 pp 1109ndash1118 1994

[14] C Winzler P Rovere M Rescigno et al ldquoMaturation stagesof mouse dendritic cells in growth factor-dependent long-termculturesrdquo The Journal of Experimental Medicine vol 185 no 2pp 317ndash328 1997

[15] J A Roake A S Rao P J Morris C P Larsen D F Hankinsand JM Austyn ldquoDendritic cell loss from nonlymphoid tissuesafter systemic administration of lipopolysaccharide tumornecrosis factor and interleukin 1rdquo The Journal of ExperimentalMedicine vol 181 no 6 pp 2237ndash2247 1995

[16] Z Chen J Lu N Srinivasan B K H Tan and S H ChanldquoPolysaccharide-protein complex from Lycium barbarum L isa novel stimulus of dendritic cell immunogenicityrdquoThe Journalof Immunology vol 182 no 6 pp 3503ndash3509 2009

[17] L Huang Y Lin G Tian and G Ji ldquo[Isolation purification andphysico-chemical properties of immunoactive constituentsfrom the fruit of Lycium barbarum L]rdquoYao Xue Xue Bao =ActaPharmaceutica Sinica vol 33 no 7 pp 512ndash516 1998

[18] L Gan S-H Zhang Q Liu and H-B Xu ldquoA polysaccharide-protein complex from Lycium barbarum upregulates cytokineexpression in human peripheral blood mononuclear cellsrdquoEuropean Journal of Pharmacology vol 471 no 3 pp 217ndash2222003

[19] L Gan S H Zhang X L Yang and H B Xu ldquoImmunomod-ulation and antitumor activity by a polysaccharide-proteincomplex from Lycium barbarumrdquo International Immunophar-macology vol 4 no 4 pp 563ndash569 2004

[20] Z Chen B Kwong Huat Tan and S H Chan ldquoActivation of Tlymphocytes by polysaccharide-protein complex from LyciumbarbarumLrdquo International Immunopharmacology vol 8 no 12pp 1663ndash1671 2008

[21] J Zhu L-H Zhao X-P Zhao and Z Chen ldquoLycium barbarumpolysaccharides regulate phenotypic and functional maturationof murine dendritic cellsrdquo Cell Biology International vol 31 no6 pp 615ndash619 2007

[22] J Zhou P Cheng J-I Youn M J Cotter and D I GabrilovichldquoNotch and wingless signaling cooperate in regulation of den-dritic cell differentiationrdquo Immunity vol 30 no 6 pp 845ndash8592009

[23] P Cheng Y Nefedova C A Corzo and D I GabrilovichldquoRegulation of dendritic-cell differentiation by bone marrowstroma via different Notch ligandsrdquo Blood vol 109 no 2 pp507ndash515 2007

[24] X-M Li ldquoProtective effect of Lycium barbarum polysaccha-rides on streptozotocin-induced oxidative stress in ratsrdquo Inter-national Journal of Biological Macromolecules vol 40 no 5 pp461ndash465 2007


[25] X M Li Y L Ma and X J Liu ldquoEffect of the Lycium barbarumpolysaccharides on age-related oxidative stress in aged micerdquoJournal of Ethnopharmacology vol 111 no 3 pp 504ndash511 2007

[26] H Wu H Guo and R Zhao ldquoEffect of Lycium barbarum poly-saccharide on the improvement of antioxidant ability and DNAdamage in NIDDM ratsrdquo Yakugaku Zasshi vol 126 no 5 pp365ndash371 2006

[27] F Mao B Xiao Z Jiang J Zhao X Huang and J Guo ldquoAnti-cancer effect of Lycium barbarum polysaccharides on coloncancer cells involves G0G1 phase arrestrdquoMedical Oncology vol28 no 1 pp 121ndash126 2011

[28] D Jung S K Jeong C-M Lee et al ldquoEnhanced efficacy oftherapeutic cancer vaccines produced by Co-treatment withmycobacterium tuberculosis heparin-binding hemagglutinin anovel TLR4 agonistrdquo Cancer Research vol 71 no 8 pp 2858ndash2870 2011

[29] K TNoh S J Shin KH Son et al ldquoThemycobacterium aviumsubsp paratuberculosis fibronectin attachment protein a toll-like receptor 4 agonist enhances dendritic cell-based cancervaccine potencyrdquo Experimental amp Molecular Medicine vol 44no 5 pp 340ndash349 2012

[30] M Jutel M Akdis F Budak et al ldquoIL-10 and TGF-120573 cooperatein the regulatory T cell response to mucosal allergens in normalimmunity and specific immunotherapyrdquo European Journal ofImmunology vol 33 no 5 pp 1205ndash1214 2003

[31] S J George ldquoWnt pathway A new role in regulation of inflam-mationrdquo Arteriosclerosis Thrombosis and Vascular Biology vol28 no 3 pp 400ndash402 2008

[32] S Rutz M Janke N Kassner T Hohnstein M Krueger and AScheffold ldquoNotch regulates IL-10 production by T helper 1 cellsrdquoProceedings of the National Acadamy of Sciences of the UnitedStates of America vol 105 no 9 pp 3497ndash3502 2008

[33] M Saraiva and A OrsquoGarra ldquoThe regulation of IL-10 productionby immune cellsrdquoNature Reviews Immunology vol 10 no 3 pp170ndash181 2010

[34] J M Carthy F S Garmaroudi Z Luo and B M McManusldquoWnt3a induces myofibroblast differentiation by upregulatingTGF-120573 signaling through SMAD2 in a 120573-catenin-dependentmannerrdquo PLoS ONE vol 6 no 5 Article ID e19809 2011

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


of DC by microbial products (such as LPS) or inflammatorycytokines (such as TNF) [13ndash15] is toxic and applicationlimited Therefore exploration of nontoxic vehicles that arecapable of inducing DC maturation and immunogenicity isimperative

Lycium barbarum L is a well-recognized East Asia herbalmedicine used for treatment and prevention of disease suchas insomnia liver dysfunction diabetes visual degenerationand cancer [16] The bioactivity of Lycium barbarum L ismainly attributed to its polysaccharide-protein complex [17ndash19] The major component of Lycium barbarum polysaccha-ride (LBP) consists of six monosaccharides (galactose glu-cose rhamnose arabinose mannose and xylose) and 18amino acids [16] Recent studies infer that LBP can also en-hance the immune function of patients [18 19] Verified byseveral studies administration of LBP can induce the pheno-typic and functional maturation and immunogenicity of DCsas well as activating T lymphocytes [16 20 21] Neverthelesseven with the confirmation of the potency of LBP in promot-ing efficiency of immunotherapy the underlying mechanismthroughwhich LBPmodulates the function of DCs and CTLsremains unrevealed

Thus in the current study the impact of LBP on thephenotype maturation and immunogenicity of DCs isolatedfrom mice was investigated To reveal the pathways involvedin the regulating effect of LBP on DCs the expression ofNotch pathway members was quantified The Notch pathwayis reported to be a key signaling transduction in the differ-entiation of DCs [22 23] Thereafter the influence of LBP oncytotoxicity of DC-mediated CTLs to CT26-WT colon cellswas further assessed Findings outlined in the current studyshowed that administration of LBP promoted the maturationand immunogenicity of DCs via Notch signaling whichstrengthened the cytotoxicity ofDC-mediatedCTLs to tumorcells

2 Materials and Methods

21 Chemicals Cell Culture and Animals LBPs (purity8432) for clinical application with an endotoxin contentlt 01 Eumg were purchased from Pharmagenesis Inc (New-town PAUSA) Antibody againstNotchwas purchased fromAbcam (Catl number ab52627 Cambridge MA) Antibodyagainst Jagged was purchased from Bioss (Catl number bs-1448R Beijing China) Murine colon cancer cell line CT26-WT was obtained from Chi Scientific (Shanghai China) andcultured in DMEM supplemented with 10 FBS and peni-cillinstreptomycin at 37∘C in an atmosphere of 5 CO

2and

95 air until a density that allowed cell division throughoutthe course of the experiment BALBc mice were purchasedfrom Changsheng Biotechnology (Liaoning China) andmaintained in cages at room temperature (20ndash25∘C) with aconstant humidity (55plusmn5) with access to food and water adlibitum in a 12 12-h light-dark cycle All animal experimentswere conducted in accordance with the Guide for the Careand Use of Laboratory Animals published by the NationalResearch Council and under IACUC approval The use ofanimals was reviewed and approved by the Animal CareReview Committee ofThe First Affiliated Hospital of JinzhouMedical University

22 Collection of Bone Marrow Cells (BMCs) and Inductionof DCs Themice were anesthetized with overdose pentobar-bital sodium and sacrificed by neck removing method andsterilized with 75 alcohol for 10min Thereafter humerusand tibia were removed and administrated with 75 alcoholfor 2min Then the marrow cavities of tissues were washedwith 1640 medium and BMCs in the suspensions were sep-arated with 70 120583m Cell Strainers and centrifuged with Tris-NH4Cl Lysis Buffer to collect BMCs for induction ofDCsThe

collected BMCs incubated with 1640 medium supplementedwith 10 fetal bovine serum (FBS) and with 20 ngml mGM-CSF and 20 ngml rmIL-4 at 37∘C in an atmosphere of5 CO

2and 95 air for 3 days Afterwards the suspension

was replaced with fresh medium and suspended cells werediscarded After another 5 days the DCs were collectedcentrifuged and resuspended in 1640medium for subsequentassays

23 LBP Administration For assessing the effect of LBP onthe phenotype maturation and immunogenicity of DCs5ml healthy DCs (5 times 105ml) were cultured in 60mm platesadministrated with different doses of LBP (0120583gml 1120583gml10 120583gml and 100 120583gml) for different time courses (24 hand 48 h) The treatment combination with the strongestDCmaturation inducing effect was employed for subsequentassays

24 Isolation of T Lymphocytes from Mice and Induction ofCTLs by DCs T lymphocytes were isolated from spleen ofmice spleen tissues were grinded and cells were suspendedby PBS The mixture was filed with 74-120583m filter gauze andthen subjected to centrifugation for at 1000 rpm for 10minto discard supernate Then the precipitation was added with2ml RBC Lysis Buffer and incubated at room temperaturefor 5min After being subjected to centrifugation at 1000 rpmfor 10min the precipitation was resuspended using 1640medium Afterwards the suspension was mixed with equalvolume of lymphocyte separation medium and then cen-trifuged at 2500 rpm for 25minThe interlayer of the suspen-sionwas collected resuspended using PBS and centrifuged at1000 rpm for 5minThe precipitation was resuspended using1640 medium and incubated with DCs or LBP treated DCs ata ratio of 5 1 (2 times 106 DCs and 1 times 107 T lymphocytes) in solu-tion containing 500 IUml IL-2 and 50 ngml CD3 for 4 days[9] Then the CD8+ CTLs were purified by positive selectionusing Cell Isolation Kit (Catl number 130-049-401 MiltenyiBiotec China) according to the manufacturersrsquo instructionThe phenotypic characteristics of CD8+ CTLs were assessedby detecting expression status of CD3 and CD8 by flowcytometry as described below

25 Cytotoxicity Assessment of CTLs on CT26-WT CellsCT26-WT cells were employed as target cells for assessmentof cytotoxicity of different CTLs (effector cells) For ELISAdetection of IFN-120574 two groups were classified (A) T +E group CT26-WT cells treated with CTLs (effector-targetratio 100 1) for 20 h and (B) T + E + LBP group CT26-WT cells treated with CTLs (effector-target ratio 100 1) and100 ngml LBP for 20 h For flow cytometry detection of cell













3 Results



(a)

Mea

n flu

ores

cent

inte

nsity

15000

10000

5000

0CD80 CD86

lowast

lowast



(b)

Mea

n flu

ores

cent

inte

nsity

15000

10000

5000

0CD80 CD86


(c)






Leve

l of I

L-12

(pg

mL)

300

200

100

0DC DC + LBP

lowastlowast

(a)

400

500

300

200

100

0DC DC + LBP

lowastlowast

Leve

l of I

L-10

(pg

mL)

(b)

400

300

200

100

0DC DC + LBP

lowastlowast

Leve

l of T

GF-

(p

gm

L)

(c)





4 Discussion








lowastlowast

lowastlowast

lowastlowast

lowastlowast



Rela

tive e

xpre

ssio

n of

Not

ch3

2

1

0

Rela

tive e

xpre

ssio

n of

Jagg

ed

25

20

15

10

05

00

Rela

tive e

xpre

ssio

n of

Hes1

4

3

2

1

0

Rela

tive e

xpre

ssio

n of

Hes5

3

2

1

0

(a)

DC DC + LBP


DC DC + LBP


Rela

tive e

xpre

ssio

n of

Not

ch

4

3

2

1

0

Rela

tive e

xpre

ssio

n of

Jagg

ed

25

20

15

10

05

00


125 kDa

43 kDa

Notch

GAPDH

134 kDa

43 kDa

Jagged

GAPDH

(b)



T + ECD

8-A

1072

106

105

104

103

1015

CD8

-A

1072

106

105

104

103

1015

101 102 103 104 105 106 1072

R 542 R 816

T + E + LBP

CD3-A101 102 103 104 105 106 1072

CD3-A(a)

T + E T + E + LBP

Leve

l of I

NF-

(p

gm

gpro

t)

400

300

200

100

0

lowastlowast

(b)




5 Conclusions



Apop

tosis

rate

()

80

60

40

20

0



T + E + LBPT + E

T +

E +

LBP

FL2-

A

FL1-A

T +

E

10 1 20 1 40 1

10 1 20 1 40 1

lowast

lowastlowast

101101

102

102

103

103

104

104

105

105

106

106

1072

FL1-A101 102 103 104 105 106 1072

FL1-A101 102 103 104 105 106 1072

FL1-A101 102 103 104 105 106 1072

FL1-A101 102 103 104 105 106 1072

FL1-A101 102 103 104 105 106 1072

1072

FL2-

A

101

102

103

104

105

106

1072

FL2-

A

101

102

103

104

105

106

1072

FL2-

A

101

102

103

104

105

106

1072

FL2-

A

101

102

103

104

105

106

1072

FL2-

A

101

102

103

104

105

106

1072

Q1-UL01

Q1-UR64

Q1-LL774

Q1-LR160

Q1-UL01

Q1-UR79

Q1-LL693

Q1-LR228

Q1-UL02

Q1-UR186

Q1-LL532

Q1-LR280

Q1-UL02

Q1-UR127

Q1-LL571

Q1-LR301

Q1-UL04

Q1-UR198

Q1-LL492

Q1-LR307

Q1-UL12

Q1-UR332

Q1-LL365

Q1-LR291

(a)

NK

activ

ity

10

08

06

04

02

00


T + E + LBPT + E

10 1 20 1 40 1

lowast

lowastlowast

(b)








Acknowledgments


References








































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of































3 Results



(a)

Mea

n flu

ores

cent

inte

nsity

15000

10000

5000

0CD80 CD86

lowast

lowast



(b)

Mea

n flu

ores

cent

inte

nsity

15000

10000

5000

0CD80 CD86


(c)






Leve

l of I

L-12

(pg

mL)

300

200

100

0DC DC + LBP

lowastlowast

(a)

400

500

300

200

100

0DC DC + LBP

lowastlowast

Leve

l of I

L-10

(pg

mL)

(b)

400

300

200

100

0DC DC + LBP

lowastlowast

Leve

l of T

GF-

(p

gm

L)

(c)





4 Discussion








lowastlowast

lowastlowast

lowastlowast

lowastlowast



Rela

tive e

xpre

ssio

n of

Not

ch3

2

1

0

Rela

tive e

xpre

ssio

n of

Jagg

ed

25

20

15

10

05

00

Rela

tive e

xpre

ssio

n of

Hes1

4

3

2

1

0

Rela

tive e

xpre

ssio

n of

Hes5

3

2

1

0

(a)

DC DC + LBP


DC DC + LBP


Rela

tive e

xpre

ssio

n of

Not

ch

4

3

2

1

0

Rela

tive e

xpre

ssio

n of

Jagg

ed

25

20

15

10

05

00


125 kDa

43 kDa

Notch

GAPDH

134 kDa

43 kDa

Jagged

GAPDH

(b)



T + ECD

8-A

1072

106

105

104

103

1015

CD8

-A

1072

106

105

104

103

1015

101 102 103 104 105 106 1072

R 542 R 816

T + E + LBP

CD3-A101 102 103 104 105 106 1072

CD3-A(a)

T + E T + E + LBP

Leve

l of I

NF-

(p

gm

gpro

t)

400

300

200

100

0

lowastlowast

(b)




5 Conclusions



Apop

tosis

rate

()

80

60

40

20

0



T + E + LBPT + E

T +

E +

LBP

FL2-

A

FL1-A

T +

E

10 1 20 1 40 1

10 1 20 1 40 1

lowast

lowastlowast

101101

102

102

103

103

104

104

105

105

106

106

1072

FL1-A101 102 103 104 105 106 1072

FL1-A101 102 103 104 105 106 1072

FL1-A101 102 103 104 105 106 1072

FL1-A101 102 103 104 105 106 1072

FL1-A101 102 103 104 105 106 1072

1072

FL2-

A

101

102

103

104

105

106

1072

FL2-

A

101

102

103

104

105

106

1072

FL2-

A

101

102

103

104

105

106

1072

FL2-

A

101

102

103

104

105

106

1072

FL2-

A

101

102

103

104

105

106

1072

Q1-UL01

Q1-UR64

Q1-LL774

Q1-LR160

Q1-UL01

Q1-UR79

Q1-LL693

Q1-LR228

Q1-UL02

Q1-UR186

Q1-LL532

Q1-LR280

Q1-UL02

Q1-UR127

Q1-LL571

Q1-LR301

Q1-UL04

Q1-UR198

Q1-LL492

Q1-LR307

Q1-UL12

Q1-UR332

Q1-LL365

Q1-LR291

(a)

NK

activ

ity

10

08

06

04

02

00


T + E + LBPT + E

10 1 20 1 40 1

lowast

lowastlowast

(b)








Acknowledgments


References








































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















(a)

Mea

n flu

ores

cent

inte

nsity

15000

10000

5000

0CD80 CD86

lowast

lowast



(b)

Mea

n flu

ores

cent

inte

nsity

15000

10000

5000

0CD80 CD86


(c)






Leve

l of I

L-12

(pg

mL)

300

200

100

0DC DC + LBP

lowastlowast

(a)

400

500

300

200

100

0DC DC + LBP

lowastlowast

Leve

l of I

L-10

(pg

mL)

(b)

400

300

200

100

0DC DC + LBP

lowastlowast

Leve

l of T

GF-

(p

gm

L)

(c)





4 Discussion








lowastlowast

lowastlowast

lowastlowast

lowastlowast



Rela

tive e

xpre

ssio

n of

Not

ch3

2

1

0

Rela

tive e

xpre

ssio

n of

Jagg

ed

25

20

15

10

05

00

Rela

tive e

xpre

ssio

n of

Hes1

4

3

2

1

0

Rela

tive e

xpre

ssio

n of

Hes5

3

2

1

0

(a)

DC DC + LBP


DC DC + LBP


Rela

tive e

xpre

ssio

n of

Not

ch

4

3

2

1

0

Rela

tive e

xpre

ssio

n of

Jagg

ed

25

20

15

10

05

00


125 kDa

43 kDa

Notch

GAPDH

134 kDa

43 kDa

Jagged

GAPDH

(b)



T + ECD

8-A

1072

106

105

104

103

1015

CD8

-A

1072

106

105

104

103

1015

101 102 103 104 105 106 1072

R 542 R 816

T + E + LBP

CD3-A101 102 103 104 105 106 1072

CD3-A(a)

T + E T + E + LBP

Leve

l of I

NF-

(p

gm

gpro

t)

400

300

200

100

0

lowastlowast

(b)




5 Conclusions



Apop

tosis

rate

()

80

60

40

20

0



T + E + LBPT + E

T +

E +

LBP

FL2-

A

FL1-A

T +

E

10 1 20 1 40 1

10 1 20 1 40 1

lowast

lowastlowast

101101

102

102

103

103

104

104

105

105

106

106

1072

FL1-A101 102 103 104 105 106 1072

FL1-A101 102 103 104 105 106 1072

FL1-A101 102 103 104 105 106 1072

FL1-A101 102 103 104 105 106 1072

FL1-A101 102 103 104 105 106 1072

1072

FL2-

A

101

102

103

104

105

106

1072

FL2-

A

101

102

103

104

105

106

1072

FL2-

A

101

102

103

104

105

106

1072

FL2-

A

101

102

103

104

105

106

1072

FL2-

A

101

102

103

104

105

106

1072

Q1-UL01

Q1-UR64

Q1-LL774

Q1-LR160

Q1-UL01

Q1-UR79

Q1-LL693

Q1-LR228

Q1-UL02

Q1-UR186

Q1-LL532

Q1-LR280

Q1-UL02

Q1-UR127

Q1-LL571

Q1-LR301

Q1-UL04

Q1-UR198

Q1-LL492

Q1-LR307

Q1-UL12

Q1-UR332

Q1-LL365

Q1-LR291

(a)

NK

activ

ity

10

08

06

04

02

00


T + E + LBPT + E

10 1 20 1 40 1

lowast

lowastlowast

(b)








Acknowledgments


References








































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















Leve

l of I

L-12

(pg

mL)

300

200

100

0DC DC + LBP

lowastlowast

(a)

400

500

300

200

100

0DC DC + LBP

lowastlowast

Leve

l of I

L-10

(pg

mL)

(b)

400

300

200

100

0DC DC + LBP

lowastlowast

Leve

l of T

GF-

(p

gm

L)

(c)





4 Discussion








lowastlowast

lowastlowast

lowastlowast

lowastlowast



Rela

tive e

xpre

ssio

n of

Not

ch3

2

1

0

Rela

tive e

xpre

ssio

n of

Jagg

ed

25

20

15

10

05

00

Rela

tive e

xpre

ssio

n of

Hes1

4

3

2

1

0

Rela

tive e

xpre

ssio

n of

Hes5

3

2

1

0

(a)

DC DC + LBP


DC DC + LBP


Rela

tive e

xpre

ssio

n of

Not

ch

4

3

2

1

0

Rela

tive e

xpre

ssio

n of

Jagg

ed

25

20

15

10

05

00


125 kDa

43 kDa

Notch

GAPDH

134 kDa

43 kDa

Jagged

GAPDH

(b)



T + ECD

8-A

1072

106

105

104

103

1015

CD8

-A

1072

106

105

104

103

1015

101 102 103 104 105 106 1072

R 542 R 816

T + E + LBP

CD3-A101 102 103 104 105 106 1072

CD3-A(a)

T + E T + E + LBP

Leve

l of I

NF-

(p

gm

gpro

t)

400

300

200

100

0

lowastlowast

(b)




5 Conclusions



Apop

tosis

rate

()

80

60

40

20

0



T + E + LBPT + E

T +

E +

LBP

FL2-

A

FL1-A

T +

E

10 1 20 1 40 1

10 1 20 1 40 1

lowast

lowastlowast

101101

102

102

103

103

104

104

105

105

106

106

1072

FL1-A101 102 103 104 105 106 1072

FL1-A101 102 103 104 105 106 1072

FL1-A101 102 103 104 105 106 1072

FL1-A101 102 103 104 105 106 1072

FL1-A101 102 103 104 105 106 1072

1072

FL2-

A

101

102

103

104

105

106

1072

FL2-

A

101

102

103

104

105

106

1072

FL2-

A

101

102

103

104

105

106

1072

FL2-

A

101

102

103

104

105

106

1072

FL2-

A

101

102

103

104

105

106

1072

Q1-UL01

Q1-UR64

Q1-LL774

Q1-LR160

Q1-UL01

Q1-UR79

Q1-LL693

Q1-LR228

Q1-UL02

Q1-UR186

Q1-LL532

Q1-LR280

Q1-UL02

Q1-UR127

Q1-LL571

Q1-LR301

Q1-UL04

Q1-UR198

Q1-LL492

Q1-LR307

Q1-UL12

Q1-UR332

Q1-LL365

Q1-LR291

(a)

NK

activ

ity

10

08

06

04

02

00


T + E + LBPT + E

10 1 20 1 40 1

lowast

lowastlowast

(b)








Acknowledgments


References








































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of






















lowastlowast

lowastlowast

lowastlowast

lowastlowast



Rela

tive e

xpre

ssio

n of

Not

ch3

2

1

0

Rela

tive e

xpre

ssio

n of

Jagg

ed

25

20

15

10

05

00

Rela

tive e

xpre

ssio

n of

Hes1

4

3

2

1

0

Rela

tive e

xpre

ssio

n of

Hes5

3

2

1

0

(a)

DC DC + LBP


DC DC + LBP


Rela

tive e

xpre

ssio

n of

Not

ch

4

3

2

1

0

Rela

tive e

xpre

ssio

n of

Jagg

ed

25

20

15

10

05

00


125 kDa

43 kDa

Notch

GAPDH

134 kDa

43 kDa

Jagged

GAPDH

(b)



T + ECD

8-A

1072

106

105

104

103

1015

CD8

-A

1072

106

105

104

103

1015

101 102 103 104 105 106 1072

R 542 R 816

T + E + LBP

CD3-A101 102 103 104 105 106 1072

CD3-A(a)

T + E T + E + LBP

Leve

l of I

NF-

(p

gm

gpro

t)

400

300

200

100

0

lowastlowast

(b)




5 Conclusions



Apop

tosis

rate

()

80

60

40

20

0



T + E + LBPT + E

T +

E +

LBP

FL2-

A

FL1-A

T +

E

10 1 20 1 40 1

10 1 20 1 40 1

lowast

lowastlowast

101101

102

102

103

103

104

104

105

105

106

106

1072

FL1-A101 102 103 104 105 106 1072

FL1-A101 102 103 104 105 106 1072

FL1-A101 102 103 104 105 106 1072

FL1-A101 102 103 104 105 106 1072

FL1-A101 102 103 104 105 106 1072

1072

FL2-

A

101

102

103

104

105

106

1072

FL2-

A

101

102

103

104

105

106

1072

FL2-

A

101

102

103

104

105

106

1072

FL2-

A

101

102

103

104

105

106

1072

FL2-

A

101

102

103

104

105

106

1072

Q1-UL01

Q1-UR64

Q1-LL774

Q1-LR160

Q1-UL01

Q1-UR79

Q1-LL693

Q1-LR228

Q1-UL02

Q1-UR186

Q1-LL532

Q1-LR280

Q1-UL02

Q1-UR127

Q1-LL571

Q1-LR301

Q1-UL04

Q1-UR198

Q1-LL492

Q1-LR307

Q1-UL12

Q1-UR332

Q1-LL365

Q1-LR291

(a)

NK

activ

ity

10

08

06

04

02

00


T + E + LBPT + E

10 1 20 1 40 1

lowast

lowastlowast

(b)








Acknowledgments


References








































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















T + ECD

8-A

1072

106

105

104

103

1015

CD8

-A

1072

106

105

104

103

1015

101 102 103 104 105 106 1072

R 542 R 816

T + E + LBP

CD3-A101 102 103 104 105 106 1072

CD3-A(a)

T + E T + E + LBP

Leve

l of I

NF-

(p

gm

gpro

t)

400

300

200

100

0

lowastlowast

(b)




5 Conclusions



Apop

tosis

rate

()

80

60

40

20

0



T + E + LBPT + E

T +

E +

LBP

FL2-

A

FL1-A

T +

E

10 1 20 1 40 1

10 1 20 1 40 1

lowast

lowastlowast

101101

102

102

103

103

104

104

105

105

106

106

1072

FL1-A101 102 103 104 105 106 1072

FL1-A101 102 103 104 105 106 1072

FL1-A101 102 103 104 105 106 1072

FL1-A101 102 103 104 105 106 1072

FL1-A101 102 103 104 105 106 1072

1072

FL2-

A

101

102

103

104

105

106

1072

FL2-

A

101

102

103

104

105

106

1072

FL2-

A

101

102

103

104

105

106

1072

FL2-

A

101

102

103

104

105

106

1072

FL2-

A

101

102

103

104

105

106

1072

Q1-UL01

Q1-UR64

Q1-LL774

Q1-LR160

Q1-UL01

Q1-UR79

Q1-LL693

Q1-LR228

Q1-UL02

Q1-UR186

Q1-LL532

Q1-LR280

Q1-UL02

Q1-UR127

Q1-LL571

Q1-LR301

Q1-UL04

Q1-UR198

Q1-LL492

Q1-LR307

Q1-UL12

Q1-UR332

Q1-LL365

Q1-LR291

(a)

NK

activ

ity

10

08

06

04

02

00


T + E + LBPT + E

10 1 20 1 40 1

lowast

lowastlowast

(b)








Acknowledgments


References








































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















Apop

tosis

rate

()

80

60

40

20

0



T + E + LBPT + E

T +

E +

LBP

FL2-

A

FL1-A

T +

E

10 1 20 1 40 1

10 1 20 1 40 1

lowast

lowastlowast

101101

102

102

103

103

104

104

105

105

106

106

1072

FL1-A101 102 103 104 105 106 1072

FL1-A101 102 103 104 105 106 1072

FL1-A101 102 103 104 105 106 1072

FL1-A101 102 103 104 105 106 1072

FL1-A101 102 103 104 105 106 1072

1072

FL2-

A

101

102

103

104

105

106

1072

FL2-

A

101

102

103

104

105

106

1072

FL2-

A

101

102

103

104

105

106

1072

FL2-

A

101

102

103

104

105

106

1072

FL2-

A

101

102

103

104

105

106

1072

Q1-UL01

Q1-UR64

Q1-LL774

Q1-LR160

Q1-UL01

Q1-UR79

Q1-LL693

Q1-LR228

Q1-UL02

Q1-UR186

Q1-LL532

Q1-LR280

Q1-UL02

Q1-UR127

Q1-LL571

Q1-LR301

Q1-UL04

Q1-UR198

Q1-LL492

Q1-LR307

Q1-UL12

Q1-UR332

Q1-LL365

Q1-LR291

(a)

NK

activ

ity

10

08

06

04

02

00


T + E + LBPT + E

10 1 20 1 40 1

lowast

lowastlowast

(b)








Acknowledgments


References








































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of

























Acknowledgments


References








































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























of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















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