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1、The Protection of Crocin AgainstUlcerative Colitis and ColorectalCancer via Suppression ofNF-B-Mediated InflammationShanshan Teng1,Jie Hao1,Hui Bi2,Congcong Li1,Yongfeng Zhang1,Yaqin Zhang1,Weiwei Han1*and Di Wang1*1School of Life Sciences,Jilin University,Changchun,China,2Department of Anesthesiolo
2、gy,Hospital of Stomatology,JilinUniversity,Changchun,ChinaBackground:In China,the incidence of ulcerative colitis(UC)is increasing every year,butthe etiology of UC remains unclear.UC is known to increase the risk of colorectal cancer(CRC).The aim of this study was to investigate the protective effec
3、ts of crocin against UCand CRC in mouse models.Methods:Crocin was used totreat the dextran sodium sulfate(DSS)-induced UC mice for3weeks,and ApcMinC/Gpt mice with colorectal cancer for 8weeks.Proteomics screeningwas used to detect changes in the protein profiles of colon tissues of UC mice.Enzyme-li
4、nked immunosorbent assays and western blot were used to verify these changes.Results:Crocin strongly reduced the disease activity index scores of UC mice,andimproved the pathological symptoms of the colonic epithelium.The anti-inflammatoryeffects of crocin were indicated by its regulation of the act
5、ivity of various cytokines,such asinterleukins,via the modulation of nuclear factor kappa-B(NF-B)signaling.CrocinsignificantlysuppressedtumorgrowthinApcMinC/Gptmiceandamelioratedpathological alterations in the colon and liver,but had no effects on spleen and kidney.Additionally,crocin significantly
6、decreased the concentrations of interleukins and tumornecrosis factor-in the sera and colon tissues,suggesting its anti-inflammatory effectsrelated to NF-B signaling.Finally,12-h incubation of SW480 cells with crocin caused cellcycle arrest,enhanced the apoptotic rate,promoted the dissipation of mit
7、ochondrialmembrane potential,and the over-accumulation of reactive oxygen species.From thetheoretical analyses,phosphorylated residues on S536 may enhance the protein-proteininteractions which may influence the conformational changes in the secondary structure ofNF-B.Conclusion:The protective effect
8、s of crocin on UC and CRC were due to its suppressionof NF-B-mediated inflammation.Keywords:crocin,ulcerative colitis,colorectal cancer,anti-inflammation,antitumor,NF-BEdited by:Sabine Grsch,Goethe University Frankfurt,GermanyReviewed by:Claudio Ferrante,University of Studies GdAnnunzioChieti and Pe
9、scara,ItalySatish Ramalingam,SRM Institute of Science andTechnology,India*Correspondence:Weiwei HDi WThese authors have contributedequally to this work.Specialty section:This article was submitted toInflammation Pharmacology,a section of the journalFrontiers in PharmacologyReceived:09 December 2020A
10、ccepted:11 February 2021Published:18 March 2021Citation:Teng S,Hao J,Bi H,Li C,Zhang Y,Zhang Y,Han W and Wang D(2021)The Protection of Crocin AgainstUlcerative Colitis and ColorectalCancer via Suppression of NF-B-Mediated Inflammation.Front.Pharmacol.12:639458.doi:10.3389/fphar.2021.639458Frontiers
11、in Pharmacology|www.frontiersin.orgMarch 2021|Volume 12|Article 6394581ORIGINAL RESEARCHpublished:18 March 2021doi:10.3389/fphar.2021.639458INTRODUCTIONUlcerative colitis(UC)is a chronic and relapsing inflammatoryintestinal disease,and typical UC symptoms are bloody diarrhea,mucus discharge,acute pa
12、in and weight loss(Baumgart andSandborn,2007;Wu and Chen,2019).Notably,the etiology ofUC remains unclear.UC is known to increase the risk ofcolorectalcancer(CRC)(Zhaoetal.,2018),aspro-inflammatory cytokines in UC can promote tumorigenesis bytriggering the formation and metastasis of tumor blood vess
13、els.In China,the incidence of UC is increasing every year,statistical data show that the prevalence rates of Heilongjiang,Xian,Guangzhou,Zhongshan,Hong Kong and Taiwan are 1.77/100,000,0.42/100,000,3.44/100,000,3.14/100,000,3.06/100,000and 4.59/100,000(Lu and Zhao,2020).With most UC patientshaving r
14、epeated attacks(Pu et al.,2019).Genetic factors,immunologicelementsandenvironmentalfactorsareconsideredtocontributetothedevelopmentofUC(Yamamoto-Furushoetal.,2019).Inaddition,lowconcentrations of peroxisome proliferator-activated receptor(PPAR-)have been noted in the colon cells of UC patients(Scaio
15、li et al.,2017),and PPAR-suppresses colitis by negativelyregulating nuclear factor kappa-B(NF-B)(Dubuquoy et al.,2003),this finding suggests that there is an important causalrelationship between UC and NF-B-mediated inflammatorypathways.Another link between these conditions is evidencedby the fact t
16、hat colonic epithelial cells may become cancerous andeventually lead to CRC,which is a common complication of UC.The activation of NF-B is responsible for the expression ofinterleukin(IL)-6(Qiu et al.,2015),which has been recognized asthe key promoter in the tumorigenesis of early colitis-associated
17、cancer(Grivennikov et al.,2009).UC patients have a 2.4-foldincreasedriskofCRC(Yangetal.,2016),andUC-associatedCRCconstitutes 1%of all CRC cases(Bopanna et al.,2017).At present,surgery,radiotherapy and chemotherapy are thecommon therapeutic strategies for CRC(Lin et al.,2018).Inaddition,aminosalicyli
18、c acid preparations,glucocorticoids andimmunosuppressive agents are the three major types of drugsused clinically in the treatment of UC;however,these drugs haveside effects,some of which are serious,which greatly limits theirapplication(Wu and Chen,2019).Similarly,5-fluorouracil iscommonly used for
19、 treating CRC,but resistance to this drug canoccur during chemotherapy(Wang et al.,2017b).Therefore,theaim of this study was to explore an agent that may have thepotential to treat UC and CRC and to prevent the development ofCRC from UC.AmongthetherapeuticagentsapprovedbytheFoodandDrugAdministration
20、(FDA),40%are natural ingredients or theirderivatives(Al-Hrout et al.,2018).Due to the low toxicity andhigh efficacy of natural products,natural products have beenextensively studied and used totreat various diseases(Amin etal.,2012;Hamza et al.,2016;Alaa et al.,2017;Al-Dabbagh et al.,2018;Hamzaetal.
21、,2018;Al-Dabbaghetal.,2019;El-Dakhlyetal.,2020).Crocin(C44H64O24),themainbiologicallyactivecompoundofsaffron,has been reported to show various pharmacologicalactivities(Ashktorab et al.,2019),including anti-inflammatoryand anti-cancer activities(Khorasany and Hosseinzadeh,2016).Saffron threads are t
22、he dried stigmas of Crocus sativus L(Iridaceae)and constitute one of the most expensive andvaluable spices in the world,leading to it being dubbed“redgold”.Saffron is cultivated in Iran;Mediterranean regions such asItaly,Spain and Greece;Northern Africa India(Kashmir)andsome other countries in Europ
23、e and Asia(Yilmaz et al.,2010).Notably,saffron was used by the ancient Egyptians to treatgastrointestinal diseases(Khorasany and Hosseinzadeh,2016).In modern-day studies,administration of crocin has shown toinhibit dextran sodium sulfate(DSS)-induced UC and suppressthe expression of various cytokine
24、s such as tumornecrosis factor-(TNF-)and NF-B(Kawabata et al.,2012).Crocin also hasanti-inflammatory effects in DSS-induced colitis mice,such thatit can be used to prevent or treat colitis(Rezaei et al.,2019),andthis activity was shown to be related to its regulation of NF-B(Khorasany and Hosseinzad
25、eh,2016).Moreover,the anti-CRCeffects of crocin may be related to its anti-proliferative and pro-apoptoticactivities(KhorasanyandHosseinzadeh,2016;Amerizadehetal.,2018).However,theunderlyingmechanisms of the protective effects of crocin against UC andCRC have not been systematically investigated.In
26、this study,the anti-UC and anti-CRC effects of crocinwere successfully confirmed by experiments in a human colonadenocarcinoma cell line,SW480,and in DSS-induced UCmice and ApcMinC/Gpt mice,where the latter were obtainedby point-mutating the adenomatous polyposis coil(APC)gene.Ourexperimentsconfirme
27、dthattheanti-inflammatory effects of crocin are related to its regulationof NF-B signaling.MATERIALS AND METHODSCell CultureSW480 cells,a human colon adenocarcinoma cell line(catalognumber:SCSP-5033;Chinese Academy of Sciences,Shanghai,China),were cultured in Dulbeccos modified Eagles medium(DMEM)co
28、ntaining10%fetalbovineserum(FBS),1%100 g/mlstreptomycinand100 units/mlpenicillin(ThermoFisherScientific,Inc.,Waltham,MA,United States)at 37 C in a CO2incubator.Cell Viability AssaySW480 cells were seeded into 96-well plates at a density of 8 103cells/well.After incubation at 37 C overnight,cells wer
29、e treatedwith 0.4,0.8,2,3 and 6 mM of crocin(catalog number:B21336;Shanghai Yuanye Biological Technology Co.,Ltd.,Shanghai,China)for 24 h at 37 C.Thereafter,5 l of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazoliumbromide(MTT)(Shanghai Yuanye Biological Technology Co.,Ltd.,Shanghai,China)was
30、added to each well,to obtain a final concentration of5 mg/ml MTT.The plates were then incubated at 37 C for afurther 4 h,at which point the supernatant was aspirated,100 lof dimethyl sulfoxide(catalog number:DH105-2;BeijingDingguo Changsheng Biotechnology Co.,Ltd.,Beijing,China)was added to each wel
31、l,and finally the absorbance of the platesFrontiers in Pharmacology|www.frontiersin.orgMarch 2021|Volume 12|Article 6394582Teng et al.Natural Compounds for Treating Inflammationwas measured at 490 nm using a Synergy4 Microplate Reader(Biotek,Winooski,Vermont,United States).Cell Cycle,Cell Apoptosis
32、andMitochondrial Membrane Potential(MMP)DetectionSW480cellswereseededintosix-wellplatesatadensityof2105cells/well,and the plates were incubated at 37 C overnight.Thereafter,cells were treated with 2.5 mM or 5 mM crocinand incubated at 37 C for a further 12 h.For cell cycle detection,collected cells
33、were washed with pre-cooled phosphate buffered saline(PBS)and then incubated with70%pre-cooled ethanol for more than 3 h.The cells were thenexposed to MuseCell Cycle Reagent(catalog number:MCH100106;EMDMilliporeCorp.,BillericaMA,United States)at room temperature for 30 min in darkness.AMuseCell Anal
34、yzer(EMD Millipore Corp.,Billerica MA,United States)was then used to detect the cell cycle conditionsof the cells.For cell apoptosis detection,collected cells were resuspendedin DMEM containing 1%FBS and 1%bovine serum albumin(BSA)at a final concentration of 1 106cells/ml.Cells wereincubated with th
35、e MuseAnnexin V and Dead Cell Reagent(catalog number:MCH100105;EMD Millipore Corp.,BillericaMA,United States)at room temperature for 20 min in darkness.A Musecell analyzer was then used to detect the apoptoticstatus of the cells.For MMP detection,collected cells were treated withmitopotential workin
36、g fluid at a final concentration of 10 M(catalog number:MAK160;Sigma-Aldrich,St.Louis,Missouri,United States)and then incubated at 37 C in darkness for 20 min.Thereafter,cells were washed three times with PBS,and then theMMP changes of SW480 cells were analyzed using a Musecellanalyzer.Animal Experi
37、mental ProtocolThe experiment was approved by the Institutional Animal EthicsCommittee of Jilin University(SY201905009 and SY201905004).C57BL/6 J mice(male,78 weeks old,2225 g)(catalog number:cs-005)purchased from Liaoning Changsheng BiotechnologyCo.,Ltd(Liaoning,China)and ApcMinC/Gpt mice(male,48 w
38、eeks old,2025 g)(catalog number:T001457)purchasedfrom GemPharmatech Co.,Ltd.(Jiangsu,China)were kept in atemperature-and humidity-controlled room at 22 1 C and5065%humidity,with no convective wind and guaranteedsunshine for 12 h each day.Mice were provided with cleanwater and food each day,and their
39、 litter was cleaned daily.Sixty C57BL/6 J mice were adaptively fed for 1 week,and thenrandomly divided into five groups(n?12 per group).All48 treatment-group mice were allowed ad libitum access to3.0%solution of DSS in water(catalog number:S14048;Shanghai Yuanye Biological Technology Co.,Ltd.,Shangh
40、ai,China)for 1 week.Subsequently,model mice(n?12)wereorally treated with normal saline,positive control mice(n?12)were orally treated with 0.6 g/kg of sulfasalazine(SASP)(catalognumber:BP779;Sigma-Aldrich,St.Louis,Missouri,United States),and crocin-treated mice were orally treatedwith 10 mg/kg(n?12)
41、and 30 mg/kg(n?12)crocin onceper day for 3 weeks.All mice continued to have ad libitum accessto 3.0%DSS every second day for a further 3 weeks.The controlmice(n?12)(entitled CTRL)were orally treated with normalsaline once per day for 3 weeks.Twenty ApcMinC/Gpt mice were properly fed for 9 weeks,andt
42、hen randomly divided into two groups(n?10 per group).Oneof these was designated the control group,and these mice weretreated with normal saline(0 mg/kg of crocin);the other wasdesigned the treatment group,and these mice were treated orallywith 30 mg/kg of crocin once per day for 8 weeks.At the concl
43、usion of the experiment,all mice were euthanizedby sodium barbiturate injection.Blood samples were collectedfrom mouse caudal veins,and organs(colon,liver,kidney,andspleen)werecollectedforsubsequentbiochemicalandpathological analysis.Disease Activity Index(DAI)Score of MiceWith UCThe body weight,sto
44、ol consistency and bleeding of mice with UCwere recorded daily,and their DAI scores were measuredaccording to the Hamamoto standard(Hamamoto et al.,1999).ProteomicsA protein sample solution was obtained from the colon of micewith UC,quantified by using a bicinchoninic acid(BCA)kit,andthen hydrolyzed
45、 with lyase.The resulting peptide samples weredetached and then analyzed by liquid chromatography-tandemmass spectrometry,and the results were processed usingMaxQuant(1.5.6.0).TheUNIPROTdatabase(Uniprot_mouse_1206_09)wasusedtoobtainproteinsequences,and the protein sequences and their respectiverever
46、se bait sequences were used in the MaxQuant search.Corresponding differentially expressed proteins were identifiedfrom statistical analysis of standardized quantitative results.Proteins with different expression folds(ratio A/B 1.5 orratio A/B 0.66)were defined as significantly different.Finally,Gen
47、e Ontology(GO)and Kyoto Encyclopedia ofGenesandGenomes(KEGG)pathwayanalyseswereperformed to determine protein interactions.Enzyme-Linked Immunosorbent Assay(ELISA)The colon tissues of mice with UC and CRC were homogenizedwith physiological saline,and the protein concentration ofhomogenates was measu
48、red using a PierceBCA ProteinAssayKit(catalognumber:23225;ThermoScientific,Shanghai,China).TheconcentrationsofIL-1(catalognumber:KT2040-A),IL-2(catalog number:KT2795-A),IL-4(catalog number:KT2165-A),IL-6(catalog number:KT2163-A),IL-15(catalog number:FY2172-A),IL-17(catalog number:KT2170-A),IL-18(cat
49、alog number:KT2169-A),induciblenitric oxide synthase(iNOS)(catalog number:KT2454-A),cyclooxygenase-2(COX-2)(catalognumber:KT2356-A),Frontiers in Pharmacology|www.frontiersin.orgMarch 2021|Volume 12|Article 6394583Teng et al.Natural Compounds for Treating InflammationTNF-(catalog number:KT2132-A)and
50、interferon-(IFN-)(catalog number:FY2366-A;Jiangsu Kete Biotechnology Co.,Ltd.,Yancheng,Jiangsu,China)in the colon and serum samplesfrom mice with UC and CRC were determined according to themanufacturers instructions.Hematoxylin and Eosin(H&E)StainingFresh samples of colon,liver,kidney,and spleen tis