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1、ORIGINAL RESEARCHPerturbed Mitochondrial Dynamics Is a Novel Feature of ColitisThat Can Be Targeted to Lessen DiseaseNicole L.Mancini,1,aLuke Goudie,2,aWarren Xu,2Rasha Sabouny,3Sruthi Rajeev,1Arthur Wang,1Nicolas Esquerre,4Ala Al Rajabi,1Timothy S.Jayme,1Erik van Tilburg Bernandes,1,5Yasmin Nasser,
2、4Jos G.P.Ferraz,4Timothy Shutt,3Jane Shearer,6and Derek M.McKay11Gastrointestinal Research Group(GIRG)and Inflammation Research Network,Department of Physiology and Pharmacology,Calvin,Joan and Phoebe Snyder Institute for Chronic Diseases,Cumming School of Medicine,University of Calgary;2Department
3、of Biomedical Engineering,Schulich School of Engineering,University of Calgary;3Department of MedicalGenetics,Alberta Childrens Hospital Research Institute,University of Calgary;4GIRG,Division of Gastroenterology,Department of Medicine,Cumming School of Medicine,University of Calgary;5Department of
4、Pediatrics,Cumming School ofMedicine,University of Calgary;and6Department of Biochemistry and Molecular Biology,Faculty of Kinesiology,University ofCalgary,Calgary,Alberta,CanadaSUMMARYMitochondrial dysfunction has been described in inflam-matory bowel disease.Systemic delivery of P110,a drugdesigne
5、d to prevent fragmentation of the mitochondrialnetwork,significantly reduced the severity of disease(eg,clinical signs,histopathology,and pain)in murine models ofcolitis.BACKGROUND&AIMS:Mitochondria exist in a constantlyremodelling network,and excessive fragmentation can bepathophysiological.Mitocho
6、ndrial dysfunction can accompanyenteric inflammation,but any contribution of altered mito-chondrial dynamics(ie,fission/fusion)to gut inflammation isunknown.We hypothesized that perturbed mitochondrial dy-namics would contribute to colitis.METHODS:Quantitativepolymerasechainreactionformarkers of mit
7、ochondrial fission and fusion was applied totissue from dextran sodium sulfate(DSS)-treated mice.An in-hibitor of mitochondrial fission,P110(prevents dynaminrelated protein Drp-1 binding to mitochondrial fission 1protein Fis1)was tested in the DSS and di-nitrobenzene sul-fonic acid(DNBS)models of mu
8、rine colitis,and the impact ofDSS P110 on intestinal epithelial and macrophage mito-chondria was assessed in vitro.RESULTS:Analysis of colonic tissue from mice with DSS-colitisrevealed increased mRNA for molecules associated with mito-chondrial fission(ie,Drp1,Fis1)and fusion(optic atrophyfactor 1)a
9、nd increased phospho-Drp1 compared with control.Systemic delivery of P110 in prophylactic or treatment regi-mens reduced the severity of DSS-or DNBS-colitis and thesubsequent hyperalgesia in DNBS-mice.Application of DSS toepithelial cells or macrophages caused mitochondrial frag-mentation.DSS-evoked
10、 perturbation of epithelial cell energeticsand mitochondrial fragmentation,but not cell death,wereameliorated by in vitro co-treatment with P110.CONCLUSIONS:We speculate that the anti-colitic effect ofsystemic delivery of the anti-fission drug,P110,works at leastpartially by maintaining enterocyte a
11、nd macrophage mito-chondrial networks.Perturbed mitochondrial dynamics can bea feature of intestinal inflammation,the suppression of which isa potential novel therapeutic direction in inflammatory boweldisease.(Cell Mol Gastroenterol Hepatol 2020;10:287307;https:/doi.org/10.1016/j.jcmgh.2020.04.004)
12、Keywords:inflammation;epithelium;macrophage;DNBS;DSS.Perturbed gut function can occur in response tomitochondrial dysfunction.1,2Analyses of colitis inmice and epithelial cell culture models of barrier functionhave provided mechanistic underpinnings for how per-turbed mitochondrial activity can cont
13、ribute to entericinflammation.38Furthermore,abnormal mitochondrial ul-trastructure,reduced levels of adenosine triphosphate(ATP)synthase,antioxidants,and Krebs cycle enzymes,aswell as increased oxidative stress have been observed incolonic biopsies from patients with inflammatory boweldisease(IBD).9
14、12Indeed,a focus on epithelial mitochon-drial dysfunction is compatible with the consensus on IBDetiology;disease occurs in genetically susceptible in-dividuals by hyperactive immune responses triggered byenvironmental stimuli involving the gut microbiota.13In thiscontext,mitochondria are important
15、in the regulation ofepithelial permeability,14and infection with bacterial path-ogens can impair mitochondrial function.15,16Ulcerativecolitis has been suggested as a disease of energy defi-ciency,17and in accordance,recent transcriptomic analysisrevealed a mitochondriopathy in ulcerative colitis.18
16、Mitochondria exist as a network that is constantlyremodelling by the processes of fusion and fission that iscritical to cellular homeostasis.19Fusion facilitates subcel-lular targeting of ATP and reactive oxygen species(ROS)and equalizes the distribution of proteins,metabolites,andmitochondrial DNA
17、throughout the network.20Fission al-lows for mitochondrial partitioning during cell division andthe removal of damaged segments of mitochondria bymitophagy;excessive fragmentation of the network can be astimulus for apoptosis.21The guanosine triphosphatase(GTPase)dynamin family members,dynamin-relat
18、ed pro-tein 1(Drp1)and optic atrophy factor 1(OPA1),are majorplayers in mitochondrial fission and fusion,respectively.19Imbalanced mitochondrial dynamics play a role in manypathologic conditions including neurodegenerative disease,heart disease,metabolic syndrome,and cancer.22,23All oftheaforementio
19、nedconditionshaveaninflammatorycomponent,and yet mitochondrial dynamics are unstudiedinthecontextofentericinflammation.Furthermore,blocking excessive fission has proven beneficial in animalmodels of myocardial infarction,pulmonary arterial hyper-tension,ischemic-reperfusion injury,multiple sclerosis
20、,andHuntingtons disease.2325Consequently,we hypothesized that excessive mitochon-drial fission would contribute to the pathogenesis of colitis,potentially by resulting in reduced barrier function andreduced viability of the epithelium.After uncovering evi-dence for altered mitochondrial dynamics in
21、tissues frommice with colitis,systemic administration of a selective in-hibitor of fission,P110(inhibits Drp1 binding to mitochon-drial fission 1 protein Fis1)25reduced disease severity indextransodiumsulfate(DSS)-ordinitrobenzenesulfonicacid(DNBS)-treated mice.Furthermore,DSS induced fragmenta-tion
22、 of macrophage and gut epithelial mitochondria in vitro,andtheaccompanyingreductioninbioenergeticsinthelatterwas partially abrogated by co-treatment with P110.Thesedata suggest that perturbed mitochondrial dynamics inenterocytes and macrophages could be a component ofenteric inflammatory disease,and
23、 that reducing mitochon-drial dysfunction due to excessive fragmentation could pro-mote intestinal homeostasis in individuals who showevidence of enhanced mitochondrial fission.ResultsQuantitative Polymerase Chain ReactionIndicates Altered Expression of MitochondrialDynamics Molecules in ColitisQuan
24、titative polymerase chain reaction(qPCR)on mu-rine colonic tissue revealed increased expression of mRNAfor Drp1,Fis1,OPA1,mitofusin(MFN)1,and MFN2 by 5days after exposure to DSS(Figure 1AE).Immunoblottingrevealed an increase in phospho-Ser616 Drp1(a stimulusfor Drp1 to translocate to mitochondria)in
25、 tissue extractsfrom mice treated with DSS for 5 days(Figure 1F and G).Systemic Administration of P110 ReducesDisease Severity in Murine Models of ColitisP110 is a selective inhibitor of Drp1-Fis1 driven mito-chondrial fission.24P110(1 mmol/L)activity was confirmedin vitro by its ability to reduce D
26、rp1 recruitment to mito-chondriaandROSgenerationbycarbonylcyanideaAuthors share co-first authorship.Abbreviations used in this paper:ATP,adenosine triphosphate;DNBS,di-nitrobenzene sulfonic acid;Drp1,dynamin related protein-1;DSS,dextran sodium sulfate;Fis1,mitochondrial fission protein-1;GM-CSF,gra
27、nulocyte-macrophage colony-stimulating factor;GTPase,guano-sine triphosphatase;IBD,inflammatory bowel disease;IEC,intestinalepithelial cell;IL,interleukin;LPS,lipopolysaccharide;MFN,mitofusin;OPA1,optic atrophy factor 1;qPCR,quantitative polymerase chainreaction;ROS,reactive oxygen species;TER,trans
28、epithelial electricalresistance;TNF,tumornecrosisfactor;TUNEL,deoxyuride-50-triphosphate biotin nick end labeling.Most current article2020 The Authors.Published by Elsevier Inc.on behalf of the AGAInstitute.This is an open access article under the CC BY-NC-NDlicense(http:/creativecommons.org/license
29、s/by-nc-nd/4.0/).2352-345Xhttps:/doi.org/10.1016/j.jcmgh.2020.04.004288Mancini et alCellular and Molecular Gastroenterology and Hepatology Vol.10,No.2ABCGFED2020Colitis and Mitochondrial Dynamics289ABDCEFigure 2.Systemic administration of P110 reduces severity of DSS-induced colitis.(A)Male BALB/c m
30、ice were given 5%DSS drinking water for 5 days,followed by 3-day water recovery phase daily P110(intraperitonal;3 mg/kg)as shown.Onnecropsy,(B)macroscopic disease activity scores were calculated,(C)colon length was measured,and(D)just beforenecropsy,colonic motility was assessed in anesthetized anim
31、als(s,seconds).(E)Histopathology scoring was performed onH&E stained sections of mid-colon in a blinded fashion(mean standard error of the mean;n 821 mice from 2 to 5experiments;*P .05 compared with control,#P .05 compared with DSS;(B and E)Kruksal-Wallis and then Dunn multiplecomparison test;(C and
32、 D)one-way analysis of variance and then Tukey multiple comparison test.Figure 1.(See previous page).Colitis evokes increased expression of mRNA of molecules that regulate mitochondrialdynamics.(AE)qPCR performed on segments of colonic tissue from DSS(5%)treated male BALB/c mice revealed increasedex
33、pression of Drp1,mitochondrial Fis1,OPA1,and MFN1 and MFN2 mRNA(data normalized to18S rRNA as a housekeeping geneHSG).Expression levels in each tissue extract were compared with the mean of the control group that was set at 1(*P .05compared with control;one-way analysis of variance,Dunnett multiple
34、comparison test).(F)Densitometric analysis for phos-phorylated Drp1(p-Drp1 Ser616):total Drp1 ration normalized to actin in murine colonic extracts(day 5,5%DSS),with(G)illus-trating a representative immunoblot(mean standard error of the mean;*P .05,t test)(each dot represents an individual mouse).29
35、0Mancini et alCellular and Molecular Gastroenterology and Hepatology Vol.10,No.2m-chlorophenyl hydrazine(10 mmol/L)treatment of T84epithelial cells(data not shown;n 4).Mice treated withthe TAT protein only(data not shown)or P110 only(Figure 2)showed no signs of ill health,whereas DSS-treated mice ha
36、d shortened colons,increased macroscopicdisease scores,increased colonic transit time,and histopa-thology(ie,loss of architecture,goblet cell depletion,in-flammatory cell infiltration,and ulceration)(Figure 2).Macroscopic disease scores were reduced by w50%in theDSS P110 group compared with DSS(Figu
37、re 2B),andmice receiving P110 had a longer colon and colonic transittime not different from control mice(Figure 2C and D).Incontrast,colonichistopathologywasnotappreciablydifferent between DSS-and DSS P110treated mice(Figure 2E).Bacterial dysbiosis occurred in DSS-treatedmice,and this was unaffected
38、 by co-treatment with P110(Table 1,Figure 3).Treatment of BALB/c mice with DNBS resulted in severecolitis,with w21%of mice(5/24)reaching a pre-determinedillhealthendpointrequiringeuthanasia(Figure 4A and B).Systemic delivery of P110 in a prophy-lactic regimen resulted in less weight loss(Figure 4C),
39、withonly 1 mouse of 27(1%)requiring euthanasia(Figure 4B).Similarly,the P110-treated mice had longer colons andlower disease activity scores compared with DNBS-onlytreated mice(Figure 4D and E).Mice treated with DNBS P110 had significantly less colonic histopathology,display-ing improved architectur
40、e and crypt morphology andreduced inflammatory cell infiltrate(Figure 4F and G).Positive deoxyuride-50-triphosphate biotin nick end labeling(TUNEL)staining mirrored the damage observed on H&Esections,with numerous dead cells(and debris)throughoutsections from DNBS-treated mice and less so in the DNB
41、S P110 group(Figure 4G).Assessment of epithelial prolifer-ation revealed Ki67cells toward the base of crypts incontrol tissue,and this extended further up the crypt insections from DNBS P110treated mice,likely reflectingongoing tissue regeneration/recovery after the pro-coliticstimulus(Figure 4G).Co
42、lon from DNBS-treated mice hadincreased granulocyte-macrophage colony-stimulating fac-tor(GM-CSF),interleukin(IL)6,and tumor necrosis factor(TNF)a protein,the levels of which were lower in colonfrom DNBS P110treated mice(Figure 5A).In addition,P110 treatment improved gut barrier function as assessed
43、Table 1.Analysis of Fecal/Cecal Bacterial Composition in Dextran Sodium SulfateTreated Mice Systematic Treatment Withthe P110 Inhibitor of Mitochondrial FissionTreatmentControlP110DSSDSS P110Bacteroides6.54 0.086.64 0.076.77 0.08a6.80 0.07aClostridium coccoides(cluster XIV)6.50 0.096.50 0.066.22 0.0
44、9a6.25 0.08aClostridium leptum(cluster IV)5.71 0.065.78 0.805.22 0.10a5.23 0.06aClostridium cluster XI1.68 0.061.33 0.081.53 0.081.50 0.16Clostridium cluster I1.50 0.111.51 0.101.08 0.15a0.97 0.10aRoseburia hominis2.75 0.192.81 0.311.38 0.08a1.35 0.04aFaecalibacterium prausnitzii3.68 0.193.87 0.302.
45、30 0.08a2.29 0.04aLactobacillus spp.1.76 0.081.78 0.121.51 0.09a1.56 0.09aBifidobacterium spp.3.63 0.083.79 0.043.54 0.09a3.49 0.08aMethanobrevibacter spp.2.02 0.111.95 0.051.94 0.081.88 0.11Enterobacteriacea2.15 0.112.17 0.101.74 0.15a1.63 0.10aAkkermansia muciniphila1.72 0.182.02 0.101.57 0.251.41
46、 0.19NOTE.Data are mean 16s RNA gene/mg fecal-cecal material standard error of the mean;n 4 samples/group.aP .05 compared with controls.Figure 3.P110 treatment does not correct DSS-induceddysbiosis.To discern changes to the bacterial populationcaused by DSS P110(Figure 2),total DNA was extractedfrom
47、 fecal/cecal samples and analyzed with qPCR for 16SrRNA copy number.Data were assessed by Metaboanalyst4.0,and principal component analysis(PCA)was plotted.Each dot represents data from an individual animal,and areaoutlined in corresponding color depicts the 95%confidenceregion.P110 only did not alt
48、er bacterial composition ascompared with control and did not affect dysbiosis caused byDSS.2020Colitis and Mitochondrial Dynamics291ABCDEFFigure 4.Prophylactic administration of P110 reduces severity of DNBS-induced colitis.Male BALB/c mice were treatedwith DNBS(3 mg,intrarectal)P110(intraperitoneal
49、,3 mg/kg)as shown in(A).(B)and(C)Numbers of mice requiring humaneeuthanasia because of disease severity and daily weight change,respectively.On necropsy,colon length was measured(D),macroscopic disease score was calculated(E),and a piece of mid-colon was processed for H&E staining and assessment ofh
50、istopathology in a blinded fashion(F)(mean standard error of the mean;n 1127 mice from 3 to 6 experiments).(G)Representative histologic images of the colon stained with H&E,an antibody against the proliferation-associated antigen,Ki67,and the TUNEL assay for apoptosis(n 911 mice,2 experiments;m,musc