Flavaglines Crohn Colitis Prohibitins

ORIGINAL ARTICLE
Flavaglines Ameliorate Experimental Colitis and Protect Against

Intestinal Epithelial Cell Apoptosis and Mitochondrial Dysfunction
Jie Han, MS,* Qian Zhao, MS, Christine Basmadjian, PhD,† Laurent Désaubry, PhD,†
and Arianne L. Theiss, PhD*
Downloaded from https://academic.oup.com/ibdjournal/article/22/1/55/4561782 by guest on 24 December 2023

Background: Flavaglines are a family of natural compounds shown to have anti-inflammatory and cytoprotective effects in neurons and
cardiomyocytes. Flavaglines target prohibitins as ligands, which are scaffold proteins that regulate mitochondrial function, cell survival, and
transcription. This study tested the therapeutic potential of flavaglines to promote intestinal epithelial cell homeostasis and to protect against a model of
experimental colitis in which inflammation is driven by epithelial ulceration.
Methods: Survival and homeostasis of Caco2-BBE and IEC-6 intestinal epithelial cell lines were measured during treatment with the flavaglines FL3 or FL37
alone and in combination with the proinflammatory cytokines tumor necrosis factor (TNF) a and interferon g. Wild-type mice were intraperitoneally injected
with 0.1 mg/kg FL3 or vehicle once daily for 4 days during dextran sodium sulfate–induced colitis to test the in vivo anti-inflammatory effect of FL3.
Results: FL3 and FL37 increased basal Caco2-BBE and IEC-6 cell viability, decreased apoptosis, and decreased epithelial monolayer permeability. FL3
and FL37 inhibited TNFa- and interferon g–induced nuclear factor kappa B and Cox2 expression, apoptosis, and increased permeability in Caco2-BBE
cells. FL3 and FL37 protected against TNFa-induced mitochondrial superoxide generation by preserving respiratory chain complex I activity and
prohibitin expression. p38-MAPK activation was essential for the protective effect of FL3 and FL37 on barrier permeability and mitochondrial-derived
reactive oxygen species production during TNFa treatment. Mice administered FL3 during dextran sodium sulfate colitis exhibited increased colonic
prohibitin expression and p38-MAPK activation, preserved barrier function, and less inflammation.
Conclusions: These results suggest that flavaglines exhibit therapeutic potential against colitis and preserve intestinal epithelial cell survival,
mitochondrial function, and barrier integrity.
(Inflamm Bowel Dis 2016;22:55–67)
Key Words: barrier function, prohibitin, intestinal epithelium, TNFa, colitis
immune system, thereby aggravating the inflammatory condition.1

I nflammatory bowel diseases (IBD), the most common forms
being Crohn’s disease and ulcerative colitis, are associated with
disturbed intestinal epithelial cell (IEC) homeostasis. IECs structur-
Disruption of the epithelial barrier can be manifested by increased
epithelial cell apoptosis not equal to epithelial proliferation (ulcera-
ally provide host defense by forming a single-cell barrier between tion) and/or by alteration of permeability at the tight junctions that
luminal contents and the underlying intestinal tissue. Epithelial bar- establish a semipermeable barrier between epithelial cells, restricting
rier dysfunction is an early event in the pathogenesis of IBD, result- passage of large molecules.2 Restoring and maintaining the epithe-
ing in increased exposure of intraluminal contents to the mucosal lial barrier are critical to limit mucosal inflammation and promote
healing.1 Identification of agents that act to promote epithelial cell
Supplemental digital content is available for this article. Direct URL citations
homeostasis and barrier integrity in the context of tissue injury or
appear in the printed text and are provided in the HTML and PDF versions of this IBD is critical for the development of therapeutic or preventative
article on the journal’s Web site (www.ibdjournal.org). strategies to promote or maintain mucosal healing.
Received for publication July 29, 2015; Accepted August 4, 2015.
Flavaglines comprise a family of compounds found in
From the *Department of Internal Medicine, Division of Gastroenterology, Baylor
Research Institute, Baylor University Medical Center, Dallas, Texas; and †Therapeutic medicinal plants of Southeast Asia that have shown anticancer,
Innovation Laboratory, CNRS/Université de Strasbourg, Illkirch, France. anti-inflammatory, and cytoprotective activities.3 Flavaglines have
Supported by National Institutes of Health grants R03-DK098229 (A. L. been reported to reduce neurotoxicity in a mouse model of Parkin-
Theiss) and funds from the Baylor Research Institute. L. Désaubry was supported
son’s disease4 and protect cardiomyocytes from doxorubicin-
by the “Association pour la Recherche sur le Cancer.” C. Basmadjian and Q. Zhao
received fellowships from AAREC Filia Research and ANRT. induced cardiac toxicity.5 In addition, at nanomolar concentrations,
The authors have no conflicts of interest to disclose. flavaglines inhibit the production of interferon (IFN) g, TNFa,
Reprints: Arianne L. Theiss, PhD, Department of Internal Medicine, Division interleukin 2, and interleukin 4 by T lymphocytes and proinflam-
of Gastroenterology, 1053 Wadley Tower, 3600 Gaston Avenue, Baylor Research matory mediators from microglia and endothelial cells.4,6 It is well
Institute, Baylor University Medical Center, Dallas, TX 75246 (e-mail: arianne.
theiss@baylorhealth.edu). established that flavaglines target prohibitins (PHBs) as their
Copyright © 2015 Crohn’s & Colitis Foundation of America, Inc. molecular ligands.3 PHB (B-cell receptor–associated protein 32)
DOI 10.1097/MIB.0000000000000592 and PHB2 (repressor of estrogen receptor activity, B-cell
Published online 18 September 2015. receptor–associated protein 37) are highly conserved proteins
Inflamm Bowel Dis Volume 22, Number 1, January 2016 www.ibdjournal.org | 55
Copyright © 2015 Crohn’s & Colitis Foundation of America, Inc. Unauthorized reproduction of this article is prohibited.
Han et al Inflamm Bowel Dis Volume 22, Number 1, January 2016
with diverse functions, including regulation of cell cycle progres- (50 -CAGAAUGUCAACAUCACACUGCGCA-30 ) or Stealth
sion, apoptosis, and transcription, depending on their posttransla- RNAi Negative Control Med GC (Life Technologies, Carlsbad,
tional modifications and subcellular localization.7 The best CA) at 20 mm concentration using Amaxa electroporation with
characterized function of PHB and PHB2 is their role in main- Nucleofector kit T (Lonza, Basel, Switzerland).
taining the structure and function of mitochondria, including res-
piration and protein metabolism, while residing in the inner Induction of Colitis in Mice
mitochondrial membrane as heterodimers.8–10 In IECs, PHB is Eight-week-old wild-type (C57BL/6) male and female mice
predominantly localized in the mitochondria, where it has been were administered orally DSS (molecular weight, 50,000; MP
shown to be required for optimal activity of complexes I and IV of Biomedicals, Solon, OH) at 2.5% (wt/vol) in tap water ad libitum
the electron transport chain (ETC).11–14 During IBD, expression of for 6 days. Controls were administered normal tap water throughout
PHB is decreased in uninvolved and inflamed epithelium.12,14 the treatment period. Mice were intraperitoneally injected with

Epithelial PHB is protective against intestinal inflammation as 0.1 mg/kg FL3 or vehicle (veh) once daily on days 0 to 4. We
evidenced by less severe experimental colitis in transgenic mice chose this dose because it proved to be adequate in our previous
with IEC-specific PHB overexpression.15,16 Gene silencing of assays of cardioprotection in mice.5 Mean DSS water consumption,
PHB in cultured IECs induces mitochondrial membrane depolar- body weight, and clinical signs of inflammation were assessed daily
ization and cellular stress pathways, including intracellular reac- during the treatment period. All mice were group housed in stan-
tive oxygen species (ROS) generation and apoptosis.17 dard cages under a controlled temperature (258C) and photoperiod
Furthermore, cultured IECs overexpressing PHB exhibit less (12-hour light/dark cycle) and were allowed standard chow and tap
intracellular ROS and apoptosis,17 suggesting that relative levels water ad libitum. All experiments were approved by the Baylor
of PHB modulate epithelial cell homeostasis. Research Institute Institutional Animal Care and Use Committee.
In this study, we investigated the anti-inflammatory activity
of the flavaglines FL3 and FL37, which are among the most Sodium Dodecyl Sulfate Polyacrylamide Gel
potent of flavaglines tested in a wide variety of pharmacological Electrophoresis and Western
assays,18 in cultured IECs and the dextran sodium sulfate (DSS) Immunoblot Analysis
model of colitis. We also elucidated the mechanism of FL3 and Total protein was isolated from cultured cells or distal colon
FL37 protection in IECs. mucosa from wild-type mice. The samples were separated by
sodium dodecyl sulfate polyacrylamide gel electrophoresis using
Laemmli’s 2· sodium dodecyl sulfate sample buffer and AnyKD
MATERIALS AND METHODS gradient polyacrylamide gels (Bio-Rad, Hercules, CA) followed
by electrotransfer to nitrocellulose membranes (Bio-Rad). Mem-
Cell Culture branes were incubated with primary antibodies at 48C overnight
Flavaglines FL3 and FL37 were synthesized in our labora- and subsequently incubated with corresponding peroxidase-
tory as previously described.19,20 The Caco2-BBE human colonic conjugated secondary antibodies. Membranes were washed and
adenocarcinoma epithelial cell line and the nontransformed IEC-6 immunoreactive proteins were detected using Amersham ECL
rat small IEC line were used as in vitro models of polarized intes- Plus reagent (GE Healthcare, Piscataway, NJ). PHB antibody
tinal epithelium. Both cell lines were obtained from the American was purchased from Thermo Fisher (Waltham, MA); PHB2,
Type Culture Collection (ATCC, Manassas, VA). Cells were Stat3, and ERK1/2 from Santa Cruz Biotechnology (Santa Cruz,
grown in Dulbecco’s modified Eagle’s medium (Caco2-BBE) sup- CA); phospho-p38-MAPK, p38-MAPK, pERK1/2, pS727-Stat3,
plemented with penicillin (40 mg/L), streptomycin (90 mg/L), and pY705-Stat3, pSAPK/JNK, SAPK/JNK, pAKT, AKT, and
10% fetal bovine serum. Caco2-BBE cells and IEC-6 cells were cleaved caspase 3 from Cell Signaling Technology (Danvers,
plated on permeable supports (pore size, 0.4 mm; transwell-clear MA); proliferating cell nuclear antigen (PCNA) from Abcam
polyester membranes; Corning, Tewksbury, MA) and cultured for 8 (Cambridge, MA); p65 from BD Biosciences (San Jose, CA);
days to allow the cells to polarize. All experiments performed on and Cox2 from Cayman Chemicals (Ann Arbor, MI). Blots were
Caco2-BBE cells were between passages 32 and 45 and IEC-6 cells reprobed with b-tubulin or b-actin (Sigma-Aldrich Corp.,
were between passages 10 and 18. FL3 and FL37 were adminis- St. Louis, MO) antibody as a loading control.
tered at 1, 10, and 50 nM because previous studies showed that
flavaglines exhibit cardioprotective and neuroprotective effects at 1 Measuring Mitochondrial ROS
to 10 nM.18 Caco2-BBE or IEC-6 cells were treated with 10 ng/mL Cells were incubated with Hank’s balanced salt solution
recombinant human or rat TNFa or 50 ng/mL recombinant human with 5 mM MitoSOX Red Mitochondrial Superioxide Indicator
or rat IFNg, respectively (R&D Systems, Minneapolis, MN). dye (Life Technologies) for 10 minutes at 378C. Cells were
washed twice with warm Hank’s balanced salt solution, and fluo-
PHB Knockdown rescent intensity was measured at 510 nm excitation/580 nm
To achieve PHB knockdown, Caco2-BBE cells were emission. For experiments using complex I or III inhibitors, 15 mi-
transiently transfected with Stealth RNAi against PHB1 nutes before collection, cells were treated with 5 mM rotenone
56 | www.ibdjournal.org
Inflamm Bowel Dis Volume 22, Number 1, January 2016 Flavaglines Ameliorate Experimental Colitis
(Sigma-Aldrich), a complex I inhibitor, or 1 mM antimycin A Clinical Score Assessment

(Sigma-Aldrich), a complex III inhibitor. For experiments using A clinical activity score was generated using body weight
p38-MAPK inhibitor, cells were incubated with 20 mM p38- loss, stool consistency, and the presence of occult blood by
MAPK inhibitor SB203580 (Sigma-Aldrich) for 1 hour before a guaiac test (Hemoccult Sense; Beckman Coulter, Fullerton, CA)
treatment with FL3, FL37, and TNFa. as described previously.16 The scores for each parameter were
added to get a clinical activity score with 12 being the maximal
Measuring ATP Concentration score.
The concentration of ATP was determined using the
Enlighten ATP Assay Bioluminescence Detection kit (Promega, Histological Damage Score
Madison, WI) according to the manufacturer’s protocol. Distal colon was fixed in formalin and stained with
hemotoxylin and eosin for histology. Sections were coded for

Detection of Mitochondrial Complex I Activity blind microscopic assessment of inflammation. Histological
The activity of complex I was measured using the Complex scoring was performed on the basis of 3 parameters: the severity
I Dipstick Assay kit (Abcam) according to the manufacturer’s of inflammation, crypt damage, and ulceration as described
protocol using 20 mg of protein. previously.16 These values were added to give a maximal histo-
logical score of 11.
Cytotoxicity Test
Lactate dehydrogenase cytotoxicity detection kit (Clontech, Myeloperoxidase Activity
Mountain View, CA) was used to measure cell viability. An Neutrophil infiltration into the distal colon was quantified
aliquot of 100 mL of culture media was added to 100 mL of lactate by measuring myeloperoxidase activity. Briefly, a portion of
dehydrogenase reagent, and percent cytotoxicity and percent via- colon or cecum was homogenized in 1:20 (wt/vol) 50 mmol/L
ble cells were measured according to the manufacturer’s protocol. phosphate buffer (pH 6.0) containing 0.5% hexadecyltrimethyl
ammonium bromide on ice by using a Polytron homogenizer. The
Measuring Cell Apoptosis homogenate was sonicated for 10 seconds, freeze thawed 3 times,
Percentage of apoptotic cells was measured using the Cell and centrifuged at 14,000 rpm for 15 minutes. Supernatant was
Death Detection ELISA Plus kit (Roche, Indianapolis, IN) as added to 1 mg/mL of o-dianisidine hydrochloride and 5 · 1024%
described by the manufacturer. As a second measure of apoptosis, hydrogen peroxide, and the change in absorbance at 460 nm was
cells or colon sections were stained for terminal deoxynucleotidyl measured. One unit of myeloperoxidase activity was defined as
transferase–mediated deoxyuridine triphosphate nick-end labeling the amount that degraded 1 mmol of peroxidase per minute at
(TUNEL) as described by the manufacturer’s protocol (Roche). 258C.
The nuclei of cells were stained with 4’,6-diamidino-2-phenylindole
(Life Technologies). The number of TUNEL-positive cells were In Vivo Permeability Assay
quantitated using a fluorescent microscope across 20 fields per Barrier function was assessed using an FITC-labeled
treatment for in vitro experiments or across 20 well-oriented crypts dextran method. Briefly, on day 6 of DSS administration, mice
per animal for in vivo experiments. were gavaged with permeability tracer (60 mg/100 g body weight
Colonic epithelial cells were isolated from mice as pre- of FITC-labeled dextran, FD-4, Mr 4000; Sigma-Aldrich). Serum
viously described.21 Total protein was extracted and analyzed by was collected retro-orbitally 4 hours after FD-4 gavage, and fluo-
Western blotting for cleaved caspase 3. rescence intensity of each sample was measured (excitation, 492
nm; emission, 525 nm). FITC-dextran concentrations were deter-
Measurement of Transepithelial Electrical mined from standard curves generated by serial dilution of FITC-
Resistance and Macromolecular Permeability dextran and normalized to total protein.
In Vitro
Transepithelial electrical resistance was measured with an Measuring In Vivo Oxidative Damage
epithelial voltohmmeter (Millicell-ers; Millipore, Billerica, MA). Five-micrometer paraffin-embedded sections of colon were
For permeability assays, cells were incubated in Hank’s balanced analyzed for 4-hydroxynoneal (4-HNE) staining as a marker of
salt solution. Fluorescein isothiocyanate (FITC)-dextran (10 mg/ lipid peroxidation. Sections were deparaffinized in xylene, rehy-
mL) (molecular weight 4 kDa; Sigma-Aldrich) was added to the drated in ethyl alcohol gradient, incubated in 0.3% H2O2 for 30 mi-
apical chamber. The apical and basolateral chambers were sam- nutes, and treated with 10 mM sodium citrate buffer (pH 6.0) at
pled at 30 minutes, 1 hour, and 2 hours after the addition of 1108C for 20 minutes in a pressure cooker. Sections were blocked
FITC-dextran to the apical chamber. FITC-dextran concentration with 5% normal goat serum and incubated with 4-HNE antibody
was quantified through spectrofluorimetry (excitation, 492 nm, (Abcam) overnight at 48C. After washing with phosphate-buffered
emission, 510 nm). Values are shown as rate (nanograms per saline, sections were incubated with biotinylated secondary anti-
milliliter per minute) of FITC-dextran translocation to the baso- bodies for 30 minutes at room temperature, and color development
lateral reservoir. was performed using the Vectastain ABC kit (Vector Laboratories)
www.ibdjournal.org | 57
and 3,3’-Diaminobenzidine (Dako, Carpinteria, CA). Sections were epithelial proliferation. To determine whether very low (nanomolar)
counterstained with hematoxylin. doses of FL3 or FL37 affect IEC viability, polarized Caco2-BBE or
The oxidative damage to proteins was assessed using IEC-6 cell monolayers were treated with increasing concentrations
a protein carbonyl assay kit (Cayman Chemicals) according to of FL3 or FL37 for 16 hours and markers of cell proliferation or
the manufacturer’s protocol. apoptosis were measured. Ten and 50 nanomolar of FL3 or FL37
enhanced cell viability in Caco2-BBE cells as measured by lactate
Statistical Analyses dehydrogenase release (Fig. 1A). IEC-6 nontransformed cells were
Values are expressed as mean 6 SEM. Comparisons used as a second in vitro model of IECs. FL3 and FL37 increased
between FL3 or FL37 treatment versus vehicle control were ana- viability of IEC-6 cells at the higher doses tested (see Fig. A,
lyzed by unpaired Student’s t test. Comparisons between FL3 or Supplemental Digital Content 1, http://links.lww.com/IBD/B116).
FL37 combined with TNFa or IFNg treatment were analyzed by To determine whether increased cell viability was associated with

2-way analysis of variance, and subsequent pairwise comparisons changes in cell proliferation, PCNA protein expression was mea-
used Bonferroni post hoc tests to test for significant differences sured. FL3 and FL37 did not affect PCNA protein expression in
between 2 particular groups. P value ,0.05 was considered sta- Caco2-BBE cells, suggesting that FL3 and FL37 do not increase
tistically significant in all analyses. cell proliferation (Fig. 1B). FL3 and FL37 decreased Caco2-BBE
cell apoptosis measured by enzyme-linked immunosorbent assay
(Fig. 1C) and TUNEL staining (Fig. 1D). The number of apoptotic
RESULTS cells was decreased to approximately 5% during treatment with
FL3 or FL37 compared with 10% in vehicle-treated cells (Fig. 1D).
FL3 and FL37 Decrease IEC Apoptosis
An intact intestinal epithelial barrier that prevents the FL3 and FL37 Increase Protein Expression of
translocation of intraluminal contents and subsequent immune cell Known Flavagline Targets PHB and PHB2
activation is an initial event in suppressing inflammation deeper in in IECs
the bowel wall.1 Disruption of the epithelial barrier can be man- To determine whether FL3 or FL37 alters IEC expression
ifested by increased epithelial cell apoptosis not balanced with of PHB or PHB2, which are established flavagline targets,3
FIGURE 1. FL3 and FL37 decrease apoptosis in Caco2-BBE cells. Cells were treated with increasing concentrations of FL3 or FL37 for 16 hours A,
Cell viability using lactate dehydrogenase assay; *P , 0.05 versus vehicle; n ¼ 8 per treatment across 2 separate experiments. B, Representative
Western blots of PCNA protein expression, a marker of cell proliferation. C, Apoptosis measured by enzyme-linked immunosorbent assay; *P ,
0.05 versus vehicle; n ¼ 4 per treatment. D, TUNEL-positive cells were quantified across 20 fields; *P , 0.05 versus vehicle.
Caco2-BBE cells were treated with increasing doses of FL3 or

FL37. Treatment with FL3 or FL37 at very low (nanomolar)
concentrations increased PHB and PHB2 protein levels in Ca-
co2-BBE (Fig. 2A). FL3 and FL37 increased PHB protein expres-
sion rapidly, as early as 15 minutes, with peak induction for FL3
between 1 and 2 hours and for FL37 between 15 minutes and 1
hour (Fig. 2B). The induction of PHB2 by FL3 and FL37 showed
an identical pattern as PHB. FL3- and FL37-induced PHB and
PHB2 protein expression was associated with p38-MAPK activa-
tion (Fig. 2B), but not activation of Stat3, SAPK/JNK, ERK, or
AKT (see Fig., Supplemental Digital Content 2, http://links.lww.

com/IBD/B117). FL3 and FL37 induced PHB and PHB2 protein
expression and activation of p38-MAPK in IEC-6 cells in a similar
pattern as induction in Caco2-BBE cells (see Fig. B, Supplemen-
tal Digital Content 1, http://links.lww.com/IBD/B116).
Pretreatment with FL3 or FL37 Decreases

TNFa- or IFNg-induced Expression of Nuclear
Factor Kappa B p65 and Cox2 in IECs
To determine whether flavaglines exhibit anti-inflammatory
action in IECs, Caco2-BBE cells were pretreated with increasing
concentrations of FL3 or FL37 followed by treatment with tumor
necrosis factor (TNF) a or IFNg, 2 proinflammatory cytokines
upregulated during intestinal inflammation. Western blotting re-
vealed that FL3 and FL37 prevented TNFa- or IFNg-induced
nuclear factor kappa B p65 and Cox2 protein expression, which
FIGURE 3. Pretreatment with FL3 or FL37 decreases TNFa- or IFNg-induced
are 2 key proinflammatory pathways in the intestine. This effect expression of nuclear factor kappa B (NFkB) p65 and Cox2 in Caco2-BBE
of FL3 and FL37 was associated with sustained PHB expression, cells. Cells were pretreated with increasing concentrations of FL3 (A) or FL37
which is decreased by TNFa or IFNg (Fig. 3A, B). Similar results (B) for 1 hour, followed by treatment with 10 ng/mL TNFa or 50 ng/mL
were evident in IEC-6 cells, in which 1 and 10 nM FL3 or FL37 IFNg for 16 hours. Total protein was isolated for Western blotting for
pretreatment prevented TNFa-induced nuclear factor kappa B p65 expression of NFkB p65, Cox2, PHB, and b-actin (loading control).
FIGURE 2. FL3 and FL37 increase protein expression of known flavagline targets PHB and PHB2 in IECs. A, Polarized Caco2-BBE cells were treated
with increasing concentrations of FL3 or FL37 for 2 hours. Representative Western blots are shown for PHB, PHB2, and b-actin (loading control). B,
Caco2-BBE cells were treated with 10 nM FL3 or FL37 for increasing time. Representative Western blots are shown for PHB, PHB2, phospho-p38-
MAPK, total p38-MAPK, and b-tubulin (loading control).
and Cox2 induction (see Fig. A, Supplemental Digital Content 3, associated with increased cell proliferation. TUNEL staining re-
http://links.lww.com/IBD/B118). All further experiments were vealed that pretreatment with FL3 or FL37 prevented TNFa- or
performed with FL3 or FL37 at a concentration of 10 nM because IFNg-induced Caco2-BBE apoptosis (Fig. 4C).
this was the lowest most effective dose at preventing TNFa- or
IFNg-induced loss of PHB protein expression. Pretreatment with FL3 or FL37 Protects
Against Cytokine-induced Barrier Dysfunction
Pretreatment with FL3 or FL37 Prevented IEC in Caco2-BBE Cell Monolayers
Apoptosis Induced by TNFa or IFNg Ten nanomolar of FL3 or FL37 increased transepithelial
We next determined whether FL3 or FL37 affected electrical resistance (Fig. 5A) and decreased translocation of FITC-
cytokine-induced proliferation and/or apoptosis in IECs. TNFa dextran between Caco2-BBE monolayers from the apical chamber
or IFNg significantly decreased Caco2-BBE cell viability in to the basolateral chamber (Fig. 5B). Pretreatment with 10 nM FL3

vehicle-treated cells, with TNFa reducing cell viability by 24% or FL37 prevented TNFa- or IFNg-induced reduction in transepi-
and IFNg by 11% (Fig. 4A). Pretreatment with 10 nM FL3 or thelial electrical resistance (Fig. 5A) and increase in FITC-dextran
FL37 prevented the reduction in cell viability by TNFa or IFNg across Caco2-BBE cells (Fig. 5B). These results suggest that FL3
(Fig. 4A). Similar results were evident in IEC-6 cells, in which and FL37 enhance intestinal epithelial barrier function basally and
pretreatment with 10 nM FL3 or FL37 increased cell viability protect from barrier dysfunction induced by TNFa and IFNg.
during TNFa or IFNg treatment (see Fig. B, Supplemental Digital
Content 3, http://links.lww.com/IBD/B118). Pretreatment with 10 FL3 and FL37 Preserve Mitochondrial
nM FL3 or FL37 did not affect PCNA protein expression when Function During TNFa Treatment
normalized to b-actin expression during TNFa or IFNg treatment PHB and PHB2, known targets of flavaglines, are pre-
(Fig. 4B). These results suggest that the protection of cell viability dominantly localized to the inner mitochondrial membrane of
by FL3 and FL37 during TNFa or IFNg treatment is not IECs.14 Because treatment with FL3 or FL37 decreased basal and
FIGURE 4. Pretreatment with FL3 or FL37 prevented Caco2-BBE apoptosis induced by TNFa or IFNg. Cells were pretreated with 10 nM FL3 or FL37
for 1 hour, followed by treatment with 10 ng/mL TNFa or 50 ng/mL IFNg for 16 hours. A, Cell viability using lactate dehydrogenase assay; n ¼ 8 per
treatment across 2 separate experiments. B, Representative Western blots of PCNA protein expression, a marker of cell proliferation. C, TUNEL-
positive cells were quantified across 20 fields; *P , 0.05, **P , 0.01 versus no tx + vehicle; #P , 0.01 versus TNFa + vehicle; †P , 0.05 versus IFNg
+ vehicle. No tx, no treatment.
Previous studies have shown that TNFa promotes cellular

injury predominantly through mitochondrial ROS production re-
sulting from decreased activity of ETC complex I.34 To determine
whether FL3 or FL37 suppresses ROS production induced by
complex I, mitoSOX fluorescence was measured in Caco2-BBE
cells pretreated with 10 nM FL3 or FL37 followed by TNFa
treatment and rotenone (5 mM; complex I inhibitor). The protec-
tive effect of FL3 or FL37 on TNFa-induced mitochondrial ROS
production was sustained during the addition of rotenone, sug-
gesting that complex I activity is resistant to rotenone inhibition in
FL3- or FL37-treated cells (Fig. 6C). FL3 or FL37 did not protect

against ROS production induced by the complex III inhibitor
antimycin A (1 mM) during TNFa treatment. These results sug-
gest that FL3 and FL37 preserve complex I activity, but not
complex III activity, during TNFa treatment. Complex I activity
was then measured by the Mitochondrial Dipstick Assay kit. FL3
or FL37 did not alter basal complex I activity in Caco2-BBE cells
compared with vehicle control cells (Fig. 6D). TNFa treatment
decreased complex I activity in vehicle-treated cells, whereas pre-
treatment with FL3 or FL37 prevented TNFa inhibition of com-
plex I (Fig. 6D).
FIGURE 5. Pretreatment with FL3 or FL37 protects against cytokine- Because FL3 or FL37 treatment increases expression of
induced permeability changes in IECs. Monolayers of polarized Caco2- PHB in Caco2-BBE cells (Fig. 2) and because PHB is crucial for
BBE cells were pretreated with 10 nM FL3 or FL37 for 1 hour, followed complex I assembly and function,11 we next determined whether
by treatment with 10 ng/mL TNFa or 50 ng/mL IFNg for 16 hours. A, PHB was necessary for FL3 or FL37 protection against TNFa-
Transepithelial electrical resistance. B, Macromolecular permeability as induced mitochondrial ROS production (Fig. 6E). During loss of
measured by rate of 4 kDa FITC-dextran translocation from apical to PHB expression by small interfering RNA transfection, FL3 or
basolateral chamber (nanograms per milliliter per minute); *P , 0.05, FL37 did not protect against mitochondrial ROS production
**P , 0.01 versus no tx + vehicle; #P , 0.01 versus TNFa + vehicle; induced by TNFa, suggesting that PHB is necessary for FL3
†P , 0.05 versus IFNg + vehicle.
and FL37 protection against TNFa-induced mitochondrial
dysfunction.
TNFa- or IFNg-induced apoptosis, we next assessed mitochon-
drial function, which is known to be dysregulated during IBD and p38-MAPK Activation Is Necessary for
in animal models of colitis22–28 and plays a central role in cell fate Protective Effect of FL3 and FL37 on TNFa-
decisions, especially apoptosis.29 Blockade of forward electron induced Mitochondrial-derived ROS and
flow through inhibition of ETC complexes leads to electrons Increased Permeability in Caco2-BBE Cells
accumulating at upstream complexes, ROS generation, and Because FL3- and FL37-induced PHB and PHB2 protein
reduced ATP production.30 Ten nanomolar of FL3 or FL37 expression was associated with p38-MAPK activation (Fig. 2B),
increased basal ATP levels, but this did not reach statistical signif- but not activation of Stat3, SAPK/JNK, ERK, or AKT (see Fig.,
icance (Fig. 6A). TNFa decreased ATP levels in vehicle control Supplemental Digital Content 2, http://links.lww.com/IBD/B117),
cells as shown previously31–33; pretreatment with 10 nM FL3 or we next determined whether p38-MAPK activation was essential
FL37 prevented TNFa-induced decrease in ATP (Fig. 5A). IFNg for FL3 and FL37 protection against TNFa-induced epithelial
did not affect ATP production and pretreatment with FL3 or FL37 mitochondrial and barrier dysfunction. SB203580, a p38-MAPK
before IFNg treatment affected ATP levels similar to treatment with inhibitor, abolished the protective effect of FL3 or FL37 to inhibit
the flavaglines alone (Fig. 5A). For this reason, remaining experi- TNFa-induced mitochondrial-derived ROS (Fig. 7A) and
ments assessed the effect of FL3 or FL37 on changes to mitochon- increased permeability in Caco2-BBE cells (Fig. 7B).
drial function induced by TNFa and not IFNg. SB203580 alone had no effect on mitochondrial ROS production
To determine mitochondrial ROS generation, we used the or barrier function.
mitochondrial superoxide detection dye MitoSox Red. Ten nano-
molar of FL3 or FL37 did not significantly affect basal FL3 Protects Against DSS-induced Colitis
mitochondrial ROS production (Fig. 6B). TNFa significantly To determine the therapeutic potential of FL3 to reduce
increased mitoSOX fluorescence in Caco2-BBE cells, as shown acute colitis, wild-type mice were intraperitoneally injected with
previously.32,33 Pretreatment with 10 nM FL3 or FL37 prevented 0.1 mg/kg FL3 or vehicle once daily on days 0 to 4 and
TNFa-induced mitochondrial ROS production. administered DSS on days 0 to 6, which is a well-characterized

FIGURE 6. FL3 and FL37 preserve mitochondrial function during TNFa treatment. Caco2-BBE cells were pretreated with 10 nM FL3 or FL37 for 1
hour, followed by treatment with 10 ng/mL TNFa or 50 ng/mL IFNg for 16 hours. A, ATP concentration. B, Mitochondrial ROS levels were measured
using mitoSOX dye; n ¼ 8 per treatment across 2 separate experiments. C, 15 minutes before collection, cells were treated with 5 mM rotenone,
a complex I inhibitor, or 1 mM antimycin A, a complex III inhibitor. Mitochondrial ROS levels were measured using mitoSOX dye; n ¼ 8 per
treatment. D, Mitochondrial complex I activity was determined using the Mitochondrial Dipstick Assay kit. Activity of no tx + vehicle control cells
was set to 100%. E, Before pretreatment with FL3 or FL37, cells were transfected with siPHB or siNegative control (siNC) for 72 hours. Mitochondrial
ROS levels were measured using mitoSOX dye. Total protein was analyzed by Western blotting to ensure efficiency of PHB knockdown by siPHB;
*P , 0.05 versus no tx + vehicle; #P , 0.01 versus TNFa + vehicle; †P , 0.05 versus TNFa + vehicle + rotenone.
model of colonic epithelial ulceration.35 Mice given vehicle during distal colon the most severely affected. Hemotoxylin and eosin–
DSS administration showed significant weight loss starting on day stained sections of distal colon of water control mice treated with
5 of DSS treatment (Fig. 8A). In contrast, FL3-treated mice main- FL3 showed similar histology to mice given water and vehicle
tained their body weight during the course of DSS treatment and (Fig. 8C). Distal colon sections of DSS-treated mice given vehi-
exhibited body weights similar to control mice given water. On day cle showed severe inflammatory infiltration, complete crypt loss
6, the mice treated with DSS were assigned a clinical score con- in focal areas, and increased ulceration. In contrast, mice treated
sisting of severity of body weight loss, stool consistency, and the with FL3 during DSS showed moderate inflammatory infiltra-
presence of gross bleeding or blood in the stool. FL3-treated mice tion, less crypt loss, and less ulceration compared with mice
given DSS exhibited a significantly lower clinical score compared given vehicle (Fig. 8C). Histological scoring of inflammation
with vehicle-treated mice given DSS (Fig. 8B). revealed that FL3-treated mice given DSS exhibited a signifi-
DSS-induced colitis is histopathologically characterized cantly less damage (severity of inflammatory infiltration, ulcer-
by infiltration of inflammatory cells into the mucosa and ation, and crypt damage) compared with vehicle-treated mice
submucosa, epithelial ulceration, and crypt damage, with the given DSS (Fig. 8B).
To determine whether in vivo effects of FL3 corroborate

our in vitro results in cultured IECs, oxidative damage and colonic
epithelial apoptosis were measured. FL3 decreased 4-HNE staining
(Fig. 9A) and protein carbonyl content (Fig. 9B), markers of lipid
peroxidation and oxidative damage to protein, respectively, in the
colon of DSS-treated mice compared with vehicle. FL3 did not
affect 4-HNE staining or generation of protein carbonyls in
water-treated control mice. FL3 treatment abolished cleaved cas-
pase 3 protein expression during DSS colitis in isolated colonic
epithelial cells (Fig. 9C), suggesting that FL3 protects against
DSS-induced epithelial apoptosis. This was further supported by

TUNEL staining of distal colon, which demonstrated that FL3
treatment significantly decreased the number of TUNEL-positive
epithelial cells per crypt during DSS colitis (Fig. 9D, E). FL3 did
not significantly alter epithelial cleaved caspase 3 expression or the
number of TUNEL-positive cells in water-treated control mice.
To determine whether the protective effects of FL3
treatment on DSS-induced colitis were associated with increased
expression of PHB or p38-MAPK activation, total protein from
distal colon was isolated and assessed by Western blotting. DSS-
treated mice given FL3 exhibited increased PHB expression and
p38-MAPK activation (Fig. 9F), but not activation of Stat3,
SAPK/JNK, ERK, or AKT (see Fig., Supplemental Digital Con-
tent 4, http://links.lww.com/IBD/B119).
DISCUSSION
Our study is the first to characterize the therapeutic potential
of flavaglines in the intestinal epithelium during inflammation. We
FIGURE 7. p38-MAPK activation is necessary for protective effect of show that the flavaglines FL3 and FL37 protect against apoptosis
FL3 and FL37 on TNFa-induced mitochondrial-derived ROS and and barrier dysfunction in cultured IECs basally and during
increased permeability. Caco2-BBE monolayers were pretreated with proinflammatory cytokine treatment. Protective effects of FL3 were
20 mM p38-MAPK inhibitor SB203580 for 1 hour, then treated with 10 corroborated in vivo, with FL3 administration reducing the severity
nM FL3 or FL37 for 1 hour, and finally treated with 10 ng/mL TNFa for of DSS-induced colitis and barrier permeability.
16 hours A, Mitochondrial ROS levels were measured using mitoSOX Since the first flavagline, rocaglamide, was first isolated in
dye; n ¼ 8 per treatment. B, Rate of 4 kDa FITC-dextran translocation 1992, more than 60 natural flavaglines have been identified.20,36
from apical to basolateral chamber (nanograms per milliliter
Crude extracts from leaves and flowers of different Aglaia (family
per minute); n $ 6 per treatment; *P , 0.05 versus no tx; #P , 0.05
Meliaceae) plants, from which flavaglines are extracted, are used
versus TNFa.
in several countries of southeast Asia as traditional medicine for
the treatment of inflammatory skin diseases and allergic inflam-
A reduction in colon length is a gross indicator of disease matory disorders, such as asthma.6 However, these plants contain
severity in the DSS model of colitis. All animals treated with DSS many classes of pharmacologically active agents, and the above-
showed reduced colon length compared with water controls; cited activities may not involve flavaglines but other classes of
however, shrinkage was less severe in FL3-treated mice compared drugs. Interest in flavaglines as therapeutic compounds stems
with vehicle controls (Fig. 8D). In addition, FL3 treatment signif- from their anticancer, anti-inflammatory, and cytoprotective prop-
icantly reduced myeloperoxidase activity, a marker of neutrophil erties. At very low nanomolar concentrations, flavaglines enhance
infiltration, in the distal colon during DSS colitis (Fig. 8E). Intes- survival of neurons and cardiomyocytes when challenged with
tinal permeability was measured using translocation of 4 kDa numerous stressors.3 In this study, we assessed the effect of
FITC-dextran into serum in DSS-treated mice. Barrier dysfunc- FL3 and FL37 in IECs because both compounds were shown to
tion is one of the earliest events in DSS-induced colitis that pre- display in vivo anticancer and cardioprotective effects. As an
cedes evident inflammation or mucosal damage. Therefore, we in vitro model of inflammation, cultured IECs were challenged
measured intestinal permeability after 3 days of DSS treatment. with TNFa and IFNg, 2 proinflammatory cytokines involved
FL3-treated mice showed decreased FITC-dextran translocation in IBD pathogenesis.37,38 Similar to results in neurons and
compared with vehicle-treated mice during DSS colitis (Fig. 8F). cardiomyocytes, FL3 and FL37 exhibited prosurvival and

FIGURE 8. FL3 protects against DSS-induced colitis. Mice were given 2.5% DSS for 6 days and intraperitoneally injected with 0.1 mg/kg FL3 or
vehicle (veh) once daily on days 0 to 4. Control mice were given regular drinking water throughout the protocol; n ¼ 6 per treatment group across
2 separate experiments with similar results. A, Percent change in body weight; *P , 0.05 versus DSS + FL3. B, Clinical and histological damage
score; *P , 0.05 versus DSS + vehicle. C, Representative photomicrographs of paraffin-embedded hemotoxylin and eosin–stained sections of distal
colon. Original magnification, ·20. D, Colon length measured on day 6 of DSS treatment. Photos of representative colons from DSS-treated mice;
*P , 0.05. E, Neutrophil infiltration into the colon, quantified by measuring myeloperoxidase activity; *P , 0.05. F, On day 3 of DSS treatment, mice
were gavaged with 4 kDa FITC-dextran. Translocation of fluorescent FITC-dextran across the intestinal epithelium was measured in serum col-
lected 4 hours after gavage; *P , 0.05.
anti-inflammatory effects in IECs at nanomolar concentrations mitochondrial stress participates in the pathology of IBD.22,24,41
during TNFa and IFNg treatment. FL3 and FL37 inhibited TNFa Our results suggest that FL3 and FL37 protection of mitochon-
and IFNg downstream proinflammatory signaling as evidenced by drial function in IECs is associated with decreased apoptosis,
reduced nuclear factor kappa B and Cox2 expression, maintained enhanced cell viability, and sustained epithelial barrier function
cell viability, reduced TNFa- and IFNg-induced apoptosis, and during TNFa-induced damage. FL3 and FL37 also protected
prevented TNFa- and IFNg-increased epithelial permeability. We against IFNg-induced apoptosis and epithelial barrier dysfunction,
went on to demonstrate that mitochondrial dysfunction, as char- but unlike TNFa, these effects of IFNg were not associated with
acterized by increased mitochondrial-derived ROS, reduced activ- altered mitochondrial function, suggesting that FL3 and FL37
ity of ETC complexes and ultimately decreased ATP production, cytoprotective effects against IFNg involve signaling pathways
was induced by TNFa33 but not IFNg, and was prevented by FL3 beyond those regulating mitochondrial function.
or FL37. Importantly, multiple studies have reported mitochon- FL3 and FL37 rapidly induced expression of PHB and
drial dysfunction in the epithelium during IBD and experimental PHB2 in IECs, which are established flavagline targets.3 Our
models of colitis.12,25,27,28,39,40 Recent studies indicate that mito- study demonstrated that PHB expression was essential for FL3
chondria integrate cellular homeostasis signaling and that or FL37 inhibition of TNFa-induced mitochondrial-derived ROS

FIGURE 9. FL3 decreased colonic oxidative damage and epithelial apoptosis induced by DSS colitis, and these protective effects were associated
with increased PHB and phospho-p38 MAPK expression. A, Immunohistochemistry staining of 4-HNE. B, Protein carbonyl content in the distal
colon; *P , 0.05, n ¼ 6 per treatment. C, Representative Western blots showing total and cleaved caspase 3 levels in isolated colonic epithelial
cells. b-actin is included as a loading control. D, TUNEL staining (green) of colonic sections from mice treated with FL3 or vehicle (veh) during DSS
colitis or control (water). Sections were stained with 4’,6-diamidino-2-phenylindole to visualize nuclei (blue). E, Number of TUNEL-positive cells per
crypt in well-oriented crypts; *P , 0.05, **P , 0.01; n ¼ 5 per treatment with a minimum of 20 crypts counted per animal. F, Representative
Western blots showing PHB, phospho-p38-MAPK, p38-MAPK, and b-actin in total protein isolated from whole colon.
production in IECs. Furthermore, we show that FL3 and FL37 pattern of p38-MAPK activation during a time course of FL3 or
preserve ETC complex I activity during TNFa treatment. Com- FL37 treatment was similar to that of PHB and PHB2 induction.
plex I of the ETC is a predominant site of PHB binding, resulting Activation of p38-MAPK by flavaglines has been demonstrated in
in optimal activity of complex I and the respiratory chain.11,13 lymphocytes, which causes immunosuppression through selective
Previous studies have shown that TNFa reduces PHB expression inhibition of the transcription factor nuclear factor of activated T
in IECs,32,42 which we also confirm in the current study (Fig. 3). cells (NFAT).6 NFAT is expressed in the intestinal epithelium where
Therefore, our results suggest that FL3 and FL37 promote mito- it regulates differentiation, cell cycle, and apoptosis.43 It is not
chondrial function in IECs during stress induced by TNFa by known whether PHBs mediate activation of p38-MAPK or NFAT
sustaining PHB expression and ETC complex I activity, which by flavaglines.7 We show that p38-MAPK activation is essential for
are targets of TNFa-induced damage.33,42 the protective effects of FL3 and FL37 on IEC barrier permeability
FL3 and FL37 rapidly induced activation of p38-MAPK in and mitochondrial-derived ROS production during TNFa treatment.
IECs, but not activation of Stat3, SAPK/JNK, ERK, or AKT. The Future studies will elucidate whether p38-MAPK activation by FL3
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Flavaglines Crohn Colitis Prohibitins

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ORIGINAL ARTICLE

Flavaglines Ameliorate Experimental Colitis and Protect Against

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immune system, thereby aggravating the inﬂammatory condition.1

Inﬂamm Bowel Dis Volume 22, Number 1, January 2016 www.ibdjournal.org | 55

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(Sigma-Aldrich), a complex I inhibitor, or 1 mM antimycin A Clinical Score Assessment

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Caco2-BBE cells were treated with increasing doses of FL3 or

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Pretreatment with FL3 or FL37 Decreases

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Previous studies have shown that TNFa promotes cellular

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To determine whether in vivo effects of FL3 corroborate

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or FL37 is downstream of PHBs and whether p38-MAPK activation REFERENCES

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