ORIGINAL ARTICLE

Mitigation of Colitis with NovaSil Clay Therapy

Katherine E. Zychowski • Sarah E. Elmore • Kristal A. Rychlik •

Hoai J. Ly • Felipe Pierezan • Anitha Isaiah • Jan S. Suchodolski •

Aline Rodrigues Hoffmann • Amelia A. Romoser • Timothy D. Phillips

Received: 23 July 2014 / Accepted: 6 September 2014 / Published online: 21 September 2014

� Springer Science+Business Media New York 2014

Abstract

Background/Aims Five million people currently live with

Crohn’s disease (CD) or ulcerative colitis, the two major

forms of inflammatory bowel disease. Available treatments

frequently result in side effects that compromise the immune

health of the patient. Consequently, alternative therapies that

cause fewer systemic effects are needed. Dioctahedral

smectite clays have been utilized to treat medical conditions,

including diarrheal and enteric disease. Herein, we report the

ability of a refined dioctahedral smectite (NovaSil, NS) to

sorb inflammatory proteins and reduce inflammation in a

TNBS (2,4,6-trinitrobenzenesulfonic acid) mouse model of

CD. We also investigated whether NS could rescue gut

microbial diversity in TNBS-induced mice.

Methods ELISA, X-ray diffraction, and transmission

electron microscopy were employed to characterize the NS–

cytokine interaction in vitro. A TNBS mouse colitis model

was utilized to study the efficacy of NS supplementation for

4 weeks. The three treatment groups included control,

TNBS, and TNBS ? NS. DNA was extracted from feces and

sorted for bacterial phylogenetic analysis.

Results Results suggest that NS binds TNFa in vitro. In

TNBS-treated mice, supplementation with NS significantly

reduced weight loss, and serum proinflammatory cytokine

levels (IL-2, IL-6, and IL-12, TNFa, IFNc) compared with

the TNBS group. TNBS-treated mice demonstrated a sig-

nificant reduction in gut microbiota species richness when

compared with the TNBS ? NS group and control group.

Conclusions NovaSil mitigated the effects of TNBS-

induced colitis based on reduction in systemic markers of

inflammation, significant improvement in weight gain, and

intestinal microbial profile.

Keywords Montmorillonite � Colitis � Crohn’s �Inflammation � Cytokine � Microbiota

Introduction

Crohn’s disease (CD) and ulcerative colitis are chronic

inflammatory diseases that affect nearly 0.1–16 per

Electronic supplementary material The online version of thisarticle (doi:10.1007/s10620-014-3360-7) contains supplementarymaterial, which is available to authorized users.

K. E. Zychowski � S. E. Elmore � K. A. Rychlik �H. J. Ly � F. Pierezan � A. Isaiah � J. S. Suchodolski �A. R. Hoffmann � A. A. Romoser � T. D. Phillips (&)

College of Veterinary Medicine and Biomedical Sciences, Texas

A&M University, TAMU 4458, College Station,

TX 77843-4458, USA

e-mail: tphillips@cvm.tamu.edu

K. E. Zychowski

e-mail: kzychowski@cvm.tamu.edu

S. E. Elmore

e-mail: selmore@cvm.tamu.edu

K. A. Rychlik

e-mail: kabrown@cvm.tamu.edu

H. J. Ly

e-mail: hjly@utmb.edu

F. Pierezan

e-mail: fpierezan@cvm.tamu.edu

A. Isaiah

e-mail: aisaiah@cvm.tamu.edu

J. S. Suchodolski

e-mail: jsuchodolski@cvm.tamu.edu

A. R. Hoffmann

e-mail: arodrigues@cvm.tamu.edu

A. A. Romoser

e-mail: aromoser@cvm.tamu.edu

123

Dig Dis Sci (2015) 60:382–392

DOI 10.1007/s10620-014-3360-7

100,000 and 0.5–24.5 per 100,000 people worldwide,

respectively. CD is characterized by painful ulceration that

can occur along the gastrointestinal tract, from the mouth

to the anus, as opposed to ulcerative colitis (UC) which is

restricted to the colon [1]. Similar to other autoimmune

diseases, the majority of cases occur in the developed

world [2]. Although the etiology of CD is complex, genetic

polymorphisms, alterations in intestinal microbiota, and

modulated immune response have all been attributed to

possible causes of the disease. Additionally, cigarette

smoking, excess stress, and environmental factors are

known to play a role in the progression of the disease [3–5].

Treatments for CD can be expensive and often cause

undesirable side effects [6, 7]. Common pharmaceutical

treatments include aminosalicylates, antibiotics, cortico-

steroids, and biologics (anti-TNFa agents) [8]. Immuno-

suppressive treatments often involve increased risk of

infection and certain types of cancer, such as lymphoma

[9]. Due to various risks associated with these medications,

there is a need to develop alternative therapies. Diarrhea

caused by various gastrointestinal (GI) conditions can be

mitigated by dietary clays. Historically, dioctahedral

smectite clays have been utilized effectively for the treat-

ment of diarrhea caused by infectious diseases [10–12].

From these studies, it can be easily speculated that clay

treatments are capable of pathogen or toxin sorption.

However, dioctahedral smectites have also been shown to

increase mucosal barrier integrity against pepsin and TNFaexposures in vivo [13, 14] and to provide protection from

immune system disturbances induced in guinea pigs sen-

sitized to cow’s milk [15]. Moreover, inflammation

occurring as a result of acute hapten exposures decreased

following treatment with dietary clays [16].

Although the specific mechanisms of action have yet to

be reported in the case of chronic diarrhea, several theories

have been proposed that may support the therapeutic nature

of dioctahedral smectite clays, including the possibility of

intestinal mucus barrier reinforcement (reducing penetra-

tion of luminal antigens) and modulation of proinflamma-

tory cytokine production and effects. Of particular

importance is the intestinal barrier, which is affected by the

presence of luminal inflammatory cytokines, such as IL-6,

IL-1b, and TNFa [17]. In the absence of bacterial or viral

infection, malfunction of the intestinal barrier is pivotal in

diseases causing chronic diarrhea, such as inflammatory

bowel disease (IBD) [18]. Additionally, dioctahedral

smectite clays have demonstrated the potential to shift the

population of intestinal flora from a pathological to a bal-

anced state [19–21], which is important since the gastro-

intestinal microbiome is known to play a role in the

etiology and management of IBD [22, 23]. Furthermore,

in vitro and in vivo studies have indicated that dioctahedral

smectite clays can form aggregates with E. coli [24, 25].

Copper-bearing montmorillonite, a type of smectite clay,

decreased E. coli and Clostridium counts in the intestine of

male broilers and also improved intestinal mucosal mor-

phology [26]; however, no studies have profiled the intes-

tinal microbiome in smectite-supplemented animals.

NovaSil (NS) is a type of dioctahedral smectite with a

negatively charged interlayer that has been administered as

a supplement both in animal feeds and in clinical inter-

vention trials throughout the world to reduce dietary

mycotoxin bioavailability [27]. Currently, little informa-

tion is available concerning the potential NS anti-inflam-

matory properties or its impact on gut microbiota. Similar

clays have been reported to possess anti-inflammatory

properties, but the mechanism remains unclear [16, 28, 29].

For this reason, we investigated the ability of NS to interact

with proinflammatory cytokines, protect the intestinal

microbiota, and mitigate colitis.

The pH of the normal colon ranges between 6.5 and 7.6.

Comparatively, the colonic pH in an individual with CD is

approximately 5.3 [30], but can drop to a pH of 0.6 in a

patient with severe disease [31]. Based on the fact that the

isoelectric point of TNFa is 6.4 ± 0.3 [32], it is expected

that TNFa is protonated in individuals living with CD. This

suggests that negatively charged NovaSil could sorb pro-

tonated proinflammatory cytokines at a low pH. NovaSil

has a long-standing record of safety and efficacy and does

not interfere with serum vitamin or nutrient levels.

The purpose of this research was twofold: (1) to char-

acterize the NS–cytokine interaction in vitro and (2) to

determine the ability of NS to reduce colitis-related effects

and counteract dysbiosis in a TNBS mouse model.

Materials and Methods

ELISA-Based Assessment of In Vitro Binding Affinity

Deionized H2O was adjusted to pH 5 to simulate the intes-

tinal pH that would likely be present in diseased intestine.

Recombinant TNFa (Sigma-Aldrich, St. Louis, MO) was

added to the pH-adjusted H2O in borosilicate glass vials,

resulting in a protein concentration of 210 pg/mL in each

vial, to simulate a relevant level of TNFa produced in CD

tissue [33–35]. Additionally, NS ranging from 0 to 400 lg/

mL was added to the vials. To determine whether protein was

primarily bound to internal or external clay surfaces, inter-

action with both intact and heat-collapsed NS was investi-

gated. Heat-collapsed NS was synthesized according to a

previously published method [36]. The interlayer, the

internal binding surface, contributes to the majority of neg-

ative charge of the NS structure [37]. Collapsing the structure

of NS results in elimination of H2O from the interlayer as

well as dehydroxylation of the clay. Briefly, NS was heated

Dig Dis Sci (2015) 60:382–392 383

123

in a furnace for 30 min at 200 �C and then at 800 �C for 1 h.

The intact and heat-collapsed NS were then separately added

to individual vials. The protein (210 pg TNFa/vial) was

adjusted to a total volume of 1 mL with H2O. Controls

included one vial of H2O only, diluted protein only, and

H2O ? each concentration of NS. The vials were incubated at

room temperature on an orbital shaker operating at 100 rpm

for 1 h. The vials were then removed from the shaker and

centrifuged at 2,000 rpm for 20 min. The supernatant frac-

tion was used to measure TNFa levels via ELISA. The NS

pellet was then washed in H2O, incubated, and centrifuged as

described above and the supernatant subjected to TNFadetection by ELISA for a second time. This procedure was

used to determine the amount of unbound protein in the

supernatant fraction. The amount of bound protein was

determined by subtracting the amount in the supernatant

fraction from the original concentration (210 pg/ mL). Pro-

tein concentrations were calculated based on a standard

calibration curve. Experiments were performed in triplicate.

Transmission Electron Microscopy (TEM) and Powder

X-ray Diffraction (XRD)

A samples containing 1 lg TNFa/100 lg NS was prepared

for TEM in order to further characterize the protein–NS

binding interaction. This concentration was selected for

optimal visualization and characterization of NS–TNFacomposites. Samples (NS and TNFa ? NS) were dehy-

drated using an ethanol series followed by propylene oxide.

Samples were further embedded in epoxy resin, sealed with

epoxy, and cured at room temperature for 24 h, as descri-

bed by Kolman et al. [38]. Samples were sectioned into 60-

to 100-nm slices. All images were captured with a Mor-

gagni (FEI) Transmission Electron Microscope at 80 kV

(FEI Company, Hillboro, OR). Diffractograms were

recorded for NS, heat-collapsed NS, and NS ? TNFasamples. A 1-cm-diameter o-ring was coated with a thin

layer of petroleum jelly and suctioned onto a custom-made

zero-background holder. Samples (1 lg protein/100 lg

NS) were drop-casted onto the holder. A Bragg-Brentano

powder short-arm diffractometer (Bruker Coorperation,

Billerica, MA) was used for all diffraction patterns

(k = 0.1540 nm) within the 2h range of 2� to 20�, with a

0.014� step size. Bragg’s Law (nk = 2dsinh) was used to

calculate the d spacing between the smectite layers.

TNBS Induction of Crohn’s Colitis in Mice

Five-week-old female BALB/c mice were purchased from

the Jackson Laboratory (Bar Harbor, Maine) and housed at

the Comparative Medicine Program (CMP) facility at

Texas A&M University. To increase animal comfort fol-

lowing the TNBS induction process, extra bedding was

added to the cages, and powdered feed was moistened with

H2O and made accessible in shallow ceramic bowls. A

12:12 light/dark cycle, a stable temperature (23 �C), and %

relative humidity (30–70%) were maintained in the room

where the animals were housed.

TNBS induction of colitis, an established model for CD,

was utilized as previously described [39] with slight

modifications. Thirty mice were equally and randomly

allocated into three different treatment groups: control

(control, n = 10), TNBS-induced (TNBS, n = 10), and

TNBS induction with 4 % NS supplementation

(TNBS ? NS, n = 10). Based on the results from a pre-

vious 1-week pilot study (data not shown), mice in the

TNBS ? NS group were conditioned with the 4 % NS diet

for 1 week prior to the beginning of the 4-week trial. One

hundred milliliters of 1:1 TNBS/ethanol was intrarectally

administered to the TNBS and TNBS ? NS groups with a

4-cm plastic gavage tip. The control group received 100 lL

of 1:1 phosphate-buffered saline (PBS)/ethanol using the

same technique on a weekly basis. To ensure retention of

all solutions throughout the colon, mice were secured

vertically in a recovery chamber for 1 min while anesthe-

tized. Additionally, mice that were severely symptomatic

(bloody diarrhea, lethargy, impaired motor skills) or

exhibited severe weight loss ([25 % initial body weight)

were immediately euthanized. Mice were induced on a

weekly basis and killed at the end of 4 weeks via CO2

asphyxiation. This research was approved by the Institu-

tional Animal Care and Use Committee at Texas A&M

University, College Station, TX (IACUC 2013-0030). This

study was carried out in accordance with the recommen-

dations in the Guide for the Care and Use of Laboratory

Animals of the National Institutes of Health [40].

Weight Gain and Final Somatic Indexes

Mice were weighed on an individual basis twice per week

for 4 weeks and monitored on a daily basis. Following

euthanasia, the liver and colon were collected. Additionally,

animal weights and colonic length were recorded. Colon

weight/length ratio and hepatosomatic index (HSI) ((liver

weight (g)/weight of the mouse (g))*100) were calculated.

Final n values were between 6 and 10 mice/group (control:

n = 10, TNBS: n = 6, TNBS ? NS: n = 6).

Serum Cytokines

Blood (1 mL) was collected from each animal via cardiac

puncture immediately following CO2 euthanasia. Whole

blood was allowed to separate for approximately 3 h at

4 �C. Blood was subsequently centrifuged and serum was

stored at -20 �C. Circulating levels of inflammatory pro-

teins were examined using a Mouse Th1/Th2/Th17

384 Dig Dis Sci (2015) 60:382–392

123

cytokine Multi-Analyte ELISArray Kit (MEM-003A,

Qiagen, Valencia, CA, USA). Serum was pooled according

to the treatment group (6 mice/treatment), and relative

expression of the following cytokines was assayed in

triplicate: IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IL-

17A, IL-23, IFNc, TNFa, TGF-b1.

C-Reactive Protein

Serum C-reactive protein (CRP) levels were quantified for

all treatment groups. C-reactive protein levels rise in the

serum as a result of inflammation. Serum CRP was

detected using an ELISA kit (Genway Biotech Inc., San

Diego, CA, USA). Serum was analyzed using two repli-

cates per mouse.

DNA Extraction and Gut Microbiota Sequencing

Samples were thawed and genomic DNA was extracted

using a Power Soil DNA isolation kit (MoBio Laborato-

ries). Six randomly selected fecal samples were selected

from the control, TNBS, and TNBS ? NS groups. The V4

region of the 16S rRNA gene was amplified with primers

515F (50-GTGCCAGCMGCCGCGGTAA-30) and 806R

(50-GGACTACVSGGGTATCTAAT-30) at the MR DNA

Laboratory (Shallowater, TX, USA). PCR amplification

products were verified on 2 % agarose gels, and samples

were purified using calibrated Ampure XP beads. The

Illumina TruSeq DNA Library was used to prepare a DNA

library and sequenced at MR DNA on an Illumina MiSeq

instrument.

Microbiome Data Analysis

Quantitative Insights Into Microbial Ecology (QIIME,

v.1.8) software was used to phylogenetically characterize

the fecal samples procured from 6 randomly selected mice

from each treatment group. The raw sequence data were

demultiplexed by barcodes, and low-quality reads were

filtered using the QIIME database’s default parameters. A

total of 824,894 (median: 45,474; range 37,692–54,975

sequences per sample) were obtained. For further analysis,

each sample was normalized to an even sequencing depth

of 32,700 sequences per sample to adjust for uneven

sequencing depth across all samples. Sequences were then

clustered into operational taxonomic units (OTUs) using a

closed-reference OTU picking protocol at the 97 %

sequencing identity level using UCLUST [41] against the

Greengenes database [42] pre-clustered at 97 % sequence

identity [43]. Proportions of bacterial taxa (% of total

sequences) were statistically evaluated using a Kruskal–

Wallis test where appropriate and corrected by multiple

comparisons using the Benjamini & Hochberg’s False

Discovery Rate. P values B0.05 were considered statisti-

cally significant. Observed species richness, Chao 1, and

Shannon indexes were all determined using alpha-diversity

parameters within QIIME.

Beta-diversity analysis was determined using principal

coordinate analysis (PCoA) plots and unweighted Unifrac

distance metrics. Statistical significance of the resulting

distance matrix was tested by analysis of similarities

(ANOSIM) using the QIIME software [44]. All of the data

were deposited into the NCBI-SRA (sequence read

archive). The accession number is SRP041186. The Bio-

Project ID is PRJNA244160 (http://www.ncbi.nlm.nih.gov/

bioproject/?term=PRJNA244160).

Statistical Analysis

Aside from microbiome data, all other data were subject to

a one-way ANOVA followed by a Student’s t test for

parametric data. Nonparametric data were subject to Wil-

coxon rank-sum test. All statistics were analyzed with the

assistance of JMP software (SAS Institute, Cary, NC,

USA). Values were considered significant at P B 0.05.

Data in graphs are expressed as the mean ± SE.

Results

In Vitro Cytokine–NS Interaction

Results indicate that 200 lg NS sorbed 90 % of TNFa(190 pg/mL) (Fig. 1a). Heat-collapsed NS sorbed only

21 % of TNFa (44 pg/mL). The highest concentration of

NS (400 lg/mL) did not bind more TNFa than the 200 lg/

mL concentration (189 pg TNFa/mL). Heat-collapsed NS

at concentrations ranging from 50 to 400 lg/mL sorbed

equal amounts of TNFa.

XRD and TEM

X-ray diffraction yielded results (Fig. 1b) that are consis-

tent with Fig. 1a, suggesting apparent expansion of NS

interlayers in the presence of TNFa. X-ray diffraction was

used to measure the distance (d spacing) between each

atomic plane in the clay mineral. Results indicated that

TNFa ? NS (d = 13.6) exhibited increased d spacing

when compared to the NS sample (d = 13.13). Heat-col-

lapsed NS did not exhibit the typical montmorillonite peak

upon XRD analysis, confirming a lack of interlayers in the

structure. Transmission electron microscopy images of the

intact clay indicated a very tight interlayer structure

(Fig. 1c, d), compared with TNFa ? NS. Images taken

from the TNFa ? NS samples suggest that the interlayers

Dig Dis Sci (2015) 60:382–392 385

123

within the clay structure expand in the presence of this

protein (Fig. 1c, d).

Weight Gain and Final Somatic Indexes, In Vivo

After the first induction (Fig. 2a), body weight decreased

significantly in the TNBS-treated mice (Fig. 2b). NovaSil

inclusion improved weight gain starting at the second week

compared with the TNBS group, and the greatest difference

in weights between the treatment groups was observed

after the final induction, at the beginning of week 4

(Fig. 2a, b). The TNBS group exhibited a statistically

significant weight loss in the fourth week of the study,

compared with the other treatment groups (P = 0.01).

Colon weight/length ratio did not significantly differ

between the TNBS and control groups (P = 0.17); how-

ever, the TNBS group had the greatest weight/length ratio

(Fig. 2c). Hepatosomatic index also increased in TNBS-

treated mice when compared with the other groups; how-

ever, these values were not significantly different (Fig. 2d).

Serum Cytokines

Serum cytokine levels, which indicate CD inflammation,

were evaluated upon termination of the study (Fig. 3).

Compared with the TNBS group, relative expression of IL-

2, IL-4, IL-6, and IL-12 was significantly decreased in

NS ? TNBS-treated mice. Other proinflammatory cyto-

kines including IFNc, IL-23, and TNFa also decreased

with dietary inclusion of NS; however, levels were not

statistically different from the TNBS group. No significant

differences in IL-5, IL-10, IL-13, and IL-17A levels were

detected between the TNBS and TNBS ? NS groups.

C-Reactive Protein Expression

TNBS-treated mice exhibited increased CRP levels relative

to the other treatment groups (P = 0.0002). Moreover,

there was a decrease in CRP levels in the TNBS ? NS

treatment group compared with the TNBS group; however,

this result was not statistically significant (data not shown).

Fig. 1 In vitro characterization of NS–protein interaction. a TNFasorbed onto the surface of NS and heat-collapsed NS (0–400 lg) as

determined by ELISA. The supernatant fraction was used to

determine amount of TNFa bound to NS surfaces. b X-ray diffraction

of TNFa ? NS (1 lg TNFa/ 100 lg NS), NS, and heat-collapsed NS.

Samples were prepared and drop-casted onto a zero-background

holder. XRD results indicate d spacing, or spacing in between silicate

layers (in brackets []). Bragg’s Law nk = 2dsinh was used to

determine the d spacing. Transmission electron microscopy (TEM) of

c NS and d NS ? TNFa. TEM samples were prepared using epoxy

embedment and sectioned into 60- to 100-nm slices. Images displayed

are the most representative from the samples

386 Dig Dis Sci (2015) 60:382–392

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16S Bacterial rRNA Analysis

Diversity can be described using alpha-diversity or beta-

diversity metrics [45]. Beta diversity indicates between-

sample taxonomic diversity, while alpha diversity

describes within-sample taxonomic diversity. Because beta

diversity indicates how taxa are shared between groups,

clustering can be visualized using a principal coordinate

analysis (PCoA) plot. Alpha diversity, which accounts for

species richness (number) and/or evenness, can be

Fig. 2 Weight change and

somatic indexes. a Week-by-

week weight change over

4 weeks. Individual mice were

weighed twice per week, and

values were averaged by group.

b Final weight according to

treatment. Starred values (*) are

significantly different from

other treatment groups

(P B 0.05) c Colonic weight to

length ratio (mg of colon/cm of

colon) 9 100. d Hepatosomatic

index (HSI) (g of liver/g of body

weight) 9 100. Columns that

do not share the same letter are

significantly different

(P B 0.05)

Fig. 3 Relative expression of serum cytokines. Relative expression

of select cytokines from the TNBS and TNBS ? NS treated mice.

Mouse serum was pooled by group at the end of the study and assayed

in triplicate using an ELISA array (6 mice/treatment group). Starred

values are significantly different (P B 0.05)

Dig Dis Sci (2015) 60:382–392 387

123

described using the estimated observed species, the Chao1

index, and the Shannon index [46]. The observed species

metric, a measure of species richness, is based on the

number of operational taxonomical units (OTUs). The

Chao1 index also estimates species richness and is calcu-

lated based on the number of singletons (species recovered

once in the sample) and doubletons (species recovered

twice in the sample) detected. The Shannon index is yet

another estimation that takes into account both species

richness and evenness [47].

There was a significant clustering in the TNBS group

when compared with the TNBS ? NS group according to

the principal coordinate analysis (PCoA) for unweighted

Unifrac distances [P = 0.02, R statistic = 0.324 (Fig. 4)].

Rarefaction analysis revealed a significant decrease in

species richness in the TNBS group compared with the

TNBS ? NS and control groups at 37,200 sequences,

based on the estimated observed species (P = 0.03)

(Fig. 5). The TNBS group exhibited decreased, although

not statistically significant, Chao 1 index compared with

the control and TNBS ? NS groups (P = 0.09). The

Shannon index, which indicates the evenness and abun-

dance of species, did not differ significantly between

groups, most likely due to the reduction in the number of

rare taxa.

The Weissella genus was significantly more abundant in

the TNBS group compared with the other two treatments

(Table S1). None of the other genera varied significantly

between treatments. Prevalent bacteria in all three treat-

ment groups included Clostridiales, S 24-7, Oscillospira,

and Aneroplasm.

Discussion

In vitro results (Fig. 1) suggest that TNFa becomes bound to

NS, based on the remaining cytokine levels in the superna-

tant fraction (Fig. 1a) and change in structural morphology

of the clay (Fig. 1b–d). Figure 1 also suggests that this

protein is primarily sorbed to the interlayer surfaces of the

clay, indicating that it is attracted to the structural portion of

the clay with the greatest negative charge. Multiple research

groups have characterized interactions between proteins and

clays [38, 48], yet none have explored the potential for

smectites to sorb proinflammatory cytokines. Others have

explored the potential for montmorillonites to selectively

remove proteins from mucosal fluids, and these clays have

been described to sorb proteins such as lysozyme in the blood

[48]. Additionally, similar silicate materials have been sat-

urated with a drug in vitro to be explored for therapeutic

measures [49–51]. Previous research indicates that as a sil-

icate structure becomes increasingly saturated with protein,

the layered structure becomes separated, or exfoliated [52].

Similarly, the results from this research indicate that the

interlayers of NS become ‘‘propped open’’ in the presence of

a proinflammatory cytokine [53].

Fig. 4 Principal coordinate analysis for control, TNBS, and

TNBS ? NS. Principal coordinate analyses (PCoA) of fecal samples

were collected from mice at the end of the 4-week study. Feces were

collected from the colon following euthanasia. Samples were ‘‘flash-

frozen’’ in liquid nitrogen and subsequently transferred to a -80 �C

freezer until further use. Graphical representation of b diversity for

control (red, n = 6), TNBS (black, n = 6), and TNBS ? NS (aqua

blue, n = 6) groups. R values closer to 0 indicate no difference

between treatments, while values closer to 1 indicate differences

between groups. Clustering differences were observed between the

TNBS and TNBS ? NS groups for unweighted Unifrac distances of

16S rRNA genes (R = 0.324)

388 Dig Dis Sci (2015) 60:382–392

123

NovaSil is ingested orally and subsequently travels

through the GI tract. Because NS is never absorbed through

the gastrointestinal wall, it is ultimately excreted in the

feces. Therefore, NovaSil’s anti-inflammatory effect would

presumably occur at the site of ulceration in a colitis

model. Our results from the colitis induction trial provide

some evidence for the efficacy of NS to alleviate some of

the factors associated with GI tract inflammation in vivo. It

should be noted that, similar to other studies, TNBS-treated

mice exhibited the largest variation in weight. The

response to TNBS induction was not uniform throughout

the mice in each treatment group, which is typical for this

model. Additionally, previous studies have reported an

increase in colon weight/length ratio as a result of CD and

UC induction in mice [54–56]. This study indicates that

colon weight/length ratios were not significantly different

between treatments; however, the TNBS group exhibited

the highest colon weight/length ratio. There was a trend

toward recovery with the addition of NS in the feed in

TNBS-induced mice, suggesting that NS may have pre-

vented some inflammation and subsequent shortening of

the colon in the affected areas.

Crohn’s disease is typically characterized by a Th1/

Th17 immune response and is associated with upregulation

of cytokines such as IL-2, IL-12, IFNc, TNFa, IL-6, and

IL-1b. Enzyme-linked immunosorbent assay results indi-

cate that NS prevented upregulation of inflammatory

cytokines associated with TNBS-induced colitis in mice.

Typically, decreased levels of pleiotropic IL-4 in the

lamina propria are associated with CD. However, we

detected higher expression of IL-4 in the TNBS group than

in the TNBS ? NS group. This may be explained by the

Fig. 5 Alpha diversity analysis of control, TNBS, and TNBS ? NS

groups a Rarefaction curves for 16S ribosomal RNA gene sequences

for control (n = 6), TNBS (n = 6), and TNBS ? NS (n = 6) from

fecal samples. b Alpha diversity measured at 37,000 sequences in the

control, TNBS, and TNBS ? NS treatment groups. The Y axis

represents the number of observed species, Chao 1 index and Shannon

index. c Summary of alpha-diversity data. Number of observed

species in the TNBS group was significantly lower than in the control

or TNBS ? NS groups (P B 0.05)

Dig Dis Sci (2015) 60:382–392 389

123

fact that early CD lesions produce higher levels of IL-4 in

the early stages of the disease; however, expression of this

cytokine is reduced as the disease progresses [57]. Due to

the subchronic timeframe (4 week) of the study, the

increased expression of IL-4 may indicate that the mice

were between the early and late stages of CD-like colitis.

The presence of NS in the feed did not affect the majority

of measured anti-inflammatory proteins, including IL-10

and TGF-b1, suggesting that NS did not inhibit the anti-

inflammatory response.

Numerous studies have focused on the impact of the

microbiome in CD. Research suggests that an abnormal

immune response to the body’s endogenous flora in a

genetically susceptible individual can trigger inflammation

in the GI tract [58]. Furthermore, dysbiosis has been shown

to instigate the initial inflammatory response [59]. There

are currently only a few studies that have reported the

effects of clay supplementation on gastrointestinal micro-

biota [60–62], despite the fact that clays have been

administered as dietary supplements in both humans and

animals for many years [21, 63, 64]. It has been suggested

that certain ‘‘probiotic’’ microorganisms, such as Lacto-

bacilli, Bifidobacteria, and Saccharomyces, are beneficial

for CD patients [65–67]. In this study, levels of Lactoba-

cillus were nonsignificantly decreased in the TNBS group

(P = 0.24), compared with the control and TNBS ? NS

groups and Bifidobacteria levels remained unchanged in all

three treatment groups. Metagenomic analysis has also

determined that CD patients have reduced intestinal flora

diversity compared with healthy controls [68]. Alterations

in the gut microbiome in active CD are most likely due to

inflammation, resulting in permanent alterations in flora,

even during remission [69]. In agreement with these find-

ings, we detected a reduction in microbial diversity in the

TNBS-treated mice (Figs. 4, 5). Importantly, the addition

of NS into the feed of TNBS-treated animals resulted in a

bacterial population that was more closely related to that in

the control group. One genus (Weissella) was significantly

different in the TNBS group. There were some other bac-

terial genera, such as Blautia sp., that decreased in the

TNBS group, although not quite significantly (P = 0.09).

A decrease in Blautia has been observed in other species

with IBD-like or other gastrointestinal inflammation [44,

70]. UniFrac distances, a metric of community dissimilar-

ity, are used to measure beta diversity in the PCoA plots.

Based on ANOSIM, our results revealed a significant

cluster in the TNBS-treated mice compared with the

TNBS ? NS mice, as depicted in the PCoA plot. Because

this is the first study to explore the impact of clay sup-

plementation on the colonic microbiome, further charac-

terization is needed to fully understand the mechanism by

which NS can prevent colitis-induced changes in intestinal

flora.

In summary, dietary NS inclusion mitigated several

TNBS-induced colitis effects, including inflammation,

weight loss, and microbial dysbiosis. One potential mech-

anism for these beneficial effects is NS–cytokine binding,

which is supported by the capability of NS to bind proin-

flammatory cytokines in vitro. Another possible mecha-

nism in which our data support is the protective effect that

NS has on the gastrointestinal environment which may

promote healthy intestinal flora. Alternatively, changes in

the gut microbiome may be a direct result of NovaSil’s

amelioration of TNBS-induced colitis. Due to these find-

ings, we report that NS has potential as an alternative or

supplemental therapy for CD. Long-term studies are war-

ranted to further investigate the effects of NS on GI tract

inflammation.

Acknowledgments The authors would like to thank Dr. Harold

Ross Payne (Veterinary Pathobiology Department, Texas A&M

University) for assistance with electron microscopy imaging. This

research was supported by Texas A&M University College of Vet-

erinary Medicine and Biomedical Sciences Graduate Student and

Postdoctoral Trainee grants.

Conflict of interest None.

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