The animal protocol used in this study was based on the ethical procedures for scientific care, and reviewed and approved by the Pusan National University-Institutional Animal Care and Use Committee (PNU-IACUC; Approval Number PNU-2015-0949). Adult SD rats were purchased from SamTacho BioKorea (Osan, Korea), and handled at the Pusan National University Laboratory Animal Resources Center accredited by the Korea Food and Drug Administration (FDA)(Accredited Unit Number-000231) and AAALAC International (Accredited Unit Number; 001525). All rats were provided with standard irradiated chow (Purina Mills, Seoungnam, Korea) ad libitum, and were maintained in a specific pathogen-free (SPF) state under a strict light cycle (lights on at 06:00 h and off at 18:00 h) at 22±2℃ and a relative humidity of 50±10%.

The model for constipation was followed as previously described [79]. Briefly, the 8-week-old SD rats (Samtako BioKorea Co., Osan, Korea) (n=28) were assigned to either a non-constipation group (No group, n=14) or a constipation group (n=14). Constipation was induced by subcutaneous injection of Lop (4 mg/kg weight) prepared in 0.5% Tween 20 in saline, twice a day for 3 days, whereas the non-constipation group was injected with 0.5% Tween 20 in saline. The non-constipation group was further divided into a No treated group (n=7) and a Urd treated group (n=7). The No treated group was untreated during the experimental period, whereas the Urd treated group received a single dose of 100 mg/kg Urd (Sigma-Aldrich Co., Saint Louis, MO, USA) (Figure 1A). Additionally, the constipation group was further divided into a Lop+Vehicle treated group (n=7) and Lop+Urd treated group (n=7). The Lop+Urd treated groups were orally administered a single dose of 100 mg/kg body weight Urd, while the Lop+Vehicle treated group received the same volume of 1× PBS under the same pattern. At 24 h after Urd treatment, all SD rats were euthanized using CO₂ gas, and the tissue samples were acquired and stored in Eppendorf tubes at 70℃ until assay.

Throughout the experimental period, alterations in food intake and water intake were measured daily at 10:00 am, using an electrical balance (METTLER-TOLEDO, Greifensee, Switzerland) and a measuring cylinder, respectively. All measurements were performed three times to ensure accuracy.

To avoid any contamination, all SD rats in the subset groups were bred in individual metabolic cages (Daejong Ltd., Seoul, Korea) during the experimental period. The stools and urine excreted from each SD rat were collected at 10:00 am. Stool weight was measured three times per sample using an electric balance (METTLER-TOLEDO), whereas the water content was determined as the difference between the wet and dry weights of the stool, as previously described [89].

Transverse colons were prepared for histological analysis as previously described [9]. Briefly, transverse colons collected from SD rats were fixed with 10% formalin for 12 h, embedded in paraffin wax, and sectioned into 4 ìm thick slices that were then stained with hematoxylin & eosin (H&E, Sigma-Aldrich Co.). Morphological features of these sections were observed by light microscopy, after which the mucosa layer thickness and muscle thickness were measured using the Leica Application Suite (Leica Microsystems, Heerbrugg, Switzerland).

To detect the transcript level of ER stress related genes, frozen transverse colons were chopped with scissors and homogenized in RNAzol B solution, according to the manufacturer's protocol (Tet-Test Inc., Friend-swood, TX, USA). The concentration of isolated RNA was then determined by UV-spectroscopy. The expression of the genes was subsequently examined by RT-PCR using 5 µg of the total RNA from each tissue. Briefly, 500 ng of oligo-dT primer [Gibco BRL, Uxbridge, Middlesex, UK] was annealed with template RNA at 70℃ for 10 min. The comple-mentary DNA, which served as the template for subsequent amplification, was then synthesized by adding dATP, dCTP, dGTP and dTTP with 200 units of reverse transcriptase and 10 pmole of sense and antisense primers. Next, the amplification was conducted by subjecting the samples to 28 cycles of 30 sec at 94℃, 30 sec at 62℃, and 45 sec at 72℃, in a T100TM Thermal Cycler (BIORAD, Hercules, CA, USA). In each case, negative-RT controls were included to differentiate the DNA and RNA products. RT-PCR was also performed using primers specific to β-actin to ensure the RNA integrity. The primer sequences used to evaluate XBP-1 expression were as follows: sense primer, 5′-AAACA GAGTA GCAGC ACAGA CTGC-3′; antisense primer, 5′-CTTCC AGCTT GGCTG ATGAG GTCC-3′. Additionally, the primer sequences used to evaluate GADD34 expression were as follows: sense primer, 5′-TAGAG AGCAA GAAGT GGAGC ACACA GC-3′; antisense primer, 5′-CGTCA TCTTC TTCTT CTGTG TCCTC-3′. The sequences of the β-actin sense and antisense primers were 5′-TGGAA TCCTG TGGCA TCCAT GAAAC-3′ and 5′-TAAAA CGCAG CTCAG TAACA GTCCG-3′, respectively. The level of the PCR products was quantified using a Kodak Electrophoresis Documentation and Analysis System 120 and 1% agarose gels.

To investigate an alteration in the expression of ER stress marker proteins, total proteins collected from the transverse colons of No-, Lop+Vehicle and Lop+Urd treated rats were separated by 4–20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for 3 h, after which the resolved proteins were transferred to nitrocellulose membranes for 2 h at 40 V. Each membrane was subsequently separately incubated overnight at 4℃, with the following primary antibodies: eIF2α (Cell Signaling Technology Inc., Cambridge, MA, USA), p-eIF2α (Cell Signaling Technology Inc.), IRE1β (Santa Cruz Biotechnology, Santa Cruz, CA, USA), p- IRE1β (Santa Cruz Biotechnology) and anti-β-actin (Cell Signaling Technology Inc.). The membranes were then washed with washing buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄, and 0.05% Tween 20) and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Zymed Laboratories, South San Francisco, CA, USA) at a dilution of 1:1,000 at room temperature for 2 h. Finally, the membrane blots were developed using Chemiluminescence Reagent Plus kits (Pfizer, New York, NY, USA and Pharmacia, New York, NY, USA). The chemiluminescence signals that originated from specific bands were detected using FluorChemi®FC2 (Alpha Innotech Co., San Leandro, CA, USA).

The number of cells with DNA fragmentation was measured in situ using the BrdU-Red DNA fragmentation (TUNEL) detection kit (BD Bioscience, Franklin Lakes, NJ, USA), according to the manufacturer's protocol (with minor modifications). After the collection of transverse colon from SD rats, tissues were embedded in paraffin; serial sections 4 µm thick were placed onto slides and stained with hematoxylin and eosin. For the TUNEL assay, sections of the transverse colon were deparaffinized, and apoptotic cells were detected using the specific antibody for fragmented DNA assay kit. The percentage of FITC-BrdU positive cells and mean green fluorescence intensity were detected using Moticam pro 285A (Motic, Xiamen, China). All experiments were performed in duplicate.

One-way ANOVA was used to identify significant differences between the No and Lop treated groups (SPSS for Windows, Release 10.10, Standard Version, Chicago, IL, USA). Additionally, differences between the Lop+Vehicle treated group and the Lop+Urd treated group were evaluated by a post hoc test (SPSS for Windows, Release 10.10, Standard Version) of the variance and significance levels. All values are expressed as the means±SD. A P value of <0.05 is considered significant.

To confirm the laxative effects of Urd in a Lopinduced constipation model, an alteration in the feeding behaviors, excretion parameters and histological structure were measured in No, Lop+Vehicle and Lop+Urd treated SD rats. Although the level of food intake and water consumption were maintained constant after Urd treatment, the decrease in the number, weight and water contents of stools in the Lop+Vehicle treated group were almost recovered in the Lop+Urd treated groups, relative to those in the No treated group (Figure 1B-F). A significant recovery was also observed in the histological structure of the transverse colon. The thickness of the mucosa layer and muscle significantly decreased in the Lop+Vehicle treated group, relative to the No treated group. However, these levels were dramatically enhanced by 44 and 121% after Lop+Urd co-treatment, as compared with the Lop+Vehicle treated group (Figure 2). Taken together, these results indicate that Urd treatment symptomatically improves chronic constipation in Lop-induced SD rats, without any changes in the feeding behavior.

We next investigated whether the laxative effects of Urd are correlated with the subsequent abolition of the ER stress response. To achieve this, an alteration in the levels of key proteins of the PERK/eIF2-ATF4 pathway and IRE1α/XBP pathway were evaluated in the transverse colon of Lop-induced constipation rats treated with Urd. In the PERK/eIF2-ATF4 pathway, the levels of eIF2α phosphorylation and GADD34 transcripts were higher (43 and 29%, respectively) in the Lop+Vehicle treated group than the No treated group. However, these levels were almost restored in the Lop+Urd treated group, although the decreased levels were less than those of No treated group (Figure 3).

A similar change was observed in the IRE1α/XBP pathway. The greatest increase in the phosphorylation of IRE1α and XBP-1 transcript was observed in the Lop+Urd treated group, when compared with the Lop+Vehicle treated group (Figure 4). These results indicate that Lop-induced ER stress can be successfully abolished by the laxative effect of Urd treatment in the chronic constipation model.

The prolonged unfolded protein response (UPR) activation is known to promote apoptosis [10]. In order to investigate the correlation between the laxative effects of Urd and ER stress-induced apoptosis, an alteration in the number of apoptotic cells and protein expression were analyzed in Lop+Urd treated rats. After Lop treatment, an increase in the number of apoptotic cells with high fluorescence was observed in tissue sections of the transverse colon. However, there was a significant disappearance in the fluorescence in the Lop+Urd treatment group when compared to the Lop+Vehicle treated group (Figure 5A). Similar results were observed for Bax/Bcl-2 expression; the increase of Bax and Bcl-2 expression levels in the Lop+Vehicle treated group recovered in the Lop+Urd treated group (Figure 5B). These results suggest that the laxative effect of Urd improves the ER stress-induced apoptosis.