Lab Anim Res.  2017 Dec;33(4):298-307. 10.5625/lar.2017.33.4.298.

Correlation between laxative effects of uridine and suppression of ER stress in loperamide induced constipated SD rats

Affiliations
  • 1Department of Biomaterials Science, College of Natural Resources & Life Science/Life and Industry Convergence Research Institute, Pusan National University, Miryang 50463, Korea. dyhwang@pusan.ac.kr

Abstract

A correlation between endoplasmic reticulum (ER) stress and laxative effects was first reported in a constipation model treated with an aqueous extract of Liriope platyphylla (AEtLP) roots. To investigate the correlation between the laxative effect of uridine (Urd) and ER stress response, alterations in the key parameters for ER stress were measured in loperamide (Lop) induced constipation Sprague Dawley (SD) rats treated with Urd. The efficacy of the laxative effect of Urd was notable on the symptoms of chronic constipation, including alteration of stool parameters and structure of the transverse colon, in Lop induced constipated SD rats. In the PERK/eIF2-ATF4 pathway of ER stress response, the levels of eukaryotic initiation factor 2 alpha (eIF2α) phosphorylation and DNA damage-inducible protein (GADD34) transcripts were significantly enhanced in the Lop+Vehicle treated group. However, the levels were restored in the Lop+Urd treated group, although few differences were detected in the decrease rate. Similar changes were observed for levels of inositol-requiring enzyme 1 beta (IRE1β) phosphorylation and X-box binding protein 1 (XBP-1) transcript in the IRE1α/XBP pathway. Furthermore, the number of ER stress-induced apoptotic cells and Bax and Bcl-2 expression were recovered in the Lop+Urd treated group compared to the Lop+Vehicle treated group. The results of the present study therefore provide first evidence that the laxative effects of Urd may be tightly correlated with the recovery of ER stress response in constipation models.

Keyword

Uridine; ER stress response; Constipation; Laxative effects; Loperamide; apoptosis

MeSH Terms

Animals
Apoptosis
Carrier Proteins
Colon, Transverse
Constipation
DNA
Endoplasmic Reticulum
Eukaryotic Initiation Factor-2
Loperamide*
Phosphorylation
Rats*
Uridine*
Carrier Proteins
DNA
Eukaryotic Initiation Factor-2
Loperamide
Uridine

Figure

  • Figure 1 Evaluation of the feeding behavior and stool parameters in Lop+Urd treated SD rats. (A) Chemical structure of Urd. (B and C) Food intake and water consumption were measured at every day. (D–F) Key stool parameters, including weight, number and water contents, were analyzed as described in Materials and Methods. Data represents the mean±SD from three replicates. *P<0.05 compared to the No treated group. #P<0.05 compared to the Lop+Vehicle treated group.

  • Figure 2 Alteration of the histological structure in transverse colons. (A) Hematoxylin and eosin (H&E) stained sections of transverse colons from the No treated group, Lop+Vehicle treated group or Lop+Urd treated group were observed at two different magnifications (100× and 400×) using a light microscope. (B) The thickness of the mucosa and muscular layer are presented as graphs. Data represents the mean±SD from three replicates. *P<0.05 compared to the No treated group. #P<0.05 compared to the Lop+Vehicle treated group.

  • Figure 3 Expression of marker proteins in the PERK/eIF2-ATF4 pathway. (A) The expression levels of eIF2α and p-eIF2α in total proteins were detected with specific antibodies. The level of β-actin is also shown as an endogenous control. The band intensity of these proteins was determined using an imaging densitometer, and the relative levels of each protein were calculated based on the intensity of actin protein as an endogenous control. (B) The levels of GADD34 transcripts in total mRNA of transverse colons were measured by RT-PCR using specific primers. After the intensity of each band was determined using an imaging densitometer, the relative levels of GADD34 transcripts were calculated based on the intensity of actin transcripts. Data represent the means±SD from three replicates. *P<0.05 compared to the No treated group. #P<0.05 compared to the Lop+Vehicle treated group.

  • Figure 4 Expression of marker proteins in IRE1α/XBP pathway. (A) The expression levels of IRE1α and p- IRE1α in total proteins were detected with specific antibodies. The level of β-actin is also shown as an endogenous control. The band intensity of the proteins was determined using an imaging densitometer, and the relative levels of each protein were calculated based on the intensity of actin protein as an endogenous control. (B) The levels of XBP-1 transcripts in total mRNA of transverse colons were measured by RT-PCR using specific primers. After the intensity of each band was determined using an imaging densitometer, the relative levels of XBP-1 transcripts were calculated based on the intensity of actin transcripts. Data represents the means±SD from three replicates. *P<0.05 compared to the No treated group. #P<0.05 compared to the Lop+Vehicle treated group.

  • Figure 5 Level of apoptotic cells and proteins. (A) Representative images show staining of TUNEL in transverse colon treated with Lop+Vehicle or Lop+Urd as indicated. The transverse colon of normal (No) rats were used as controls. (B) Total tissue lysates were prepared from the transverse colon of Lop-injected SD rats treated with Vehicle or Urd, as described in the Materials and Methods. A total of 50 µg of protein per sample was immunoblotted with antibodies for each protein. Three samples were assayed in triplicate by western blotting. Data represents the means±SD of three replicates. *P<0.05 compared to the No treated group. #P<0.05 compared to the Lop+Vehicle treated group.


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