Rhubarb tannins extract inhibits the expression of aquaporins 2 and 3 in magnesium sulphate-induced diarrhoea model

Abstract

Tannins, a group of major active components of Chinese rhubarb and widely distributed in nature, have a significant antidiarrhoeal activity. Aquaporins (AQPs) 2 and 3 play important roles in regulating water transfer during diarrhoea. The present study aims to determine the effect of the total tannins extract of rhubarb on aquaporins (AQPs) 2 and 3 in diarrhoea mice and HT-29 cells both induced by magnesium sulphate (MgSO4). Our results showed that rhubarb tannins extract (RTE) significantly decreased the faecal water content in colon and evaluation index of defecation of diarrhoea mice. Interestingly, RTE could markedly reduce the mRNA and protein expression levels of AQPs 2 and 3 in apical and lateral mucosal epithelial cells in the colons of diarrhoea mice and HT-29 cells both induced by MgSO4 in a dose-dependent manner. Furthermore, RTE suppressed the production of cyclic monophosphate- (cAMP-) dependent protein kinase A catalytic subunits α (PKA C-α) and phosphorylated cAMP response element-binding protein (p-CREB, Ser133) in MgSO4-induced HT-29 cells. Our data showed for the first time that RTE inhibit AQPs 2 and 3 expression in vivo and in vitro via downregulating PKA/p-CREB signal pathway, which accounts for the antidiarrhoeal effect of RTE.

Figure 1

HPLC profile (UV chromatograms at 280 nm) of rhubarb tannins extract (RTE). (a) HPLC fingerprint of RTE. (1)—RTE. (2)—standards. (b) Structure of identified components of RTE. (A)—gallic acid. (B)—procyanidin-B1. (C)—catechin. (D)—procyanidin-B2. (E)—epicatechin. (F)—epigallocatechin gallate.

Figure 2

Rhubarb tannins extract (RTE) has the antidiarrhoeal activity in magnesium sulphate- (MgSO₄-) induced diarrhoea mice. Mice were orally administrated with RTE (125, 250, and 500 mg/kg, resp.) or water daily for 3 days before MgSO₄-induced diarrhoea. During the experiment, fecal water content and evacuation index (EI) of mice defecation were evaluated. (a) Doses of 125~500 mg/kg RTE significantly decreased the fecal water content dose-dependently compared with vehicle-treated diarrhoea mice. (b) Doses of 125~500 mg/kg RTE significantly decreased the EI of defecation in a dose-dependent manner compared with vehicle-treated diarrhoea mice. (c) Macroscopic evidence of watery stool in colon was markedly observed in vehicle-treated diarrhoea mice, while dose of 125~500 mg/kg RTE significantly alleviated the watery stool of diarrhoea mice. (d) Histological structure of colon in mice induced by MgSO₄ appeared edematous while RTE treatment seemed to reduce edema. Data are represented as the mean ± SD. ^### P < 0.001 versus normal control group. *P < 0.05, **P < 0.01, and ***P < 0.001 versus vehicle group, respectively. n = 6 in each group and each assay was repeated 3 times.

Figure 3

Rhubarb tannins extract (RTE) reduces the protein expression of aquaporins (AQPs) 2 and 3 in the colons of magnesium sulphate-induced diarrhoea mice by fluorescent immunohistochemistry. ((a) and (b)) Localization of AQPs 2 and 3 in both the apical and lateral mucosal epithelial cells in the proximal colons of normal control, vehicle-treated diarrhea, and RTE- (500 mg/kg) treated diarrhoea mice. Negative controls in which the AQPs 2 and 3 antibodies were replaced with normal rabbit immunoglobulin (IgG) at identical concentrations resulted in a lack of specific staining. Tissue is colocalized with DAPI to demonstrate the location of cell nuclei (blue). The images in merge' panel are the big magnification in merge panel, respectively. ((c) and (d)) Positive expression levels of AQPs 2 and 3 in the colons of normal control, vehicle-treated diarrhea, and RTE- (125, 250, and 500 mg/kg) treated diarrhoea mice. 10 microscopic fields were selected randomly and AQPs 2 and 3 positive cells were counted. The AQPs 2 and 3 positive expression levels of the control were taken as 100%. Data are represented as the mean ± SD. ^### P < 0.001 versus normal control group. *P < 0.05, **P < 0.01, and ***P < 0.001 versus vehicle group, respectively. n = 6 in each group and each assay was repeated 3 times.

Figure 4

Rhubarb tannins extract (RTE) inhibits the protein and gene expression of aquaporins (AQPs) 2 and 3 in the colons of magnesium sulphate-induced diarrhoea mice by western blot and real-time Reverse Transcription-Polymerase Chain Reaction. ((a) and (b)) Expression levels of AQPs 2 and 3 protein in the colons of normal control, vehicle-treated diarrhea, and RTE- (125, 250, and 500 mg/kg) treated diarrhoea mice. ((c) and (d)) Expression levels of AQPs 2 and 3 mRNA in the colons of mice. Data are represented as the mean ± SD. ^### P < 0.001 versus normal control group. *P < 0.05, **P < 0.01, and ***P < 0.001 versus vehicle group, respectively. n = 6 in each group and each assay was repeated 3 times.

Figure 5

Rhubarb tannins extract (RTE) decreases the protein expression of aquaporins (AQPs) 2 and 3 in HT-29 cells induced by magnesium sulphate (MgSO₄) by fluorescent immunohistochemistry. Cells were placed in 24-well plate or 100-mm dishes for 24 h in the presence of MgSO₄ with or without RTE (control, MgSO_4, and RTE- (20, 40, and 80 μg/mL) treatment groups, resp.). HT-29 cells were fixed, stained by immunofluorescence, and scanned in 10 random fields. ((a) and (b)) Immunolocalization of AQPs 2 (green) and 3 (red) in HT-29 cells. Negative controls in which the AQPs 2 and 3 antibodies were replaced with normal rabbit immunoglobulin (IgG) at identical concentrations resulted in a lack of specific staining. Nuclei were stained with DAPI (blue). ((c) and (d)) Positive expression levels of AQPs 2 and 3 in HT-29 cells. 10 microscopic fields were selected randomly and AQPs 2 and 3 positive cells were counted. The AQPs 2 and 3 positive expression levels of the control were taken as 100%. Data are represented as the mean ± SD. ^### P < 0.001 versus normal control group. *P < 0.05, **P < 0.01, and ***P < 0.001 versus vehicle group, respectively. n = 3 in each group and each assay was repeated 3 times.

Figure 6

Rhubarb tannins extract (RTE) reduces the protein expression of aquaporins (AQPs) 2 and 3 in magnesium sulphate- (MgSO₄-) induced HT-29 cells by western blot. Cells were placed in 24-well plate or 100-mm dishes for 24 h in the presence of MgSO₄ with or without RTE (control, MgSO₄, and RTE- (20, 40, and 80 μg/mL) treatment groups, resp.). ((a) and (b)) Expression levels of AQPs 2 and 3 protein in HT-29 cells treated by RTE (80 μg/mL) for different time (0, 3, 6, 12, and 24 h, resp.). ((c) and (d)) Expression levels of AQPs 2 and 3 proteins in HT-29 cells treated by RTE at the dosages of 20, 40, and 80 μg/mL for 24 h, respectively. Data are represented as the mean ± SD. ^### P < 0.001 versus normal control group. *P < 0.05, **P < 0.01, and ***P < 0.001 versus vehicle group, respectively. n = 3 in each group and each assay was repeated 3 times.

Figure 7

Rhubarb tannins extract (RTE) downregulates the expression of magnesium sulphate- (MgSO₄-) activated PKA and p-CREB in HT-29 cells without effect on cell viability. Cells were placed in 100-mm dishes for 24 h in the presence of MgSO₄ with or without RTE (control, MgSO_4, and RTE- (20, 40, and 80 μg/mL) treatment groups, resp.). HT-29 cells were collected to detect the expression levels of PKA (a) and p-CREB (b) protein by western blot analysis. (c) No effect of RTE (20, 40, 80 μg/mL, resp.) on the cell viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. Cell viability of the control was taken as 100%. Data are represented as the mean ± SD. ^### P < 0.001 versus normal control group. *P < 0.05, **P < 0.01, and ***P < 0.001 versus vehicle group, respectively. n = 3 in each group and each assay was repeated 3 times.