Naringenin induces laxative effects by upregulating the expression levels of c-Kit and SCF, as well as those of aquaporin 3 in mice with loperamide-induced constipation

Abstract

Constipation is a common affliction which causes discomfort and affects the quality of life of affected individuals. Naringenin (NAR), a natural flavonoid widely found in citrus fruits and tomatoes, has been reported to exhibit various pharmacological effects, such as anti-inflammatory, anti-atherogenic, anti-mutagenic, hepatoprotective and anticancer effects. Increasing evidence has indicated that NAR has potential for use in the treatment of constipation. Thus, the aim of this study was to evaluate the laxative effects of NAR in mice with loperamide-induced (Lop-induced) constipation. The data indicated that NAR relieved Lop-induced constipation in mice based on the changes of fecal parameters (numbers, weight and water content), the intestinal charcoal transit ratio and the histological alteration. ELISA revealed that NAR regulated the production levels of gastrointestinal metabolic components, such as motilin (MTL), gastrin (Gas), endothelin (ET), substance P (SP), acetylcholinesterase (AChE) and vasoactive intestinal peptide (VIP) in serum. The expression levels of enteric nerve-related factors, glial cell line-derived neurotrophic factor (GDNF), transient receptor potential vanilloid 1 (TRPV1), nitric oxide synthase (NOS), c-Kit, stem cell factor (SCF) and aquaporin 3 (AQP3) were examined by western blot analysis and RT-PCR analysis. The results of this study suggest that NAR relieves Lop-induced constipation by increasing the levels of interstitial cells of Cajal markers (c-Kit and SCF), as well as AQP3. Thus, NAR may be effective as a candidate in patients suffering from lifestyle-induced constipation.

Figure 1

Body weight and fecal parameters following intragastric administration with or without NAR in mice with Lop-induced constipation. (A) Body weight, (B) fecal number, (C) fecal weight and (D) water contents were measured at the same time during the experiment. Six mice per group were assayed in triplicate for body weight and fecal parameters analysis. Data represent the means ± SD from 3 replicates. ^**P<0.01 compared to the No group; ^#P<0.05 and ^##P<0.01 compared to Lop + Vehicle group. Lop, loperamide; NAR, naringenin. The mouse groups were as follows: No, negative control, mice treated with saline; Lop + Vehicle, mice treated with Lop and the vehicle; Lop + NAR75, mice treated with Lop and NAR at 75 mg/kg body weight; Lop + NAR150, mice treated with Lop and NAR at 150 mg/kg body weight; Lop + NAR300, mice treated with Lop and NAR at 300 mg/kg body weight.

Figure 2

Effects of NAR on GI transit in mice with Lop-induced constipation. Consitipation was induced by the intragastric administration of Lop followed by treatment with or without NAR. The length of charcoal from stomach in the intestine was measured after the administration of charcoal. Charcoal transit ratio (%) = (distance travelled by the charocal)/(total length of small intestine) ×100%. ^**P<0.01 compared to the No group; ^##P<0.01 compared to Lop + Vehicle group. NAR, naringenin; GI, gastrointestinal; Lop, loperamide. The mouse groups were as follows: No, negative control, mice treated with saline; Lop + Vehicle, mice treated with Lop and the vehicle; Lop + NAR75, mice treated with Lop and NAR at 75 mg/kg body weight; Lop + NAR150, mice treated with Lop and NAR at 150 mg/kg body weight; Lop + NAR300, mice treated with Lop and NAR at 300 mg/kg body weight.

Figure 3

Effects of NAR on serum parameters in mice with Lop-induced constipation. Consitipation was induced by the intragastric administration of Lop followed by treatment with or without NAR. The serum was collected separately for the analysis of hte concentrations of (A) MTL, (B) Gas, (C) ET, (E) AChE, (D) SP and (F) VIP in mice with Lop-induced constipation by ELISA. ^**P<0.01 compared to the No group; ^#P<0.05 and ^##P<0.01 compared to Lop + Vehicle group. NAR, naringenin; Lop, loperamide; MTL, motilin; Gas, gastrin; AChE, acetylcholinesterase; SP, substance P; VIP, vasoactive intestinal peptide. The mouse groups were as follows: No, negative control, mice treated with saline; Lop + Vehicle, mice treated with Lop and the vehicle; Lop + NAR75, mice treated with Lop and NAR at 75 mg/kg body weight; Lop + NAR150, mice treated with Lop and NAR at 150 mg/kg body weight; Lop + NAR300, mice treated with Lop and NAR at 300 mg/kg body weight.

Figure 4

Histological alterations and the expression levels of enteric nerve-related factors in the colons of mice with Lop-induced constipation. (A) H&E-stained sections of colon tissues of mice from the No, Lop + Vehicle, Lop + NAR75, Lop + NAR150 and Lop + NAR300 groups were observed using a light microscope. Scale bar, 400 µM. (B) NAR regulated the mRNA levels of enteric nerves-related factors in the colons of Lop-induced constipated mice. Consitipation was induced by the intragastric administration of Lop followed by treatment with or without NAR. The mRNA levels of TRPV1, GDNF, BDNF and NOS in colons of mice iwth Lop-induced constipation were analyzed by RT-PCR. (C) The expression levels of enteric nerve-related factors in the colons of mice with Lop-induced constipation. (C) The expression levels of TRPV1, GDNF, BDNF and NOS were examined by western blot analysis and the quantitative analysis of gray intensity was calculated and shown in (D). ^**P<0.01 compared to the No group; ^#P<0.05 and ^##P<0.01 compared to Lop + Vehicle group. Lop, loperamide; NAR, naringenin; TRPV1, transient receptor potential cation channel subfamily V member 1; GDNF, glial cell line-derived neurotrophic factor; BDNF, brain-derived neurotrophic factor; NOS, nitric oxide synthase. The mouse groups were as follows: No, negative control, mice treated with saline; Lop + Vehicle, mice treated with Lop and the vehicle; Lop + NAR75, mice treated with Lop and NAR at 75 mg/kg body weight; Lop + NAR150, mice treated with Lop and NAR at 150 mg/kg body weight; Lop + NAR300, mice treated with Lop and NAR at 300 mg/kg body weight.

Figure 5

Effect of NAR on the expression levels of c-Kit and SCF in the colons of mice with Lop-induced constipation. (A) The mRNA levels of c-Kit and SCF in colons of mice with Lop-induced constipation were analyzed by RT-PCR. (B and C) The expression levels of c-Kit and SCF in the colons of mice with Lop-induced constipation. (B) The expression levels of c-Kit and SCF were examined by western blot analysis and the quantitative analysis of gray intensity was calculated and shown in (C). ^**P<0.01 compared to the No group; ^#P<0.05 and ^##P<0.01 compared to Lop + Vehicle group. NAR, naringenin; Lop, loperamide; SCF, stem cell factor. The mouse groups were as follows: No, negative control, mice treated with saline; Lop + Vehicle, mice treated with Lop and the vehicle; Lop + NAR75, mice treated with Lop and NAR at 75 mg/kg body weight; Lop + NAR150, mice treated with Lop and NAR at 150 mg/kg body weight; Lop + NAR300, mice treated with Lop and NAR at 300 mg/kg body weight.

Figure 6

NAR increases the production of AQP3 in the colons of mice with of Lop-induced constipation. (A) NAR upregulated the mRNA levels of AQP3 which were decreased by Lop in the colons of constipated mice. The mRNA level of AQP3 in colons of mice with Lop-induced constipation mice was examined by RT-PCR analysis. (B) The expression level of AQP3 in the colons of Lop-induced constipated mice. (C) The expression level of AQP3 was examined by western blot analysis and the quantitative analysis of gray intensity was calculated and shown in (C). (D) Protein expression of AQP3 in the colons of mice Lop-induced constipation detected by immunohistochemistry. Scale bar, 400 µM. ^**P<0.01 compared to the No group; ^#P<0.05 and ^##P<0.01 compared to Lop+Vehicle group. Lop, loperamide; NAR, naringenin; AQP3, aquaporin 3. The mouse groups were as follows: No, negative control, mice treated with saline; Lop + Vehicle, mice treated with Lop and the vehicle; Lop + NAR75, mice treated with Lop and NAR at 75 mg/kg body weight; Lop + NAR150, mice treated with Lop and NAR at 150 mg/kg body weight; Lop + NAR300, mice treated with Lop and NAR at 300 mg/kg body weight.