The flavonoid compound apigenin prevents colonic inflammation and motor dysfunctions associated with high fat diet-induced obesity

Abstract

Background and purpose: Apigenin can exert beneficial actions in the prevention of obesity. However, its putative action on obesity-associated bowel motor dysfunctions is unknown. This study examined the effects of apigenin on colonic inflammatory and motor abnormalities in a mouse model of diet-induced obesity.

Experimental approach: Male C57BL/6J mice were fed with standard diet (SD) or high-fat diet (HFD). SD or HFD mice were treated with apigenin (10 mg/Kg/day). After 8 weeks, body and epididymal fat weight, as well as cholesterol, triglycerides and glucose levels were evaluated. Malondialdehyde (MDA), IL-1β and IL-6 levels, and let-7f expression were also examined. Colonic infiltration by eosinophils, as well as substance P (SP) and inducible nitric oxide synthase (iNOS) expressions were evaluated. Motor responses elicited under blockade of NOS and tachykininergic contractions were recorded in vitro from colonic longitudinal muscle preparations.

Key results: When compared to SD mice, HFD animals displayed increased body weight, epididymal fat weight and metabolic indexes. HFD mice showed increments in colonic MDA, IL-1β and IL-6 levels, as well as a decrease in let-7f expression in both colonic and epididymal tissues. HFD mice displayed an increase in colonic eosinophil infiltration. Immunohistochemistry revealed an increase in SP and iNOS expression in myenteric ganglia of HFD mice. In preparations from HFD mice, electrically evoked contractions upon NOS blockade or mediated by tachykininergic stimulation were enhanced. In HFD mice, Apigenin counteracted the increase in body and epididymal fat weight, as well as the alterations of metabolic indexes. Apigenin reduced also MDA, IL-1β and IL-6 colonic levels as well as eosinophil infiltration, SP and iNOS expression, along with a normalization of electrically evoked tachykininergic and nitrergic contractions. In addition, apigenin normalized let-7f expression in epididymal fat tissues, but not in colonic specimens.

Conclusions and implications: Apigenin prevents systemic metabolic alterations, counteracts enteric inflammation and normalizes colonic dysmotility associated with obesity.

Fig 1. In HFD mice, apigenin counteracted the increase in body and epididymal fat weight.

(A) Body weight gain (%) in mice fed with SD, SD plus treatment with apigenin (10 mg/Kg/day), HFD or HFD plus treatment with apigenin (10 mg/Kg/day) for 8 weeks. Values are means ± SEM, n = 5. ^aP<0.05 significant difference vs SD at the respective weeks; *P<0.05 significant difference vs HFD at the respective weeks. (B) Epididymal fat weight in mice fed with SD, SD plus treatment with apigenin (10 mg/Kg/day), HFD or HFD plus treatment with apigenin (10 mg/Kg/day) for 8 weeks. Values are means±SEM, n = 5. ^cP<0.001, significant difference vs SD; ^$P <0.01 significant difference vs HFD.

Fig 2. In HFD mice, apigenin decreased MDA, IL-1β and IL-6 colonic levels.

MDA (A), IL-1β (B) and IL-6 (C) levels in colonic tissues from mice fed with SD, SD plus treatment with apigenin (10 mg/Kg/day), HFD or HFD plus treatment with apigenin (10 mg/Kg/day). Values are means±SEM, n = 5. ^aP < 0.05, ^cP < 0.001 significant difference vs SD; *P <0.05, ^#P<0.001 difference difference vs HFD.

Fig 3. In HFD mice, apigenin normalized let-7f expression in epididymal fat tissues but not in colonic specimens.

let-7f expression in colonic (A) and epididymal fat (B) tissues from SD, SD plus treatment with apigenin (10 mg/Kg/day), HFD or HFD plus treatment with apigenin (10 mg/Kg/day). Values are means±SEM, n = 5.^cP < 0.001 significant difference vs SD, ^bP<0.01 significant difference vs SD, ^*P<0.05 significant difference vs HFD.

Fig 4. Apigenin decrease eosinophil infiltration in colonic tissues from HFD-mice.

Representative microscopic pictures of haematoxylin/eosin-stained colonic sections from mice fed with SD, SD plus treatment with apigenin (10 mg/Kg/day), HFD or HFD plus treatment with apigenin (10 mg/Kg/day). The magnification within the boxed area shows the presence of eosinophils (green arrows) and neutrophils (yellow arrows). Scale bars = 50 μm; 20 μm (inset). The column graphs display the mean value of eosinophil density per square millimeter of tunica mucosa/submucosa areas (cell/mm²) ± SEM, n = 5. ^aP<0.05 significant difference vs SD; *P<0.05 significant difference vs HFD.

Fig 5. Apigenin reduced SP and iNOS expression in colonic myenteric ganglia from HFD mice.

Representative pictures of SP and iNOS immunostaining of cross-sections from full-thickness mice colonic specimens from SD, SD plus treatment with apigenin (10 mg/Kg/day), HFD or HFD plus treatment with apigenin (10 mg/Kg/day). Scale bar = 20μm. Column graphs display the quantitative assessment of SP and iNOS in myenteric ganglion area. Each column represents the mean value of PPP±SEM, n = 5. ^aP<0.05 significant difference vs SD; *P<0.05 significant difference vs HFD.

Fig 6. In HFD mice, apigenin normalized electrically evoked contractions elicited under NOS blockade or mediated by tachykininergic stimulation.

Electrically evoked (ES, 10 Hz) contractile activity of colonic longitudinal smooth muscle preparations isolated from mice fed with SD, SD plus treatment with apigenin (10 mg/Kg/day), HFD, or HFD treated with apigenin (10 mg/Kg/day). (A) Colonic tissues maintained in standard Krebs solution. (B) Colonic tissues maintained in Krebs solution containing guanethidine (10 μM) and L-NAME (100 μM). (C) Colonic tissues maintained in Krebs solution containing guanethidine (10 μM), L-NAME (100 μM), GR159897 (1 μM), SB218795 (1 μM) and atropine (1 μM). Values are means±SEM, n = 5. ^aP<0.05, ^bP<0.01 significant difference vs SD; *P<0.05, ^$P<0.01 significant difference vs HFD.