Effects of aged garlic extract on aging?related changes in gastrointestinal function and enteric nervous system cells

Abstract

Dysmotility of the gastrointestinal (GI) tract is commonly seen in elderly individuals, where it causes significant morbidity and can lead to more severe conditions, including sarcopenia and frailty. Although the precise mechanisms underlying aging-related GI dysmotility are not fully understood, neuronal loss or degeneration in the enteric nervous system (ENS) may be involved. Aged garlic extract (AGE) has been shown to have several beneficial effects in the GI tract; however, it is not known whether AGE can improve GI motility in older animals. The aim of the present study was to examine the effects of AGE on the ENS and gut motility in older mice and elucidate potential mechanisms of action. An AGE-formulated diet was given to 18-month-old female mice for 2 weeks. Organ bath studies and cell culture demonstrated that AGE: i) Altered gut contractile activity; ii) enhanced viability of ENS cells; and iii) exhibited neuroprotective effects on the ENS via reduction in oxidative stress. These findings suggest that AGE could be used to develop novel dietary therapeutics for aging-related GI dysmotility by targeting the associated loss and damage of the ENS.

Keywords: aged garlic extract; aging; enteric nervous system; intestinal motility; neuronal nitric oxide synthase; neuroprotection; oxidative stress.

Figure 1

Effects of AGE on aging-related colorectal dysfunction in old mice. (A) Experimental overview. (B) GI transit time, (C) rectal bead expulsion time, (D) fecal pellet output and (E) fecal water content were measured to determine the GI motility in ‘Young’, ‘Old’ and ‘Old + AGE’ mice. (F) No significant difference in food intake was observed in each group. Results are shown as mean ± SEM, n=6/group. ^*P<0.05, ^**P<0.01, ^***P<0.001, ^****P<0.0001. AGE, aged garlic extract; GI, gastrointestinal.

Figure 2

Effects of AGE on colonic smooth muscle activity. Representative traces of contractile activities of colonic smooth muscle in (A) spontaneous (double head arrows indicate range in which area under curve was quantified) and (B) in response to EFS (double head arrows indicate quantified maximum amplitude). Red and black arrows represent addiction of AGE or L-NAME, and application of EFS, respectively. (C) Area under curve (n=12/group) and (D) maximum amplitude of muscle contraction (n=7/group) were measured and compared between in the absence or presence of AGE. (E) L-NAME added to the organ bath abolished the effects of AGE (n=5/group). Results are shown as mean ± SEM. ^*P<0.05. EFS, electric field stimulation; L-NAME, NG-Nitroarginine methyl ester; AGE, aged garlic extract.

Figure 3

Effects of AGE on cultured ENS cells. (A) Significant reduction in viability of cells isolated from ‘Old’ mice. AGE improved viability of (B) ‘Young’- or (C) ‘Old’-derived ENSCs. (D-I) ENS cells were isolated from Plp1^GFP; Baf53b-tdT mice. (F, G and J) AGE increased the number of Plp1-GFP positive glia/neural progenitors (yellow arrows) and (D, E and K) Baf-tdT positive neurons (white arrows), respectively (dashed box enlarged in inset). Scale bars, 50 µm; magnification, x20. Results are shown as mean ± SEM, n=3/group. ^*P<0.05, ^**P<0.01, ^***P<0.001, ^****P<0.0001. AGE, aged garlic extract; ENS, enteric nervous system; ENSC, enteric neural stem cells.

Figure 4

Effects of AGE on proliferation of cultured enteric neural cells. Cultured enteric neural cells were treated with PBS as vehicle control and immunostained for (A) Hu (neuron), (C) P75 (glia/neural progenitor cell) and (E) EdU, with the merged image shown in (G), (dashed box enlarged in inset). Treatment of 1 mg/ml AGE for cultured enteric neural cells was performed, followed by immunocytochemistry for (B) Hu, (D) P75 and (F) EdU, with the merged image presented in (H) (dashed boxes indicate enlarged insets). White arrows indcate Hu+/EdU+ neurons by treatment of (A and E) vehicle control and (B and F) 1 mg/ml AGE, respectively. (D and F) P75+/EdU+ cells were marked by yellow arrows in presence of 1 mg/ml AGE. Both the number of (I) Hu+/EdU+ neurons and (J) P75+/EdU+ cells significantly increased in the presence of 1 mg/ml AGE. Scale bars, 50 µm; magnification, x20, Results are shown as mean ± SEM, n=3/group. ^**P<0.01. EdU; 5-ethynyl-2'-deoxyuridine; AGE, aged garlic extract.

Figure 5

Effects of AGE on oxidative stress in myenteric plexus of old mice. MitoSOX staining of colonic myenteric layer of (A) ‘Young’ and (B) ‘Old’ and (C) ‘Old + AGE’ mice. (D) Enlarged image of white box is shown in (B). (E) AGE-fed old mice showed significantly less ROS in myenteric layer. Scale bars, 100 µm; magnification, x20. Results are shown as mean ± SEM, n=4/group. ^*P<0.05, ^***P<0.001. ROS, reactive oxygen species; MFI, mean fluorescence intensity; AGE, aged garlic extract.

Figure 6

Neuroprotective effects of AGE on oxidative stress-induced enteric neural cells in culture. (A and B) Fluorescence images showed neurons and glial/neural progenitor cells labeled with Baf53b-tdT and Plp1GFP, respectively in the presence of PBS as a vehicle control, exhibiting normal morphology, and (C) is the merged image of (A) and (B). (D) The tdT-labeled neurons exposed to 100 µM hydroperoxide display neuronal damage. (E) GFP-labeled glial/neural progenitor cells appeared to a reduction of cell number, and (F) is the merged image of (D) and (E). The tdT-labeled neurons and GFP-labeled glial/neural progenitor cells were co-treated with 100 µM hydroperoxide and (G and H) 0.5 mg/ml or (J and K) 1 mg/ml AGE, which exhibited neuroprotective effects. (I) Presents the merged image of (G) and (H), and (L) presents the combined image of (J) and (K). The (M) number of neurons and (N) glial/neural progenitors, (O) neurite length and (P) cell viability were measured. Scale bars, 50 µm; magnification, x10. Results are shown as mean ± SEM, n=3/group. ^*P<0.05, ^**P<0.01, ^***P<0.001, ^****P<0.0001. AGE, aged garlic extract.