博士 / 國立臺灣大學 / 解剖學暨細胞生物學研究所 / 103 / Arecoline, a major alkaloid in areca nuts, is involved in the pathogenesis of inflammatory oral diseases, cancers and in the developmentally toxic effect of lowering weight and retarding growth of the embryo.
Experiment I：Mammalian taste buds are the basic structural unit for detecting taste stimuli in the oral cavity, however, the effects of arecoline on the taste bud are poorly understood. We injected arecoline intraperitoneally into C57BL/6 mice twice daily for 1 - 4 weeks and aimed to revealthe taste bud morphology, life span and gustatory functional activity by immunohistochemistry (IHC) and electron microscopy.
At end of arecoline treatment, the animals were sacrificed through perfusion, and the vallate papillae were excised and processed for electron microscopy and immunohistochemistry (IHC) analysis of taste receptor proteins (T1R2, T1R3, T1R1 and T2R) and taste associated proteins (α-gustducin, PLCβ2 and SNAP25). Expression levels of c-fos were detected in the solitary nucleus and gustatory cortex. Body weight, food intake and water consumption were recorded during the treatment period every other day. After arecoline treatment, a two-bottle preference test between water and 1 % sucrose was performed for 4 weeks. For study of taste bud cells life-span, mice were also injected with BrdU (50 mg/kg i.p.) at end of arecoline treatment and then perfused at day 1 to day 14 following BrdU administration.
The results demonstrated that (1) arecoline treatment did not change the number and size of the taste buds, and the number of taste bud cells was not affected, (2) electron microscopy revealed the swollen mitochondria, dilated endoplasmic reticulum cisternae, and numerous irregular autophagosomes accumulated in type II cells, (3) IHC demonstrated a decrease in the number of taste receptor T1R2- and T1R3-expressing cells, (4) the level of c-fos expression was decreased in the solitary nucleus and gustatory cortex, (5) the body weight and food intake amount were markedly reduced, (6) the sweet preference behaviour was-reduced, (7) the number of BrdU-labeled taste bud cells was significant reduced at 1, 3, 7 and 14 days, and (8) PCR array experiments showed that the expression of cyclin B2 and E2F1, was markedly downregulated, but the expression of p53 and bax was markedly upregulated by arecoline in the circumvallate.
We conclude that the long-term arecoline injection alters the morphology of type II taste bud cells, retards the growth of mice from puberty to adulthood, affects gustatory discrimination competencies, also inhibits taste progenitor cells proliferation and shorten life span of renew taste bud cells.
Experiment II：The effects of arecoline on birth defects have been explored in many species, including chicken, mice, and zebrafish. The effects of arecoline on embryos after long-term exposure are well established, however, the effects of short-term arecoline exposure to embryos are not fully understood. Using zebrafish as an animal model, we studied the effects of short-term exposure of arecoline on zebrafish embryos to mimic the areca nut-chewing woman during early pregnancy. Arecoline, at concentrations from 0.001 to 0.04%, was administered to zebrafish embryos from 4 to 24 hpf (hours post fertilization). The morphological changes, hatching and survival rates, body length, and somitic skeletal muscle fiber structure were investigated by immunohistochemistry (IHC), confocal microscopy, and conventional electron microscopy.
With exposure of zebrafish embryos to the increasing concentrations of arecoline, we observed a significant decline in the hatching and survival rates, general growth retardation, lower locomotor activity and impairment of swimming ability. Immuno-fluorescence staining demonstrated a loose arrangement of myosin heavy chains, and ultrastructural observations revealed an altered arrangement of somitic myofibril and swelling of the mitochondria. In addition, the results from flow-cytometry, JC-1 staining to assay mitochondria activity, and RT-PCR analysis of mitochondrial functional gene expression, revealed mitochondrial dysfunctions after exposure to arecoline.
We confirmed that short-term arecoline exposure resulted in retarded embryonic development and decreased locomotor activity due to defective somitic skeletal muscle development and mitochondrial dysfunction.
| Identifer | oai:union.ndltd.org:TW/103NTU05391012 |
| Date | January 2015 |
| Creators | Wei-Hao Peng, 彭偉豪 |
| Contributors | Kuo-Shyan Lu, 盧國賢 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 130 |
Page generated in 0.0157 seconds