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Outline
To study the effect of arecoline on rat embryo cartilage development in vitro, so as to explore the embryonic teratogenicity of arecoline.
The embryonic limb buds of gestation day 14 timed-pregnant (GD14) rats were taken, and the forelimb buds were cultured in micromass (MM). The teratogenicity of arecoline was determined by the half-proliferation inhibitory concentration (IC50-P), the half-differentiation inhibitory concentration (IC50-D) and their ratios. The hindlimb buds were cultured in organ culture and randomly divided into five experimental groups, i.e., blank control (0 mg·mL-1 arecoline), low-dose (0.1 mg·mL-1 arecoline), middle-dose (1 mg·mL-1 arecoline), high-dose (10 mg·mL-1 arecoline), and positive control group (10 ng·mL-1 5-fluorouracil). They were cultured in vitro for 24, 72, and 120 h, respectively, and then harvested. Morphological scoring was performed under a stereo microscope, and followed by paraffin embedding, sectioning, HE staining and toluidine blue staining to observe the morphology cartilage cells. The effects of arecoline on the expression of bone morphogenetic protein 2 (BMP-2) in each group were studied by immunohistochemistry.
In MM model, according to the ECVAM prediction model, it was determined that arecoline was a developmental toxic chemical and a non-specific weak teratogen. In the limb bud organ model, 1 mg·mL-1 (medium dose) and above concentrations of can cause developmental toxicity arecoline to limb bud organs. Arecoline at 10 mg·mL-1 (high dose) can produce significant toxicity after 24 h of administration. The developmental status of each dose group has a significant measurement correlation after 72 h of culture. It can be seen from the pathological section results that arecoline further affected the proliferation and differentiation of proliferating chondrocytes and hypertrophic chondrocytes, thereby affecting the development of limb buds, palms and limbs. In the immunohistochemical analysis, the expression intensity of BMP-2 was related to the dose of arecoline. The larger the dose of arecoline used, the lower degree of positive expression obtained, reflecting the slowdown of chondrocyte proliferation and differentiation.
Arecoline at concentration over 1 mg·mL-1 inhibited the growth of rat embryonic limb buds and cartilage development in vitro. It can be preliminarily believed that arecoline has weak teratogenicity on embryonic development, and arecoline affected the expression of BMP-2 to inhibit the proliferation and differentiation of chondrocytes directly or indirectly.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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