| Objectives1 Effects of ractopamine(RAC) and zilpaterol(ZIL) on the expression levels of p-p38 MAPK and leptin in the 3T3-L1 adipocytes were detected, and the mechanism that RAC and ZIL promoted the synthesis of leptin via p38 MAPK signaling pathway was verified, which was used to investigate the potential role of RAC and ZIL in the endocrine disorder of adipocytes and its related diseases.2 Effects of RAC and ZIL on mouse embryo development rate were discussed by using mouse 2-cell embryos in vitro with adding different doses of RAC and ZIL,which provided experimental basis for the research of RAC and ZIL on female reproductive health.Materials and Methods1 Materials1.1 3T3-L1 pre-adipocytes3T3-L1 pre-adipocytes were purchased from Shanghai Cell Bank, Chinese Academy of Science.1.2 AnimalsSpecific pathogen-free female Kunming mice(4-6 weeks) and male Kunming mice(9-10 weeks) were purchased from Guangdong animal experimental center, and raised under the condition of temperature from 22 to 25 ℃ and 12 h light/dark cycle.All mice were free to water and feed. Before the experiment, the mice were allowedto acclimate for one week.2 Grouping and administration2.1 3T3-L1 pre-adipocytesAdipocytes were cultured with different doses of RAC, CLB and ZIL,respectively with or without pre-incubation with the inhibitor SB203580(10 μmol/L),and the adipocytes were harvested after 12 h and 24 h to detect cell viabilities and the expression levels of leptin and p-p38 MAPK. The grouping is shown as tables 1 and 2.2.2 mouse embryosThe 2-cell embryos were acquired from Kunming mice. All of mouse embryos were added with 0, 0.5, 5, 50 μmol/L RAC and 50 μmol/L clenbuterol(CLB)respectively, or 0, 0.1, 1, 10 μmol/L ZIL and 10 μmol/L CLB respectively. Each group had approximately 80 mouse embryos. Embryo development and morphologic variation were observed by microscopy.3 Statistical analysisThe cell data were presented as mean±standard deviation(SD), the mouse embryo experimental data were expressed as percentage. The statistical software program version SPSS 16.0 was used to analyze the results. Cell data were determined by t test and one-way analysis of variance(One-Way ANOVA). The Pearson chi-square test was used to compare the embryo blastocyst developmental rate. A P value<0.05 was considered to be statistical significant.Results1 Viability and expression levels of p-p38 MAPK and leptin of 3T3-L1 adipocytes were treated with RAC in vitroAfter added 12 h of RAC and CLB, the cell viabilities decreased compared with negative control group(P<0.05), however, the cell viabilities of 50 μmol/L RAC and CLB groups were significantly increased when SB203580 was added(P<0.01). The RAC and CLB groups up-regulated expression levels of p-p38 MAPK after 12 h compared with negative control group(P<0.05), however, the expression levels of p-p38 MAPK were significantly decreased when SB203580 was added(P<0.01). The RAC and CLB groups up-regulated expression levels of leptin after 12 h compared with negative control group(P<0.05), however, the expression levels of leptin were significantly decreased when SB203580 was added(P<0.01). The expression levels of p-p38 MAPK and leptin were no significant difference between 12 h and24 h groups(P>0.05).2 Viability and expression levels of p-p38 MAPK and leptin of 3T3-L1 adipocytes were treated with ZIL in vitroAfter 12 h, the cell viabilities of ZIL and CLB groups decreased compared with negative control group(P<0.05), however, the cell viabilities of all ZIL and CLB groups were significantly increased when SB203580 was added(P<0.01). The cell viabilities of 10 μmol/L ZIL and CLB were decreased after 24 h compared with 12 h(P<0.05). The expression levels of p-p38 MAPK with 1, 10 μmol/L ZIL and CLB groups were up-regulated after 12 h or 24 h compared with those of negative controlgroup(P<0.05), however, the expression levels of p-p38 MAPK were significantly decreased when SB203580 was added(P<0.01). The ZIL and CLB groups up-regulated expression levels of leptin after 12 h compared with those of negative control group(P<0.05), the expression levels of leptin with 1, 10 μmol/L ZIL and CLB groups were up-regulated after 24 h compared with those of negative control group(P<0.05), however, the expression levels of leptin were significantly decreased when SB203580 was added(P<0.01).3 Effects of RAC on the in vitro development of mouse embryoA significant decrease was showed in the blastocyst developmental rate of the2-cell embryos in the 5 μmol/L RAC(52.7%) and 50 μmol/L RAC groups(47.1%)compared with negative control group(74.7%, P<0.01). Moreover, embryos cultured with RAC showed more granules, fragments, and degeneration than negative control group.4 Effects of ZIL on the in vitro development of mouse embryoWith increasing doses of ZIL, the inhibitory effects on embryo development were significantly increased, and blastocyst developmental rate in 0.1, 1, 10 μmol/L ZIL and CLB were 57.5%, 55.0%, 51.3% and 25.7%, respectively. The inhibitory effects on embryo development were significantly increased in the 0.1, 1 and 10μmol/L ZIL compared with negative control group(74.7%, P<0.01). Embryos cultured with ZIL showed more granules, fragments, and degeneration than negative control group. Blastocyst developmental rate were no significant difference between0.5 μmol/L RAC and 0.1 μmol/L ZIL, 5 μmol/L RAC and 1 μmol/L ZIL or 50 μmol/L RAC and 10 μmol/L ZIL(P>0.05).Conclusions1 The experiment of 3T3-L1 adipocytes treated with RAC and ZIL in vitro showed that RAC and ZIL could up-regulate the expression levels of p-p38 MAPK and leptin in 3T3-L1 adipocytes. Upon inhibiting the phosphorylation of p38 MAPK, the expression levels of leptin protein reduced, which might lead to IR, and might cause female reproductive endocrine disorders such as the pathogenesis of PCOS. Thissuggested that RAC and ZIL could trigger the p38 MAPK signaling pathway to stimulate adipocytes to express more leptin protein. The viability rates of cells,p-p38 AMPK and leptin expression levels were not significantly different between 12 h and 24 h after RAC added. After ZIL added 24 h, the viability rates of cells were lower, p-p38 AMPK and leptin expression levels increased compared with 12 h. It showed that the biological effect of ZIL was greater than that of RAC, and the effects of ZIL on human body was serious.2 The experiment of mouse embryos treated with RAC and ZIL in vitro showed that RAC and ZIL could inhibit development of embryo in vitro. Embryos cultured with RAC and ZIL showed more granules, fragments than negative control group. This suggested that RAC and ZIL could hinder the development of embryo by increasing the fragments and coarse particles of the embryo, which would affect the female reproductive health. Comparison of the high and low doses between ZIL and RAC on the same effect on embryonic development, the concentration of ZIL was lower. |
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