桉叶油联合维甲酸对SD胎鼠脑组织Pax3和缝隙连接蛋白43表达的影响

Effects of eucalyptus oil with retinoic acid on the expression of Pax3 and connexin 43 in the fetal rat brain tissue

目的探讨桉叶油联合维甲酸（RA）对SD胎鼠脑组织转录因子Pax3、缝隙连接蛋白43（Cx43）表达的影响。方法 SD孕鼠42只,随机分为6组,每组7只,正常对照组,RA组,溶剂对照组（花生油＋RA）,3个实验组（桉叶油高、中、低剂量＋RA）。正常对照组自由摄食饮水,溶剂对照组于孕第7～14天每只花生油2ml灌胃,每天1次,孕第10天40mg/kg RA灌胃1次。桉叶油3个剂量组于孕第7～14天分别桉叶油300、200和100mg/kg灌胃,每天1次,孕第10天40mg/kg RA灌胃1次。RA组于孕第10天40mg/kg RA灌胃1次。各组于孕21天处死孕鼠取胚胎,Western blotting、免疫组织化学技术检测胎鼠脑组织的Pax3、Cx43蛋白的表达。Real-time PCR检测脑组织Pax3、Cx43 mRNA表达。阿利新蓝和茜素红进行骨骼染色,体视显微镜下观测椎骨发育,脊柱间隙。结果维甲酸灌胃各组胎鼠椎骨数量异常的胎鼠数与正常对照组比较差异无显著性,但在维甲酸灌胃各组中,胎鼠椎骨形态异常的异常率和胎鼠椎骨分裂的百分率最低值分别为55%（桉叶油高剂量＋RA组）和45.8%（桉叶油低剂量＋RA组）,较正常对照组高（0）（P〈0.05）。在维甲酸灌胃各组中,桉叶油各组胎鼠椎骨形态异常的异常率和胎鼠椎骨分裂的百分率最高值分别为62.5%（桉叶油低剂量＋RA组）和55.0%（桉叶油高剂量＋RA组）,较维甲酸组（73.7%,73.7%）和溶剂对照组低（68.2%,63.6%,P〈0.05）。与正常组胎鼠比较,维甲酸灌胃各组大脑组织的Pax3、Cx43蛋白及其mRNA的表达较正常组高（P〈0.05）,在维甲酸灌胃各组中,桉叶油灌胃各组胎鼠脑组织Pax3、Cx43蛋白及其mRNA的表达较单纯RA灌胃组低,其差异有统计学意义（P〈0.05）。结论桉叶油对维甲酸引起胎鼠神经管缺陷有一定的拮抗作用。其机制可能与桉叶油拮抗维甲酸上调胎鼠神经组织的Pax3、Cx43蛋白过度表达有关。

Objective To investigate the effect of eucalyptus oil with retinoic acid on the expression of paired box3（ Pax3） and connexin（ Cx43） in the fetal rat brain tissue. Methods Forty-two pregnant SD rats were randomly divided into 6 groups（ 7 rats per group）,normal group,retinoic acid group（ RA）,solvent group（ peanut oil ＋ RA） and 3experimental groups（ according to eucalyptus oil of high,medium,low dose ＋ RA）,normal group with free diet. RA group lavaged with retinoic acid（ 40 mg / kg） at gestation 10 th day. Three experiment groups and solvent group were lavaged from the 7th to 14 th day with eucalyptus oil in 300 mg / kg,200 mg / kg,and 100 mg / kg and peanut oil 2ml per animal per day,respectively. Each of them was lavaged with retinoic acid（ 40 mg / kg） at gestation 10 th day. All pregnant rats were sacrificed at the 21 st day and the live-fetus was collected. Expression of Pax3,Cx43 protein and it＇s mRNA in fetal rat brain tissue were detected by Western blotting,immunohistochemistry and Real-time PCR. The vertebraedevelopment and paravertebral space of fetus were observed under a stereoscope by staining with alician blue and alizarin red S. Results The number of fetal rats with abnormal vertebrae in lavaged retinoic acid groups were not significantly different from those in normal group,but the lowest percentage of vertebrae form abnormal（ 55%,eucalyptus 300 mg / kg＋ RA 40 mg / kg） and vertebrae the division（ 45. 8% eucalyptus 100 mg / kg ＋ RA40 mg / kg） were higher than those in normal group（ 0,0）（ P〈0. 05）. In lavaged retinoic acid groups,the highest percentage of vertebrae form abnormal（ 62. 5%,eucalyptus 100 mg/kg ＋ RA 40mg/kg） or vertebrae the division（ 55. 5% eucalyptus 300 mg/kg ＋ RA40mg/kg） of fetal rats in eucalyptus oil groups were lower than those in retinoic acid group（ 73. 7%,73. 7%） and solvent control group（ 68. 2%,63. 6%）,（ P〈0. 05）. Compared with normal group,the expression of brain tissue Pax3、Cx43 protein and mRNA in graged retinoic acid groups were significantly higher than those in normal group（ P〈0. 05）. In lavaged retinoic acid groups,the expression of fetal brain tissue Pax3,Cx43 protein and mRNA in intragastric eucalyptus oil groups were significantly lower than those in pure graged retinoic acid group（ P〈0. 05）. Conclusion Eucalyptus oil can antagonize the neural tube defects caused by RA. Its mechanism may be related to eucalyptus oil antagonize Pax3 and Cx43 protein overexpression which caused by RA.