江西建昌帮阴附片和阳附片对阳虚小鼠棕色脂肪组织的影响

目的:观察棕色脂肪组织(BAT),细胞,蛋白以及相应基因在大黄混悬液所致阳虚模型小鼠中的表达情况,探讨建昌帮特色附子炮制品的产热机制。方法:随机选取20只小鼠,雌雄各半,作为正常雌、雄组,其余80只小鼠灌服大黄混悬液(质量浓度0. 25 g·mL~(-1))建立阳虚模型,造模结束后随机分为模型雌、雄组,生附片雌、雄组,阴附片雌、雄组以及阳附片雌、雄组,每组10只。按照小鼠组别给予相应药液,灌胃2周(1. 54 g·kg~(-1)·d~(-1)),正常组和模型组给予等体积蒸馏水。灌胃结束后,采集小鼠肩胛处BAT,进行苏木素-伊红(HE)染色观察BAT细胞变化;采用蛋白免疫印迹法(Western blot)和实时荧光定量聚合酶链式反应(Real-time PCR)检测解偶联蛋白1(UCP1)及其mRNA的表达。结果:与同性别正常组比较,模型组的BAT比重显著降低(P0. 01);雌性小鼠中生附片组和阴附片组的BAT比重较模型组显著增加(P0. 01),而雄性小鼠各给药组与模型组相比则无显著性差异。与同性别正常组比较,模型组小鼠BAT的细胞有许多散在分布的空泡,由于空泡较大,可观察到的细胞较少;与同性别模型组比较,生附片组小鼠BAT细胞可见细胞空泡减少,细胞较小并且排列紧密,阳附片组中BAT细胞的密度也明显增加,阴附片组BAT细胞中空泡相对减少,但细胞无显著增加。同性别小鼠之间比较,模型组与正常组的UCP1表达水平相比具有统计学意义(P0. 05,P0. 01);在雌性小鼠中,阳附片组较模型组的UCP1表达水平显著升高(P0. 05),雄性小鼠的各给药组较同性别模型组均有显著差异(P0. 05),其中阳附片的显著性最优。模型组较同性别正常组的UCP1 mRNA相对表达量显著下降(P0. 05,P0. 01);在雌性小鼠中,与模型组比较,阳附片组、生附片组和阴附片组的UCP1 mRNA相对表达量显著升高(P0. 05,P0. 01),与阳附片组比较,生附片组和阴附片组的UCP1 mRNA相对表达量有显著性差异(P0. 05);在雄性小鼠中,与模型组比较,阳附片组的UCP1 mRNA相对表达量显著升高(P0. 01),生附片组、阴附片组则无显著性差异,生附片组和阴附片组的UCP1 mRNA相对表达量与阳附片组比较有显著性差异(P0. 05)。结论:生附片、阴附片和阳附片对大黄所致的阳虚证均有明显改善作用,机制可能为促进UCP1蛋白及其mRNA的表达、增强BAT的活性,且阳附片效果最好。

Effect of Yinfupian and Yangfupian on Brown Adipose Tissue of Yang Deficiency Mice

Objective To observe the expression of brown adipose tissue (BAT), cells, proteins and corresponding genes in Yang deficiency model mice induced by Rhei Radix et Rhizoma suspension, and to explore the thermogenesis of processed products of Aconiti Lateralis Radix Praeparata with Jianchang faction characteristics.Method Twenty mice, half male and half female, were randomly selected as the normal female and male groups. And the other 80 mice were administrated with Rhei Radix et Rhizoma suspension (the content of 0.25 g·mL^-1) to establish Yang deficiency model, after the model was established, they were randomly divided into the model female and male groups, female and male groups of Shengfupian, female and male groups of Yinfupian, female and male groups of Yangfupian, 10 mice in each group. Mice were intragastric administrated with corresponding medical solution for two weeks (1.54 g·kg^-1·d^-1) according to groups. Normal group and model group were given equal volume distilled water. After administration, BAT of scapular region of mice was collected and the changes of BAT cells were observed by hematoxylin-eosin (HE) staining. The expression of uncoupling protein 1 (UCP1) and its mRNA were detected by Western blot and real-time fluorescence quantitative polymerase chain reaction (Real-time PCR).Result Compared with the normal group of the same sex, the proportion of BAT in the model group decreased significantly (P<0.01). Compared with the model group of the same sex, the proportion of BAT in female mice from Shengfupian and Yinfupian groups increased significantly (P<0.01), while there was no significant difference between each administration group and model group in the male mice. Compared with normal mice of the same sex, there were many scattered vacuoles in BAT cells of the model group, and fewer cells could be observed due to larger vacuoles. Compared with the model group of the same sex, BAT cells in mice from the Shengfupian group showed fewer vacuoles, smaller cells and tight arrangement, the density of BAT cells in mice from the Yangfupian group also increased significantly, while the vacuoles in BAT cells of mice from the Yinfupian group decreased relatively and the cells did not increase significantly. Compared with the same sex mice, the expression level of UCP1 in the model group and the normal group was statistically significant (P<0.05, P<0.01). In the female mice, the expression level of UCP1 in Yangfupian group was significantly higher than that in the model group (P<0.05), each administration group of male mice was significantly different from that of the model group of the same sex (P<0.05), of which Yangfupian was the most significant. The relative expression of UCP1 mRNA in the model group was significantly lower than that in the normal group of the same sex (P<0.05, P<0.01). In the female mice, compared with the model group, the relative expression levels of UCP1 mRNA in Yangfupian group, Shengfupian group and Yinfupian group increased significantly (P<0.05, P<0.01), compared with Yangfupian group, the relative expression levels of UCP1 mRNA in Shengfupian and Yinfupian were also significantly different (P<0.05). In the male mice, compared with the model group, the relative expression of UCP1 mRNA in Yangfupian group was significantly increased (P<0.01), but there was no significant difference in Shengfupian group and Yinfupian group, in addition, compared with Yangfupian group, the relative expression of UCP1 mRNA in Shengfupian group and Yinfupian group had significant difference (P<0.05).Conclusion Shengfupian, Yinfupian and Yangfupian all have obvious improvement on Yang deficiency syndrome induced by Rhei Radix et Rhizoma suspension. The mechanism may be to promote the expression of UCP1 protein and its mRNA and enhance the activity of BAT. And the effect of Yangfupian is the best.