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2.4 Influence of Medulla Tetrapanacis on the expression of casein and lactalbumin of HC11 mammary epithelial cells
The fluorescence quantitative PCR is used to analyze the influence of different dosages of medulla tetrapanacis extracted solution on the mRNA expression of casein and lactalbumin in HC11 mammary epithelial cells, as shown in Table 2. The results show that the HC11 mammary epithelial cells in the low dosage and high dosage medulla tetrapanacis group can significantly increase the mRNA expression of β-casein (P <0.01), as shown in Figure 5; the low dosage and high dosage medulla tetrapanacis can significantly increase the mRNA expression of HC11 mammary epithelial cells, but the low dosage medulla tetrapanacis cannot significantly affect the mRNA expression of lactalbumin (P> 0.05), while the influence of high dosage medulla tetrapanacis on the mRNA expression of lactalbumin has the statistical significane (P <0.01), as shown in Figure 6.
 
The Western blotting is used to analyze the influence of different dosages of medulla tetrapanacis extracted solution on the casein expression of HC11 mammary epithelial cells. The results show that high dosage of medulla tetrapanacis extracted solution can significantly increase the casein expression, as shown in Figure 7.

The two groups without *has no statistical difference (P> 0.05), the comparison among others has statistical difference (P <0.01)
Table 2 Expression of Casein mRNA and lactalbumin mRNA detected by real-time PCR in control group, low-dose and high-dose medulla tetrapanacis treated group
Fig5 Expression of Casein mRNA and lactalbumin mRNA detected by real-time PCR in control group, low-dose and high-dose medulla tetrapanacis treated group

2.5 Influence of medulla tetrapanacis on STAT5 signaling pathway of HC11 mammary epithelial cells
The Western blotting is used to analyze the influence of different dosages of medulla tetrapanacis extracted solution on the expressions of STAT5 and phosphorylation STAT5 in the HC11 mouse mammary epithelial cells. There is no significant difference in the total STAT5 protein expression in the control group, low-dose group and high-dose group, but the medulla tetrapanacis can significantly increase the phosphorylation level of STAT5 with the dosage-effect relationship, as shown in Figure 8.

3. Discussion
The secretion of milk is composed by a series of complex physiological processes, regulated by a variety of hormone, and the most important hormone for the generation of milk is the prolactin secreted by the anterior pituitary [5]. Prolactin plays a central role throughout the lactation process, and it promotes the secretion of milk via the Jak/Stat pathway of mammary epithelial cells and induces the synthesis of important nutrients in the milk[6]. Currently, most western medicines to promote milk secretion are to promote the secretion of milk through the increases of prolactin levels in centre. For example, domperidone, can interfere the inhibition of dopamine to the anterior pituitary to generate prolactin, to indirectly increase the synthesis of prolactin, increasing the secretion of milk [7], but it also can cause gastrointestinal and cardiovascular side effects to the delivery women [8]. Chinese traditional medicine, medulla tetrapanacis, has unique advantage on the treatment of postpartum hypogalactia, its efficacy has been proved by long-term clinical experience, and the action is moderate with few side effects, has also been widely adopted and accepted among people. We study the pharmacological mechanism of medulla tetrapanacis through these experiments, to preliminarily explore the approaches and mechanisms of medulla tetrapanacis to promote the secretion of milk.
 
The experimental results show that, the amount of milk secretion of female mice after delivery is higher in the medulla tetrapanacis group than the negative control group, particularly there is statistical difference on the amount of milk secretion compared with the negative control group on Day 3, 8 and 13. It finds that, after the prolactin levels of mice are determined, although the prolactin level in the peripheral blood of mice is raised, compared with the control group the elevated degree has no statistical difference, so it can be seen that the medulla tetrapanacis has no effect on hypothalamic - pituitary - gonadal axis to increase the secretion of prolactin and achieve the promotion of milk secretion. After the HC11 mammary epithelial cells are treated by medulla tetrapanacis, there is no significant difference in the intracellular total STAT5 protein expression, but the level of phosphorylation of STAT5 is increased significantly, which has dosage-effect relationship. Thus, we speculate that, the medulla tetrapanacis may directly act on the breast target cells, to increase the phosphorylation level of transcription activator STAT5 in the cell signaling pathway, to enhance the intracellular signal transduction, so as to promote the secretion of milk; after the application of medulla tetrapanacis, it may increase the quantity or activity of prolactin receptor on cell surface, leading the increase of intracellular STAT5 phosphorylation level. The main influence of medulla tetrapanacis on milk is to increase the protein content of milk, especially the content of casein and lactalbumin, but reduce the content of lactose in the milk, and its causes and mechanism are required further study. This experiment is only a preliminary study about the mechanism of medulla tetrapanacis to increase the milk secretion, and some aspects are still needed further experiments, to comprehensively explain the mechanism of medulla tetrapanacis to promote lactation.
 
In short, the medulla tetrapanacis can act on the transcription activator STAT5 in the signaling pathway of cells, increase the STAT5 phosphorylation levels and strengthen the intracellular signal transduction, increase the milk secretion of mammary cells and the protein content of milk, can effectively treat the postpartum hypogalactia.