| Objective:To study the effect of phytoestrogen puerarin on lactation in postpartum hypogalactia mice and postpartum normal mice,and to explore the mechanism of promoting lactation from the two aspects of lactation-related hormones and JAK2/STAT5 signaling pathway,so as to detect the effects of puerarin on the expression of milk protein,lactose and milk fat,and to preliminarily explore the safety of puerarin on postpartum normal mice.It provides experimental basis for rational application of Radix Puerariae and puerarin to promote postpartum lactation in traditional Chinese medicine.Method:1 The effect of puerarin on lactation of postpartum hypogalactia model mice and its mechanism1.1 The effect of puerarin on the lactation of postpartum hypogalactia miceForty female mice with a difference of no more than 24 hours before and after delivery were selected and divided into blank control group,hypogalactia model group,low-dose puerarin group,high-dose puerarin group and Domperidone group by random number table method.Each group of female mice was 8,and each litter of pups is also adjusted to 8 mice.Modeling and administration were started on the 3rd day after delivery.Every morning,except for the blank control group,the other 4 groups were given bromocriptine intragastrically(1.6 mg/kg)in the morning.Every afternoon,except for the hypogalactia model group and the blank control group,the low-dose puerarin group(18 mg/kg)was given intragastric administration,the high-dose puerarin group(72 mg/kg)was given intragastric administration,and the Domperidone group(3.5 mg/kg)was given intragastric administration for 10 days.On the 11th day,the mice were weighed and sacrificed,and blood and breast tissue samples of the mother mice were collected.The body weight of the mother mice and their offspring were observed,and the lactation yield of the mother mice was evaluated.The pathological morphology of the mammary gland of the mother mice was observed by paraffin section and HE staining.1.2 Study on the mechanism of puerarin on lactation of postpartum hypogalactia miceEnzyme-linked immunosorbent assay was used to detect the serum levels of prolactin(PRL),progesterone(P4),estradiol(E2)and the PRL content in breast tissue.Immunohistochemistry was used to detect the prolactin receptor(PRLR),?-casein(?-casein).Western blotting was used to detect PRLR,Janus protein tyrosine kinase 2(JAK2),signal transduction and activation factor 5a(STAT5a),?-casein,and glucose in breast tissue The expression level of transporter 1(GLUT1)and fatty acid synthase(FAS).2 The effect of puerarin on the lactation of postpartum normal mice2.1 The effect of puerarin on the lactation yield of normal mice after delivery and its mechanismTwenty-four female mice with a difference of no more than 24 hours before and after delivery were selected and divided into normal control group,puerarin low-dose group and puerarin high-dose group by random number table method.Each group of female mice was 8,and each litter of pups is also adjusted to 8 mice.Modeling and administration were started on the 3rd day after delivery.Every morning,the puerarin low-dose group was given intragastric administration(18 mg/kg),the puerarin high-dose group was given intragastric administration(72 mg/kg)and the normal control group was given the same amount of normal saline for 10 days.On the 11th day,the mice were weighed and sacrificed,and samples of the mother mice's blood,breast tissue,liver,kidney,ovary,and uterus were collected.The body weight changes of postpartum normal mice and offspring mice were observed,and the milk yield of mice was evaluated.The pathological morphology of the mammary glands of the postpartum mice was observed by paraffin section and HE staining.2.2 The effect of puerarin on the lactation mechanism and safety of normal mice after deliveryEnzyme-linked immunosorbent assay was used to detect PRL,P4,E2 in mouse serum and PRL content in breast tissue.Immunohistochemical method was used to detect PRLR and?-casein in breast tissue.Western blotting method was used to detect PRLR and JAK2 in breast tissue,STAT5a,?-casein,GLUT1,FAS expression level.Paraffin sections and HE staining were used to observe the pathological morphology of the liver,kidney,ovary,and uterus of postpartum mice.Result:1 The effect of puerarin on lactation of postpartum hypogalactia model mice and its mechanism1.1 The effect of puerarin on the lactation of postpartum hypogalactia miceCompared with the blank control group,the weight loss of the mother mice in the lack of milk model group increased significantly(P<0.05),and the weight of the offspring,the weight of the mammary gland and the mammary gland index were significantly reduced(P<0.05 or P<0.01).From the 3rd day of the model,the lactation amount of the female mice in the hypogalactia model group decreased significantly in a single hour((P<0.05 or P<0.01).Compared with the hypogalactia model group,the weight loss of mother mice in the low-dose and high-dose puerarin groups and the Domperidone group was significantly delayed,and the weight of offspring and mammary glands increased significantly(P<0.05 or P<0.01).The low-dose puerarin group The mammary index of the Domperidone group and the Domperidone group were significantly increased(P<0.05).the single-hour lactation output of the low and high-dose puerarin groups and the Domperidone group were significantly increased((P<0.05 or P<0.01).The results of HE staining of mammary tissue showed that the breast cavity of the hypogalactia model group was smaller,the secretion in the gland cavity was significantly reduced,and the breast tissue was severely atrophy,the low and high dose groups of puerarin and the Domperidone group were significantly enlarged and the gland cavity secreted.The amount of material has increased significantly,and the atrophy of breast tissue has recovered significantly.1.2 Study on the mechanism of puerarin on lactation of postpartum hypogalactia miceCompared with the blank control group,the serum levels of PRL,P4,and E2 in the hypogalactia model group were significantly reduced(P<0.05 or P<0.01),and the PRL content in breast tissue was significantly increased and significantly decreased(P<0.01).Immunohistochemical revealed that the yellow-brown positive expression of PRLR and?-casein in the breast tissue of the hypogalactia model group was significantly reduced.Western blotting revealed that PRLR,JAK2,STAT5a,?-casein,GLUT1,FAS protein expression in the breast tissue of the hypogalactia model group were significantly reduced(P<0.01).Compared with the hypogalactia model group,the levels of PRL,P4,and E2 in the serum of the puerarin low-dose and high-dose groups and the Domperidone group were significantly increased(P<0.05 or P<0.01),and the PRL content in breast tissue was also significantly increased(P<0.01).Immunohistochemical detection found that the positive expression of PRLR and ?-casein in breast tissues of the low and high dose groups of puerarin and the Domperidone group were significantly increased.Western blot detection found that the low and high doses of puerarin expressions of PRLR,JAK2,STAT5a,?-casein,GLUT1,and FAS protein in breast tissues of the group were significantly increased(P<0.01).Only GLUT1 protein expression is better at high doses of puerarin than low doses of puerarin,and the protein expression of other indicators is better at low doses of puerarin.2 The effect of puerarin on the lactation of postpartum normal mice and its mechanism2.1 The effect of puerarin on the milk yield of normal mice after deliveryCompared with the normal control group,from the 6th day of administration,the lactation of the low-dose puerarin group was significantly increased(P<0.05),but only on the 6th day,the lactation of the high-dose puerarin group was higher than normal control group(P<0.05).The weight loss of postpartum mice and the weight gain of offspring in the low and high dose groups of puerarin were significantly higher than those in the normal control group(P<0.01).The weight of mammary glands in the low dose group of puerarin was significantly higher than normal Control group(P<0.05).The results of HE staining showed that in the low-dose and high-dose groups of puerarin,the acinars in the breast lobules were significantly enlarged,and the secretions increased significantly,the low-dose group of puerarin and the higher-dose group had more secretions and fewer lipid droplets.2.2 The effect of puerarin on the lactation mechanism and safety of postpartum normal miceCompared with the normal control group,the serum PRL content in the puerarin high-dose group was significantly increased(P<0.01).Immunohistochemical detection revealed that the positive expression of ?-casein in the low-dose puerarin group was significantly higher than that in the normal control group and the low-dose puerarin group(P<0.05).While the high dose puerarin group had a yellowish expression of PRLR and ?-casein in the breast tissues.The positive expression of brown was significantly lower than that of the normal control group and the low-dose puerarin group(P<0.01).Western blotting showed that the expression of PRLR,?-casein and FAS protein in the low-dose puerarin group was significantly higher than that of the normal control group(P<0.05).While the expression of PRLR,JAK2,?-casein and FAS protein in the high-dose puerarin group Significantly lower than the normal control group,also lower than the puerarin low-dose group(P<0.05 or P<0.01),and the expression of STAT5a in the puerarin high-dose group was significantly lower than that of the puerarin low-dose group(P<0.05).There were no pathological abnormalities in the HE staining of liver,kidney,ovary,and uterus in the low and high dose groups of puerarin,and there was no difference in tissue and organ index between the groups(P>0.05).Conclusion:Both low and high doses of puerarin can significantly promote lactation in mice with postpartum hypogalactia,but the lactation effect is better at low doses.The lactation mechanism may be related to the increase in lactation-related hormones PRL,P4,E2,and activation of the JAK2/STAT5 signaling pathway.Puerarin may also improve the milk quality of lactose,milk fat and milk protein-related protein expression.Puerarin may also promote lactation in normal postpartum mice at low doses.The lactation mechanism may be related to the increase of PRLR protein expression,but it has little effect on the JAK2/STAT5 signaling pathway.The low-dose group of puerarin may also enhance the expression of milk protein and milk fat-related protein,and improve milk quality.But puerarin has an inhibitory effect on lactation at high doses,and its expression of milk protein and milk fat is also inhibited.Under low and high doses of puerarin,no obvious pathological changes were found in the liver,kidney,ovary and uterus of mother mice,and its safety is good. |
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