Study On The Mechanism Of Prolactin Action Of Total Barley Malt Alkaloids Based On JAK2/Stat5 Pathway

Objective:Raw malt is traditionally used for strengthening the spleen and stomach,soothing the liver and promoting qi,and has the effect of lowering milk.It is also commonly used in the treatment of postpartum hypogalactia,but the specific dosage and mechanism of action of malt for postpartum hypogalactia are not yet clear.In this study,based on the previous stage of the research group,through animal experiments and cell experiments,the effective part of raw malt that exerts prolactin effect-malt total alkaloids,the effective dose and mechanism of action were explored,and the clinical use of raw malt in the treatment of postpartum hypogalactia was carried out.Symptoms provide data reference and theoretical basis.The total barley malt alkaloids were made into hydrogel patches,and the stability and total alkali content of the preparations were preliminarily explored to provide the possibility of a new type of preparation for clinical use of raw malt in the treatment of postpartum lactation.Methods:（Ⅰ）The postpartum hypogalactia rat model was replicated with bromocriptine mesylate;the milk yield of the mother mice and the net growth of the litter weight of the pups were weighed and calculated,the serum PRL level was detected by Elisa（enzyme-linked immunosorbent assay）,and the bromine Whether the rat model of postpartum hypogalactia induced by crypting is successful.（Ⅱ）Postpartum hypogalactemic rats were given metoclopramide and different doses of total barley malt alkaloids by intragastric administration.Weigh and calculate the milk yield of mother mice and the net growth of litter weight;HE staining was used to observe the pathological changes of mammary gland tissue;Elisa was used to detect serum PRL,P and E2 levels;Western blot was used to detect PRL and PRLR in mammary gland tissue.β-casein,p-JAK2/JAK2,p-Stat5/Stat5 protein expression levels;Real-Time PCR（real-time fluorescence quantitative analysis）detection of PRL,PRLR,β-casein m RNA expression levels in breast tissue;immunohistochemical detection The positive expression of JAK2,Stat5,ki67 andα-SMA in breast tissue.（Ⅲ）Mouse mammary epithelial cells（HC11 cells）were cultured with total barley malt alkaloids.The cell viability was detected by CCK-8,and the protein levels of PRL andβ-casein were detected by Western bolt to determine the effective administration time of total barley malt alkaloids.HC11 cells were treated with total barley malt alkaloids and JAK2-specific inhibitor AG490,and the protein expression levels ofβ-casein,PRL,PRLR,JAK2 and Stat5 in the cells were detected by Weather bolt.（Ⅳ）Prepare total malt alkaloid hydrogel patch,optimize the prescription by orthogonal test design,and investigate the spreadability of the matrix,the uniformity of appearance,the skin followability,the repeatability of sticking,whether it is permeable,the curing time,and the initial adhesion.,sticking strength,peel strength and 12h transdermal absorption rate,the patches were scored.The best formulations were assayed using high performance liquid chromatography and their quality was controlled using the above indicators.Results:（Ⅰ）After continuous intragastric administration of 0.1 mg·m L-1bromocriptine for 12 days,the milk production of the mother mice was significantly reduced,the net growth of the litter weight of the pups and the PRL content of the mother mice were significantly reduced（P<0.05）,indicating that bromine Cryptotin has an effective inhibitory effect on lactation in postpartum female mice.（Ⅱ）After treatment with metoclopramide and different doses of total barley malt alkaloids,the milk production and the net increase of litter weight in the15g administration group increased significantly（P<0.05）.According to the results of HE staining,compared with the model group,the mammary gland tissue of the 15g group was restored,the mammary lobules and ducts were well developed,and a large amount of secretions were stored in the acini.Elisa test results showed that serum PRL,P and E2 levels in 15g group were significantly increased（P<0.05）.Western blot and Real-Time PCR results showed that compared with the model group,the protein and m RNA contents of PRL,PRLR andβ-casein in the breast tissue of the 15g group were significantly increased（P<0.05）;The protein expression of p-JAK2/JAK2,p-Stat5/Stat5 was significantly increased（P<0.05）.Immunohistochemical results showed that the positive expressions of JAK2,Stat5,ki67 andα-SMA in the breast tissue of the 15g group were significantly increased（P<0.05）.（Ⅲ）The time and dose of total barley malt alkaloids to exert their efficacy were determined by CCK-8 combined with Western bolt detection results.Cells were treated with 10.8μg/m L TBMA for 48 h or 5.4μg/m L and 10.8μg/m L TBMA for 72 h.Under the treatment time,total barley malt alkaloids can effectively promote the secretion ofβ-casein and PRL in HC11 cells.Pretreatment of cells with JAK2 specific inhibitor AG490 for 1h could significantly inhibit the protein expression of JAK2 andβ-casein（P<0.05）.After cells were pretreated with AG490 for 1 h and treated with corresponding doses of total barley malt alkaloids for corresponding time,compared with the control group,the protein expressions ofβ-casein,PRL,PRLR,JAK2 and Stat5 were significantly increased（P<0.05）.（Ⅳ）The preparation method of the malt total alkaloid hydrogel patch determined by the preliminary experiment is as follows:add NP-700,aluminum glycerol,tartaric acid and carbomer into glycerol in turn,stir well,as phase A;mix PVP-K90 and The total barley malt alkaloids are fully dissolved in pure water as phase B;pour phase B into phase A and stir well until the paste is formed.After forming a stable and uniform gel,apply it while it is still hot,cover it with a backing,and leave it at room temperature for 24 hours.Conclution:（Ⅰ）The rat model of postpartum hypogalactia was successfully replicated by continuous intragastric administration of 0.1 mg·m L^-1bromocriptine every day.（Ⅱ）The effective dose of raw malt to promote lactation is 15g,and the total barley malt alkaloids contained in it can effectively promote lactation and mammary gland development in postpartum deficient mother mice.（Ⅲ）Malt total alkaloids promote milk secretion by activating JAK2 and activating JAK2/Stat5 signaling pathway.（Ⅳ）The hydrogel patch of TBMA in compliance with the provisions of the2020 edition of the Chinese Pharmacopoeia was successfully prepared and its quality was inspected.The optimal prescription is:NP-700 2g,aluminum glycerol 0.1g,propylene glycol 5g,glycerin 31.9g,carbomer 1g,PVP-K900.4g,tartaric acid 0.25g,water 58.85g,TBMA 0.5g.