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薛秋云, 黄玉蓉, 李慧, 等. 催乳颗粒通过分泌Frizzled相关蛋白2-Wnt/β-catenin信号通路改善大鼠产后缺乳[J]. koko体育app 学报（医学版）, 2024, 55(3): 619-629. DOI:
引用本文: 薛秋云, 黄玉蓉, 李慧, 等. 催乳颗粒通过分泌Frizzled相关蛋白2-Wnt/β-catenin信号通路改善大鼠产后缺乳[J]. koko体育app 学报（医学版）, 2024, 55(3): 619-629. DOI:
XUE Qiuyun, HUANG Yurong, LI Hui, et al. Cuiru Keli Improves Postpartum Hypogalactia in Rats Through Secreted Frizzled-Related Protein 2-Wnt/β-catenin Signaling Pathway[J]. Journal of Sichuan University (Medical Sciences), 2024, 55(3): 619-629. DOI:
Citation: XUE Qiuyun, HUANG Yurong, LI Hui, et al. Cuiru Keli Improves Postpartum Hypogalactia in Rats Through Secreted Frizzled-Related Protein 2-Wnt/β-catenin Signaling Pathway[J]. Journal of Sichuan Universit✤y (Medical Sciences), 2024, 55(3): 619-629.🐓 DOI:

催乳颗粒通过分泌Frizzled相关蛋白2-Wnt/β-catenin信号通路改善大鼠产后缺乳

Cuiru Keli Improves Postpartum Hypogalactia in Rats Through Secreted Frizzled-Related Protein 2-Wnt/β-catenin Signaling Pathway

  • 摘要:
    目的 基于分泌Frizzled相关蛋白2（secreted frizzled-related protein 2, SFRP2）-Wnt/β-catenin信号通路探讨催乳颗粒（Cuiru Keli, CRKL）治疗产后缺乳的效果及其作用机制。
    方法 通过向分娩后第3天的雌鼠灌胃2 mL 1.6 mg/mL甲磺酸溴隐亭来建立产后缺乳大鼠模型。将产仔时间差小于48 h的雌性大鼠随机分为7组：正常组（不造模，不给药）；模型组；CRKL低剂量组（模型组+3 g/kg CRKL）；CRKL中剂量组（模型组+6 g/kg CRKL）、CRKL高剂量组（模型组+9 g/kg CRKL）；阳性药组（模型组+3 mg/kg 多潘立酮）；NC组（模型组+生理盐水）；每组6只。除正常组和模型组外，其余5组按分组药物和剂量连续灌胃给药，每天1次，共10 d。取7组大鼠，测量10 d仔鼠总窝质量变化、HE染色观察乳腺病理变化。取除阳性对照组之外的6组大鼠，HE染色观察垂体病理变化，检测血清中PRL含量（ELISA）、乳腺组织（mammary tissue, MT）中PRLR的表达（免疫组化染色）、MT中乳汁合成相关基因mRNA的表达（RT-qPCR），然后以网络药理学和分子对接研究CRKL对产后缺乳的治疗作用和机制，特别是其是否通过SFRP2-Wnt/β-catenin信号通路治疗产后缺乳；再通过检测相关通路基因（RT-qPCR）和蛋白（Western blot）进行验证。然后进行细胞实验验证：取鉴定后的大鼠乳腺上皮细胞（rat mammary epithelial cell, RMEC），将RMEC分为4组：正常组（原代培养的RMEC，不处理）、 SFRP2过表达组（原代培养的RMEC+SFRP2过表达载体）、 SFRP2过表达+CRKL组（SFRP2过表达组+10%含药血清）、阴性对照组（原代培养的RMEC+空载体）。RT-qPCR检测过表达SFRP2后CRKL对于乳汁合成相关基因FASN、CSN2和GLUT1 mRNA表达的影响。
    结果 与模型组相比，低、中、高剂量的CRKL均可以提高仔鼠10 d体质量增加量（P<0.05或P<0.01），均能有效增加产后缺乳大鼠泌乳量（P<0.01），增加乳腺小叶的面积，增加腺泡腔的大小和填充量，并促进产后缺乳大鼠催乳素的分泌和表达（P<0.05或P<0.01），显著促进产后缺乳大鼠乳腺乳脂、乳蛋白和乳糖合成基因的表达（P<0.05或P<0.01）。网络药理学表明，Wnt信号通路可能是CRKL治疗产后缺乳的关键通路。分子对接结果表明CRKL和CCND1、SFRP2具有良好的结合能力。与模型组相比，低、中、高剂量的CRKL均抑制体内SFRP2基因的表达（P<0.01），激活了产后缺乳大鼠乳腺中的Wnt/β-catenin信号通路中CCND1、c-Myc的基因和蛋白的表达（P<0.05或P<0.01）。细胞实验表明，过表达SFRP2后，与正常组相比，乳汁合成相关基因FASN、CSN2和GLUT1的mRNA水平也降低（P<0.01）。加入含药血清后，上述基因的表达上调（与SFRP2 过表达组相比， P<0.01）。
    结论 CRKL通过SFRP2-Wnt/β-catenin信号通路治疗产后缺乳，SFRP2可能成为产后缺乳诊断和治疗的新靶点。这揭示了CRKL治疗产后缺乳的新机制，并促进了其临床推广。
     
    Abstract:
    Objective Based on the secreted frizzled-related protein 2 (SFRP2)-Wnt/β-catenin signaling pathway, this study explored the effect and mechanism of Cuiru Keli (CRKL) in the treatment of postpartum hypogalactia.
    Methods A rat model of postpartum hypogalactia was established by gavaging 2 mL of 1.6 mg/mL bromocriptine mesylate to female rats on the third day after delivery. Female rats with a delivery time difference of less than 48 hours were selected and randomly assigned to 7 groups, including a normal group (without any modeling or medication), a model group, a CRKL low-dose group of model group model rats receiving CRKL at the dose of 3 g/kg, a CRKL medium-dose group of model rats receiving CRKL at the dose of 6 g/kg, a CRKL high-dose group of model rats receiving CRKL at the dose of 9 g/kg, a positive drug group of model rats receiving domperidone at the dose of 3 mg/kg, and a negative control (NC) group of model rats receiving normal saline. Each group contained 6 rats. Except for the normal and model groups, the remaining 5 groups were continuously administered with the respective intervention drugs at the specified doses by gavage once a day for 10 days. Changes in the total litter mass of the offspring in the 7 groups within 10 days were measured, and HE staining was performed to identify pathological changes in the mammary tissue (MT). Six groups of rats (excluding the positive control group) were used to observe the pathological changes of eosinophils in pituitary tissue. ELISA was performed to determine the content of prolactin (PRL) in serum, immunohistochemical staining was used to determine the expression of prolactin receptor (PRLR) in MT, and RT-qPCR was used to determine the mRNA expression of genes related to lactation in MT. Network pharmacology and molecular docking were used to study the therapeutic effect and mechanism of CRKL on postpartum hypogalactia, particularly whether it acted through the SFRP2-Wnt/β-catenin signaling pathway. The mechanism of CRKL treatment was further validated by detecting mRNA (RT-qPCR) and protein expression (Western blot) of related pathway genes. Cell experiments were conducted using primary culture rat mammary epithelial cells (RMEC) from rat MT. RMEC were divided into four groups, including a normal group (primary culture RMEC, untreated), SFRP2 overexpression group (primary cultured RMEC treated with SFRP2 overexpression vector), SFRP2 overexpression+CRKL group (receiving treatment for SFRP2 overexpression group plus 10% drug-containing serum), and negative control group (primary culture RMEC treated with empty vector). The effect of CRKL on the expression of lactation-related genes FASN, CSN2, and GLUT1 mRNA after SFRP2 overexpression was detected by RT-qPCR.
    Results In this study, CRKL was administered at a dose of 3 g/kg in the CRKL low-dose group, 6 g/kg in the medium-dose group, and 9 g/kg in the high-dose group (P<0.05 or P<0.01). Compared with the model group, CRKL at all doses significantly increased the total litter weight gain of the offsprings within 10 days (P<0.05 or P<0.01), and effectively increased lactation (P<0.01), the area of mammary lobules, and the size and filling of acinar cavities. CRKL at all doses also increased the number of eosinophils that secreted PRL in the pituitary gland of the postpartum hypogalactia rat model, and increased the content of PRL in the serum (P<0.05 or P<0.01). CRKL promoted the secretion and expression of PRL in postpartum hypogalactic model rats. In addition, it significantly promoted the expression of genes related to milk fat, milk protein, and lactose synthesis in MT (P<0.05 or P<0.01). Network pharmacology predicted that the Wnt signaling pathway might be a key pathway for CRKL in treating postpartum hypogalactia. The molecular docking results showed that related chemical components in CRKL had good binding ability with CCND1 and SFRP2. Compared with the model group, CRKL at all doses inhibited the expression of SFRP2 gene in vivo (P<0.01) and activated the mRNA and protein expression of CCND1 and c-Myc in the Wnt/β-catenin signaling pathway in MT (P<0.05 or P<0.01). Cell experiments showed that, compared to the normal group, SFRP2 overexpression reduced the mRNA expression of milk synthesis-related genes FASN, CSN2, and GLUT1 in RMEC (P<0.01). The CCK8 results indicated that 10% of the drug-containing serum was the effective concentration administered to cells (P<0.01). After administering drug-containing serum, the expression of the lactation-related genes FASN, CSN2, and GLUT1 were up-regulated (compared with the SFRP2 overexpression group, P<0.01).
    Conclusion CRKL alleviates postpartum hypogalactia through the SFRP2-Wnt/β-catenin signaling pathway. SFRP2 might be a potential new target for the diagnosis and treatment of postpartum hypogalactia. This reveals a new mechanism of CRKL in treating postpartum hypogalactia and promotes its clinical application.
     
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