Metabolomics and Network Pharmacology-Based Investigation into the Mechanisms Underlying the Therapeutic Effect of a New Chinese Traditional Medicine (Cui Nai Ling) on Bromocriptine-Induced Hypogalactia

As a traditional veterinary medicine to promote lactation, Cui Nai Ling (CNL) can not only increase milk supply and promote health but also improve the overall physiological function and immunity of the animals. In order to further improve CNL's effect on lactation, we have previously made a new CNL (NCNL) by adding Tetrapanacis Medulla and replacing Vaccariae Semen with fried Vaccariae Semen in CNL. We have demonstrated that the lactation-promoting effect of NCNL is better than that of CNL. However, the underlying mechanisms by which NCNL promotes lactation are unclear. In this study, we performed metabolomics, network pharmacology, and pharmacodynamic studies to explore the underlying mechanisms by which NCNL promotes lactation in rats with bromocriptine-induced hypogalactia. The results showed that NCNL significantly improved the loss of appetite in female adult rats and the weight loss of pups caused by the disorder of lactation. Biochemical analysis showed that NCNL could regulate the levels of PRL, T4, E2, Ca, UREA, GLU, ALT, AST, TCHO, and TG in serum. The pathological results showed that NCNL could promote lactation and increase the mammary gland index by improving breast acinar tissue morphology in rats with hypogalactia. Network pharmacology studies showed that NCNL promotes lactation through P13K-Akt, insulin resistance, and prolactin signaling pathways, among which the most frequently affected pathway was the P13K-Akt signaling pathway. Metabolomics studies showed that NCNL can significantly upregulate phenylalanine, tyrosine, and tryptophan biosynthesis and tyrosine metabolism pathways and downregulate cysteine and methionine metabolism pathways. NCNL can significantly increase the serum prolactin concentration, improve the glucose and lipid metabolism disorders, and regulate PI3K-Akt, insulin resistance, and prolactin pathways to affect the amino acids' metabolism in the mammary gland and ultimately exert its therapeutic effect on bromocriptine-induced postpartum hypogalactia. These findings revealed the effect and application value of NCNL on animals with postpartum hypogalactia.


Preparation of the Aqueous Extract of CNL and NCNL.
A proper amount of CNL and NCNL powder was added with 10 times of the distilled water and soaked for 30 minutes. After heating and refluxing for 1 hour, it was filtered and added with 8 times of distilled water to reflux and extract the residue for 1 h. Following filtering, the two filtrates were combined. CNL was concentrated to 1 g/mL, and NCNL was concentrated to 0.5 g/mL and 1 g/mL, respectively (the content of ferulic acid was 0.53 mg/g, the content of Senkyunolide I was 0.25 mg/g, the content of chlorogenic acid was 0.24 mg/g, and the content of ligustilide was 15.42 mg/g).

Animal Grouping and Drug
Administration. SD pregnant rats (at 15-16 days of gestation) were purchased from SPF (Beijing) Biotechnology Co., Ltd., and raised in a single cage at the condition of 12 hours of light per day, the temperature of 22 ± 3°C, and humidity of 60 ± 5% for 7 days. e female rats within 12 hours of parturition were randomly divided into 5 groups: the normal group, the control group, CNL treatment group (CNL, 1000 mg/kg), low-dose NCNL treatment group (NCNLL, 500 mg/kg), and highdose NCNL treatment group (NCNLH, 1000 mg/kg). Each group had 7 female rats, and each female adult was assigned 10 pups. Except for the normal group, rats in each group were treated with bromocriptine (1.6 mg/kg) by intragastric administration once a day to create a hypogalactia model. e female rats in the CNL group, NCNLL group, and NCNLH group were administered intragastrically with CNL (1000 mg/kg), low-dose NCNL (500 mg/kg), and high-dose NCNL (1000 mg/kg) at 8 am and 8 pm continuously for 7 days. Rats in the normal and control group were given the same volume of normal saline similarly. All animals were handled according to the animal welfare guidelines and approved by the Institutional Animal Care and Use Committee of Shanxi University of Chinese Medicine (2019LL202).

Analysis of Physical Signs.
During the first week after drug administration, the weight of each female adult and the total weight of pups in each group were measured. e food and water intake of each female adult rat was monitored daily. One week after the drug administration, the adults fasted for 12 hours and were anesthetized, and blood was collected from the abdominal aorta for later uses. e mammary gland tissues of the female adult rats were collected, the thymus tissues of each adult rat were weighed, and the mammary gland index was calculated (thymus weight/adult weight ×100%).

Histopathological Observation.
Mammary gland tissues were fixed in 4% paraformaldehyde in 0.1 M PBS, embedded in paraffin, and cut into 5 μm sections. To observe structural damage to mammary gland tissue, sections were stained with hematoxylin and eosin (H&E). e sections were visualized using light microscopy, and digital images were captured and analyzed.

Western Blotting Analysis.
Mammary gland tissues were isolated by using a T-PER tissue protein extraction reagent ( ermo Fisher Scientific, Waltham, MA, USA). e protein content was measured using a Bradford assay, separated using SDS-PAGE, and transferred to a nitrocellulose membrane [4]. e membranes were incubated in 5% skimmed milk, dissolved in Tris-buffered saline with 1% Tween-20 (TBST), pH 7.5, for 30 min at RT for blocking, and then incubated with anti-prolactin (Boster Biological Technology Co., Ltd. Wuhan, Hubei, China), albumin (Abcam, Cambridge, England, UK), and β-actin (Bioswamp Biological Pharmaceutical Co., Ltd, Hubei, Wuhan, China) antibodies at 1 : 1000 overnight at 4°C and incubated with HRP-conjugated mouse or rabbit secondary antibodies for 3 h at RT. Blots were scanned and analyzed with ChemiDoc MP Imaging System (GeneGnome XRQ Gene Co., Ltd., USA). [5]. Serum samples were thawed at 4°C on ice. en, 100 μL of the sample was taken and placed in an EP tube, extracted with 400 μL of extraction solvent (methanol: acetonitrile � 1 : 1 [v/v], containing an internal standard, L-2 chlorophenylalanine at 2 μg/mL), vortexed for 30 s, treated with ultrasound for 5 min (during incubation in ice water), and incubated for 1 h at −20°C to precipitate proteins. e solutions were then centrifuged at 12000 rpm for 15 min at 4°C. e supernatant was transferred into fresh EP tubes, and the extracts were dried in a vacuum concentrator without heating and reconstituted in 200 μL of the extraction solvent (acetonitrile: water � 1 : 1 [v/ v]). e reconstituted solution was vortexed for 30 s, sonicated for 10 min (in a 4°C water bath), and centrifuged for 15 min at 12000 rpm at 4°C. e supernatant was transferred into a fresh 2 mL LC-MS glass vial, 10 μL aliquots from each sample were collected and pooled as QC samples, and the supernatant was collected for UHPLC-QTOF-MS analysis.
e Triple TOF mass spectrometer was used for its ability to acquire MS/MS spectra on a full MS dd-ms2 during the LC-MS experiment. In this mode, the electrospray capillary voltage was 3.2 kV in positive and negative ionization modes, the atomized gas velocity was 35 L·min -1 , the auxiliary air velocity was 5 L·min -1 , the ion source temperature was 320°C, and the auxiliary heater temperature was 350oC. e NCE values of energy collision were 25, 35, and 45, respectively. e LC/Q/TOF-MS metabolomic profiling data were imported into Compound Discoverer 3.0 to perform the metabolic feature extraction by the adoption of a molecular feature extraction algorithm ( ermo Fisher, Inc., Santa Clara, CA, USA). e parameters were set as follows: mass range, 100-1, 500; mass deviation, 5 × 10 −6 ; retention time deviation, 0.05 min; SNR threshold, 3. SIMCA-P (Version 14.1, Umetrics AB, Umea, Sweden) was used for multivariate statistical analysis of the integral values obtained from LC-MS findings. e mean-centered data were used for principal component analysis (PCA). e modeling of sample classes was performed using orthogonal projection to latent structure discriminant analysis (OPLS-DA) algorithm at a unit variance-scaled modality. OPLS-DA analysis was performed between the two groups to evaluate the differences between the normal and the control and the control and the NCNLH and to generate corresponding S-plots and VIP graphs. e points at each end of the S-plot diagrams are potential deferentially abundant metabolites. Metabolites with VIP >3 and one-way ANOVA values with P < 0.05 were used to identify differences in metabolite levels between groups. e disturbed metabolites and metabolic pathways is study aimed to screen all the active ingredients in NCNL to construct an "ingredient-target-disease" network and explore the pharmacodynamic mechanisms of NCNL. erefore, the active ingredients for NCNL were not screened based on Oral Bioavailability (OB) and Druglikeness (DL).

Identification of NCNL and Lactation Overlapping
Genes. Genes related to lactation were identified from online Mendelian Inheritance in Man (OMIM, http://www. omim.org/) [6] and GeneCards ® ( e Human Gene Database) (https://www.genecards.org) [7] using relevant keywords as queries. e results of the OMIM database and GeneCards database were combined according to the relevant search conditions to obtain gene targets for lactation. Drug and disease target genes were uploaded to the online Venn program (http://bioinformatics.psb.ugent.be/ webtools/Venn) and obtain the overlapping genes related to the active ingredients and lactation.

Construction of the NCNL and Lactation Protein-Protein
Interaction (PPI) Network. PPI was constructed by the protein interaction database (STRING 11.0, https://stringdb.org) [11]. e confidence score threshold was set at 0.95 to hide unconnected nodes in the PPI network.

Pathway Analysis of Core Targets.
e common protein targets of NCNL and lactation were uploaded to the Database for Annotation (Visualization and Integrated Discovery, DAVID, https://david.ncifcrf.gov/home.jsp) [12].
2.11. Statistical Analysis. Data analyses were conducted using GraphPad Instat software (ver. 5.0; GraphPad Software, La Jolla, CA, USA). e data were expressed as the mean ± standard deviation (SD) of seven mice in each group. e significance of treatment effects was determined by oneway analysis of variance (ANOVA) followed by Tukey's post hoc analysis. e P value of 0.05 was considered to indicate statistical significance.

Effect of NCNL on the Physiological Signs of Rats with
Postpartum Hypogalactia. As shown in Table 1, there was no significant difference in the body weight of adult rats between the normal and control groups (P > 0.05). Furthermore, there was no significant difference in the body weight of adult rats between the control group and the treatment groups (CNL, NCNLL, and NCNLH) (P > 0.05).
ese results indicated that body weight of the adult rats did not change significantly after postpartum hypogalactia was induced or after CNL, NCNLL, and NCNLH administration. During the one-week drug administration period, the total weight of the pups in the control group decreased significantly from day 2 to day 7 compared with that in the normal group due to the obstruction of lactation in adult rats (P < 0.05). At day 7, the total weight of pubs in the CNL group was significantly higher than that in the control group (P < 0.05). Compared with the control group, the total weight of the pups in the NCNLL and NCNLH groups began to increase significantly at Day 5 and Day 6 (P < 0.05), respectively, after intragastric administration of NCNL. ese results indicated that NCNLL and NCNLH can significantly improve the weight loss of pups caused by the impaired lactation of the adult rats.
In addition, during the one-week drug administration, the food intake of the adult rats in the control group was significantly lower than that in the normal group (P < 0.05). Food intake of the adult rats in the CNL group was not significantly different from that in normal group (P > 0.05). Compared to the control group, food intake in NCNLL and NCNLH groups began to increase significantly on day 5 (P < 0.05). ese results indicated that NCNLL and NCNLH can significantly improve the loss of appetite in adult rats caused by lactation disorders. On days 2 and 3, the water intake of adult rats in the control group was significantly different from that in the normal group (P < 0.05). ere was no significant difference of the water intake between the CNL group and the control group (P > 0.05). Furthermore, there was no significant difference in water intake between NCNLL/NCNLH groups and the control group (P > 0.05).
ese results indicated that NCNLL and NCNLH have no Evidence-Based Complementary and Alternative Medicine 5 significant effect on the water intake of adult rats with bromocriptine-induced hypogalactia.

e Effect of NCNL on Serum Biochemical Parameters in Rats with Postpartum Hypogalactia.
To determine the effect of NCNL on bromocriptine-induced postpartum hypogalactia in adult rats, we measured the concentration of hormones in serum. One week after administration, the serum levels of GH, PRL, DA, T3, and T4 in the control group were significantly lower than those in the normal group (P < 0.05). Serum T3, T4, and DA concentrations in the CNL group were significantly higher than those in the control group (P < 0.05). e serum concentrations of PRL and T4 in the NCNLL and NCNLH groups were significantly higher than those in the control group (P < 0.05). In addition, the concentrations of E2, PROG, Ca, and UREA in the control group were significantly higher than those in the normal group due to the dysfunction of lactation. e concentrations of E2, Ca, and UREA in the CNL group were significantly lower than those in the control group (P < 0.05). e concentrations of E2, Ca, and UREA in the NCNLL group were significantly lower than those in the control group (P < 0.05). e concentrations of E2 and Ca in the NCNLL group were significantly lower than those in the control group (P < 0.05).
ese results indicated that NCNLL and NCNLH can improve the bromocriptine-induced postpartum hypogalactia by regulating the level of lactation-related hormones in the serum of adult rats.
At the same time, the concentrations of INS and IGF-1 in the control group were significantly lower than those in the normal group (P < 0.05), while the GLU concentration in the control group was significantly higher than that in the normal group. e concentrations of INS, IGF-1, and GLU were not significantly different between the CNL group and the control group (P > 0.05). Interestingly, compared with the control group, the INS, IGF-1, and GLU concentrations in the NCNLL group changed significantly (P < 0.05). Compared with the control group, GLU concentration in the NCNLH group also changed significantly (P < 0.05). ese results indicated that NCNLL and NCNLH can improve the disorders of glucose metabolism caused by postpartum hypogalactia in adult rats.
Finally, compared with the normal group, ALT, AST, TCHO, and TG levels in the control group were significantly increased (P < 0.05). After a week of intragastric administration, the levels of ALT, AST, TCHO, and TG in the CNL, NCNLL, and NCNLH groups were significantly decreased compared to those in the control group (P < 0.05). ese results indicated that NCNLL and NCNLH can regulate the lipid metabolism disorder in adult rats with hypogalactia ( Table 2).

e Effect of NCNL on the Pathological Alterations of the Mammary Glands in Rats with Postpartum Hypogalactia.
To determine the effect of NCNL on lactation disorders, we performed H&E staining for the tissues from mammary glands of the rats with postpartum hypogalactia. As shown in Figure 1(a), the mammary glands in the control group exhibited atrophy and connective tissues appeared. After drug administration, the rats in each treatment group had obvious breast acini, but there were varying degrees of acinar hyperplasia and secretion in the alveolar cavity. e mammary gland index in the control group was significantly lower than that in the normal group (P < 0.05). e mammary gland index in the CNL group was significantly higher than that in the control group (P < 0.05). However, there was no significant difference between the mammary gland index in the CNL, NCNLL, and NCNLH groups and that in the control group (P > 0.05) (Figure 1(b)). ese results indicated that NCNLL and NCNLH can improve lactation and increase mammary gland index by improving the tissue type of breast acini in rats with hypogalactia.

Network Pharmacology-Based Studies on the Mechanisms of Actions of NCNL on Postpartum Hypogalactia.
A total of 664 active ingredients and 5,421 targets were obtained. e lactation-related genes from the OMIM database and GeneCards database were combined, resulting in 3303 gene targets related to lactation. e ingredients and disease targets were uploaded to the online Venn diagram (http:// bioinformatics.psb.ugent.be/webtools/Venn/), and a total of 165 overlapping genes associated with both active ingredients and lactation were obtained. e top 20 significant overlapping genes are presented in Table 3. e other overlapping genes are shown in Supplementary Table 1. Cytoscape 3.7.1 (http://www.cytoscape.org/) software was used to construct the "ingredient-target-disease" interactive network between NCNL and lactation (Figure 2(a)). According to the "ingredient-target-disease" interactive network between NCNL and lactation, a total of 302 high-frequency common ingredients were obtained.
A protein-protein interaction database (STRING 11.0, https://string-db.org/) was constructed for CNL and lactation (Figure 2(b)), and the most significant 20 associated nodes in PPI are shown in Table 4. Other nodes in PPI are shown in Supplementary Table 2. e comparative analysis resulted in a total of 30 highfrequency common targets. e central node of the PPI network between CNL and lactation was INS. In addition, protein kinase B1 (ALB), interleukin 6 (IL6), cysteine acid aspartic protease 3 (CASP3), epidermal growth factor receptor (EGFR), vascular endothelial growth factor A (VEGFA), nucleophosphoprotein (MYC), and mitogenactivated protein kinase 8 (MAPK8) also appeared frequently.
ese common protein targets of CNL and lactation were uploaded to the DAVID database, and a total of 1583 GO entries and 17 high-frequency common KEGG pathways were obtained. e results were visualized using Cytoscape 3.7.1 (http://www.cytoscape.org/) (Figures 2(c)  and 2(d)). e results showed that the pathways involved in NCNL and lactation included the P13K-Akt, insulin resistance, and the prolactin signaling pathway, among which the most frequent pathway was the P13K-Akt signaling pathway (Figure 2(e)). ese data provided a theoretical basis for the possibility that the antagonistic activity of NCNL against DN may be 6 Evidence-Based Complementary and Alternative Medicine   ese predicted results were verified by western blotting.
Significant inhibition of prolactin (P < 0.001) was observed in the control group compared with the normal group. e administration of CNL and NCLN at low and high doses significantly increased prolactin expression (Figure 1(c)).
Significant increases in albumin (P < 0.05) occurred in the control group compared with the normal group. However, NCNLH administration significantly decreased the expression of albumin (P < 0.01) (Figure 1(d)).  Figure 1: e effect of NCNL on the mammary pathology (a), mammary gland index (b), and protein expression (c, d) in rats with hypogalactia. Tissue staining with H&E (a). All tissues were observed using a light microscope; representative images are shown (×400). Mammary gland to body weight ratio (b). Expression of prolactin and albumin was determined by western blotting (c-d). Relative expression of prolactin and albumin was calculated using β-actin for normalization. Normal: normal group. Control: bromocriptine-induced hypogalactia control group. CNL: traditional Cui Nai Ling aqueous extract 1000 mg/kg administered group. NCNLL: new Cui Nai Ling aqueous extract 500 mg/kg administered group. NCNLH: new Cui Nai Ling aqueous extract 1000 mg/kg administered group. e data are expressed as the mean ± standard deviation (n � 7). * P < 0.05, * * P < 0.01, and * * * P < 0.001 versus the normal group; # P < 0.05, ## P < 0.01, and ### P < 0.001 versus the control group. Table 3: e top 20 significant overlapping genes between NCNL and lactation.

No.
Overlapping genes 1 Prostaglandin-endoperoxide synthase 1 (PTGS1) 2 Coagulation factor VII (F7) 3 Gamma-aminobutyric acid type A receptor subunit alpha-1 (GABRA1) 4 Transient receptor potential cation channel subfamily V member 1 (TRPV1) 5 Cholinergic receptor muscarinic 2 (CHRM2) 6 RELA proto-oncogene, NF-KB subunit (RELA) 7 Proliferating cell nuclear antigen (PCNA) 8 Albumin (ALB)  9 MYC proto-oncogene, BHLH transcription factor (MYC) 10 Uncoupling protein 2 (UCP2) 11 Progesterone receptor (PGR) 12 Nuclear receptor subfamily 3 group C member 2 (NR3C2) 13 Nuclear receptor coactivator 1 (NCOA1) 14 Aldo-keto reductase family 1 member B (AKR1B1) 15 Plasminogen activator, urokinase (PLAU) 16 Alcohol dehydrogenase 1B (class I), beta polypeptide (ADH1B) 17 Telomerase-associated protein 1 (TEP1) 18 Erb-b2 receptor tyrosine kinase 2 (ERBB2) 19 Peroxisome proliferator-activated receptor gamma (PPARG) 20 Lipoprotein lipase (LPL) 8 Evidence-Based Complementary and Alternative Medicine   e PCA results showed that the normal group and the control group can be completely separated, indicating that the physiological and metabolic status of the rat with bromocriptine-induced hypogalactia changed significantly. Further analysis using the OPLS-DA pattern recognition method showed that the scatter points of the normal group and the control group can be clearly separated, which is consistent with the results of PCA analysis. ese results indicated that the hypogalactia model developed successfully in rats. e scatter points in CNL, NCNLL, and NCNLH groups can also be clearly distinguished from those in the control group, indicating that the overall metabolism in the drug treatment groups was significantly different from that in hypogalactia group. At the same time, the model verification results show that all R 2 and Q 2 values on the left were lower than the original point on the right and the intersection of the Q 2 regression line and vertical axis was less than zero, indicating that the model had high reliability (Figures 3(f )-3(i)).
We performed S-plot (Figures 3(j)-3(m)) combined with VIP value (>3) and an independent sample t-test to compare the difference in metabolites between the normal and control groups and identify metabolites that were specifically associated with bromocriptine-induced hypogalactia. We obtained 30 significantly different metabolites between different groups shown in Table 5. e contents of nine metabolites including 9-oxo-10(E), 12(E)-octadecadienoic acid, abietic acid, choline, DL-carnitine, DL-stachydrine, cytosine, hypoxanthine, methyl palmitate, and trans-3-indoleacrylic acid in the control group were significantly increased compared to those in the normal group, while the contents of 21 metabolites including 2-hydroxyphenylalanine, acetyl-L-carnitine, betaine, corticosterone, creatinine, DL-tryptophan, docosahexaenoic acid, erucamide, hexadecanamide, L-phenylalanine, L-tryptophan, methionine, N,N′-diphenylguanidine, nicotinamide, oleamide, PEG n11, PEG n12, pipecolic acid, proline, spermine, and stearamide in the control group were significantly decreased compared to those in the normal group (Figure 3(j)).    Based on the metabolic profile, the NCNLH group was significantly separated from the control group, indicating that NCNLH can effectively improve bromocriptine-induced hypogalactia. Based on the level of metabolites, NCNLH can significantly reduce the concentration of 13 metabolites including 9-oxo-10(E), 12(E)-octadecadienoic acid, adenosine, choline, DL-carnitine, DL-stachydrine, DLtryptophan, hypoxanthine, leucine, methyl palmitate, nicotinamide, oleamide, pipecolic acid, and trans-3-indoleacrylic acid. Compared with the control group, NCNLH can significantly increase the concentration of 15 metabolites including acetyl-L-carnitine, betaine, corticosterone, cytosine, docosahexaenoic acid, hexadecanamide, L-phenylalanine, L-tryptophan, L-tyrosine, N, N′-diphenylguanidine, PEG n10, PEG n11, spermine, stearamide, and taurocholic acid. NCNLH did not significantly alter the content of 2hydroxyphenylalanine, creatinine, and PEG n14 (Figure 3(l)). e abovementioned differential metabolites were imported into MetPA (http://www.metaboanalyst.ca/) for pathway analysis (Figures 3(n) and 3(o)). e metabolic pathway with Impact >0.1 was considered as the potential target pathway. Compared with the normal group, five metabolic pathways including phenylalanine, tyrosine, and tryptophan biosynthesis, phenylalanine metabolism, nicotinate and nicotinamide metabolism, tryptophan metabolism, and cysteine and methionine metabolism pathways were significantly altered in the control group. e metabolic pathways that were significantly upregulated by NCNLH included phenylalanine, tyrosine, and tryptophan biosynthesis, and tyrosine metabolism pathways. e pathways that were downregulated by NCNLH were cysteine and methionine metabolism pathways.

Discussion
Veterinary drugs refer to drugs and their related products used to maintain growth and development, prevent diseases, and promote production performance in livestock and poultry animals [16]. According to the report of the World Health Organization Joint Expert Committee on Food Additives (JECFA), there are approximately 120 veterinary drug residues in food. Veterinary drugs are mainly divided into antibiotics, hormones, and anthelmintics. Antibiotics are the main veterinary drug additives, accounting for 60% of the total veterinary drugs [17][18][19]. Extensive use of veterinary drugs not only affects the quality and safety of animal products but also damages the health of consumers due to the drug residues. Improper use of antibiotics, hormones, and chemical synthetic additives poses safety concerns to livestock products [20]. At present, there is a huge market demand for safe, effective, and low-toxic new pharmaceutical additives that are developed by traditional Chinese medicine.
Traditional Chinese medicine has a long history and has been used to prevent, diagnose, and treat diseases or regulate human physiological functions under the guidance of its unique theoretical system [21,22]. e main source of traditional Chinese medicine is derived from plants, while a small portion is derived from animals, minerals, and biological products. ere are approximately 3,000 kinds of Chinese medicines recorded in the Chinese medicine literature, and most of them were derived from wild animals and plants. ey are obtained by natural processing methods to retain their natural structure and biological activities. ey are used for humans and animals for a long time and have been verified to have therapeutic and health care effects [23,24]. Under the guidance of modern analytical technology, animal nutrition, and modern engineering technology, Chinese medicine additives are prepared with the characteristics of low toxicity, few residues, low level of drug resistance, wide ranges of sources, and low prices.
In this study, we added Tetrapanacis Medulla and replaced Vaccariae Semen with fried Vaccariae Semen in CNL described in the 2015 edition of "Chinese Veterinary Pharmacopoeia". Vaccariae Semen has been used to reduce swelling for breast carbuncle or other types of sore carbuncle and pain [25]. Frying can easily produce the effective ingredients of Vaccariae Semen with warm characteristics, strong dispersing effect, better ability to promoter blood circulation, menstruation, and lactation. Fried Vaccariae Semen is mostly used for postpartum milk retention. Zhou et al. [26,27] demonstrated that the dissolution rate of cyclic peptides, erythronine, and flavonoid glycosides in Vaccariae Semen increased significantly in water decoction after frying. Tetrapanax papyrifer is sweet, light, and slightly cold and is mostly used for edema and postpartum milk retention. Tetrapanacis Medulla can promote the stomach gas to move up and increase lactation [28]. Combined with Chuanxiong Rhizoma in CNL, it enhances the function of gas regulation and promotes blood circulation and lactation. rough the above-described ingredient adjustments, the effect of NCNL on dysmenorrhea and lactation is enhanced. e results of these studies showed that NCNLL and NCNLH can significantly improve the loss of appetite in female adult rats and significantly reduce the weight loss of pups due to lactation disorders in adults. e synthesis of milk and lactation is a complex physiological process controlled by the neuroendocrine system, including a variety of endocrine hormones [29]. It is well recognized that PRL secreted by the pituitary gland is the most important hormone in the process of lactation [30]. RPL plays an important role in the maintenance of lactation. Various other factors regulate milk synthesis and lactation through prolactin. erefore, the occurrence of postpartum hypogalactia is the process and consequence of the decreased synthesis and secretion of breast milk due to the reduced secretion of prolactin by the anterior pituitary gland or inhibition of its function. e secretion of prolactin is mainly regulated by the neuroendocrine system [31]. For example, secretion of prolactin by the prolactinsecreting cells in the pituitary gland is regulated by the signal balance between the secreted inhibitors and stimulators from the hypothalamus as well as the hormones in the peripheral blood. Among them, the prolactin inhibitory factor (PIF) secreted by the hypothalamus includes DA and r-aminobutyric acid, while the prolactin-releasing factor (PRF) includes gonadotropin-releasing hormone, thyrotropin-releasing hormone, vasoactive intestinal polypeptide, and angiotensin II. In addition, neurotransmitters in the brain, e.g., catechins and serotonin, can also promote the release of prolactin. Various internal and external factors, such as nutritional deficiency and stress, can increase the level of PIF through the neuroendocrine system. When PRF decreases, secretion of prolactin is reduced, resulting in a decrease in the concentration of prolactin in the blood and decreases or termination of milk secretion and postpartum hypogalactia or agalactia [29]. In this study, compared with the control group, the NCNLL and NCNLH groups can regulate the levels of PRL, T4, E2, Ca, UREA, GLU, ALT, AST, TCHO, and TG in the serum, indicating that NCNL at either low or high doses can improve bromocriptine-induced lactation disorder, glucose, and lipid metabolism disorder through regulating the level of lactation-associated hormones in the serum of female adult rats. Such mechanisms were further explored and verified by network pharmacology studies. INS is the core target of NCNL in bromocriptine-induced hypogalactia. INS-associated pathways include the P13K-Akt signaling, insulin resistance, and prolactin signaling pathways among which the most frequent pathway is the PI3K-Akt signaling pathway.
ere are INS and its receptors in breast tissue; thus, INS can directly act on the breast through the receptor and have a regulatory effect on the final differentiation, maturity, and milk production of the breast, as well as the growth and development of newborns. NCNL can promote the proliferation of mammary gland cells through the PI3K-Akt signaling pathway, regulate the level of insulin through the insulin resistance signaling pathway, and increase lactation through the prolactin signaling pathway.
In addition, the metabolism of mammary gland nutrients is also an important factor affecting lactation [32]. Whey protein is the most important nutrient in milk and one of the important indicators to measure milk quality [33]. As the basic unit of whey protein synthesis, amino acids are actively metabolized in the mammary gland and are closely related to lactation [34]. Most free amino acids adsorbed from blood to the mammary glands are used for whey protein synthesis. Amino acids in the mammary glands also play an important role in metabolism regulation. Amino acids enter the mammary epithelial cells through the transporter and undergo protein synthesis and degradation processes. e metabolism of amino acids in the mammary gland is complex, and the metabolic process and efficiency of various amino acid metabolism need to be further revealed. In this study, the metabolomics results showed that the metabolic pathways that are different between control and normal groups or between control and each treatment group were concentrated in amino acid metabolism. For example, compared to the normal group, five metabolic pathways including phenylalanine, tyrosine, and tryptophan biosynthesis, phenylalanine metabolism, nicotinate and nicotinamide metabolism, tryptophan metabolism, and cysteine and methionine metabolism pathways were altered significantly in the control group. Compared to the control group, phenylalanine, tyrosine, and tryptophan biosynthesis, and tyrosine metabolism pathways were upregulated, while cysteine and methionine metabolism pathways were downregulated in the NCNLH group.
ese results suggested that NCNL can increase the uptake of essential amino acids by the mammary gland and the balanced uptake of nonessential amino acids, which ultimately improves the bromocriptine-induced postpartum hypogalactia.
To the best of our knowledge, this is the first study using metabolomics coupled with network pharmacology to reveal the mechanisms underlying the therapeutic effect of an NCNL on bromocriptine-induced hypogalactia. However, we performed metabolic profiling only by nontargeted metabolomics. Future studies will focus on validation of the results by targeted metabolomics and improving the specificity enriched by network pharmacology and association with target diseases.

Conclusions
In this study, we modified CNL by adding or replacing the original component to achieve NCNL and applied NCNL in the bromocriptine-induced postpartum hypogalactia model. Our results demonstrated that NCNL can significantly increase the serum prolactin concentration in rats with bromocriptine-induced postpartum hypogalactia. Furthermore, NCNL can significantly improve the glucose and lipid metabolism disorders and affect amino acid metabolism through PI3K-Akt, insulin resistance, and prolactin pathways to achieve its therapeutic effect. ese findings revealed the effect and application value of NCNL on postpartum hypogalactia in animals. However, the safety of the milk and the growth and metabolism parameters in the pups require further evaluation in future studies.