2.4.1 Preparation of rat serum samples

The 9 female Sprague-Dawley rats were randomly divided into a blank group (B group), Raw products of Semen Cuscuta (S) group, and Semen Cuscutae processed with salt solution (Y) group, with three rats in each group. In this experiment, the medium dose of 10.8 g/kg was chosen for administration. The medium dose was 10 times the equivalent dose. The rats were orally administered twice daily for 3 days. Group B was fed normally. The blood was collected from the orbital plexus of rats at 30 and 60 min after the last administration, respectively. Then centrifuged at 3500 r/min for 10 min at 4℃ after resting for 30 min, and the supernatant was extracted to obtain serum samples.

400 μL of each serum sample was taken; 40 μL hydrochloric acid (2 mol/L) was added, vortex shaken for 1 min, the samples were incubated at 4℃ for 15 min, repeat vortex resting four times and dry by nitrogen blowing; The dried sample was dissolved in 200 μL of 80 % methanol and centrifuged at 4 °C for 10 min (12000 rpm). Finally, the collected supernatant was used to perform UHPLC-Q/TOF-MS analysis.

2.4.2 Chromatographic conditions

Sample analysis was performed on the UHPLC (Nexera UHPLC LC-30A) system was coupled. The column was Waters UPLC BEH C18 (2.1 mm × 100 mm, 1.7 μm). Flow rate of 0.4 mL/min, injection volume of 3 μL. The mobile phase consisted of 0.1 % formic acid water (A) and 0.1 % formic acid acetonitrile (B) with a gradient as follows: 0–3.5 min, 95–85 % A; 3.5–6 min, 85–70 % A; 6–6.5 min, 70 % A; 6.5–12 min, 70–30 % A; 12–12.5 min, 30 % A; 12.5–18 min, 30–0 % A; 18–22 min, 0 % A.

2.4.3 Mass spectrometer conditions

The AB 5600 Triple TOF mass spectrometer is capable of primary and secondary mass spectrometry data acquisition based on the IDA function under the control of the control software (Analyst TF 1.7, AB Sciex). In each data acquisition cycle, the most intense molecular ions with an intensity greater than 100 were selected to obtain the MS/MS data. The bombardment energy: 40 eV, collision energy difference: 20 V, 15 secondary spectra every 50 ms. The ESI ion source parameters were set as follows: nebulization air pressure (GS1): 55 psi, auxiliary air pressure: 55 psi, air curtain air pressure: 35 psi, temperature: 550 °C, spray voltage: 5500 V (positive ion mode) or −4000 V (negative ion mode).

2.4.4 UHPLC-Q/TOF-MS data analysis

The XIC Manager tool in PeakView software was used for post data acquisition analysis. A database of prototypical components of Semen Cuscutae in blood was established basis on the in vitro screened chemical compositions, containing name, molecular formula, exact molecular weight, retention time, etc. Blank serum as well as drug-containing serum samples of S/Y were imported into the XIC Manager tool in PeakView software with the methodological parameters for data processing as XIC intensity > 50 counts, plus or minus error at ± 5 ppm, and retention time within the specified time. The mol formula of the screened compounds was then imported, and the secondary fragment ions were matched to the structure of the components in the light of the literature as well as the cleavage pattern, and a match of > 80 % was considered reasonable.

2.5.1 Target prediction of Y and KDM target screening

The structural formulas of the 13 Y components absorbed into blood were downloaded from PubChem (https://pubchem.ncbi.nlm.nih.gov/), and the component potential targets were obtained from the TCMSP (https://old.tcmsp-e.com/tcmsp.php) and Swiss Target Prediction (https://www.swisstargetprediction.ch/). The targets with probability > 0.5 were selected. GeneCards Human gene database (GeneCards) (https://www.genecards.org/) and Online Mendelian Inheritance in Man (OMIM) (https://www.genecards.org/) were used to search the related genes of KDM by searching for keywords, “recurrent spontaneous abortion, abortion and miscarriages”. Finally, the above targets are standardized as GeneSymbol through UniProt database (https://www.uniprot.org/). In order to assess the interaction between the targets of SS and KDM, common targets were obtained using Venny 2.1 (https://bioinfogp.cnb.csic.es/tools/venny/), and then the component-target-disease pathway network were performed using the Cytoscape 3.7.2 software.

2.5.2 Protein-protein interaction (PPI) construction and analysis

The common targets between the components and the disease were imported into the STRING (https://string-db.org/). The parameters were set to “Homo sapiens” to obtain PPI network relationship maps and related network parameters. Import the obtained network parameters into CytoScape 3.7.2 software to construct and visualize the PPI network.

2.5.3 GO and KEGG analysis

Metascape database (https://metascape.org/) was used for performing Gene Ontology (GO) enrichment analysis including biological processes (BP), molecular function (MF), and cellular component (CC) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Correlation results were obtained with P < 0.01 as the screening condition.

2.5.4 “Ingredients–target–pathway” network construction

In order to analyze the action mechanism of SS active ingredients to improve KDM, the Cytoscape 3.7.2 software was used to construct “ingredients–target–pathway” network and determine the core targets in the pathway and the main entry components for treating KDM based on network parameters component.

2.5.5 Molecular docking

Chemical structures of compounds were available in PubChem (https://pubchem.ncbi.nlm.nih.gov/) and crystal structures of proteins were extracted from the RCSB Protein Data Bank (https://www.rcsb.org/). Through Pymol software, the solvent molecules in the target are removed, and the steps of hydrogenation, electron addition, and ROOT are performed on the target and through Autodock software. The processed protein and chemical structures were imported into AutoDockTools for docking experiments.

2.6.1 Animal model construction and grouping and morphological observation

The female and male rats were mated (mating ratio 2:1). Vaginal smears of female rats were examined daily in the early morning hours. Day 0 of pregnancy is defined by the presence of a large number of sperm on the vaginal smear or the dropping of a vaginal plug. Finally, 40 pregnant rats were screened and randomly divided into the following five groups (n = 8): Control group (C), Model group (M), Positive drug group (P), Raw products of Semen Cuscuta (S) group, Semen Cuscutae processed with salt solution (Y) group. Hydroxyurea combined with mifepristone by gavage was used to establish a KDM model (Wang et al., 2021). The model and each treatment group were given hydroxyurea (450 mg/kg) daily in the morning, and the corresponding therapeutic drug was administered in the afternoon for each treatment group (positive drug: 18 mg/kg progesterone capsules; 10.8 g/kg water extract of S; 10.8 g/kg water extract of Y) for 10 days. Except for the control group, mifepristone was given on day 11 at a dose of 3.75 mg/kg. After 24 h of mifepristone administration, serum and tissue samples were obtained from each group of rats after anesthetized by intraperitoneal injection with pentobarbital (30 mg/kg).

After anesthetizing the rats, the uterus was taken and weighed after stripping the surrounding fat and other tissues to observe the morphology of the embryo and uterus. The criteria for determining miscarriage as follows: uterine morphology, color, intrauterine changes; number of normal embryos and number of aborted embryos. Normal embryos: the uterus was thick as a string of beads, pink or light red in color. The size of the fetal mouse was seen to be consistent with gestational age after dissection. The embryo was intact and the heartbeat of the fetal mouse could be observed, and no stasis of blood was seen. Partial abortion: stasis of blood was seen in the uterine cavity and the embryo was incomplete. Complete abortion: significant weight loss in the mother, bamboo-like changes in the uterus, obvious fatigue or necrotic embryos seen in the uterine cavity, or only the embryo bed site was seen, no embryo was seen, or the embryo was dark brown, or there was vaginal bleeding.

2.6.2 Collection and preparation of samples

Pentobarbital (30 mg/kg body weight) was injected intraperitoneally to anesthetize the rats. Blood was collected from the abdominal aorta. Then, it was centrifuged at 3,500 rpm and 4 °C lasting for 15 min. Supernatant was aspirated, then stored at −80 °C.

The decidual tissue was separated from the uterine embedding site, and the entire meconium tissue was separated from the embryo and its embedding site, washed, absorbed water, and fixed with 4 % paraformaldehyde liquid fixative.

2.6.3 Measurement of embryo loss rate and uterine bleeding

Embryo loss rate (ELR): the uterus of intact pregnant rats was removed and opened longitudinally from the uterine horns. And the total number of fetuses, live fetuses and abortions in each group of pregnant rats in the uterus were recorded. The embryo loss rate was calculated. ELR = number of aborted embryos / (number of aborted embryos + number of normal embryos) × 100 %.

Uterine bleeding (UB): take 20 μL of rat venous blood, add 4 mL of 5 % NaOH and mix well. Collect the cotton ball containing the uterine blood of a rat in an EP tube, add an appropriate amount of 5 % NaOH (4 ∼ 7 mL) according to the actual situation of bleeding, the blood stain of cotton ball were soaked and scrubbed until the eluate was colorless. The absorbance (A) of the extracts and the rat venous blood were measured at 546 nm using 5 % NaOH solution as blank control. Vaginal bleeding (μL) = volume of venous blood × (A _{uterine dipstick} × V2) / (A _{venous blood} × V1), V1: dilution of venous blood; V2: amount of NaOH used for extracting uterine blood.

2.6.4 Histopathology

To evaluate histological changes in the decidual tissue, fixed in 4 % paraformaldehyde, followed by paraffin embedded sections, before being cut into 4 μm sections, and stained with hematoxylin and eosin (H&E). Pathological changes in the decidual tissues were observed by light microscope.

2.6.5 Immunohistochemical detection of decidual tissue

The decidual tissue sections were then incubated with primary antibodies at the following concentrations: VEGF antibody (1:100), PI3K antibody (1:100), AKT antibody (1:100), p-PI3K antibody (1:100), p-AKT antibody (1:100) followed by the necessary secondary antibodies according to the appropriate IHC staining protocol. These sections were then observed by light microscopy. For the negative controls, the primary antibody was replaced with 0.01 M phosphate buffered solution (PBS). The intensity of staining was determined by analyzing the integrated optical density (IOD) using an image analysis system (JD-801). large IOD values tend to be brownish-yellow, indicating dark staining and strong protein expression, while small IOD values are darker, indicating light staining and weak protein expression. IOD = area density × average optical density.

2.6.6 Detection of biochemical indices in rat serum

Enzyme-linked immunosorbent assay (ELISA) was used to test the content of progesterone (P), estradiol (E2) and vascular endothelial growth factor A (VEGF-A) in serum according to the manufacturer’s instructions. Optical density (OD) values were measured at 450 nm using a microplate reader.

3.2.1 Construction of the component-target-disease network and protein–protein interaction (PPI) network

Based on the 13 components of Semen Cuscutae processed with salt solution absorbed into blood identified in Table 1, the serum components were used to predict the targets of Y for improving KDM using a network pharmacology approach. Finally, 177 targets related to the components of Y were identified using the Swiss Target Prediction website. Using Gene Cards and the OMIM database, 2303 disease targets associated with miscarriage were obtained. Combining the targets related to active compounds of Y with the targets of KDM, and 92 potential targets for Y against KDM was obtained. (Fig. 3A). Next, 92 overlapping targets were imported into Cytoscape 3.7.2 software to construct the component-target-disease network, as shown in Fig. 3B, with 88 nodes and 145 edges, in which the orange diamond represents the Y, the yellow circle represents the blood component, and the green square represents the gene.

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Fig. 3

(A) Venn diagram of Y and kidney deficiency miscarriage targets; (B) Y-active components-disease targets; (C) protein–protein interaction (PPI) network; (D) Ingredients–target–pathway network.

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The 92 overlapping components and disease targets were imported into the STRING database to construct the PPI network. The PPI network has 91 core targets and 1099 edges (Fig. 3C), where the topological parameter degree of network analysis is the center of the node, which reflects the connectivity of the targets involved in the pathological process of the disease, meaning that the larger the value the more involved in the disease process (Cheng et al., 2022). With a degree value ≥ 39 as the screening condition, ultimately, a total of 18 key targets were obtained, including TPP53, AKT1, TNF, IL6, VEGFA, CASP3, JUN, EGFR, etc., as shown in Table 3.

3.2.2 GO and KEGG analysis

The Metascape database was used to perform GO enrichment (biological processes, cellular components and molecular functions) and KEGG pathway analysis on 92 common targets to determine the biological properties and various mechanisms of SS on KDM target genes. The results of GO enrichment analysis were obtained, in which 1181 were associated with BP, 45 were associated with CC, 117 were associated with MF as shown in Fig. 4A ∼ C. The p-value represents the enrichment degree, the lower the p, the higher the -log10 (p), the higher and more significant the enrichment and the bluer the color; the size of the circle indicates the number of genes enriched under the function. The results showed that the components of Y absorbed into blood were involved in cell growth and development, proliferation and apoptosis, and protein binding in the treatment of miscarriage. Here we found that BP enrichment was mainly involved in hormonal response, oxidative stress response, steroid hormone response; CC enrichment was mainly involved in serine/threonine protein kinase complex, cell cyclin-dependent protein kinase holoenzyme complex. Homodimerization activity of proteins, oxidoreductase activity, kinase binding was closely related to MF enrichment.

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Fig. 4

Bubble chart of GO and KEGG enrichment analysis. (A) biological process, (B) cellular component, (C) molecular function, (D) KEGG enrichment result.

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KEGG pathway analysis yielded 168 enrichment terms (p < 0.01), and the top-ranked KEGG pathways were selected based on p-value, as shown in Fig. 4D and Table 4. The significantly enriched KEGG pathways were PI3K-Akt signaling pathway, p53 signaling pathway, apoptosis, TNF signaling pathway, and HIF-1 signaling pathway. The more target genes on the pathway, the more Y may exert therapeutic effects by regulating these signaling pathways.

3.2.3 “Ingredients–target–pathway” network construction

All the targets on the pathway obtained from KEGG enrichment analysis were imported into Cytoscape 3.7.2 to map the component-target-pathway network and analyze the parameters such as degree, betweenness and closeness of components and targets. As shown in Fig. 3D, the network contains 93 nodes (10 ingredients, 29 signaling pathway, and 54 targets) and 340 edges. Each blood component corresponds to multiple targets, and each target corresponds to multiple components, reflecting the multi-component, multi-target improvement of KDM by Y.

Among them, luteolin (degree 33, medium 0.2212898, tightness 0.53488372). Therefore, luteolin was predicted to be the main active component of this blood component for the treatment of KDM, followed by kaempferol, and palmitic acid. This suggests that the pathways act in concert with each other through shared targets. Therefore, it is suggested that all the pathways in Y act synergistically in the treatment of KDM.

3.2.4 Molecular docking

The top 5 core targets TP53 (PDBID: 3D06), AKTI (PDBID: 1UNO), TNF (PDBID: 1EXT), IL6 (PDBID: 1ALU), VEGFA (PDBID: 1FLT) in the protein interaction network (PPI) degree ranking were selected as molecular docking genes based on the network pharmacology results and were molecularly docked with the compounds ranked in the top 5 degrees in the component-target network including: Luteolin, Kaempferol, Apigenin, Oleic Acid, Palmitic acid. The docking results of some representative are shown in Fig. 5A ∼ E. The stability of binding between receptors and ligands depends on the binding energy, the lower the binding energy, the more stable the binding conformation of the receptor and ligand (Liu et al., 2023).

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Fig. 5

(A) VEGFA-Luteolin, (B) VEGFA-Kaempferol, (C) AKTI-Luteolin, (D) TP53-Kaempferol, (E) AKTI-Kaempferol, (F) Heatmap shows docking scores of TP53, AKT1, TNF, IL-6, VEGFA combining to the five active ingredients of Y.

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The highest docking score was obtained for VEGFA-Luteolin (vascular endothelial growth factor-Luteolin) with a binding energy of −9.78 kcal·mol⁻¹ (Fig. 5F), and the mean value of docking binding energy for all docking results was −5.52 kcal·mol⁻¹, which indicated a good binding activity between the blood component and the target site when molecular docking was performed. Five compound-molecule pairs with docking binding energies below −7.5 kcal·mol⁻¹ were selected for docking patterns as well as pocket structure mapping. After molecular docking verification, it can be concluded that Luteolin was an important component of the serum component of Y, protein VEGFA is the core target of action of Y in the treatment of miscarriage, and other serum components of Y have certain or even strong binding ability to the targets acting on miscarriage.

3.3.1 Establishment of KDM model and general observation

The 100 female rats and 50 male rats were mated (mating ratio 2:1), and the vaginas of the rats were coated with saline using a cotton swab early each morning, and the smear results are shown in Fig. 6A. A large number of keratinized epithelial cells during estrus. Due to individual differences and the inability to standardize the estrus period among rats, 40 fertile rats were finally selected for the hydroxyurea combined with mifepristone-induced kidney deficiency abortion model. Hydroxyurea combined with mifepristone induced KDM is a well-established and highly replicated model for testing the therapeutic potential of Y. The modeling results showed that the rats in the model group were huddled and less active, their body hair was thin, their food and water intake decreased, their body weight increased slowly or even decreased, their stools were loose and their anus was stuck with sticky and thin stools; or they had vaginal bleeding during the pregnancy, which was consistent with the performance of the rats with kidney deficiency, and these results indicated that the model of abortion induced by hydroxyurea in rats with kidney deficiency was established successfully, while the rats in other groups were active as usual.

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Fig. 6

(A) Micrograph of rat vaginal smear. (Light microscopy, 10 × ): (a) Interestrus, (b) oestrus, (c) late oestrus, (d) Vaginal sperm smear in rats; (B) Morphology of rat uterus: (N) Normal; (C) Control; (M) Model; (P) Progesterone group; (S) raw products of Semen Cuscuta group; (Y) Semen Cuscutae processed with salt solution group.

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3.3.2 Morphological observation

According to Fig. 6B, the uterus of normal pregnant control (C) rats was thick as a string of beads and pink or light red, and the embryonic tissue was approximately the same in appearance; In the model group (M), the embryonic uterus of the rats showed bamboo-like changes, the uterine cavity showed obvious fatigue or necrotic embryos, or only the embryonic implantation site was seen without embryos, or the embryos were dark brown, or there was vaginal bleeding. In the positive drug group (P) the uterine of rats showed light red color, the embryos were basically uniform in size, with occasional individual unevenness, no bamboo-like changes, occasional bruising spots, and no obvious absorbed embryonic tissues were seen. The abortion of raw products of Semen Cuscuta (S) group, Semen Cuscutae processed with salt solution (Y) group were slightly improved. The uterus was light red or dark red, the size of embryonic tissues was basically the same, occasionally a small part of embryonic tissues was shrunken, no obvious bamboo-like changes were seen, and there was a small amount of bruising in the uterine cavity. Most of the embryonic tissues were not mechanized.

3.3.3 Calculation of embryo loss rate and uterine bleeding in rats with KDM

Statistical analysis of the number of embryos in each group showed that the rate of embryo loss in the M group was as high as 71.77 %, which was statistically significant compared to the C group (P < 0.01); the rate of embryo loss was greatly reduced in each administration group compared to the M group, and the therapeutic effect was better in the Y group. The amount of uterine bleeding showed that the amount of uterine bleeding in the model group was significantly higher than that in the other groups. After administration of the corresponding drugs, the groups improved significantly, with the amount of uterine bleeding in the Y group being significantly lower than that in the S group. The specific values are shown in Table 5 and Fig. 7A and B.

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Fig. 7

Embryo loss rate (A) and amout of uterine bleeding (B) in pregnant rats. E2, P, VEGF-A content in the serum (C, D, E). Compared with the C group, *P < 0.05, **P < 0.01, ***P < 0.001; Compared with the M group, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 Compare with the Y group, ^△P < 0.05, ^△△P < 0.01, ^△△△P < 0.001.

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3.3.4 Hormone e and VEGF-A levels in rat serum

As shown in Fig. 7C, D and E, compared with C group, the serum levels of estradiol, progesterone and VEGF-A were significantly lower (P < 0.001) in rats in group M; Compared with the M group, the estradiol, progesterone and VEGF-A levels in the serum of rats in the P group were significantly higher (P < 0.01), and the serum of rats in the S and Y groups were significantly elevated compared with the M group (P < 0.001), with the Y being significantly higher than the S group.

3.3.5 Histopathology

As shown in Fig. 8, the cytoplasmic staining of the decidual tissue of the rats in the C group was uniform, with clear cell boundaries, tight columns and abundant blood vessels. In M group, the metaphase cells were degenerated and lost intercellular connections, some metaphase cells were necrotic, and the interstitial blood vessels were obviously reduced. The vascular morphology of the metaphase tissue of the rats in each administration group showed a trend of improvement, with closely arranged metaphase cells, intact glandular morphology, regular arrangement of interstitial cells, clear structure, uniform cytoplasmic staining and good vascular growth, which tended to the normal group. Among them, Y group showed the most obvious improvement.

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Fig. 8

Decidual tissue sections were stained with hematoxylin and eosin (H&E) for histopathologic analysis. Scale bars = 100 μm (Light microscopy, 20 × ): (C) Control group; (M) Model group; (P) Progesterone group; (S) raw products of Semen Cuscuta group; (Y) Semen Cuscutae processed with salt solution group.

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3.3.6 Immunohistochemistry

As shown in Fig. 9, the positive expression of VEGF, PI3K, AKT, CD31, p-AKT, and p-PI3K was significantly decreased in M group compared with C group (P < 0.01). Compared with the model group, the protein expressions of all the administered groups were all back-regulated, and the expression of VEGF, PI3K, CD31, p-AKT, and p-PI3K in the Y group was higher than that in the S group, this result showed that Y had a significant improvement effect on kidney deficiency abortion rats.

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Fig. 9

The expression of (A)VEGF, (B)PI3K, (C)p-PI3K, (D)CD31, (E)AKT, and (F)p-AKT, and determined by immunohistochemical staining. Scale bars = 20 μm (Light microscopy, 40 × ). Compared with the C, *P < 0.05, **P < 0.01, ***P < 0.001; Compared with the M group, ^#P < 0.05, ^##P < 0.01, ^###P < 0.01; Compare with Y group ^△P < 0.05, ^△△P < 0.01, ^△△△P < 0.001.

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