Abstract

Purpose

   Yangjing Zhongyu Tang (YJZYT) is a classic Chinese prescription for
   infertility treatment and exerts therapeutic effects via activity on
   the thin endometrium (TE). However, the major components and underlying
   mechanisms of YJZYT actions remain to be established. The main
   objectives of this study were to clarify the effects of YJZYT on the TE
   and provide insights into the related mechanisms based on network
   pharmacology and molecular docking analyses.

Methods

   Network pharmacology was employed to explore the main bioactive
   components and targets of YJZYT. TE-related genes were obtained from
   the Genecards database and screened for intersections with YJZYT. The
   Cytoscape 3.8.2 was used to build a “compounds-disease-targets” network
   and molecular docking analysis performed on key targets. The mechanism
   of action of YJZYT was further validated in vivo using a rat model.

Results

   A total of 98 YJZYT active ingredients, 2409 thin
   endometrium-associated genes, and 186 common targets were obtained.
   Through topological analysis, 10 core objectives were screened. Data
   from the PPI network suggest that AKT1, TNF, VEGFA, IL-6, TP53, INS,
   ESR1, MMP9, ALB, and ACTB serve as key targets in the action of YJZYT
   on TE. PI3K-Akt, TNF, apoptosis, IL-17 and MAPK were established as the
   main functional pathways. Molecular docking analysis revealed high
   affinity of the active ingredients of YJZYT, specifically, ursolic
   acid, palbinone, stigmasterol, and beta-sitosterol, for TNF, VEGFA,
   IL-6, AKT, and MMP9. YJZYT improved endometrial recovery, promoted
   endometrial angiogenesis, and upregulated protein expression of VEGF,
   PI3K, AKT, and p-AKT in the TE rat model.

Conclusion

   Network pharmacological and animal studies facilitated the prediction
   and validation of the active components and key targets of YJZYT
   potentially contributing to TE. Preliminary evidence from in vivo
   experiments showed that YJZYT promotes angiogenesis and thin
   endometrial repair via regulation of the PI3K/AKT pathway, providing a
   reference for further research.

   Keywords: network pharmacology, molecular docking, Yangjing Zhongyu
   Tang, thin endometrium, PI3K/AKT signaling pathway

Introduction

   Thin endometrium (TE), a recent hot topic of research in the field of
   reproductive medicine, is a key factor affecting the clinical pregnancy
   outcomes of assisted reproductive technology (ART). This condition is
   not a disorder but a special type of imaging manifestation detected via
   ultrasound during the development of ART. The 2019 Canadian Fertility
   and Andrology Society (CFAS) guidelines stipulate that endometrial
   thickness <8 mm on the day of human chorionic gonadotropin (HCG)
   administration and <7 mm in the frozen transplant cycle represent TE,
   and cancelling of the cycle is recommended.[36]1 The pathogenesis of TE
   is significantly related to a number of factors, including
   inflammation, drugs, and iatrogenic manipulation. Insufficient
   endometrial receptivity in patients often results in embryo
   implantation failure.[37]2 However, the associated mechanisms are
   unclear and proposed to be related to thinning of the inner membrane of
   the functional layer, which leads to a much smaller distance between
   the embryo and inner membrane of the basal layer and damage to the
   embryo caused by high concentrations of oxygen.[38]3,[39]4 A previous
   study demonstrated that in addition to differences in uterine
   thickness, TE patients have vascular dysplasia, which leads to a
   significant increase in uterine artery blood flow resistance and
   consequent suppression of endometrial receptivity.[40]5

   Traditional Chinese medicine (TCM) classifies TE in the categories of
   “infertility”, “oligomenorrhea”, and “amenorrhea”. The endometrium is
   metaplasted by sperm blood and the root cause of thinness lies in the
   deficiency of sperm blood. Yangjing Zhongyu Tang (YJZYT), a treatment
   for “thin infertility” in the seed chapter of “Fuqingzhu female
   department”, is a commonly used therapeutic agent. YJZYT consists of
   four TCMs, specifically, cooked shu-di-huang (SDH) (Rehmannia
   glutinosa), dang-gui (DG) (Angelica sinensis), shan-zhu-yu (SZY)
   (Cornus Officinalis), and bai-shao (BS) (Paeonia Albiflora), and has
   several beneficial effects in terms of nourishing kidneys, filling
   essence, nourishing blood, and regulating flushes. The compound is
   clinically used for the treatment of infertility, polycystic ovary
   syndrome, thin endometrium, repeated implant failure, and premature
   ovarian insufficiency.[41]6–8 While clinical studies to date have
   confirmed that YJZYT increases endometrial thickness, improves
   endometrial receptivity, and augments the clinical pregnancy rate, its
   specific mechanisms of action are yet to be established.[42]9,[43]10
   Despite considerable research, the pharmacological interpretation of
   TCM prescriptions still faces significant challenges, which greatly
   hinders the widespread clinical application of TCM worldwide.[44]11 In
   the current study, we comprehensively explored the active ingredients,
   potential targets and molecular mechanisms of YJZYT actions based on
   network pharmacology and molecular docking experiments along with
   preliminary in vivo experiments to validate the key pathways.

Materials and Methods

Prediction of Active Compounds and Targets of YJZYT

   All chemical components and targets of YJZYT were searched in the TCMSP
   ([45]https://tcmsp-e.com/),[46]12 ETCM
   ([47]http://www.tcmip.cn/ETCM/),[48]13 and BATMAN-TCM databases
   ([49]http://bionet.ncpsb.org/batman-tcm/).[50]14 Oral bioavailability
   (OB) ≥30% and drug-likeness (DL) ≥0.18 were used as screening criteria
   to identify active ingredients based on recommendations in the TCMSP
   database.[51]15 The ETCM database was screened as moderate or good
   according to “Druglikeness Grading” and targets with a comprehensive
   prediction score >0.8 retained. Compounds with a “score cutoff” >100
   were screened from the BATMAN-TCM database according to the target
   prediction method. The active compounds obtained from all three major
   databases were removed from duplicates and the literature consulted to
   supplement the compounds with high contents and significant medicinal
   effects. Screened target proteins were imported into the UniProt
   database ([52]http://www.uniprot.org/) for protein name conversion gene
   ID[53]16 and a network for herbs-active compounds constructed.

Prediction of TE-Related Genes

   Setting the keyword as “thin endometrium”, human genes were screened in
   the Genecards ([54]https://www.genecards.org/),[55]17 OMIM
   ([56]https://omim.org/),[57]18 and DisGeNET
   ([58]http://www.disgenet.org/) databases[59]19 with the aim of
   identifying TE-related genes. All key genes identified in the databases
   were combined and duplicate and false-positive genes removed to obtain
   a final disease target dataset.

Network Construction and Pathway Enrichment

   A Venn diagram ([60]http://bioinformatics.psb.ugent.be/webtools/Venn/)
   was generated to determine the common targets of YJZYT and TE. The
   protein–protein interaction (PPI) networks of common targets were
   plotted using the STRING database ([61]https://string-db.org/),[62]20
   with species set to “Homo Sapiens” and the protein interaction
   confidence score threshold set to 0.4.[63]21 Cytoscape 3.8.2 software
   was used for network topology analysis. The “Analyze network” plug-in
   was used to find the key targets and obtain the network diagram of the
   key targets.[64]22

   Gene ontology and Kyoto Encyclopedia of Genes and Genomes (GO and KEGG,
   respectively) pathway enrichment analyses were performed using the
   DAVID database ([65]http://www.david.niaid.nih.gov).[66]23 DAVID
   software was used for GO function enrichment analysis of target
   proteins potentially involved in the therapeutic effects of YJZYT on
   TE. For GO analysis, the three categories of biological processes (BP),
   cellular composition (CC), and molecular function (MF) were selected.
   KEGG pathway enrichment analysis retention of the first 20 critical
   pathways associated with TE.

Molecular Docking

   The protein structure was obtained from the RCSB PDB database
   ([67]https://www.rcsb.org/), along with structures of compounds from
   the PubChem database ([68]https://pubchem.ncbi.nlm.nih.gov/).[69]24
   AutoDockTool 1.5.6 software was employed for docking key proteins with
   compounds to obtain the molecular docking score. Visualization of
   docking results was performed with PyMOL 2.5.[70]25

Experimental Animals and Groups

   Forty specific pathogen-free Sprague-Dawley (SD) female rats aged 56–62
   days and weighing 200–220 g were housed in the Experimental Animal
   Center of the Affiliated Hospital of Shandong University of Traditional
   Chinese Medicine in a barrier environment (12-h light/12-h dark,
   temperature of 20–22°C, humidity of 40–60%). Our experiments conformed
   to the 3R principles of the Guide for the Care and Use of Laboratory
   Animals.

   Thirty female rats with regular estrus cycles were randomly divided
   into control (n = 10), model (n = 10), and YJZYT (n = 10) groups.
   Except for the control group, rats were anesthetized by intraperitoneal
   injection of 3% sodium pentobarbital 0.1 mL/100 g. The researcher
   slowly injected 95% absolute ethanol until the uterus was full while
   holding the needle still after which the ethanol remained in the
   uterine cavity for 5 min. The uterus could be observed to change from
   red to white. As described previously, a total of 1 mL of 95% absolute
   ethanol was infused into the uterine cavity on both sides.[71]26,[72]27
   Rats were observed for two estrus cycles for successful establishment
   of the TE model. Rats in the YJZYT group received the prescription via
   gavage for three estrus cycles while those in the other two groups
   received the same volume of normal saline via gavage.

Sample Collection

   The rats were sacrificed by inhalation of CO[2]. The uterus of rats was
   weighed and one-quarter of it was fixed in the 4% paraformaldehyde
   solution. The rest of the uterus was collected on ice and stored at
   −80°C.

Experimental Drugs

   YJZYT was purchased from Guangdong Yifang Pharmaceutical Co., Ltd
   (Guangzhou, China). The composition of YJZYT was as follows: SDH 30 g
   (voucher No. 1101983), DG 15 g (voucher No. 1103203), BS 15 g (voucher
   No. 1100093), and SZY 15 g (voucher No. 1092873). The drug quality of
   Intelligent Free decoction granules was assessed by the pharmacy
   department. Adults are generally recommended 75 g YJZYT per day,
   equivalent to 18 g of formula granules. Accordingly, the daily dosage
   for rats was calculated as 1.674 g/kg. In view of our previous finding
   that the medium dose was optimal, rats were administered 1 mL/100 g per
   day via gavage. YJZYT formula granules were prepared as a suspension at
   a concentration of 0.334 g/mL.

Reagents and Instruments

   The reagents used for study included RIPA buffer (high) (R0010,
   Solarbio, Beijing, China), BCA protein assay kit (P0010, Beyotime
   Biotechnology, Shanghai, China), phosphatase inhibitor (M7528, AbMole,
   Shanghai, China), anti-CD34 antibody ([73]GB121693, Servicebio, Wuhan,
   China), anti-PI3K antibody (4249, Cell Signaling Technology (CST),
   USA), anti-AKT antibody (4691, CST, USA), anti-pAKT antibody (4060,
   CST, USA), anti-VEGF antibody (A5708, ABclonal, Wuhan, China), GAPDH
   antibody (AC033, ABclonal, Wuhan, China), goat anti-rabbit IgG (AS014,
   ABclonal, Wuhan, China) and goat anti-mouse IgG (AS003, ABclonal,
   Wuhan, China).

   The instruments employed used included a light microscope
   (AxioScope.A1, Zeiss, Germany), photomicroscope (AxioVert.A1, Zeiss,
   Germany), slice scanner (KF-PRO-005-EX, Ningbo, China), high-speed
   low-temperature tissue grinding instrument (KZ-III-F, Servicebio,
   Wuhan, China) and electrophoresis system (Mini-Protean system, BioRad,
   USA).

Hematoxylin and Eosin (H&E) Staining

   Paraffin-fixed tissues were cut into 4-μm-thick sections. After
   dewaxing, rehydration, and embedding, paraffin sections were dewaxed to
   water, stained in hematoxylin-eosin solution, rinsed with running
   water, dehydrated with gradient alcohol, and sealed with neutral gum.
   Endometrial thickness was measured with K-Viewer software (Version
   1.0.2) after scanning. The number of uterine glands per unit area was
   counted using Fiji-ImageJ software (Version 2.1.0). Five fields of view
   were randomly selected from each slice for counting the number of
   endometrial glands. Counting was performed three times for each slice
   to obtain an average value.

Immunofluorescence (IF)

   After dewaxing and hydration, paraffin sections were repaired, washed
   with PBS, and treated with BSA at room temperature for 30 min.
   Following rinsing with PBS, primary CD34 (1:500) antibody was added to
   the wet box and incubated overnight at 4°C. Sections were washed with
   PBS, followed by incubation with FITC-labeled goat anti-rabbit antibody
   for 50 min away from light. After further washes with PBS, DAPI
   staining of the nucleus was performed and sections were sealed with
   anti-fluorescence quenching sealant. Relative fluorescence intensity
   was evaluated via microscopy using Fiji ImageJ software.

Western Blot (WB) Analysis

   Uterine tissue was processed with an electric grinder and fully
   homogenized by adding RIPA lysis buffer. Lysates were centrifuged at
   12,000 rpm at 4°C for 5 min and the supernatant fraction used for
   determination of protein concentrations with the BCA assay. After
   mixing with 5X Loading Buffer, the uterine protein supernatant sample
   was placed in a metal bath at 96°C and boiled for 8 minutes. Proteins
   were separated via 7.5% sodium dodecyl sulfate-polyacrylamide gel
   electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride
   (PVDF) membrane. The PVDF membrane was blocked with rapid blocking
   solution for 15 min, incubated with antibodies (VEGF 1:2000, AKT
   1:1000, pAKT 1:2000, PI3K 1:1000, GAPDH 1:50000) specific for target
   proteins overnight at 4°C, and subsequently washed five times with
   Tris-Buffered Saline containing Tween-20 for 5 min. Following
   incubation with horseradish peroxidase (HRP)-conjugated goat anti-mouse
   or rabbit IgG (H + L) (1:5000) for 1 h, the membrane was visualized
   using an enhanced chemiluminescence (ECL) method. The gray value of the
   protein band was measured with Fiji-ImageJ software and the target
   protein/GAPDH value calculated as relative expression.

Statistical Analysis

   Statistical analyses and graph plotting were performed using GraphPad
   Prism (Version 9.4). Results are presented as mean ± SEM. Data that
   conformed to normality and homogeneity of variance were analyzed via
   one-way ANOVA. Otherwise, a non-parametric test (Mann–Whitney U) was
   used. Differences were accepted as significant at P < 0.05 (highlighted
   with different letters and asterisks).

Results

Active Components and Targets of YJZYT

   A total of 110 active components were obtained from three major Chinese
   medicine databases and earlier literature, including 9 processed
   Rehmanniae radix, 30 Cornus officinalis, 20 Radix paeoniae radix and 51
   Radix sinensis radix. After removal of repeat components, 98 active
   components remained, which yielded a total of 679 protein targets.
   After removal of duplicates, 477 protein targets remained. Based on the
   data, a network of YJZYT active components was constructed ([74]Figure
   1, [75]Supplementary Table S1).

Figure 1.

   [76]Figure 1
   [77]Open in a new tab

   Yangjing Zhongyu Tang (YJZYT) active component network.

Potential Targets of YJZYT Involved in Therapeutic Effects on TE

   A total of 2409 disease genes for TE were retrieved from the Genecards
   database while none were retrieved from the OMIM and DisGeNET
   databases, as of December 31, 2022. A Venn diagram of TE and YJZYT
   genes was generated, leading to the identification of 186 potential TE
   therapeutic targets of YJZYT ([78]Figure 2A). These targets were
   entered into the STRING database to obtain PPI ([79]Figure 2B). A
   network analyzer was used to evaluate network topology characteristics,
   sorted according to the degree value of nodes, and 40 core nodes
   subsequently determined with a degree greater than twice the average of
   the PPI network ([80]Figure 2C). Taking Degree, Betweenness Centrality,
   and Closeness Centrality as further screening parameters, a total of 10
   key targets were obtained, specifically, AKT1, tumor necrosis factor
   (TNF), VEGFA, interleukin 6 (IL-6), tumor protein 53 (TP53), insulin
   (INS), estrogen receptor 1 (ESR1), matrix metalloprotein 9 (MMP9),
   albumin (ALB), and beta actin (ACTB) as shown in [81]Figure 2D and
   [82]E. A network diagram of active YJZYT compounds and TE targets was
   further generated ([83]Figure 3). The graph included 289 nodes and 546
   edges, whereby the nodes represent target genes and edges represent
   lines connecting the nodes.

Figure 2.

   [84]Figure 2
   [85]Open in a new tab

   Protein–protein interaction (PPI) network and cluster analysis of
   disease targets. (A) Venn diagram of TE and YJZYT target genes. (B) PPI
   network of potential targets of YJZYT contributing to therapeutic
   effects on TE. (C) PPI network of significant proteins extracted from
   (B) based on network analysis. (D) Forty core proteins implicated in
   the effects of YJZYT on TE were extracted from (C). (E) A total of 10
   key targets were obtained.

Figure 3.

   [86]Figure 3
   [87]Open in a new tab

   Network of YJZYT active compounds and TE targets.

   Notes: The degree value is from large to small, and the color gradually
   changes from red to orange.

GO and KEGG Pathway Enrichment Analysis for Potential Targets of YJZYT in TE
Treatment

   GO functional enrichment analysis showed that items with P < 0.01,
   minimum count of 3, and enrichment factor >1.5 were screened, resulting
   in enrichment of a total of 956 items. The top 10 entries for BP, CC,
   and MF are presented ([88]Figure 4A, [89]Supplementary Table S2). The
   biological processes mainly included regulation of response to
   lipopolysaccharide, regulation of cell apoptosis, response to
   endogenous stimuli, active regulation of cell proliferation, and
   responses to various compounds and drugs. MF hint, enzyme binding, RNA
   polymerase II transcription factor activity, identical protein binding,
   steroid binding, protein homodimerization activity, iron ion binding,
   protein binding, steroid hormone receptor activity, oxidoreductase
   activity, and transcription factor binding. Therefore, YJZYT may exert
   beneficial effects on TE through mediating signal transduction,
   affecting cell proliferation and apoptosis, and regulating hormone
   levels, metabolic processes, and oxidative stress responses.

Figure 4.

   [90]Figure 4
   [91]Open in a new tab

   Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG)
   functional analysis. (A) Top 10 GO enrichment results. (B) Top 20 key
   signaling pathways from KEGG analysis.

   Abbreviations: BP, biological process; CC, cellular composition; MF,
   molecular function.

   KEGG pathway enrichment analysis of the 186 potential targets of YJZYT
   revealed 175 signaling pathways with significance (P < 0.01), which
   were related to cell proliferation and apoptosis, inflammatory
   response, hormone regulation, and angiogenesis ([92]Figure 4B,
   [93]Supplementary Table S3). Irrelevant pathways, such as “Lipid and
   atherosclerosis”, were subsequently removed. The top 20 pathways are
   presented in [94]Figure 4B, among which the major pathways identified
   were PI3K-Akt, TNF, apoptosis, IL-17, MAPK, and estrogen signaling.

Molecular Docking

   To further explore the potential effects of the seven active compounds
   of YJZYT on eight key hypothetical targets (AKT1, TNF, VEGFA, IL-6,
   TP53, INS, ESR1, and MMP9), we performed molecular docking analysis
   ([95]Supplementary Table S4). Binding free energies are shown in
   [96]Table 1. PyMoL software was used to visualize target proteins and
   small molecules with strong binding affinities ([97]Figure 5). Our
   docking results showed that binding between the compound and target
   protein is mainly mediated by hydrophobic interactions. The free
   binding energies between the components of YJZYT, specifically, ursolic
   acid, palbinone, beta-sitosterol and stigmasterol, and their
   corresponding targets, AKT1, VEGFA, TNF and ESR1, were relatively high.
   Thus, these compounds may represent the key pharmacological components
   of YJZYT with therapeutic activity.

Table 1.

   Molecular Docking Scores of the Seven Candidate Compounds and Eight
   Targets
   Molecular Name Molecular Docking Score
   Gene Name AKT1 (2UZT) VEGFA (4K2N) TNF (2AZ5) TP53 (1G2H) IL6 (1ALU)
   INS (7S4Y) ESR1 (3OS8) MMP9 (5TH6)
   Kaempferol −7.84 −4.48 −4.26 −4.73 −4.26 −4.1 −6.44 −4.87
   Ursolic acid −8.32 −6.93 −9.72 −7.78 −6.45 −7.2 −7.38 −8.12
   Palbinone −7.86 −6.77 −7.59 −7.12 −7.01 −6.62 −11.37 −7.38
   Beta-sitosterol −6.92 −5.7 −7.84 −6.79 −6.39 −7.6 −11.21 −7.81
   Azelaic Acid −4.45 −2.91 −4.73 −7.11 −4.89 −3.98 −3.21 −3
   Angelicin −6.01 −5.71 −6.55 −5.66 −5.55 −5.05 −6.46 −7.79
   Stigmasterol −7.44 −6.76 −7.95 −8.22 −7.38 −7.58 −9.16 −7.87
   [98]Open in a new tab

   Notes: Molecules were docked using AutoDock-Tools software. Lower
   scores are indicative of better binding.

   Abbreviations: AKT1, Protein kinase B; VEGFA, Vascular endothelial
   growth factor A; TNF, tumor necrosis factor; TP53, tumor protein 53;
   IL-6, interleukin 6; INS, insulin; ESR1, estrogen receptor 1; MMP9,
   matrix metalloprotein 9.

Figure 5.

   [99]Figure 5
   [100]Open in a new tab

   Docking results of seven active compounds of YJZYT and eight key TE
   targets. (A1) AKT1 and palbinone; (A2) AKT1 and kaempferol; (A3) AKT1
   and ursolic acid; (B1) VEGFA and palbinone; (B2) VEGFA and
   stigmasterol; (B3) VEGFA and ursolic acid; (C1) TNF and
   beta-sitosterol; (C2) TNF and stigmasterol; (C3) TNF and ursolic acid;
   (D1) TP53 and palbinone; (D2) TP53 and stigmasterol; (D3) TP53 and
   ursolic acid; (E1) IL6 and palbinone; (E2) IL6 and stigmasterol; (E3)
   IL6 and ursolic acid; (F1) INS and beta-sitosterol; (F2) INS and
   stigmasterol; (F3) INS and ursolic acid; (G1) ESR and beta-sitosterol;
   (G2) ESR and stigmasterol; (G3) ESR and palbinone; (H1) MMP9 and
   beta-sitosterol; (H2) MMP9 and stigmasterol; (H3) MMP9 and ursolic
   acid.

   Abbreviations: AKT1, protein kinase B; VEGFA, Vascular endothelial
   growth factor A; TNF, tumor necrosis factor; TP53, tumor protein 53;
   IL-6, interleukin 6; INS, insulin; ESR1, estrogen receptor 1; MMP9,
   matrix metalloprotein 9.

Histological Analysis of Rat Uterus

   In control rats, the uterine cavity was morphologically intact, with
   normal distribution of glands and blood vessels in the stroma
   ([101]Figure 6A and [102]D). In rats from the model group, the uterine
   cavity was larger, the whole layer of the uterine wall became thinner
   and wavy, and the structure was incomplete, with few glands and sparse
   capillaries ([103]Figure 6B and [104]E). The YJZYT treatment group
   displayed slightly greater endometrial thickness, mainly intact
   glandular and luminal epithelial cells, continuous endometrium,
   increased number of glands, and higher number of capillaries showing
   neat arrangement ([105]Figure 6C and [106]F). Compared with the model
   group, endometrial thickness and number of glands were significantly
   increased in the control and YJZYT groups (P<0.05, P<0.01; [107]Figure
   6G and [108]H, respectively). The uterine wet weight and uterine organ
   index of the control and YJZYT groups were higher than those of the
   model group, with significant differences (P<0.05, P<0.01; [109]Figure
   6I and [110]J, respectively).

Figure 6.

   [111]Figure 6
   [112]Open in a new tab

   Histological analysis of rat uterus. (A–C) H&E staining results of
   uterine in the control, model and YJZYT groups (magnification: 40×).
   (D–F) The red areas represent magnification of H&E staining results.
   (G–J) Rat uterine wet weight, uterine organ index, thickness of the
   endometrium and number of glands. Data are presented as mean ± SEM;
   **Compared to the Control group, **P < 0.01. ^#Compared to the Model
   group, ^#P < 0.05, ^##P < 0.01.

Expression of CD34 in Rats with TE

   CD34, a widely used marker of hematopoietic stem and progenitor cells,
   is mainly expressed in monolayer epithelium, including the cytoplasm of
   luminal and glandular epithelial cells.[113]28 Immunofluorescence
   experiments revealed expression in the cytoplasm of endometrial cavity
   epithelial and glandular epithelial cells of rats from all groups
   ([114]Figure 7A–I). Expression of CD34 in the model group was
   significantly decreased relative to that in the YJZYT and control
   groups ([115]Figure 7J).

Figure 7.

   [116]Figure 7
   [117]Open in a new tab

   Changes in angiogenesis of rat uterus. (A–I) Immunofluorescence of the
   uterine receptivity marker, CD34 (red), and nuclear marker, DAPI (blue)
   (Magnification: 400×). (J) Relative fluorescence intensity of rats in
   control, model, and YJZYT groups. Data are presented as mean ± SEM;
   *Compared to the Control group, *P < 0.05. ^#Compared to the Model
   group, ^#P < 0.05.

Protein Expression of PI3K, AKT, p-AKT and VEGF in Rats

   Compared with the control group, PI3K, AKT, p-AKT and VEGF protein
   levels in the uterus of rats in the model group were significantly
   decreased. Relative to model rats, PI3K, AKT, p-AKT and VEGF protein
   levels in the uterus of rats in the YJZYT group were increased, with
   significant differences between the groups (P<0.05, P<0.01; [118]Figure
   8A–E).

Figure 8.

   [119]Figure 8
   [120]Open in a new tab

   Protein expression patterns of rats in control, model, and YJZYT
   groups. (A) Western blot analysis. (B–E) Protein expression of PI3K,
   AKT, p-AKT and VEGF using GAPDH as an internal reference. Data are
   presented as mean ± SEM; *Compared to the Control group, *P < 0.05, **P
   < 0.01, ***P < 0.001. ^#Compared to the Model group, ^#P < 0.05, ^##P <
   0.01.

Discussion

   TE is the main cause of embryo implantation failure that can lead to
   long-term infertility and poor family outcomes.[121]29 Periodic changes
   in thickness of the lining of the uterus occur due to changes in
   steroid hormones.[122]30 At present, no uniform diagnostic standards
   for thin endometrium are available. Clinical research reports suggest
   that the minimal endometrial thickness that can facilitate pregnancy is
   3.7 mm.[123]31 Further studies have consistently highlighted that at
   endometrial thickness <6 mm, the possibility of pregnancy is extremely
   low.[124]32,[125]33 Appropriate endometrial thickness is therefore an
   important condition for successful pregnancy. The physiopathology and
   molecular mechanisms of thin endometrium remain ambiguous and current
   therapeutic options are limited and controversial due to its complex
   pathogenesis. Several treatments for thin uterine lining have been
   attempted,[126]34 such as high estrogen doses, tamoxifen to promote
   endometrial growth,[127]34–36 supplementation of growth hormone,[128]37
   intrauterine perfusion of platelet-rich plasma[129]38 and mesenchymal
   stem cells.[130]39,[131]40 However, endometrial thickness and
   implantation rates have only slightly improved, especially in patients
   with severe endometrial damage. Treatment of TE thus remains a
   considerable clinical challenge.

   TCM has become a research hotspot in recent years, and clinical
   intervention with YJZYT is reported to play a positive role in
   improving pregnancy outcomes of patients with TE. In this study, the
   network pharmacology method was used to explore the main potential
   targets and pathways of YJZYT involved in its therapeutic effects on
   TE. Screening of active ingredients and target prediction showed that
   Cornus Officinalis had the most active components and binding targets,
   indicative of an important therapeutic role. The mechanism mainly
   involved active ingredients, such as ursolic acid, palbinone,
   beta-sitosterol and stigmasterol, and many targets detected in the
   network of “YJZYT-active compounds-targets” could serve as key
   therapeutic ingredients for thin endometrium. A number of studies have
   demonstrated that inflammatory activation and abnormal autophagy are
   critical pathological links that cause endometrial epithelial
   thinning.[132]41 Endometrial vascular dysplasia has been identified as
   a major cause of endometrial thinning due to decreased VEGF in the
   glandular epithelium.[133]42 Therefore, induction of angiogenesis may
   serve as an effective strategy for clinical treatment of thin
   endometrium. The related compounds and pathways identified in this
   study, including IL-17, MAPK and estrogen, and target proteins, such as
   MMP9, ESR, TNF, INS, and Akt, warrant further research. In addition,
   the multi-target and multi-channel regulation properties of YJZYT
   support its potential as a therapeutic agent for other gynecological
   disorders, such as endometrial cancer, pelvic inflammatory and
   inflammatory diseases.

   Previous experiments have also confirmed that Bushen Huoxue recipe can
   induce endometrial damage in rats with TE.[134]43 Icariin produced an
   increase in endometrial thickness, and it may regulate the expression
   of VEGF and CD31.[135]44 Dingkun Dan combined with estradiol valerateon
   was found to increase the thickness of the endometrium via
   up-regulation of VEGF.[136]45 Our experimental results show that after
   intrauterine injection of 95% ethanol in rats, the endometrium was
   significantly damaged, significantly thinner, gland number was greatly
   reduced, and integrity was destroyed, all of which are consistent with
   previous findings.[137]46–48 Rats with TE were subject to YJZYT
   intervention for three estrous cycles. Notably, the thickness and
   number of glands in rats with TE that were treated with YJZYT increased
   with a concomitant reduction in the degree of endometrial damage. Our
   data suggest that YJZYT can effectively repair damaged endometrium,
   promote endometrial growth, and restore its shape and structure.

   Angiogenesis is an important factor for repair and proliferation of
   endometrium after injury. Expression of CD34 in rats in the YJZYT group
   was increased, indicating a positive effect of Chinese medicine on
   angiogenesis. WB results showed that compared with the model group,
   expression of PI3K, p-Akt, and VEGF proteins in the treatment group was
   significantly upregulated. Our data suggest that the PI3K/Akt signaling
   pathway occurs in both normal and thin endometrial tissues, but a
   significant difference in expression levels exists between the two
   groups. PI3K/Akt is a classic pathway that plays important roles in
   many aspects of cellular activity through phosphorylation or
   dephosphorylation, in particular, regulation of cell growth,
   proliferation, migration and vascular permeability. Previous studies
   confirmed that Ligustrazine, a TCM, induces the repair of TE in rats
   via up-regulation of the expression of VEGF and VEGFR-2 and activation
   of the PI3K/AKT signaling pathway.[138]49 Our experiments support the
   theory that YJZYT activates the PI3K/Akt signaling pathway to promote
   angiogenesis, cell proliferation, and endometrial repair, providing new
   perspectives in terms of the utility of YJZYT as a therapeutic option
   therapy for TE and guidance on the value of TCM in clinical
   applications. In addition, combination of network pharmaceutical
   forecasting and experimental verification can be effectively employed
   to elucidate the multi-target and multi-target pharmacological
   mechanisms of action of traditional Chinese medicine formulae.

   This study has a number of limitations that should be taken into
   consideration. First, while we examined the effects of YJZYT on
   angiogenesis in rats with TE, the study did not focus on analysis of
   pregnancy in rats. Future studies will thus aim to clarify the effects
   of YJZYT on biological mechanisms associated with pregnancy in the TE
   rat model. Second, evaluation of YJZYT fingerprint maps and
   pharmacology is lacking and further high-performance liquid
   chromatography (HPLC) experiments on the chemical composition of this
   TCM are warranted. In addition, because tissue samples of rats were
   obtained after the three estrous cycle treatments, evaluation of
   dynamic changes in the PI3K/AKT pathway and angiogenesis after
   administration of YJZYT is necessary. Both short- and long-term effects
   of YJZYT require further clarification.

Conclusion

   The comprehensive strategy of network pharmacological analysis and
   experimental validation in the current study led to the successful
   identification of active ingredients and molecular mechanisms
   underlying the beneficial effects of YJZYT on thin endometrium. The key
   targets and signaling channels were established through network
   pharmacology and molecular docking experiments in vitro and further
   validated in a rat model in vivo. Our collective findings indicate that
   YJZYT promotes generic vessels of the uterine endometrium, associated
   with activation of the PI3K/AKT signaling pathway and upstream VEGF
   protein expression. In the future, the active ingredients and targets
   of YJZYT will be explored, and more mechanisms of YJZYT in the
   treatment of TE will be verified.

Acknowledgments