Mechanism of Yushenhuoxue prescription in treating endometriosis based on network pharmacology and the effect on the TNF pathway

Based on CCK8 cell proliferation experiments, YSHX was found to inhibit cell proliferation in a concentration-dependent manner (low:50 μmol/L; Medium:100 μmol/L; High 200 μmol/L) ( Fig. 9 A). The Transwell assay showed that YSHX inhibited the invasion of 12z cells ( Fig. 9 B). The results of the wound healing experiment suggested that YSHX significantly reduced the migratory ability of 12z cells ( Fig. 9 C–D). Fig. 9 (A)Effect of YSHX on12z proliferation detected by CCK8; (B)The effect of YSHX on 12z invasion detected by tanswell; (C-D)Effect of YSHX on12z migration detected by wound healing. Fig. 9 (A)Effect of YSHX on12z proliferation detected by CCK8; (B)The effect of YSHX on 12z invasion detected by tanswell; (C-D)Effect of YSHX on12z migration detected by wound healing.

Jing Chen, Guohua Hu: Conceived and designed the experiments. Jiami Huang: Performed the experiments; Analyzed and interpreted the data; Wrote the paper. Xu Zhang: Performed the experiments; Wrote the paper. Jiayun Wang: Analyzed and interpreted the data. Yanan Zhang, Cancan Gu: Contributed reagents, materials, analysis tools or data.

All the experimental protocols were approved by the Research Ethics Committee (No. No:2020SHL-KYYS-102). Animal experiments were performed in accordance with the guidelines and regulations of the Centre for Laboratory Animal Care in Shanghai Traditional Chinese Medicine Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, China.

This study was supported by the 10.13039/100007836 Science and Technology Commission of the Shanghai Municipality , China (20Z21900400).

The potential targets of YSHX in EM were compared after deduplication, and 60 intersection targets were identified ( Fig. 3 A). Among them, quercetin, kaempferol, arachidonic acid, isorhamnetin, stigmasterol, beta-sitosterol, and ellagic acid had the largest number of EM targets, corresponding to 40, 26, 11, 11, 9, 8, and 7 targets, respectively, which may be the key components of YSHX in the treatment of EM( Fig. 3 B). Fig. 3 (A)Venna diagram (blue is the disease targets; red is the drug targets; in the middle are common targets); (B)The compond-target network for YSHX on Endometriosis. Fig. 3 (A)Venna diagram (blue is the disease targets; red is the drug targets; in the middle are common targets); (B)The compond-target network for YSHX on Endometriosis. PPI network analysis was performed on the 60 intersection targets using the STRING database, of which 59 genes had protein-protein interactions, and 555 edges represented protein-protein interactions ( Fig. 4 A). The top 10 key genes were IL6, VEGFA, EGFR, MAPK8, CASP3, ESR1, MYC, FOS, CCND1, and AR ( Fig. 4 B). Fig. 4 Result of core target screening.(A)PPI network (B)Core target diagram (the darker the node, the more important the target. Fig. 4 Result of core target screening.(A)PPI network (B)Core target diagram (the darker the node, the more important the target. GO function analysis of the 60 intersection targets showed that 82 biological functions were affected (P < 0.05) ( Fig. 5 A). It mainly affects nuclear receptors and transcription factor activity. Direct ligand-regulated sequence-specific DNA binding, RNA polymerase II transcription factor binding, steroid binding, steroid hormone receptor activity, and cysteine-type endopeptidase activity are involved in the apoptotic process These genes were significantly enriched in 118 pathways (P < 0.05), among which the TNF signalling pathway was the most significant (hsa 04668) ( Fig. 5 B). Fig. 5 Analysis of the enrichment histogram of GO(A) and KEGG pathway(B). Fig. 5 Analysis of the enrichment histogram of GO(A) and KEGG pathway(B). Sixty patients with EM were enrolled based on inclusion and exclusion criteria, a total of 60 EM patients were enrolled. The mean age was 31.43 ± 5.93 years (range, 23–42 years). The disease duration was 0.3–10 years, with an average of (4.81 ± 6.32) years. The EM patients’ before treatment VAS score was 4.65 ± 1.08; after 3 months of YSHX treatment, VAS score was 3.35 ± 0.23, which was significantly lower than that before treatment ( P  < 0.05) ( Table 1 ). Table 1 The visual analogue scale (VAS) scores of EM patients （ x ‾ ± s ） . Table 1 case Vas t P Before treatment 60 4.65 ± 1.08 7.93 ＜0.001 After treatment 60 3.35 ± 0.23*** Notes: *** P  < 0.001, vs.Before treatment. The visual analogue scale (VAS) scores of EM patients （ x ‾ ± s ） . Notes: *** P  < 0.001, vs.Before treatment. Before treatment the EM patients’ serum Ca125 score was 83.17 ± 3.53; after three months of YSHX treatment, Ca125 score was 36.56 ± 1.98, which was significantly lower than those before treatment ( P  < 0.05) ( Table 2 ). Table 2 The Serum Ca125 values of EM patients （ x ‾ ± s ） . Table 2 case Ca125 t P Before treatment 60 83.17 ± 3.53 12.62 ＜0.001 After treatment 60 36.56 ± 1.98*** Notes: *** P  < 0.001, vs.Before treatment. The Serum Ca125 values of EM patients （ x ‾ ± s ） . Notes: *** P  < 0.001, vs.Before treatment. EM patients Serum Inflammatory Cytokines in high level, IL-6 (40.72 ± 11.92 pg/ml), IL-1β (22.20 ± 7.52 pg/ml), TNF-α (12.89 ± 0.49 pg/ml), after 3 months YSHX treatment, Serum Inflammatory Cytokines at a normal level, IL-6 (5.02 ± 2.69 pg/ml), IL-1β(5.46 ± 0.85 pg/ml), TNF-α (5.34 ± 0.15 pg/ml) were significantly lower than those before treatment (P < 0.05) ( Table 3 ) . Table 3 The Serum Inflammatory Cytokines of EM patients （ x ‾ ± s ） . Table 3 IL-6（pg/ml) IL-1β（pg/ml) TNF-α （pg/ml) Before treatment 60 40.72 ± 11.92 22.20 ± 7.52 12.89 ± 0.49 After treatment 60 5.02 ± 2.69*** 5.46 ± 0.85*** 5.34 ± 0.15*** t 16.26 12.69 14.21 P ＜0.001 ＜0.001 ＜0.001 Notes: *** P  < 0.001, vs.Before treatment. The Serum Inflammatory Cytokines of EM patients （ x ‾ ± s ） . Notes: *** P  < 0.001, vs.Before treatment.

Patients with EM were recruited from the Shanghai Hospital of Traditional Chinese Medicine. This study was approved by the hospital's ethics committee (No:2020SHL-KYYS-102) and registered at the China Clinical Research Trial Registration Center (ChiCTR2000036994). All patients who met the inclusion criteria voluntarily participated in the clinical trials and signed an informed consent form. (Refer to the 2021 Guidelines for Diagnosis and Treatment of EM (the third edition)) [ 11 ] Conforming to the pathological diagnosis criteria of EM: The lesion site and scope can be explored laparoscopically, and lesion tissue can be obtained for histopathological diagnosis. (1) Conforming to the pathological diagnostic criteria of EM; (2) non-lactating patients aged 25–45 years; (3) regular menstrual cycle (21–35 days of menstrual cycle) (1) Patients with gynaecological diseases such as gynaecological tumours, vaginitis, cervical cancer, pelvic tumours, and pelvic abscesses; (2) pregnant women within half a year; (3) patients allergic to the test drug; (4) patients with complications from serious medical diseases; and (5) patients taking drugs similar to this experiment, before enrolment. (1)Serious adverse reactions occurred during medication; (2) medications were not taken as required during the trial; (3) patients dropped out of the study without completing the trial; and (4) combined use of other drugs. YSHX is composed of Spatholobus spatholobi 30 g (batch number:21074281), Puhuang 18 g (batch number:21071251), Yanhusuo 9 g (batch number:21071291), Bupleurum Chinense 9 g (batch number:21071491), Frankincense 3 g (batch number:20110871), Myrrh 3 g (batch number:20070771), Sanqi powder 2 g (batch number:21082091), Clematis root 18 g (batch number:21074341), Liu Jinu 9 g (batch number:20112151), Fenugreek 18 g (batch number:20110653), From Jiangyin Tianjiang Pharmaceutical Co., Ltd. Each patient took 150 mLYSHX half an hour after breakfast and dinner every day. All patients with EM were administered drugs for three months and were followed up. (1) VAS score: visual analogue scale (VAS) was used to evaluate dysmenorrhoea, with 0 end (0 point) representing "no pain" and 10 cm end (10 point) representing "unbearable pain". Patients marked their pain levels on a scale according to their feelings, and the length from 0 points to the marked point (cm reading) was the pain level. 0–0.4 cm was defined as having no pain, 0.5–4.4 cm as mild pain, 4.5–7.4 cm as moderate pain, and 7.5–10 cm as severe pain, respectively. (2) Serum CA125 And Serum Inflammatory Cytokines: Abdominal measurements were performed three days after menstruation, before and after treatment; all tests were performed in the clinical Laboratory of Shanghai Traditional Chinese Medicine Hospital.

The active ingredients of the 10 herbs in YSHX were retrieved using the Traditional Chinese Medicine Systems Pharmacology Database (TCMSP, https://old.tcmsp-e.com/ ). The oral bioavailability (OB) ≥30% and drug-like properties (DL) ≥0.18 were used as screening criteria. The predicted gene targets of the active ingredients were obtained, and the eligible target information of the active ingredients was standardised using Uniport database. GeneCards database ( https://www.genecards.org/ ) and OMIM database ( https://omim.org/ ) were searched for EM-related genes with the keyword “endometriosis”. Duplications were eliminated to extract disease-related target genes. Targets related to YSHX and disease-related targets of EM were interposed, and a network of medicine-active ingredient-target was constructed using Cytoscape 3.7. The intersection target information of drugs and diseases was imported into the String database, the species was selected as “Homo sapiens”, and the confidence level >0.4 was used as the screening condition to download the PPI information of the intersection target, and Cytoscape software was used to visualise it. GO and KEGG pathway enrichment analyses were performed using the intersection target PPI database and the species was limited to humans. Biological processes and signalling pathways with P < 0.05 were screened and visualised using R package (ggplot 2).

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Combined with network pharmacology and experimental validation, YSHX may reduce the expression of inflammatory factors and promote cell survival and proliferation through the TNF signalling pathway to treat EM.

EM is an oestrogen-dependent disease characterised by the appearance of functional endometrioid tissues outside the uterus that grow and infiltrate, causing repeated bleeding, abdominal pain, pelvic masses, infertility, and other symptoms. The incidence rate in women of childbearing age is approximately 10%–15%, with a yearly increasing trend. This rate rises to over 40–50% among women experiencing infertility and chronic pelvic pain [ 12 , 13 ]. It affects >176 million women globally. Currently, there are two clinical treatments: drugs and surgery. For ovarian EM, surgical treatment can be considered based on meeting surgical indications. However, surgery may not be suitable for all types of EM, and the postoperative recurrence rate remains high. Drug treatment includes non-steroidal anti-inflammatory drugs (NSAID), progesterone, compound oral contraceptives (COC), gonadotropin-releasing hormone agonists (GnRHa), and TCM. However, hormonal drugs are often less accepted due to their tendency to cause vaginal bleeding, perimenopausal symptoms, and other side effects. TCM is effective for the treatment of EM, has received increasing attention, and is widely used in clinical practice. Several systematic reviews have demonstrated the efficacy and safety of TCM for treating EM over extended periods [ 14 , 15 ]. Clinical studies have confirmed the effectiveness of YSHX in EM treatment, suggesting its ability to enhance endometrial adhesion function by downregulating serum CA125 levels and whole blood viscosity. This effectively controls disease progression, reduces the recurrence rates, and enhances patients' quality of life [ [16] , [17] , [18] ]. To elucidate the molecular mechanism of YSHX in the treatment of EM on the network pharmacology, 61 active ingredients, including quercetin, kaempferol, arachidonic acid, isorhamnetin, sitosterol,-sitosterol, and ellagic acid, which act on EM through key target genes, such as IL6, VEGFA, EGFR, CASP3, ESR1, MYC, and CCND1, were evaluated. It mainly affects nuclear receptor activity, transcription factor activity, direct ligand-regulated sequence-specific DNA binding, RNA polymerase II transcription factor binding, steroid binding, steroid hormone receptor activity, and cysteine-type endopeptidase activity, which are involved in apoptosis and other biological processes. The KEGG enrichment pathway was mainly involved in apoptosis, the TNF signalling pathway, and the oestrogen signalling pathway. EM are localised pelvic diseases and systemic chronic inflammatory diseases. The occurrence of EM is closely related to inflammatory factors, and the inflammatory environment is conducive to the adhesion, invasion, and angiogenesis of endometrial tissues outside the uterine cavity [ [19] , [20] , [21] ]. Therefore, this experiment was verified based on TNF signalling pathway. EM can also cause secondary pain. Previous study showed that 89.6% of EM patients were accompanied by dysmenorrhoea, and 77.2% had chronic pelvic pain, which seriously affected their quality of life [ 22 , 23 ]. In our study, 62 EM patients were recruited and treated with YSHX for three months. Before and after treatment, patients completed the dysmenorrhoea visual analogue scale (VAS), and serum tumour markers and inflammatory factors were measured. Our studies have shown that YSHX can effectively relieve pain in patients with EM without irregular vaginal bleeding, hot flashes, night sweats, or other symptoms caused by hormonal drugs. The mean VAS score was 4.65 ± 1.08 before treatment and decreased to 3.35 ± 0.23 after three months of YSHX treatment. The serum tumour marker ca125 also decreased significantly, from 83.17 ± 3.53 36.56 ± 1.98. And all the serum inflammatory factors (IL-6,IL-1β,TNF-α)were higher than normal before treatment，after YSHX treatment，they were at normal level. Endometriotic cells are similar to tumour cells in that they have obvious invasion, metastasis, proliferation, recurrence, and other malignant biological behaviour [ 24 , 25 ]. According to the pathological characteristics and pathogenesis of EM, reducing the level of inflammation in the body may be an entry point for treating daughter EM. The TNF-α/NF-κB signalling pathway is a combination of the signalling molecule TNF-α with the TNFR1 receptor on the cell membrane, resulting in the formation of trimer TNFR1, and then recruiting downstream signalling proteins to activate the NF-κB signalling pathway, thus promoting the formation of an inflammatory response [ 26 ]. TNF-α, a ligand belonging to the TNF superfamily, plays a promoting role in mediating inflammation and immune response and is involved in the chronic inflammatory response of EM [ 27 ]. TNF-α is an important risk factor for tumorigenesis, tumour progression, invasion, and metastasis. IL-6 is an important mediator of the inflammatory response in the body [ 28 ]. It plays an immunomodulatory role, induces the differentiation of T and B lymphocytes, enhances the function of monocytes and natural killer cells, and promotes the development of EM [ 29 ]. Serum levels of IL-1β stimulate the expression of genes associated with inflammation and EM and play an important role in immune regulation and inflammation. The increased activity of NF-κB P65 binding DNA is involved in the pathogenesis and proinflammatory state of EM [ 30 ]. The research explored the potential mechanisms of YSHX on EM by integrating network pharmacology and experimental verification. Clinical studies have shown that YSHX treatment in patients with EM can improve dysmenorrhoea score, decrease tumour markers ca125 and inflammatory cytokines (IL-6, IL-1β, TNF-α). In vivo validation was performed using an EM model, and the number of glandular and interstitial cells decreased. Serum levels of IL-1β and IL-6 decreased. The expression of NF-κB P65 phosphorylation and TNF-α protein in endometrial tissues was up-regulated. In vitro experiments have shown that YSHX can inhibit the proliferation, invasion, and migration of endometriotic cells. Combined with network pharmacology and experimental validation, it may reduce the expression of inflammatory factors and promote cell survival and proliferation through the TNF signalling pathway to treat EM.

Endometriosis (EM) is a prevalent and challenging gynaecological disease, estimated to affect 2–10% of women of childbearing age [ 1 ]. Oestrogen-dependent illnesses are characterised by the development of endometrial glands and stroma outside the uterine cavity, along with the continuous expansion, infiltration, and bleeding of ectopic lesions, causing pain, masses, and infertility [ 2 ]. Superficial peritoneal implantation, ovarian EM, and deep-infiltrating EM are the three different forms of EM [ 3 ]. Although it is a benign disease, it exhibits malignant biological behaviours such as invasion, metastasis, and recurrence [ 4 ]. Presently, hormonal drugs are primarily used; however, certain side effects are present, and the acceptance of these drugs among patients is low [ 5 ]. Traditional Chinese medicine (TCM) has demonstrated effective in treating EM [ 6 ]. Yushenhuoxue Decoction (YSHX) is an empirical prescription for the treatment of EM developed by Professor Hu Guohua, a renowned TCM doctor in Shanghai. Its effectiveness in the clinical treatment of EM has been confirmed [ 7 ]. Its components are complex and have multi-component, multi-target, and multi-approach characteristics [ 8 ]. Network pharmacology integrates systems biology, bioinformatics, and pharmacology. Different from the traditional research mode of "single component, single target, and single approach", its construction mechanism is consistent with the "holism" in TCM [ 9 , 10 ]. In this study, network pharmacology was used to analyse the targets of YSHX in the treatment of EM and reveal the possible mechanisms. Experiments were conducted to further validate the analytical results. Fig. 1 illustrates the design of the experimental protocol. Fig. 1 The idea and process of this research. Fig. 1 The idea and process of this research.

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Jing Chen reports was provided by The Science and Technology Commission of Shanghai Municipality.

Data associated with this study has been deposited under the accession number ChiCTR2000036994.