Purpose: Adenomyosis (AM) is a common benign uterine disorder that has deleterious effects on women’s health. However, the pathogenesis of AM is not clearly understood. We aimed to investigate the pathophysiological changes and molecular mechanism in AM. Methods: Single-cell RNA sequencing (scRNA-seq) was employed to construct a transcriptomic atlas of various cell subsets from the ectopic endometrium (EC) and eutopic endometrium (EM) of one AM patient and evaluate differential expression. The Cell Ranger software pipeline (version 4.0.0) was applied to conduct sample demultiplexing, barcode processing and mapping reads to the reference genome (human GRCh38). Different cell types were classified with markers with the “FindAllMarkers” function, and differential gene expression analysis was performed with Seurat software in R. The findings were confirmed by Reverse Transcription Real-Time PCR using samples from three AM patients. Results: We identified nine cell types: endothelial cells, epithelial cells, myoepithelial cells, smooth muscle cells, fibroblasts, lymphocytes, mast cells, macrophages and unknown cells. A number of differentially expressed genes, including CLO4A1, MMP1, TPM2 and CXCL8, were identified from all cell types. Functional enrichment showed that aberrant gene expression in fibroblasts and immune cells was related to fibrosis-associated terms, such as extracellular matrix dysregulation, focal adhesion and the PI3K-Akt signaling pathway. We also identified fibroblast subtypes and determined a potential developmental trajectory related to AM. In addition, we identified increased cell–cell communication patterns in EC, highlighting the imbalanced microenvironment in AM progression. Conclusion: Our results support the theory of endometrial–myometrial interface disruption for AM, and repeated tissue injury and repair could lead to increased fibrosis in the endometrium. Therefore, the present study reveals the association between fibrosis, the microenvironment, and AM pathogenesis. This study provides insight into the molecular mechanisms regulating AM progression.

The epithelial‑mesenchymal transition (EMT) serves vital roles in the angiogenesis, cell invasion and metastasis of various malignant tumors, including bladder cancer. Traditional Chinese medicinal herbs have been demonstrated to exhibit anticancer properties. The present study aimed to screen the sensitivity of bladder cancer to natural compounds by using six classic anti‑inflammatory and detoxifying herbs, including the ethanol extract of Paris polyphylla (PPE), Scutellaria barbata, Pulsatillae decoction, Dahuang Huanglian Xiexin decoction, Bazhengsan and Hedyotis diffusa combined with S. barbata, were used to treat bladder cancer cells in vitro. Bladder cancer was more sensitive to PPE compared with the other tested herbs, and PPE significantly suppressed bladder cancer cell migration and invasion. Thus, the present study focused on PPE. Bladder cancer cells were treated with monomer components of PPE, including polyphyllin (PP) I, PPII, PPVI and PPVII. The results demonstrated that PPII treatment significantly inhibited cancer cell migration and invasion, increased the expression level of E‑cadherin and decreased the levels of N‑cadherin, snail family transcriptional repressor 2, twist family bHLH transcription factor 1, matrix metallopeptidase (MMP) 2 and MMP9 compared with those in the control group (untreated cells). These results suggested that PPII treatment may suppress bladder cancer cell migration and invasion by regulating the expression of EMT‑associated genes and MMPs. Therefore, PPE and PPII may have antimetastatic effects and PPII may serve as a potential therapeutic option for inhibiting bladder cancer metastasis.

Background: The use of Cyclin-Dependent kinase 4 and 6 (CDK4/6) inhibitors has profoundly changed the challenge of endocrine therapy (ET) resistance in hormone receptor-positive (HR+)/HER2-negative (HER2−) breast cancer. However, there is currently no comprehensive evaluation of the evidence for the efficacy of CDK4/6 inhibitors. We conducted an umbrella review to explore the impact of CDK4/6 inhibitor combined with ET on breast cancer by summarizing and assessing the meta-analysis (MA) and systematic review (SR) evidence. Methods: Cochrane, PubMed, Embase, and Web of Science databases were searched from inception to August 1st, 2022. Eligible studies were assessed for methodological quality, report quality, and evidence quality using the AMSTAR-2 scale, PRISMA 2020, and GRADE grading systems, respectively. We summarized all efficacy outcomes of CDK4/6 inhibitors for breast cancer and reported them in narrative form. Results: Our study included 24 MAs and SRs. The strongest evidence demonstrated that CDK4/6 inhibitor combined with ET significantly improved progression-free survival (PFS), overall survival (OS) in advanced breast cancer (ABC). A large body of moderate to high evidence showed a significant association between combination therapy and objective response rate (ORR), and clinical benefit response (CBR) benefit in ABC. Low evidence suggested some degree of benefit from combination therapy in second progression-free survival (PFS2) and time to subsequent chemotherapy (TTC) outcomes in ABC and invasive disease-free survival (IDFS) outcomes in early breast cancer. Conclusions: Based on current evidence, CDK4/6 inhibitors combined with ET have great confidence in improving PFS, OS, ORR, and CBR outcomes in patients with ABC, which provides more rational and valid evidence-based medicine for CDK4/6 inhibitor promotion and clinical decision support.

The active ingredients of the Chinese medical herb Paris polyphylla, P. polyphylla ethanol extract (PPE) and polyphyllin I (PPI), potentially inhibit epithelial-mesenchymal transition (EMT) in tumors. However, the roles of these ingredients in inhibiting EMT in adenomyosis (AM) remain to be explored. The primary goal of the study was to uncover the underlying molecular processes through which PPE and PPI suppress EMT in AM, alongside assessing the safety profiles of these substances. To assess the suppressive impact of PPE on adenomyosis-derived cells (AMDCs), we employed Transwell and wound healing assays. The polyphyllins (PPI, PPII, PPVII) contained in PPE were characterized using high-performance liquid chromatography (HPLC). Then, bioinformatics techniques were performed to pinpoint potential PPI targets that could be effective in treating AM. Immunoblotting was used to verify the key proteins and pathways identified via bioinformatics. Furthermore, we examined the efficacy of PPE and PPI in treating Institute of Cancer Research (ICR) mice with AM by observing the morphological and pathological features of the uterus and performing immunohistochemistry. In addition, we assessed safety by evaluating liver, kidney and spleen pathologic features and serum test results. Three major polyphyllins of PPE were revealed by HPLC, and PPI had the highest concentration. In vitro experiments indicated that PPE and PPI effectively prevent AMDCs invasion and migration. Bioinformatics revealed that the primary targets E-cadherin, N-cadherin and TGFβ1, as well as the EMT biological process, were enriched in PPI-treated AM. Immunoblotting assays corroborated the hypothesis that PPE and PPI suppress the TGFβ1/Smad2/3 pathway in AMDCs to prevent EMT from progressing. Additionally, in vivo studies showed that PPE (3 mg/kg and 6 mg/kg) and PPI (3 mg/kg and 6 mg/kg), successfully suppressed the EMT process through targeting the TGFβ1/Smad2/3 signaling pathway. Besides, it was observed that lower doses of PPE (3 mg/kg) and PPI (3 mg/kg) exerted minimal effects on the liver, kidneys, and spleen. PPE and PPI efficiently impede the development of EMT by inhibiting the TGFβ1/Smad2/3 pathway, revealing an alternative pathway for the pharmacological treatment of AM.

Objective. To explore the molecular mechanism of the Cinnamomi ramulus and Paris polyphylla Sm. (C-P) drug pair in the treatment of adenomyosis (AM) based on network pharmacology and animal experiments. Methods. Via a network pharmacology strategy, a drug-component-target-disease network (D-C-T-D) and proteinprotein interaction (PPI) network were constructed to explore the core components and key targets of C-P drug pair therapy for AM, and the core components and key targets were verified by molecular docking. Based on the results of network pharmacology, animal experiments were performed for further verification. The therapeutic effect of the C-P drug pair on uterine ectopic lesions was evaluated in a constructed AM rat model. Results. A total of 30 components and 45 corresponding targets of C-P in the treatment of AM were obtained through network pharmacology. In the D-C-T-D network and PPI network, 5 core components and 10 key targets were identified. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that the PI3K signaling pathway was the most significantly enriched nontumor pathway. Molecular docking showed that most of the core components and key targets docked completely. Animal experiments showed that the C-P drug pair significantly ameliorated the pathological changes of endometriotic lesions in AM model rats and inhibited PI3K and Akt gene expression, and PI3K and Akt protein phosphorylation. In addition, treatment with the C-P drug pair promoted AM cell apoptosis; upregulated the protein expression of Bax, Caspase-3, and cleaved Caspase-9; and restrained Bcl-2 expression. Conclusions. We propose that the pharmacological mechanism of the C-P drug pair in the treatment of AM is related to inhibition of the PI3K/Akt pathway and promotion of apoptosis in AM ectopic lesions.

Because adenomyosis (AM) ectopic primary cells are hard to come by, have a short lifespan, and the characteristics that alter over time, their utility in AM research is constrained. This study aimed to establish a line of immortalized human adenomyosis ectopic cell (ihAMEC) to change this situation. Primary cells were obtained from AM ectopic lesion tissue and then infected with Simian Vacuolating Virus 40 Tag (SV40 T) lentivirus and screened to establish immortalized cells. We verified the main features and found that the ihAMEC could be cultured for more than 50 generations and the proliferation ability of ihAMEC was more active than that of primary cells. The cytoskeleton and cell types of ihAMEC were similar to primary cells and maintained a normal karyotype. The expression of epithelial-mesenchymal transition (EMT) markers, estrogen-metabolizing proteins, and estrogen/progesterone receptors in ihAMEC was similar to the expression seen in primary cells. In addition, the response of ihAMEC under estrogen treatment and Lipopolysaccharide intervention is similar to primary cells. The clonogenic ability of ihAMEC was lower than tumor cells and did not form tumors in tumorigenicity assays. Thus, ihAMEC can be used as in vitro cellular model for pathogenesis and drug development studies regarding AM.