[Shaofu Zhuyu Decoction attenuates fibrosis in endometriosis through regulating PTEN/Akt/mTOR signaling pathway].

The present study aimed to investigate the protective role of Shaofu Zhuyu Decoction(SFZY) against endometriosis fibrosis in mice, and decipher the underlying mechanism through the phosphatase and tensin homolog deleted on chromosome ten(PTEN)/protein kinase B(Akt)/mammalian target of rapamycin(mTOR) pathway. Eighty-five BALB/c female mice were randomly assigned into a blank group, a model group, high-, medium, and low-dose SFZY(SFZY-H, SFZY-M, and SFZY-L, respectively) groups, and a gestrinone suspension(YT) group. The model of endometriosis was induced by intraperitoneal injection of uterine fragments. The mice in different groups were administrated with corresponding groups by gavage 14 days after modeling, and the blank group and model group with equal volume of distilled water by gavage. The treatment lasted for 14 days. The body weight, paw withdrawal latency caused by heat stimuli, and total weight of dissected ectopic focus were compared between different groups. The pathological changes of the ectopic tissue were observed via hematoxylin-eosin(HE) and Masson staining. Real-time PCR was employed to measure the mRNA levels of α-smooth muscle actin(α-SMA) and collagen type Ⅰ(collagen-Ⅰ) in the ectopic tissue. The protein levels of PTEN, Akt, mTOR, p-Akt, and p-mTOR in the ectopic tissue were determined by Western blot. Compared with the blank group, the modeling first decreased and then increased the body weight of mice, increased the total weight of ectopic focus, and shortened the paw withdrawal latency. Compared with the model group, SFZY and YT increased the body weight, prolonged the paw withdrawal latency, and decreased the weight of ectopic focus. Furthermore, the drug administration, especially SFZY-H and YT(P<0.01), recovered the pathological and reduced the area of collagen deposition. Compared with the blank group, the modeling up-regulated the mRNA levels of α-SMA and collagen-Ⅰ in the ectopic focus, and such up-regulation was attenuated after drug intervention, especially in the SFZY-H and YT groups(P<0.05,P<0.01). Compared with the blank group, the modeling down-regulated the protein level of PTEN and up-regulated the protein levels of Akt, mTOR, p-Akt, and p-mTOR(P<0.01, P<0.001). Drug administration, especially SFZY-H and YT, restored such changes(P<0.01). SFZY may significantly attenuate the focal fibrosis in the mouse model of endometriosis by regulating the PTEN/Akt/mTOR signaling pathway.

Silencing of circ_0007299 suppresses proliferation, migration, and invasiveness and promotes apoptosis of ectopic endometrial stromal cells in endometriosis via miR-424-5p-dependent modulation of CREB1.

BACKGROUND: The abnormality of endometrial stromal cells (ESCs) can contribute to endometriosis pathogenesis. Circular RNAs (circRNAs) possess critical roles in endometriosis pathogenesis. Here, we defined the activity and mechanism of human circ_0007299 in the regulation of ectopic ESCs in vitro. METHODS: Circ_0007299, miR-424-5p and cAMP response element-binding protein 1 (CREB1) were quantified by qRT-PCR or immunoblotting. Cell viability, proliferation, apoptosis, invasion and motility were gauged by CCK-8, 5-Ethynyl-2'-Deoxyuridine (EdU), flow cytometry, transwell, and wound-healing assays, respectively. Dual-luciferase reporter and RNA immunoprecipitation (RIP) assays were used to verify the direct relationship between miR-424-5p and circ_0007299 or CREB1. RESULTS: Our data showed that circ_0007299 was upregulated in human ectopic endometrium tissues and ectopic ESCs. Silencing endogenous circ_0007299 impeded the proliferation, invasiveness, and motility and enhanced apoptosis of ectopic ESCs. Mechanistically, circ_0007299 regulated miR-424-5p expression. Moreover, circ_0007299 silencing impeded the proliferation, invasiveness, and motility and enhanced apoptosis of ectopic ESCs via its regulation on miR-424-5p. CREB1 was identified as a direct miR-424-5p target, and miR-424-5p overexpression suppressed ectopic ESC proliferation, migration, and invasiveness and promoted apoptosis by downregulating CREB1. Furthermore, circ_0007299 positively modulated CREB1 expression through miR-424-5p competition. CONCLUSION: Our findings establish that circ_0007299 silencing impedes the proliferation, invasiveness, and motility and promotes apoptosis of ectopic ESCs at least in part via miR-424-5p-dependent modulation of CREB1.

[Mechanism of Shaofu Zhuyu Decoction in treatment of endometriosis-associated dysmenorrhea with syndrome of cold coagulation and blood stasis based on MSK1/2].

This study aims to decipher the mechanism underlying the effect of Shaofu Zhuyu Decoction on endometriosis(EMT)-associated dysmenorrhea in rats with the syndrome of cold coagulation and blood stasis based on mitogen-and stress-activated protein kinase 1/2(MSK1/2).We employed a random number table to randomly assign SPF female non-pregnant rats into the sham group, and treated the rest rats with autologous transplantation+refrigerator freezing for the modeling of the syndrome of cold coagulation and blood stasis.The modeled rats were then randomly assigned into the control group and high-, medium-and low-dose Shaofu Zhuyu Decoction groups.The rats in the low-, medium-, and high-dose decoction groups were respectively administrated with 9, 4.5, and 2.3 g·kg~(-1) decoction through gavage once a day for 2 consecutive weeks, and those in the control group were administrated with 0.24 mg·kg~(-1) gestrinone through gavage once every 3 days for 2 weeks.After that, the size of ectopic focus in each rat was measured via laparotomy.Enzyme-linked immunosorbent assay(ELISA) was adopted to determine the expression of interleukin(IL)-6, IL-10, prostaglandin E2(PGE2), tumor necrosis factor-α(TNF-α).Western blot was employed to determine the protein levels of MSK1/2 and dual-specificity phosphatase 1(DUSP1) and real-time quantitative polymerase chain reaction(RT-PCR) to determine the mRNA levels of the two genes in rat eutopic endometrial tissue.Compared with the sham group, the model group showed increased levels of IL-6, PGE2, and TNF-α while decrease level of IL-10 in the serum(P<0.01).Compared with the model group, the high-and medium-dose decoction groups and the gestrinone group had declined levels of IL-6, PGE2, and TNF-α while risen level of IL-10 in the serum(P<0.01).The model group had lower protein levels and mRNA levels of MSK1/2 and DUSP1 in the eutopic endometrial tissue than the sham group(P<0.01). The high-and medium-dose decoction groups and the gestrinone group had higher protein and mRNA levels of MSK1/2 and DUSP1 in the eutopic endometrial tissue than the model group(P<0.01).The results indicated that Shaofu Zhuyu Decoction can regulate the abnormal expression of pro-inflammatory cytokines TNF-α, IL-6, and PGE2 and anti-inflammatory cytokines IL-10 and DUSP1 via MSK1/2 to alleviate EMT-associated dysmenorrhea in rats with the syndrome of cold coagulation and blood stasis.

Identification of immune- and autophagy-related genes and effective diagnostic biomarkers in endometriosis: a bioinformatics analysis.

Background: To identify autophagy- and immune-related hub genes affecting the diagnosis and treatment of endometriosis. Methods: Gene expression data were downloaded from the Gene Expression Omnibus (GEO) (GSE11691 and GSE120103 for training, and GSE7305 for validation). By overlapping the differentially expressed genes (DEGs), Weighted gene co-expression network analysis (WGCNA) module genes, and autophagy-related genes (ARGs), and immune-related genes (IRGs) separately, hub genes were identified using the least absolute shrinkage and selection operator (LASSO)and support vector machine recursive feature elimination (SVM-RFE). The hub genes were analyzed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. A hub gene-prediction model was constructed and assessed using five-fold cross-validation via five supervised machine-learning algorithms: random forest, the sequential minimal optimization (SMO), K-nearest neighbours (IBK), C4.5 decision tree (J48), and logistics regression. The area under the receiver operating characteristic curve (AUC) was adopted to assess the identification ability of characteristic genes. Results: 1,116 DEGs were obtained from the training cohort, and 22 endometriosis-related IRGs were identified by overlapping the 1,116 DEGs, 3,222 module genes, and 1,793 IRGs. Meanwhile, 45 endometriosis-related ARGs were obtained (1,928 ARGs). Subsequently, nine IRG hub genes (BST2, CCL13, CD86, CSF1, FAM3C, GREM1, ISG20, PSMB8, and S100A11) and nine ARG hub genes (GSK3A, HTR2B, RAB3GAP1, ARFIP2, BNIP3, CSF1, MAOA, PPP1R13L, and SH3GLB2) were obtained by LASSO and SVM-RFE. GO analysis indicated that the ARG hub genes responded to the regulation of autophagy and mitochondrial outer membrane permeabilization, and KEGG enrichment analysis involved serotonergic and dopaminergic synapses. GO analysis also indicated that the IRG hub genes responded to the regulation of leukocyte proliferation and mononuclear cell migration, and KEGG analysis showed enrichment involved in viral protein interaction with cytokines and cytokine receptors. The AUC of the random-forest algorithm of ARGs was 0.975 in the training cohort and 0.940 in the validation cohort, and the AUC of the SMO algorithm of IRGs was 0.907 in the training cohort and 0.8 in the validation cohort. Conclusions: Seventeen hub genes are closely associated with endometriosis. These genes are potential autophagy- and immune-related biomarkers for diagnosis and treatment of endometriosis.

[Molecular mechanism of Shaofu Zhuyu decoction in treating endometriosis dysmenorrhea based on GPER2/MAPK/STAT1 axis].

The aim of this paper is to study the molecular mechanism of Shaofu Zhuyu decoction in treating dysmenorrhea of endometriosis based on GPER2/MAPK/STAT1 axis. In this study, HE staining was used to observe the pathological changes of the rats in each group. The levels of TNF-α and IL-6 were detected by ELISA assay. The mRNA expressions of neurotransmitter receptor (NK1) and GPER were detected by qPCR. The protein contents of MAPK and STAT1 were detected by Western blot. According to the results, compared with the model group, Shaofu Zhuyu decoction could significantly improve the inflammation of the ectopic uterine cavity tissue, decrease the contents of TNF-α and IL-6 in the uterine cavity, the mRNA expressions of NK1 and GPER, and the protein expressions of MAPK and STAT1. In conclusion, Shaofu Zhuyu decoction could effectively inhibit the expressions of GPER2, MAPK and STAT1, decrease the levels of TNF-α, IL-6 and NK1 mRNA and relieve the inflammatory pain in patients with endometriosis.

[Effects of Radix Hedysari, Radix Astragalus and compatibility of Angelica Sinensis on blood deficiency model mice induced by cyclophosphamide].

OBJECTIVE: To study the intervention effects of Radix Hedysari, Radix Astragalus and compatibility of Angelica Sinensis on blood deficiency model mice induced by cyclophosphamide (CTX). METHODS: The mice were randomly divided into 7 groups, 10 mice each group. The blood deficiency model was established by CTX. The blank group and model group were treated with saline by gavage, while mice in positive group were administered with Lvjiaobuxue granule. Four dosage group were administered with Radix Hedysari, Radix Hedysari-Radix Angelica Sinensis(5:1), Radix Astragalus and Radix Astragalus-Radix Angelica Sinensis(5:1) water decoction. All the drugs were administered to mice for consecutive 7 d. The contents of red blood cell (RBC), lymphocyte(LYM), hematocrit (HCT), white blood cell (WBC), platelet (PLT) were detected by hematology analyzer, while thymus index(TI), spleen index(SI), reticulocyte (RC), marrow karyocyte (MK) were calculated, and the femur by pathological section were observed by microscope. RESULTS: Compared with blank group, the contents of RBC, WBC, HCT, PLT, LYM were decreased in model group (P<0.05). Compared with model group, the contents of RBC, WBC, HCT, PLT, LYM, RC and marrow karyocyte were increased in Hedysari-Angelica Sinensis(5:1) and Astragalus Angelica Sinensis(5:1) (P<0.05), at the same time, the pathological damage of femur could be improved. CONCLUSIONS: The effect of enrichment blood on blood deficiency model mice in Hedysari-Angelica Sinensis (5:1) and Astragalus-Angelica Sinensis(5:1) were superior to Hedysari and Astragalus.