OBJECTIVES: To investigate the active ingredients in Hedyotis diffusa-Scutellaria barbata D. Don and the main biological processes and signaling pathways mediating their inhibitory effect on primary hepatocellular carcinoma (HCC). METHODS: The core intersecting genes of HCC and the two drugs were screened from TCMSP, Uniport, Genecards, and String databases using Cytoscape software, and GO and KEGG enrichment analyses of the intersecting genes were conducted. Molecular docking between the active ingredients of the drugs and the core genes was carried out using Pubcham, RCSB and Autoduckto to identify the active ingredients with the highest binding energy, whose inhibitory effect on HepG2 cells was verifies using CCK-8 assay, flow cytometry and Western blotting. RESULTS: TP53 and ESR1 were identified as the core genes of HCC and the two drugs. GO and KEGG analyses showed that the two genes were mainly involved in regulation of apoptotic signaling pathway, cell population proliferation, methane raft, and protein kinase activity, and participated in the signaling pathways of apoptosis, proteoglycans in cancer, PI3K Akt signaling pathway, and hepatitis B. Molecular docking studies showed that the active ingredients of the drugs could be docked with TP53 and ESR1 genes under natural conditions, and ursolic acid had the highest binding energy to ESR1 (-4.98 kcal/mol). The results of CCK-8 assay, flow cytometry and Western blotting all demonstrated significant inhibitory effect of ursolic acid on HepG2 cells. CONCLUSIONS The inhibitory effect of Hedyotis diffusa-scutellariae barbatae on HCC is mediated by multiple active ingredients in the two drugs.
Humans ; Molecular Docking Simulation ; Liver Neoplasms/drug therapy* ; Hep G2 Cells ; Network Pharmacology ; Carcinoma, Hepatocellular/drug therapy* ; Hedyotis/chemistry* ; Signal Transduction/drug effects* ; Cell Proliferation/drug effects* ; Tumor Suppressor Protein p53/metabolism* ; Apoptosis/drug effects* ; Estrogen Receptor alpha/metabolism* ; Drugs, Chinese Herbal/pharmacology*

Tianxiangdan (TXD), a traditional Chinese herbal remedy, demonstrates efficacy in mitigating myocardial ischemia-reperfusion (I/R)-induced damage. This study employed network pharmacology to evaluate the therapeutic targets and mechanisms of TXD in treating I/R. High-performance liquid chromatography-mass spectrometry (HPLC-MS) identified 86 compounds in TXD. Network pharmacological analysis predicted potential target genes and their modes of action. Cardiac function, ischaemic ST changes, lactate dehydrogenase (LDH), malondialdehyde (MDA), superoxide dismutase (SOD) activity, myocardial fiber, and infarct size were assessed using in vivo and in vitro I/R injury models. Estrogen receptor alpha (ERα) protein expression and estradiol (E2) levels were measured to confirm TXD's impact on estrogen levels and ERα expression. To examine if TXD reduces I/R injury through ERα, an AZD group (300 nmol·L^-1 AZD9496 and 15% TXD serum) was compared to a TXD group (15% TXD serum). The study hypothesized that TXD upregulates the ERα-mediated iron metamorphosis pathway. I/R injury-induced ferroptosis was identified using a Fer-1 group (1.0 μmol·L^-1 Fer-1 and 15% TXD serum) to elucidate the potential association between ferroptosis and ERα proteins. A DCFH-DA probe detected reactive oxygen species (ROS) and Fe²⁺, while Western blotting assessed target protein expression. Both in vitro and in vivo experiments demonstrated that TXD attenuated I/R injury by reducing elevated ST-segment levels, improving cardiac injury biomarkers (LDH, MDA, and SOD), alleviating pathological features, and preventing I/R-induced loss of cell viability in vitro. The effects and mechanisms of TXD on I/R injury-associated ferroptosis were investigated using I/R-induced H9c2 cells. The TXD group showed significantly decreased ROS and Fe²⁺ levels, while the AZ group (treated with AZD9496) exhibited increased levels. The TXD group demonstrated enhanced expression of ERα and glutathione peroxidase 4 (GPX4), with reduced levels of P53 protein and ferritin-heavy polypeptide 1 (FTH1). The AZ group exhibited contrasting effects on these expression levels. The literature indicated a novel connection between ERα and ferroptosis. TXD activates the ERα signaling pathway, promoting protection against I/R-induced myocardial cell ferroptosis. This study provides evidence supporting TXD use for myocardial ischemia treatment, particularly in older female patients who may benefit from its therapeutic outcomes.
Animals ; Ferroptosis/drug effects* ; Estrogen Receptor alpha/genetics* ; Myocardial Reperfusion Injury/genetics* ; Drugs, Chinese Herbal/pharmacology* ; Male ; Mice ; Humans ; Mice, Inbred C57BL ; Estradiol/metabolism*

Humans ; Female ; Receptors, Estrogen/metabolism* ; Breast Neoplasms/metabolism* ; Receptors, Progesterone/metabolism* ; Immunohistochemistry ; Estrogen Receptor alpha

This study aimed to investigate the therapeutic effect of Leonuri Herba aqueous decoction on primary dysmenorrhea(PD) and explore the underlying mechanism in conjunction with untargeted metabolomics. Forty adult female rats were randomly divi-ded into a normal group, a model control group, ibuprofen(0.12 g·kg~(-1)) group, and high-and low-dose Leonuri Herba aqueous decoction(5 and 2.5 g·kg~(-1)) groups, with eight rats in each group. The PD rat model was prepared using intramuscular injection of estradiol benzoate combined with intraperitoneal injection of pitocin. Drugs were administered by gavage from the 4th day of modeling for 7 d. After the last administration, pitocin was injected intraperitoneally, and the writhing latency and writhing times within 30 min were recorded. The uterine and ovarian coefficients were determined. Estradiol(E_2), progesterone(Prog), oxytocin(OT), cyclooxyge-nase 2(COX-2), prostaglandin E_2(PGE_2), prostaglandin F_(2α)(PGF_(2α)), and Ca~(2+) levels in uterine tissues were measured by ELISA and biochemical kits. Morphological changes in uterine and ovarian tissues were observed by hematoxylin-eosin(HE) staining. The protein expression of oxytocin receptor(OTR), prostaglandin E_2 receptor 3(EP3), and estrogen receptor alpha(ERα) in uterine tissues was detected by immunohistochemistry. The mRNA expression of OTR, PGE_2 receptors 1-4(EP1, EP2, EP3, and EP4), and PGF_(2α) receptor(FP) in uterine tissues was detected by quantitative real-time PCR. Untargeted metabolomics analysis was performed by ultra-high-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry(LC-QTOF-MS) technology to screen potential biomarkers and enrich metabolic pathways. The results showed that Leonuri Herba was able to significantly reduce the writhing times in PD rats(P<0.05 or P<0.01), significantly reduce the uterine and ovarian coefficients(P<0.01), and improve their histomorphology. After treatment with Leonuri Herba, PGE_2 content was significantly increased(P<0.05), COX-2, PGF_(2α) and Ca~(2+) content, and PGF_(2α)/PGE_2 was significantly decreased(P<0.05 or P<0.01), and OT content was decreased, while E_2 and Prog content tended to further increase in uterine tissues of PD rats. Correspondingly, OTR and EP3 protein expression was significantly downregulated(P<0.05 or P<0.01) and ERα protein expression was upregulated(P<0.05) in uterine tissues. The mRNA expression of FP and EP4 in uterine tissues was significantly downregulated(P<0.01), and the mRNA expression of EP1, EP3, and OTR showed a decreasing trend. The untargeted metabolomics results showed that 10 differential metabolites were restored in the plasma of PD rats after Leonuri Herba treatment. The results indicate that Leonuri Herba is effective in the prevention and treatment of PD, and the underlying mechanism may be attributed to the regulation of PGs synthesis and corresponding receptor binding.
Humans ; Rats ; Female ; Animals ; Estrogen Receptor alpha ; Oxytocin ; Dysmenorrhea/metabolism* ; Cyclooxygenase 2 ; Dinoprostone ; RNA, Messenger/metabolism* ; Dinoprost

ERα-36 is a novel subtype of estrogen receptor α which promotes tumor cell proliferation, invasion and drug resistance, and it serves as a therapeutic target. However, only small-molecule compounds targeting ERα-36 are under development as anticancer drugs at present. Gene therapy approach targeting ERα-36 can be explored using recombinant adenovirus armed with decoy receptor. The recombinant shuttle plasmid pDC316-Ig κ-ERα-36-Fc-GFP was constructed via genetic engineering to express an Ig κ-signaling peptide-leading secretory recombinant fusion protein ERα-36-Fc. The recombinant adenovirus Ad-ERα-36-Fc-GFP was subsequently packaged, characterized and amplified using AdMax^TM adenovirus packaging system. The expression of fusion protein and functional outcome of Ad-ERα-36-Fc-GFP transduction were further analyzed with triple-negative breast cancer MDA-MB-231 cells. Results showed that the recombinant adenovirus Ad-ERα-36-Fc-GFP was successfully generated. The virus effectively infected MDA-MB-231 cells which resulted in expression and secretion of the recombinant fusion protein ERα-36-Fc, leading to significant inhibition of EGFR/ERK signaling pathway. Preparation of the recombinant adenovirus Ad-ERα-36-Fc-GFP provides a basis for further investigation on cancer gene therapy targeting ERα-36.
Adenoviridae/genetics* ; Cell Proliferation ; Estrogen Receptor alpha/metabolism* ; Recombinant Proteins ; Transfection

A growing number of studies have identified sex differences in response to general anesthesia; however, the underlying neural mechanisms are unclear. The medial preoptic area (MPA), an important sexually dimorphic structure and a critical hub for regulating consciousness transition, is enriched with estrogen receptor alpha (ERα), particularly in neuronal clusters that participate in regulating sleep. We found that male mice were more sensitive to sevoflurane. Pharmacological inhibition of ERα in the MPA abolished the sex differences in sevoflurane anesthesia, in particular by extending the induction time and facilitating emergence in males but not in females. Suppression of ERα in vitro inhibited GABAergic and glutamatergic neurons of the MPA in males but not in females. Furthermore, ERα knockdown in GABAergic neurons of the male MPA was sufficient to eliminate sex differences during sevoflurane anesthesia. Collectively, MPA ERα positively regulates the activity of MPA GABAergic neurons in males but not in females, which contributes to the sex difference of mice in sevoflurane anesthesia.
Anesthesia ; Animals ; Estrogen Receptor alpha/metabolism* ; Female ; Male ; Mice ; Preoptic Area ; Sevoflurane/pharmacology* ; Sex Characteristics

OBJECTIVES: To study the effects of 17β-estradiol (E2) on the regulation of the proliferation of condylar chondrocytes and provide a preliminary discussion on the role of phosphorylate-mammalian target of rapamycin (p-mTOR) in this regulatory process. METHODS: Condylar chondrocytes were isolated from 6-week-old female rats for primary culture. Drug treatment with different concentrations of E2 and/or rapamycin (RAPA) was carried out on second-generation cells. Cell Counting Kit 8 was used to measure the cell viability of condylar chondrocytes after culture for 24, 48, or 72 h, and reverse transcription-polymerase chain reaction (RT-PCR) was applied to detect the relative gene expression of estrogen receptor alpha (ERα), estrogen receptor beta (ERβ), collagen type Ⅱ (COLⅡ), autophagy-related gene 6 (Beclin-1), and autophagy-related gene 5 (ATG-5). Western blot was employed to determine the relative protein expression of ERα, ERβ, Beclin-1, lipid-modified light chain 3B (LC3-Ⅱ), and p-mTOR. RESULTS: E2 could significantly promote the proliferation of chondrocytes cultured CONCLUSIONS At a concentration of 10
Animals ; Autophagy ; Cell Proliferation ; Chondrocytes ; Estradiol ; Estrogen Receptor alpha/metabolism* ; Estrogen Receptor beta ; Female ; Phosphorylation ; Rats

BACKGROUND: DNA methylation is involved in numerous biologic events and associates with transcriptional gene silencing, playing an important role in the pathogenesis of endometrial cancer. ESR1/PGR frequently undergoes de novo methylation and loss expression in a wide variety of tumors, including breast, colon, lung, and brain tumors. However, the mechanisms underlying estrogen and progesterone receptors (ER/PR) loss in endometrial cancer have not been studied extensively. The aims of this study were to determine the expression of DNA (cytosine-5)-methyltransferase 3A/3B (DNMT3A/3B) in endometrial cancer to investigate whether the methylation catalyzed by DNMT3A/3B contributes to low ER/PR expression. METHODS: The clinicopathologic information and RNA-Seq expression data of DNMT3A/3B of 544 endometrial cancers were derived from The Cancer Genome Atlas (TCGA) uterine cancer cohort in May 2018. RNA-Seq level of DNMT3A/3B was compared between these clinicopathologic factors with t-test or one-way analysis of variance. RESULTS: DNMT3A/3B was overexpressed in endometrioid carcinoma (EEC) and was even higher in non-endometrioid carcinoma (NEEC) (DNMT3A, EEC vs. NEEC: 37.6% vs. 69.9%, t = -7.440, P < 0.001; DNMT3B, EEC vs. NEEC: 42.4% vs. 72.8%, t = -6.897, P < 0.001). In EEC, DNMT3A overexpression was significantly correlated with the hypermethylation and low expression of the ESR1 and PGR (P < 0.05). The same trend was observed in the DNMT3B overexpression subgroup. In the ESR1/PGR low-expression subgroups, as much as 83.1% of ESR1 and 59.5% of PGR were hypermethylated, which was significantly greater than the ESR1/PGR high-expression subgroups (31.3% and 11.9%, respectively). However, the above phenomena were absent in NEEC, while DNMT3A/3B overexpression, ESR1/PGR hypermethylation, and low ER/PR expression occurred much more often. In univariate analysis, DNMT3A/3B overexpressions were significantly correlated with worse prognosis. In multivariate analysis, only DNMT3A was an independent predictor of disease-free survival (P < 0.05). CONCLUSIONS DNMT3A/3B expression increases progressively from EEC to NEEC and is correlated with poor survival. The mechanisms underlying low ER/PR expression might be distinct in EEC vs. NEEC. In EEC, methylation related to DNMT3A/3B overexpression might play a major role in ER/PR downregulation.
Adult ; Aged ; Aged, 80 and over ; Carcinoma, Endometrioid ; genetics ; metabolism ; pathology ; DNA (Cytosine-5-)-Methyltransferases ; genetics ; metabolism ; DNA Methylation ; genetics ; Endometrial Neoplasms ; genetics ; metabolism ; pathology ; Estrogen Receptor alpha ; genetics ; metabolism ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Immunohistochemistry ; Male ; Middle Aged ; Prognosis

OBJECTIVE: To investigate the effect of estradiol (E2)/estrogen receptor 1 (ESR1) on the proliferation of human chondrocytes and explore the molecular mechanism. METHODS: The Ad-Easy adenovirus packaging system was used to construct and package the ESR1-overexpressing adenovirus Ad-ESR1. Western blotting and qPCR were used to detect the expression of ESR1 protein and mRNA in human chondrocyte C28I2 cells. In the cells treated with different adenoviruses, the effects of E2 were tested on the expressions of proteins related with cell autophagy and apoptosis and the phosphorylation of ERK signaling pathway using Western blotting. Immunofluorescence assay was used to observe the intracellular autophagic flow, flow cytometry was performed to analyze the cell apoptosis rate and the cell cycle changes, and qPCR was used to detect the expressions of PCNA, cyclin B1 and cyclin D1 mRNAs. The inhibitory effect of the specific inhibitor of ERK on the expressions of autophagy- and apoptosis-related genes at both the protein and mRNA levels were detected using Western blotting and qPCR. RESULTS: Transfection with the recombinant adenovirus overexpressing ESR1 and E2 treatment of C28I2 cells significantly enhanced the expressions of autophagy-related proteins LC3, ATG7, promoted the colocalization of LC3 and LAMP1 in the cytoplasm, increased the expressions of the proliferation-related marker genes PCNA, cyclin B1 and cyclin D1, and supressed the expressions of cleaved caspase-3, caspase-12 and pERK. RNA interference of ESR1 obviously lowered the expression levels of autophagy-related proteins in C28I2 cells, causing also suppression of the autophagic flow, increments of the expressions of apoptosis-related proteins and pERK, and down-regulated the expressions of the proliferation marker genes. Blocking ERK activation with the ERK inhibitor obviously inhibited the effects of E2/ESR1 on autophagy, proliferationrelated gene expressions and cell apoptosis. CONCLUSIONS The targeted binding of E2 with ESR1 promotes the proliferation of human chondrocytes possibly by inhibiting the activation of ERK signaling pathway to promote cell autophagy and induce cell apoptosis.
Adenoviridae ; metabolism ; Apoptosis ; Autophagy ; Autophagy-Related Protein 7 ; metabolism ; Cell Line ; Cell Proliferation ; Chondrocytes ; cytology ; metabolism ; Estradiol ; metabolism ; Estrogen Receptor alpha ; metabolism ; Humans ; Lysosome-Associated Membrane Glycoproteins ; metabolism ; MAP Kinase Signaling System ; Microtubule-Associated Proteins ; metabolism ; Transfection

To study the correlation between the spatial cognitive impairment and different subtypes of estrogen receptor &alpha; (ER&alpha;) of hippocampus in diabetic mice, we used alloxan (intraperitoneal injection) to induce type 1 diabetes in male Kunming mice and compared the spatial cognitive ability of the model mice with that of control mice through Morris water maze test. Meanwhile, using Western blot, we detected the protein expressions of ER-&alpha;36, ER-&alpha;66, caveolin-1, PKC&alpha;, cAMP-response element binding protein 2 (CREB2), and synaptophysin (Syn) in the hippocampus of the mice. The results showed that on the 3rd and 5th days of training, the ability of spatial learning and memory in the diabetic mice was significantly inferior to that of the control mice (P < 0.05). In the diabetic mice, the protein expressions of caveolin-1 and PKC&alpha; were decreased (P < 0.05), but ER-&alpha;66 expression was unaffected, while ER-&alpha;36 and CREB2 expressions were significantly increased (P < 0.05) compared with those of the control mice. The results suggest that abnormal expression of ER-&alpha;36 and related signal molecules may be important factors for diabetes-induced spatial cognitive impairment.
Animals ; Caveolin 1 ; metabolism ; Cognitive Dysfunction ; Cyclic AMP Response Element-Binding Protein ; metabolism ; Diabetes Mellitus, Experimental ; physiopathology ; Estrogen Receptor alpha ; metabolism ; Hippocampus ; metabolism ; physiopathology ; Male ; Maze Learning ; Memory ; Mice ; Protein Kinase C-alpha ; metabolism ; Synaptophysin ; metabolism