ABSTRACT
Objective:To investigate the effects of ursolic acid (UA) on proliferation, migration and iron death of ectopic endometrial stromal cells (EESCs) and its mechanism.Methods:Mouse model of endometriosis was established and the primary EESCs were isolated. The cells were treated with UA at different concentrations (0, 2.5, 5, 10, 20, 40, 50, 80, 100, 200 μmol/L). The cells were divided into Control group (normal culture), 2.5 μmol/L UA group (2.5 μmol/L UA treatment), 5.0 μmol/L UA group (5.0 μmol/L UA treatment), 10.0 μmol/L UA group (10 μmol/L UA treatment), and UA+DUSP19 group (10 μmol/L UA+50 μmol/L JAK2/STAT3 signal pathway activator DUSP19 treatment). Cell survival rate was detected by CCK-8 method. Cell proliferation was detected by plate cloning method. Transwell chamber assay was used to detect cell migration. The levels of Fe 2+ and the contents of malondialdehyde (MDA), reactive oxygen species (ROS) and superoxide dismutase (SOD) were detected by kit. Protein expression levels of Ki67, PCNA, CyclinD1, p-JAK2, p-STAT3, JAK2 and STAT3 were detected by western blot. Results:The number of clones in Control, 2.5 μmol/L UA, 5.0 μmol/L UA and 10.0 μmol/L UA groups were as follows: 152.22±15.47, 121.22±11.54, 92.00±5.54, 66.44±6.88; Ki67 protein expression was 1.08±0.10, 0.73±0.07, 0.61±0.06, 0.45±0.02, respectively; The expression of PCNA protein was 0.85±0.07, 0.64±0.05, 0.41±0.03, 0.31±0.05, respectively; CyclinD1 protein expression levels were 0.98±0.11, 0.65±0.06, 0.51±0.05, 0.42±0.07, respectively. The migration numbers were 92.78±6.27, 62.22±2.20, 50.22±4.59 and 39.11±4.33, respectively; Fe 2+ levels were (1.06±0.07) μmol/g, (1.21±0.11) μmol/g, (1.33±0.08) μmol/g, (1.47±0.09) μmol/g, respectively; MDA content was (0.48±0.06) μmol/g, (0.65±0.07) μmol/g, (0.85±0.08) μmol/g, (1.03±0.11) μmol/g, respectively; ROS contents were (19.85±1.21) %, (24.83±2.79) %, (29.04±1.86) %, (33.87±2.45) %, respectively; SOD content were (36.41±3.56) U/mg, (31.03±2.81) U/mg, (25.63±2.84) U/mg, (19.62±1.67) U/mg, respectively; p-JAK2 protein expression was 0.85±0.10, 0.75±0.06, 0.53±0.05, 0.31±0.03, respectively; p-STAT3 protein expression was 1.08±0.11, 0.79±0.06, 0.63±0.07, 0.42±0.03, respectively. The p-JAK2 protein content in UA group and UA+DUSP19 group was 0.38±0.05 and 0.75±0.08, respectively; p-STAT3 protein expression was 0.46±0.04 and 0.80±0.03, respectively; The cell survival rates were (52.55±2.44) % and (82.18±4.72) %, respectively; Fe 2+ levels were (1.57±0.06) μmol/g and (1.21±0.13) μmol/g, respectively. The differences in the above indicators between the Control group and the 2.5 μmol/L UA group, 5.0 μmol/L UA group and 10.0 μmol/L UA group were statistically significant ( P<0.05). There were statistically significant differences among 2.5 μmol/L UA group, 5.0 μmol/L UA group and 10.0 μmol/L UA group ( P<0.05). There were statistically significant differences in p-JAK2, p-STAT3, cell survival rate and Fe 2+ levels between UA group and UA+DUSP19 group ( P<0.05) . Conclusion:Ursolic acid can inhibit the proliferation and migration of EESCs cells and induce iron death by regulating JAK2/STAT3 signaling pathway, thus playing a protective role in endometriosis.
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Based on the Janus kinase 2/signal transducer and activator of transcription 3(JAK2/STAT3) signaling pathway, this study investigated the effect of medicated serum of Sparganii Rhizoma(SR) and Curcumae Rhizoma(CR) on the proliferation, apoptosis, migration, and secretion of inflammatory factors of ectopic endometrial stromal cells(ESCs). Specifically, human ESCs were primary-cultured. The effect of different concentration(5%, 10%, 20%) of SR-, CR-, and SR-CR combination-medicated serum, and AG490 solution(50 μmol·L~(-1)) on the proliferation of ESCs was detected by methyl thiazolyl tetrazolium(MTT) assay, and the optimal dose was selected accordingly for further experiment. The cells were classified into normal serum(NS) group, SR group(10%), CR group(10%), combination(CM) group(10%), and AG490 group. The apoptosis level of ESCs was detected by flow cytometry, and the migration ability was examined by wound healing assay. The secretion of interleukin(IL)-1β, IL-6, and tumor necrosis factor(TNF)-α was determined by enzyme-linked immunosorbent assay(ELISA). The protein levels of cysteinyl aspartate specific protei-nase-3(caspase-3), B-cell lymphoma(Bcl-2), and Bcl-2-associated X protein(Bax) and the levels of phosphorylated(p)-JAK2 and p-STAT3 were detected by Western blot. The results showed that the viability of ESCs cells was lowered in the administration groups compared with the blank serum group(P<0.01), especially the 10% drug-medicated serum, which was selected for further experiment. The 10% SR-medicated serum, 10% CR-medicated serum, and 10% CM-medicated serum could increase the apoptosis rate(P<0.01), up-regulate the protein expression of caspase-3 and Bax in cells(P<0.05 or P<0.01), down-regulate the expression of Bcl-2(P<0.01), decrease the cell migration rate(P<0.05 or P<0.01), and reduce the secretion levels of IL-1β, IL-6, and TNF-α(P<0.05 or P<0.01), and levels of p-JAK2 and p-STAT3(P<0.05 or P<0.01). Compared with the SR and CR groups, CM group showed low cell viability(P<0.01), high protein expression of caspase-3 and Bax(P<0.05 or P<0.01), and low protein expression of Bcl-2 and p-JAK2(P<0.05). After incubation with CM, the apoptosis rate was higher(P<0.05) and the migration rate was lower(P<0.01) than that of the CR group. The p-STAT3 protein level of CM group was lower than that of the RS group(P<0.05). The mechanism of SR, CR, and the combination underlying the improvement of endometriosis may be that they blocked JAK2/STAT3 signaling pathway, inhibited ESC proliferation, promoted apoptosis, weakened cell migration, and reduced the secretion of inflammatory factors. The effect of the combination was better than that of RS alone and CR alone.
Subject(s)
Female , Humans , Janus Kinase 2 , Caspase 3 , bcl-2-Associated X Protein , Interleukin-6/genetics , Apoptosis , Signal Transduction , Cell Proliferation , STAT3 Transcription Factor/geneticsABSTRACT
ObjectiveThis study investigated the mechanism of Wenjingtang in the prevention and treatment of endometriosis （EMT） from the perspective of regulating hypoxia stress and mitochondrial function. MethodPrimary human endometrial stromal cells （ESCs） form ectopic endometrial tissues were isolated and cultured， the cells were divided into control group （Control）， 5% control serum group （5% KBXQ）， 10% control serum group （10% KBXQ）， 5% Wenjingtang serum group （5% WJTXQ） and 10% Wenjingtang serum group （10% WJTXQ）. ESCs in different groups were detected for proliferation by methyl thiazolyl tetrazolium （MTT） assay， mRNA and protein expression of hypoxia inducible factor-1α （HIF-1α） by real-time fluorescent quantitative polymerase chain reaction （Real-time PCR） and Western blot analysis， mitochondrial ultrastructure by transmission electron microscope， mitochondrial function ［mitochondrial membrane potential， mitochondrial membrane potential（MMP） and cytochrome C（Cyt C） content］ and apoptosis （cell membrane permeability， nuclear fluorescence intensity， nuclear size and cell counts） by high content screening （HCS） assay， apoptosis rate by flow cytometry， and proteins of B-cell lymphoma-2 （Bcl-2） associated X （Bax）， Bcl-2 and cleaved cysteinyl aspartate specific proteinase-3 （cleaved Caspase-3） by Western blot. ResultCompared with Control group， the 5% KBXQ and 10% KBXQ groups showed increased cell viability （P<0.01）， there was no significant change in HIF-1α mRNA and protein expression， transmission electron microscopy showed that the mitochondrial cristae were obvious and the inner and outer membranes of mitochondria were clear， HCS multichannel fluorescence staining showed that there were no significant changes in the expression of MMP， Cyt C and cell membrane permeability， and the nuclei showed uniform light staining， there were no significant changes in apoptosis rate， cleaved Caspase-3 protein expression and Bax/Bcl-2 ratio. Compared with Control group and corresponding concentration KBXQ group， the 5% WJTXQ and 10% WJTXQ group showed decreased cell viability （P<0.01） and HIF-1α mRNA and protein levels （P<0.05，P<0.01）， the ultrastructure of mitochondria was destroyed， some mitochondria were swollen and the cristae were blurred， moreover， decreased MMP and up-regulated Cyt C release （P<0.05，P<0.01）， increased cell membrane permeability （P<0.01）， and apoptosis characteristics included nuclear pyknosis， DNA agglutination in nucleus and decrease of cell numbers were observed （P<0.05，P<0.01）， increased apoptosis rate （P<0.01）， which was consistent with the results of HCS analysis， and up-regulated expression levels of cleaved Caspase-3 protein and Bax/Bcl-2 ratio （P<0.05，P<0.01）. ConclusionIn conclusion， the results suggest that Wenjingtang can improve hypoxia stress via down-regulating HIF-1α expression in ectopic ESCs， and inhibit cell proliferation， reduce mitochondrial biological activity and induce apoptosis， which might be the internal mechanism of Wenjingtang in preventing and treating EMT.
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BACKGROUND: Provision of optimal endometrial stromal cells is essential in uterine tissue engineering. Culture of these cells is significantly influenced by gonadotropin hormones. This investigation attempted to define the proliferation profiles of murine uterine endometrial stromal cells during in vitro culture with recombinant follicle stimulating hormone (rFSH), urinary follicle stimulating hormone (uFSH), and human chorionic gonadotropin (hCG). METHODS: Murine uterine endometrial stromal cells were collected from 8-week-old mice and cultured in vitro up to 72 h, with rFSH, uFSH, or hCG. Cell cycles were analyzed by BrdU assay, and cyclin D1 expression was evaluated according to dose and duration of gonadotropin treatment. RESULTS: BrdU assay showed a further inhibitory effect on murine uterine endometrial stromal cell proliferation when cultured with rFSH compared to uFSH, and a similar inhibitory proliferation profile when cultured with hCG at a specific range of concentrations. The expression of cyclin D1 of murine uterine endometrial stromal cells was down-regulated when cultured with rFSH, uFSH, or hCG, compared to control. CONCLUSIONS: FSH may inhibit the proliferation of murine uterine endometrial stromal cells during in vitro culture. rFSH may have more significant inhibitory effects on the proliferation of endometrial stromal cells than uFSH. Establishing an optimal endocrine milieu is necessary using more advanced combination of female hormones for in vitro culture of this type of cells.
Subject(s)
Animals , Female , Humans , Mice , Bromodeoxyuridine , Cell Cycle , Chorionic Gonadotropin , Cyclin D1 , Follicle Stimulating Hormone , Gonadotropins , In Vitro Techniques , Stromal Cells , Tissue Engineering , UterusABSTRACT
Background & objectives: CD9 and CD146 are important adhesion molecules that play a role in the implantation of an embryo. This study was undertaken to correlate the expression of these markers in fertile and infertile women's endometrial stromal cells. Methods: Human endometrial stromal cell culture from endometrial biopsies of fertile (n=50) and infertile females (n=50) was performed and primary cell lines were established. Expression of CD9 and CD146 was studied for all the 100 cell lines with the help of flow cytometry. Gene expression of CD9 and CD146 was performed by real-time polymerase chain reaction. Results: There was a significant difference in endometrial stromal cells of fertile and infertile females. Flow cytometric results revealed significantly lower expression of CD9 (P=0.0126) and CD146 (P=0.0006) in the infertile endometrial stromal cells as compared to fertile endometrial stromal cells. These results were comparable with real-time data. Interpretation & conclusions: This study showed that endometrial stromal cells from infertile females had lower expression of adhesion molecules, CD9 and CD146. Our findings suggest that CD9 and CD146 may have a role in infertility. Infertile female's endometrial stromal cells have decreased expression of CD9 and CD146 which can be the cause of infertility related to implantation failure.
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IGF-1, a high potent mitogenic factor with a dual function is believed to participate with female steroids and other growth factors to prepare endometrium receptivity for successful embryo implantation and further development. Stromal cells forms the functional receptive layer of endometrium structure. Analysis of the conditioned/defined medium of pure cultured human endometrial stromal cells have revealed that stromal cells in a monolayer culture secrete and produce three different types of proteins in a varying amounts. These proteins exhibit a high specificity as well as a high binding affinity for the radiolabeled IGF-1 peptide. The molecular weights of these proteins have been determined by SDS-Page electrophoresis after cross-linking with the radiolegand IGF-1 and then detected with autoradiography. By comparison to the migration of high molecular weights protein markers, these proteins have been identified to correspond to the IGFBP-1 (31 KDa), IGFBP-2 (36 KDa) and IGFBP-3 (45 KDa). The secretion of these binding proteins (IGFBP-1, -2, -3) by endometrial stromal cells may support the view of their biological importance in controlling the delivery and bioavailability of the high mitogen IGF-1 peptide to their nominative type-1 IGF-receptor on cell surface, thereby modulating its action. It seems likely that these IGFBPs may play a key role in switching on/off IGF-1 peptide action , thereby avoiding the uncontrolled proliferation effect of the IGF-1 that favor endometrium cancer development.
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OBJECTIVE: This study was performed to compare the expression of CD44 in endometrial stromal cells (ESCs) of women with and without endometriosis and to evaluate the role of CD44 in the adherence of ESCs to peritoneal mesothelial cells (PMCs). METHODS: A PMC adherence assay was performed to evaluate the adherence of ESCs to PMCs in women with and without endometriosis. The expression of CD44 mRNA was measured by reverse transcription-polymerase chain reaction. CD44 protein was evaluated by Western blot analysis. RESULTS: There were no significant differences in the expression of CD44 mRNA and protein in ESCs or in the binding of ESCs to PMCs between patients with endometriosis and controls. Although the expression of CD44 protein was decreased in both women with endometriosis and controls after anti-CD44 antibody treatment, there was no effect on binding of ESCs to PMCs. Treatment of ESCs with peritoneal fluid from endometriosis patients resulted in a significant increase in binding of ESCs to PMCs compared to untreated ESCs in the endometriosis group. CONCLUSION: This study demonstrates that the expression of CD44 protein in ESCs from women with endometriosis might not be directly associated with adherence to PMCs.
Subject(s)
Female , Humans , Ascitic Fluid , Blotting, Western , Endometriosis , RNA, Messenger , Stromal CellsABSTRACT
Integrins are heterodimeric glycoproteins that have been found to undergo dynamic temporal and spatial changes in the endometrium during the menstrual cycle and in early pregnancy. Specificity of integrins is known to be different in human endometrial stromal cells and decidual cells. These shifts of integrins suggested to play an important role in embryo implantation and can be modulated by progesterone, cAMP derivatives, and cytokines. The mechanisms of decidualization and its precise physiological role are still not clearly understood and in vitro systems could provide an alternative that overcomes limitations of studying such complex biological phenomena in vivo at the time of implantation. This study was undertaken to establish an in vitro model system for human decidualization using 8-bromo-cAMP and to investigate the characteristics of stromal integrin expression in vitro by 8-Br-cAMP. Endometrial stromal cells were isolated and cultured, and then were induced to decidualize by 0.5 mM 8-Br-cAMP for 15 days. Immunofluorescence staining and flow cytometric analyses of the integrin subunits (alpha1, alpha4, alpha5, alpha6, beta1 and alpha v beta3) were performed at day 9. In the presence of 8-Br-cAMP, the staining intensity of alpha v beta3 was significantly higher than control and measurements for alpha1, alpha4, alpha5, alpha6, and beta1 were similar. Immunofluorescent localization of the integrins reflected the differences obtained from the flow cytometric analyses described above. In summary, the expression of alpha;avbeta;b3 integrin increased in stromal cells in vitro decidualized by 8-Br-cAMP and this up-regulation of alpha v beta3 integrin expression during decidualization might influence on human implantation.
Subject(s)
Female , Humans , 8-Bromo Cyclic Adenosine Monophosphate/pharmacology , Cell Size , Cells, Cultured , Decidua/cytology , Flow Cytometry , Integrins/analysis , Prolactin/analysis , Stromal Cells/cytologyABSTRACT
OBJECTIVES: To test the hypothesis that the endometrial stromal cells from patients with endometriosis responds differently to the insulin-like growth factor (IGF) compared with those from patients without endometriosis. METHODS: IGFs in peritoneal fluid (PF) from patients with endometriosis(n=18) and without endometriosis(n=12;control patients) were measured by radioimmunoassay. Endometrial stromal cells from patients with endometriosis and control patients were cultured in serum free media(SFM) in the presence or absence of PF or IGF-I(0.25-25 ng/ml) or IGF-II(5-50 ng/ml) and the proliferation of endometrial stromal cells were evaluated by [3H] thymidine incorporation test. All statistics were performed by ANOVA test and student's t-test. RESULTS: When added to SFM, IGF-I(1-25 ng/ml) increased thymidine incorporation in both endometrial stromal cells from patients with endometriosis and control patients in dose dependent manner and IGF-II(5-25 ng/ml) gave similar response in latter cells but not in former cells. Within low IGF-I(less than 100 ng/ml) PF group or high IGF-I(more than 100ng/ml) PF group, the type of endometrial stromal cells did not result in any difference in thymidine incorporation. However, regardless of the source of stromal cells, high IGF-I PF group produced a greater extent of thymidine incorporation than low IGF-I PF group in patients with endometriosis but not in control patients. Also, thymidine incorporation was higher in high IGF-I PF group of former patients than in the same group of latter patients. PF induced higher thymidine incorporation in endometrial stromal cells than the same levels(0.25-2.5 ng/ml) of IGF-I directly added to SFM. CONCLUSIONS: The effects of IGF-I in PF on endometrial stromal cells are similar regardless of their source and IGF-I is one of several growth factors that may participate in the growth of endometrial stromal cells in pelvic endometriosis.
Subject(s)
Female , Humans , Ascitic Fluid , DNA , Endometriosis , Insulin-Like Growth Factor I , Intercellular Signaling Peptides and Proteins , Radioimmunoassay , Somatomedins , Stromal Cells , ThymidineABSTRACT
Parenchymal pulmonary endometriosis has unique symptoms such as hemoptysis, dyspnea and chest pain in associated with menstruation. We experienced a case of parenchymal pulmonary endometriosis with the complaints of catamenial hemoptysis. Bronchoalveolar lavage(BAL) was performed when catamenial hemoptysis occurred. The BAL fluid showed many erythrocytes, hemosiderin-laden macrophages, and sheets of endometrial stromal cells.