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1.
Med Sci Monit ; 25: 4250-4263, 2019 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-31175269

RESUMO

BACKGROUND Ovarian cancer has the highest mortality rate among all female genital tumors because of its insidious onset and drug resistance. Hypoxia-inducible factor 1alpha (HIF-1alpha), one of the best-studied oncogenes, plays an important part in tumor adaptation to microenvironmental hypoxia and was found to be overexpressed in several malignancies, including ovarian cancer. Previous studies found that the effect of HIF-1alpha on cancers may be correlated with autophagy and some signaling pathways, such as PI3K/AKT/mTOR, in several tumors. However, the function and potential mechanism have not been clearly defined. MATERIAL AND METHODS The expression of HIF-1alpha in ovarian cancer tissues were detected by immunohistochemistry. HIF-1alpha was knocked down by siRNA transfection. Cell viability was examined by CCK8 and colony formation assay. Apoptosis and autophagy were detected with flow cytometry, transmission electron microscopy, and laser scanning confocal microscopy, respectively. The proteins related to autophagy and PI3K/AKT/mTOR were detected through Western blot analysis. RESULTS HIF-1alpha was expressed at higher levels in epithelial or metastatic ovarian cancer tissue than in normal fallopian tube tissue. When HIF-1alpha was knocked down by siRNA in A2780 and SKOV3 cells, the viability of ovarian cancer cells was weakened, but the apoptosis and autophagy were strengthened. Accordingly, autophagosome formation increased and the expression of autophagy-related proteins LC3 and P62 increased in HIF-1alpha knockdown cells. The PI3K/Akt/mTOR signaling pathway was also found to be inactivated in HIF-1alpha knockdown cells. CONCLUSIONS These findings show that knockdown of HIF-1alpha promoted autophagy and inhibited the PI3K/AKT/mTOR signaling pathway in ovarian cancer cells.

2.
Mol Med Rep ; 19(6): 4727-4734, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31059065

RESUMO

The aim of the present study was to investigate the protective effect of integrin ß1 in the treatment of stress urinary incontinence (SUI) by electrical stimulation, and the underlying mechanisms by which electrical stimulation regulates the collagen metabolism of female vaginal wall fibroblasts (FVWFs). FVWFs obtained from the vaginal wall tissue of patients with (Ingelman­Sundberg scale; grade II, n=8; grade III, n=10) or without (n=8) SUI during gynecological operations were isolated by enzymatic digestion and subsequently identified by immunocytochemistry. Following this, cultured FVWFs were treated with an inhibitor of integrin ß1, recombinant human integrin ß1 and electrical stimulation (100 mv/mm, 2 h, 20 Hz), followed by total mRNA and protein extraction. mRNA and protein expression levels of integrin ß1, transforming growth factor (TGF)­ß1 and collagen (COL) I and III in FVWFs were quantified by reverse transcription­quantitative PCR (RT­qPCR) and western blot analysis respectively. Integrin ß1, TGF­ß1 and COL I and III expression levels were decreased in patients with SUI compared with healthy controls, and the grade III group had lower levels than the grade II group. Following electrical stimulation treatment, the expression levels of TGF­ß1, COL I and III were enhanced in the grade II group, but not in the grade III group. Nevertheless, the inhibitor of integrin ß1 reduced the protective effect of electrical stimulation in the grade II group. In addition, electrical stimulation combined with recombinant human integrin ß1 could also protect cells from SUI in the grade III group. The present study provides evidence for the increased degradation of the extracellular matrix and integrin ß1 in the vaginal wall tissues of patients with SUI, and the protective effect of electrical stimulation against SUI via integrin ß1. These results provide a novel mechanism for the treatment of SUI using electrical stimulation.


Assuntos
Estimulação Elétrica/métodos , Integrina beta1/farmacologia , Integrina beta1/uso terapêutico , Incontinência Urinária por Estresse/tratamento farmacológico , Colágeno Tipo I/metabolismo , Colágeno Tipo III/metabolismo , Matriz Extracelular/metabolismo , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Fator de Crescimento Transformador beta1 , Incontinência Urinária , Vagina/metabolismo , Vagina/patologia
3.
Oxid Med Cell Longev ; 2019: 2039856, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30962861

RESUMO

Apoptosis and oxidative damage are involved in the pathogenesis and progression of stress urinary incontinence (SUI). Our previous results indicate that cell apoptosis and oxidative damage increase in a mouse model of mechanical injury-induced SUI and in fibroblasts treated with excessive mechanical strain. Nuclear factor erythroid-2-related factor 2 (Nrf2) is a well-characterized global antioxidant gene inducer that can reduce oxidative damage and apoptosis. Therefore, we predicted that Nrf2 may have a protective role in mechanical trauma-induced SUI. To test this hypothesis, a mouse model of vaginal distension- (VD-) induced SUI was established. Leak point pressure (LPP); levels of apoptosis, apoptosis-related proteins, and peroxidation products; and the activities of antioxidative proteins in the anterior vaginal wall were measured in wild-type (Nfe2l2+/+) C57BL/6 mice and Nrf2-knockout mice (Nfe2l2-/-). The results showed that Nrf2 knockout aggravated VD-induced reduction in LPP, increase in cell apoptosis and peroxidation product levels, decrease in antioxidative protein activities, and alterations in apoptosis-related protein levels in the vaginal walls of mice. To further confirm the role of Nrf2 in mechanical trauma-induced apoptosis and SUI, VD was performed on mice overexpressing Nrf2 via in vivo transfection of LV-Nfe2l2. The results showed that Nrf2 overexpression significantly alleviated VD-induced abnormalities in the anterior vaginal wall. Taken together, our data suggested that Nrf2 is a potential protective factor in mechanical trauma-induced apoptosis in a mouse model of SUI. Antioxidative therapy may be a promising treatment for mechanical trauma-related SUI.


Assuntos
Fator 2 Relacionado a NF-E2/uso terapêutico , Incontinência Urinária por Estresse/tratamento farmacológico , Incontinência Urinária por Estresse/etiologia , Vagina/lesões , Animais , Apoptose , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Fator 2 Relacionado a NF-E2/farmacologia , Estresse Oxidativo , Transfecção , Incontinência Urinária por Estresse/patologia
4.
Cell Signal ; 59: 141-151, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30940604

RESUMO

Stress urinary incontinence (SUI) is a public health issue attributed to weakened pelvic supporting tissues. Electrical stimulation (ES) is one of the first-line conservative treatments for SUI. However, the underlying mechanism of ES in the treatment of SUI is not clear. Here, we show that ES suppresses cell apoptosis and upregulates collagen expression by functioning as a cell growth inducer to activate the calpain 2/talin 1/integrin ß1/transforming growth factor (TGF)-ß1 axis. Specifically, ES promoted Ca2+ to flow into the cytoplasm through the calcium channel, Cav 3.2, thereby activating calpain 2. Then, the activated calpain 2 cleaved talin 1, which induced the activation of integrin ß1 and upregulated the TGF-ß1-mediated transcription of collagen I and III. Notably, blocking Cav 3.2 suppressed calcium influx and inhibited the activation of downstream proteins. Furthermore, the knockdown of calpain 2 resulted in the reduction of cleaved talin 1, and the shRNA-integrin ß1 treatment downregulated the level of activated integrin ß1 and the expression of TGF-ß1-induced collagen I and III. An association of the ES-modulated collagen I and III upregulation with the therapeutic effect of the ES-Ca2+/calpain 2/talin 1/integrin ß1/TGF-ß1 axis was demonstrated in mouse fibroblast and mouse SUI models established through vaginal distension (VD). This outcome provides insight into clinical diagnosis and treatment.

5.
Sci Rep ; 9(1): 4206, 2019 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-30862846

RESUMO

Electrical stimulation (ES) therapy has good effects in patients with nervous system injury-related diseases. ES promotes nerve cell regeneration and stimulates Schwann cells to express neurotrophic factors. The incidence of stress urinary incontinence (SUI) among elderly people is increasing. Some studies suggest that damage to the pudendal nerve is closely related to the pathogenesis of SUI. It has also been found that pelvic ES can reduce SUI symptoms in a rat model of SUI caused by pudendal nerve injury. Clinically, pelvic floor electrical stimulation is effective in patients with mild to moderate SUI. These studies indicate that ES may ameliorate damage to the pudendal nerve and thus achieve the goal of SUI treatment, although the mechanism of action of this treatment remains unclear. Therefore, the purpose of the present study was to clarify the relationships among ES, neural cells and Schwann cells at the cellular level. We applied ES to nerve cells at 100 mV/mm or 200 mV/mm for 0, 0.5, 1, or 2 h to investigate changes in nerve cell activity. We then co-cultured the nerve cells with Schwann cells to explore the influence of single-culture and co-culture conditions on the nerve cells. Compared to non-ES, ES of the nerve cells increased their activity. Compared to those in single culture, co-cultured nerve cells exhibited an additional increase in activity. We also found that Schwann cell derived exosomes could promote the activity of nerve cells, with glutamate and calcium ions playing a potential role in this process. These results suggest that the mutual regulation of neural cells and Schwann cells plays an important role in the process by which ES ameliorates neurological function, which may provide a basis for subsequent studies.

6.
Talanta ; 195: 306-312, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30625547

RESUMO

Construction of convenient and effective method for enantiomer identification is of vital significance for biochemistry and medical science. Herein, we design an effective sensor for chiral recognition of tryptophan (Trp) enantiomers, and self-assembly of Cu2+-modified ß-cyclodextrin on poly-L-arginine/multi-walled carbon nanotubes (Cu-ß-CD/PLA/MWCNTs) is studied. Meanwhile, Cu2+ acts as a cap to prevent the release of the high energy water and compel Trp enantiomer into the smaller opening of ß-cyclodextrin. Recognition of L-Trp is accomplished by the formation of hydrogen bonds between the amino of L-Trp and the high energy water confined in cavity of Cu-ß-CD. Compared with D-Trp, the sensor exhibits favorable chiral recognition toward L-Trp with a separation coefficient of 3.37. And the chiral sensor presents admirable enantiomers determination with excellent sensitivity, providing a good linear correlation in the range of 1 × 10-6 M~5.5 × 10-5 M, and the detection limit can reach 3.3 × 10-7 M (S/N = 3). Besides, the proposed sensor has been able to predict the percentage of D-Trp in the racemic mixture, suggesting its potential applications in the enantiomer recognition field.

7.
Mol Med Rep ; 17(2): 2705-2711, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29207080

RESUMO

The aim of the present study was to investigate the protective effect of puerarin on pelvic organ prolapse (POP) and the underlying mechanisms that regulate the metabolism of human parametrial ligament fibroblasts (HPLFs). HPLFs obtained from the pelvic tissue of patients with (n=10) or without (n=8) POP during hysterectomy were isolated by enzymatic digestion and subsequently identified by immunocytochemistry in a previous study of the authors. Following this, cultured HPLFs were treated with 0.01, 0.10 or 1.00 mmol/l puerarin, followed by detection of proliferation rate by Cell Counting kit­8 assay. Following incubation with puerarin for 48 h, mRNA and protein expression levels of tissue inhibitor of metalloproteinase­1 (TIMP­1), matrix metalloproteinase (MMP)­2 and ­9, and collagen (COL)I and III in HPLFs were quantified by reverse transcription­quantitative polymerase chain reaction, and western blot and gelatin zymography analyses, respectively. MMP­2 and ­9 expression levels were increased, whereas expression levels of TIMP­1, and COL I and III were decreased, in patients with POP compared with healthy controls. Following puerarin treatment, the expression levels of TIMP­1, and COL I and III were enhanced, whereas MMP­2 and ­9 were inhibited. In conclusion, the present study demonstrated evidence increased degradation of the extracellular matrix in pelvic tissues of patients with POP compared with controls, and the protective effect of puerarin against POP via its anti­degradation effect on collagen. These results provide evidence for puerarin as a novel approach for the treatment of POP.


Assuntos
Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Isoflavonas/farmacologia , Prolapso de Órgão Pélvico/metabolismo , Vasodilatadores/farmacologia , Biomarcadores , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Feminino , Expressão Gênica , Humanos , Metaloproteinases da Matriz/metabolismo , Diafragma da Pelve , Prolapso de Órgão Pélvico/tratamento farmacológico , Prolapso de Órgão Pélvico/etiologia , Pelve
8.
Oxid Med Cell Longev ; 2017: 8524353, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29109834

RESUMO

Stress urinary incontinence (SUI) is a common hygienic problem affecting the quality of women's life worldwide. In this research, we revealed the involvement and regulation of extracellular matrix (ECM) remodeling, oxidative damage, and TGF-ß1 signaling in the pathological mechanisms of mechanical trauma-induced SUI. We found that excessive mechanical strain significantly increased apoptosis rate, decreased cell viability and ECM production, and broke the balance of MMPs/TIMPs compared with the nonstrain control (NC) group. The expression levels of TGFß1, p-Smad3, Nrf2, GPx1, and CAT were downregulated, the production of ROS, 8-OHdG, 4-HNE, and MDA was increased, and the nuclear translocation of Smad2/3 was suppressed after 5333 µstrain's treatment. Both mTGF-ß1 pretreatment and Nrf2 overexpression could reverse mechanical injury-induced TGFß1/Smad3 signaling inhibition and ECM remodeling, whereas mTGF-ß1 had no effect on Nrf2 expression. Nrf2 overexpression significantly alleviated mechanical injury-induced ROS accumulation and oxidative damage; in contrast, Nrf2 silencing exhibited opposite effects. Besides, vaginal distention- (VD-) induced in vivo SUI model was used to confirm the in vitro results; Nrf2 knockout aggravates mechanical trauma-induced LPP reduction, ECM remodeling, oxidative damage, and TGF-ß1/Smad3 suppression in mice. Therefore, we deduce that mechanical injury-induced ECM remodeling might be associated with Nrf2/ARE signaling suppression mediating TGF-ß1/Smad3 signaling inhibition. This might reflect a new molecular target for SUI researches.


Assuntos
Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Incontinência Urinária por Estresse/fisiopatologia , Animais , Feminino , Humanos , Camundongos , Transdução de Sinais , Transfecção
9.
PLoS One ; 12(8): e0181896, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28783735

RESUMO

Gestation and delivery can increase intra-abdominal pressure, which are well-known risk factors for Pelvic Organ Prolapse (POP). But the pathogenesis mechanism of POP remains unclear. Our previous research showed that the expression of glutathione peroxidase type 1 (GPX1) decreased in pelvic floor ligaments from POP patients, implying that oxidative stress (OS) may be related to POP. The aim of this study was to figure out the role of GPx1 regulation in the pathogenesis of POP. Women (>45 years) who received hysterectomy surgery were enrolled in this research, identified by screening. We applied mechanical strain of 0µ, 5333 µ to GPX1-overexpressing human uterosacral ligament fibroblasts (hUSLFs) isolated from menopausal women without POP respectively for 4 hours, in order to investigate the changes of cell apoptosis, oxidative status and ECM metabolism when cytomechanics model loaded on GPX1-overexpressing hUSLFs. Comparing with the non-transfection and mock-vehicle groups, we found that GPX1 not only protects hUSLFs from cell apoptosis, oxidative damage, but also improves the remodeling of ECM induced by mechanical stimulation. These results suggested that mechanical strain caused abnormalities of ECM metabolism via OS pathway in hUSLFs, which was involved in the pathogenesis of POP, and that GPx1 played a significant role in regulating mechanical strain induced POP.


Assuntos
Glutationa Peroxidase/metabolismo , Prolapso de Órgão Pélvico/metabolismo , Prolapso de Órgão Pélvico/patologia , Apoptose/genética , Apoptose/fisiologia , Western Blotting , Células Cultivadas , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Citometria de Fluxo , Glutationa Peroxidase/genética , Humanos , Ligamentos/citologia , Ligamentos/metabolismo , Pessoa de Meia-Idade , Estresse Oxidativo/genética , Estresse Oxidativo/fisiologia , Prolapso de Órgão Pélvico/genética , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
10.
Mol Med Rep ; 16(2): 1439-1444, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28586031

RESUMO

Natural botanical drugs have attracted attention due to their cancer chemopreventive and chemotherapeutic properties in cancer. Punicalagin (PUN) is the major bioactive component of pomegranate peel, and has been shown to have antioxidant, anti-inflammatory, antiviral, antiproliferation and anticancer properties. PUN has been shown to induce apoptosis in several cancer cell lines. The aim of the present study was to investigate the effect of PUN on HeLa human cervical cancer cells in vitro. The viability of the HeLa cells was assessed following treatment with PUN (0, 12.5, 25, 50, 100 and 200 µM) for 24, 36 and 48 h using a Cell Counting Kit­8 assay. In addition, the cell cycle distribution, protein expression levels of B­cell lymphoma 2 (Bcl­2)­associated X protein (Bax), Bcl­2, tissue inhibitor of metalloproteinase (TIMP)-2, TIMP­3 and the ß­catenin pathway, and the activities of matrix metalloproteinase (MMP)­2 and MMP­9 were analyzed following treatment with PUN (0, 25, 50 and 100 µM) for 36 h using cell cycle analysis, western blot analysis and gelatin zymography, respectively. In addition, a wound­healing assay was used to detect cell migration. PUN led to a number of effects on the HeLa cells, including the inhibition of cell proliferation and cell migration, downregulation of MMP­2 and MMP­9, upregulation of TIMP­2 and TIMP­3, cell­cycle arrest in the G1 phase, induction of apoptosis via alterations of Bcl­2 and Bax, and downregulation of ß­catenin and its downstream proteins, cyclin D1 and c-myc. These results suggested that PUN may have chemopreventive and chemotherapeutic effects against cervical cancer in humans through inhibition of the ß-catenin signaling pathway.


Assuntos
Taninos Hidrolisáveis/farmacologia , Transdução de Sinais/efeitos dos fármacos , Neoplasias do Colo do Útero/patologia , beta Catenina/metabolismo , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Feminino , Fase G1/efeitos dos fármacos , Células HeLa , Humanos , Taninos Hidrolisáveis/química , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Fase S/efeitos dos fármacos , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Inibidor Tecidual de Metaloproteinase-3/metabolismo , Neoplasias do Colo do Útero/enzimologia , Proteína X Associada a bcl-2/metabolismo
11.
Int J Mol Med ; 40(2): 347-356, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28656271

RESUMO

The present study aimed to reveal the metabolic alterations of the extracellular matrix (ECM) in uterosacral ligament (USL) with pelvic organ prolapse (POP) and to explore the role of transforming growth factor­ß1 (TGF­ß1) in pathogenesis of POP. For this purpse, 60 participants who underwent hysterectomy for benign indications were enrolled, 30 of which had symptomatic POP (grade II, III or IV) and composed the POP group, and the other 30 had asymptomatic POP (grade I or less) and served as the controls. Collagen fibers, elastin,matrix metalloproteinase (MMP)­2/9, tissue inhibitor of matrix metalloproteinases (TIMP)­2 and TGF­ß1 were examined by Masson's trichrome staining, immunohistochemistry and RT-qPCR using USL biopsies. In vitro, human USL fibroblasts (hUSLFs) were primary cultured, pre-treated with recombinant TGF­ß1 (0, 5, or 10 ng/ml) and then subjected to cyclic mechanical stretching (CMS; 0 or 5,333 µÎµ strain). Changes in the expression levels of collagen type I/III, elastin, TIMP­2, MMP­2/9 and Smad were detected. Our results revealed that at the tissue level, the expression of collagen fibers, elastin, TIMP­2 and TGF­ß1 was significantly reduced in the POP group, while the activities of MMP­2/9 were significantly upregulated, compared with the control group. Statistical analysis indicated that the mRNA expression of TGF­ß1 inversely correlated with the severity of POP partially. Our in vitro experimental data demonstrated that a CMS of 5333 µÎµ strain promoted the degradation of ECM proteins, inhibited the synthesis of TIMP­2, and upregulated the proteolytic activities of MMP­2/9. Pre-treatment with TGF­ß1 attenuated the loss of ECM by stimulating the synthesis of TIMP­2 and inhibiting the activities of MMP­2/9 through the TGF­ß1/Smad3 signaling pathway. On the whole, our data indicate that the reduced anabolism and increased catabolism of ECM proteins in USL are the pathological characteristics of POP. TGF­ß1 not only has a specific value in predicting the severity of POP, but should also be considered as a novel therapeutic target for POP.


Assuntos
Matriz Extracelular/patologia , Prolapso de Órgão Pélvico/patologia , Transdução de Sinais , Fator de Crescimento Transformador beta1/metabolismo , Células Cultivadas , Colágeno/análise , Colágeno/metabolismo , Elastina/análise , Elastina/metabolismo , Matriz Extracelular/metabolismo , Feminino , Humanos , Metaloproteinase 2 da Matriz/análise , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/análise , Metaloproteinase 9 da Matriz/metabolismo , Pessoa de Meia-Idade , Prolapso de Órgão Pélvico/metabolismo , Prolapso de Órgão Pélvico/terapia , Proteólise , Proteínas Smad/análise , Proteínas Smad/metabolismo , Inibidor Tecidual de Metaloproteinase-2/análise , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Fator de Crescimento Transformador beta1/análise , Fator de Crescimento Transformador beta1/uso terapêutico
12.
Mol Med Rep ; 15(5): 3278-3284, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28339064

RESUMO

Pelvic organ prolapse (POP) is a global health problem that may seriously impact the quality of life of the sufferer. The present study aimed to investigate the potential mechanisms underlying alterations in extracellular matrix (ECM) metabolism in the pathogenesis of POP, by investigating the expression of ECM components in human parametrial ligament fibroblasts (hPLFs) subject to various mechanical strain loads. Fibroblasts derived from parametrial ligaments were cultured from patients with POP and without malignant tumors, who underwent vaginal hysterectomy surgery. Fibroblasts at generations 3­6 of exponential phase cells were selected, and a four­point bending device was used for 0, 1,333 or 5,333 µ mechanical loading of cells at 0.5 Hz for 4 h. mRNA and protein expression levels of collagen type I α 1 chain (COL1A1), collagen type III α 1 chain (COL3A1), elastin, matrix metalloproteinase (MMP) ­2 and ­9, and transforming growth factor (TGF)­ß1 were detected by reverse transcription­quantitative polymerase chain reaction and western blotting, respectively. Under increased mechanical strain (5,333 µ), mRNA and protein expression levels of COL1A1, COL3A1 elastin and TGF­ß1 decreased, particularly COL1A1; however, mRNA and protein expression levels of MMP­2 and ­9 were significantly increased, compared with the control group (0 µ strain). Following 1,333 µ mechanical strain, mRNA and protein expression levels of COL1A1, COL3A1 elastin and MMP­2 increased, and MMP­9 decreased, whereas no significant differences were observed in TGF­ß1 mRNA and protein expression levels. In conclusion, ECM alterations may be involved in pathogenesis of POP, with decreased synthesis and increased degradation of collagen and elastin. Furthermore, the TGF­ß1 signaling pathway may serve an important role in this process and thus may supply a new target and strategy for understanding the etiology and therapy of POP.


Assuntos
Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Prolapso de Órgão Pélvico/patologia , Estresse Mecânico , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Colágeno Tipo III/genética , Colágeno Tipo III/metabolismo , Elastina/genética , Elastina/metabolismo , Fibroblastos/citologia , Humanos , Ligamentos/citologia , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Neoplasias/complicações , Neoplasias/diagnóstico , Prolapso de Órgão Pélvico/complicações , Prolapso de Órgão Pélvico/metabolismo , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo
13.
Urology ; 104: 45-51, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28188756

RESUMO

OBJECTIVE: To investigate the therapeutic effect and underlying molecular mechanism of electrical stimulation (ES) in a murine stress urinary incontinence (SUI) model. MATERIALS AND METHODS: Sixty female C57BL/6 mice were divided into 4 groups: CON group, no intervention; VD group, vaginal distension (VD) with an 8-mm dilator for 1 hour; VD + ES 20 group, 20 Hz ES for 0.5 hour for 7 days after VD; and VD + ES 50 group, 50 Hz ES for 7 days after VD. For functional studies, assessments of urodynamics and sneezing test were performed; then, anterior vaginal wall specimens were collected. Pathological changes were validated by Masson's trichrome and Van Gieson staining, and the expressions of collagen, transforming growth factor (TGF)-ß1-Smad2/3 pathway components, and T-type calcium channels were detected by Western blotting and reverse transcription polymerase chain reaction. RESULTS: ES significantly increased maximum bladder capacity, leak point pressure, and sneezing positive rate in SUI mice. The staining results showed that collagen was disorganized in the VD group but became organized after ES, especially at 50 Hz. The same results were found for collagens I and III. The expression of TGF-ß1, p-Smad2 and p-Smad3 significantly decreased in the VD group and significantly increased in the VD + ES groups, especially in the VD + ES 50 group. The expression of 2 T-type calcium channel subtypes (Cav 3.1 and Cav 3.2) decreased in the VD group compared with the CON group, but increased in the VD + ES group compared with the VD group. CONCLUSION: Dysregulation of collagen metabolism is involved in the pathogenesis of SUI. ES can ameliorate the symptoms of SUI by activating collagen regeneration through the TGFß1-Smad2/3 pathway. T-type calcium channels might be involved in these processes.


Assuntos
Terapia por Estimulação Elétrica , Incontinência Urinária por Estresse/terapia , Animais , Canais de Cálcio Tipo T/metabolismo , Colágeno/metabolismo , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Incontinência Urinária por Estresse/metabolismo , Urodinâmica , Vagina/patologia
14.
Mol Med Rep ; 15(1): 423-430, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27922684

RESUMO

The regulation of the extracellular matrix (ECM) by mechanical stress is of interest as the ECM is essential in the development of pelvic organ prolapse. In the present study, the effect of overexposure to mechanical stress on the ECM, and the probable underlying mechanisms in cultured human uterosacral ligament fibroblasts (hUSLFs), was explored. Mechanical stress has an effect on oxidation­antioxidation products in parametrial ligament fibroblasts. Thus, hUSLFs were incubated with different concentrations of hydrogen peroxide to elucidate any potential interactions. Excess mechanical stress and H2O2 inhibited cell proliferation, and decreased mRNA and protein expression levels of ECM components, collagen 1, collagen 3 and elastin. Further analysis revealed that the mRNA expression level of matrix metalloproteinase­2 (MMP­2) was increased and TIMP metallopeptidase inhibitor 2 (TIMP­2) decreased, and in addition the MMP2/TIMP2 mRNA ratio was increased, which may facilitate the degradation of the ECM. Due to the key role of the transforming growth factor ß1 (TGF­ß1)/mothers against decapentaplegic homolog 2 (Smad2) signaling pathway in fibrosis, the present study investigated the effect of excess mechanical stress and H2O2 on TGF­ß1/Smad2 signaling. The results indicated that excess mechanical stress and H2O2 treatment suppressed phosphorylated Smad2 expression and decreased the levels of TGF­ß1. Activation of the TGF­ß1 signaling pathway by either mechanical stress or H2O2 was demonstrated to attenuate cell proliferation and ECM components, and also increased the MMP2/TIMP2 mRNA ratio. These findings suggested that mechanical stress and H2O2 overexposure inhibit cell proliferation and remodel the ECM network via regulation of the TGF­ß1 signaling pathway.


Assuntos
Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Peróxido de Hidrogênio/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Transdução de Sinais , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Proliferação de Células , Células Cultivadas , Matriz Extracelular/genética , Feminino , Fibroblastos/citologia , Regulação da Expressão Gênica , Humanos , Ligamentos/citologia , Ligamentos/metabolismo , Metaloproteinase 2 da Matriz/genética , Estresse Oxidativo , RNA Mensageiro/genética , Proteína Smad2/metabolismo , Estresse Mecânico , Inibidor Tecidual de Metaloproteinase-2/genética
15.
Mol Med Rep ; 15(2): 853-858, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28000871

RESUMO

The present study aimed to investigate damage to human parametrial ligament fibroblasts by detecting cell proliferation, cytoskeletal structure, cellular alterations and senescence. Uterosacral and cardinal ligaments were obtained from 10 patients with cervical intraepithelial neoplasia grade II­III, who had received total vaginal hysterectomies, and fibroblasts were derived from this tissue. Fibroblasts were stretched using a four­point bending system with a force of 0 (control), 1,333 µ strain (1 mm) or 5,333 µ strain (4 mm) for 4 h. The present study revealed that mechanical force significantly reduced cell proliferation and increased cell senescence. As mechanical force increased, the mitochondria of fibroblasts began to exhibit vacuolization, and the cell cytoskeleton began to depolymerize and rearrange. In conclusion, the present study demonstrated that mechanical forces within a certain range may induce cell damage via mitochondrial injury, cytoskeletal alterations and increased cell senescence, resulting in decreased cell viability of pelvic fibroblasts.


Assuntos
Sobrevivência Celular , Fibroblastos/citologia , Peritônio/citologia , Estresse Mecânico , Proliferação de Células , Células Cultivadas , Senescência Celular , Citoesqueleto/patologia , Citoesqueleto/ultraestrutura , Feminino , Fibroblastos/patologia , Fibroblastos/ultraestrutura , Humanos , Ligamentos/citologia , Mitocôndrias/patologia , Mitocôndrias/ultraestrutura , Peritônio/patologia
16.
Int J Gynecol Cancer ; 26(9): 1557-1563, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27540692

RESUMO

AIM: The aim of this study was to investigate the effects of punicalagin, a polyphenol isolated from Punica granatum, on human A2780 ovarian cancer cells in vitro. METHODS: The viability of human A2780 ovarian cells was evaluated using Cell Counting Kit-8 assay. Cell cycle was detected with flow cytometry analysis. The protein expression levels of Bcl-2, Bax, ß-catenin, cyclin D1, survivin, tissue inhibitor of metalloproteinase (TIMP)-2, and TIMP-3 were measured using Western blot analysis. Matrix metalloproteinase (MMP)-2 and MMP-9 activity was determined with gelatin zymography. Wound healing assay was used to determine cell migration. RESULTS: Punicalagin inhibited the cell viability of A2780 cells in a dose- and time-dependent manner, and the cell cycle of A2780 cells was arrested in G1/S phase transition. The treatment also induced apoptosis as shown by the up-regulation of Bax and down-regulation of Bcl-2. On the other hand, punicalagin treatment increased the expressions of TIMP-2 and TIMP-3, decreased the activities of MMP-2 and MMP-9, and inhibited cell migration. In addition, the ß-catenin pathway was suppressed as shown by the down-regulations of ß-catenin and its downstream factors including cyclin D1 and survivin. CONCLUSIONS: Punicalagin may have cancer-chemopreventive as well as cancer-chemotherapeutic effects against human ovarian cancer in humans through the inhibition of ß-catenin signaling pathway.


Assuntos
Carcinoma/tratamento farmacológico , Taninos Hidrolisáveis/uso terapêutico , Neoplasias Ovarianas/tratamento farmacológico , Carcinoma/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Taninos Hidrolisáveis/farmacologia , Metaloproteinases da Matriz/metabolismo , Neoplasias Ovarianas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Inibidores Teciduais de Metaloproteinases/metabolismo , Proteína X Associada a bcl-2/metabolismo , beta Catenina/metabolismo
17.
Mol Med Rep ; 14(1): 243-53, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27176043

RESUMO

Mechanical loading on pelvic supports contributes to pelvic organ prolapse (POP). However, the underlying mechanisms remain to be elucidated. Our previous study identified that mechanical strain induced oxidative stress (OS) and promoted apoptosis and senescence in pelvic support fibroblasts. The aim of the present study is to investigate the molecular signaling pathway linking mechanical force with POP. Using a four­point bending device, human uterosacral ligament fibroblasts (hUSLF) were exposed to mechanical tensile strain at a frequency of 0.3 Hz and intensity of 5333 µÎµ, in the presence or absence of LY294002. The applied mechanical strain on hUSLF resulted in apoptosis and senescence, and decreased expression of procollagen type I α1. Mechanical strain activated phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt signaling and resulted in downregulated expression of glutathione peroxidase 1 and Mn­superoxide dismutase, and accumulation of intracellular reactive oxygen species. These effects were blocked by administration of LY294002. Furthermore, it was demonstrated that PI3K/Akt was activated in the uterosacral ligaments of POP patients, and that OS was increased and collagen type I production reduced. The results from the present study suggest that mechanical strain promotes apoptosis and senescence, and reduces collagen type I production via activation of PI3K/Akt-mediated OS signaling pathway in hUSLF. This process may be involved in the pathogenesis of POP as it results in relaxation and dysfunction of pelvic supports.


Assuntos
Fenômenos Mecânicos , Prolapso de Órgão Pélvico/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Idoso , Apoptose , Senescência Celular , Colágeno/biossíntese , Feminino , Fibroblastos/metabolismo , Humanos , Ligamentos/citologia , Pessoa de Meia-Idade , Estresse Oxidativo , Prolapso de Órgão Pélvico/diagnóstico , Prolapso de Órgão Pélvico/etiologia , Espécies Reativas de Oxigênio/metabolismo
18.
Mol Med Rep ; 13(4): 2999-3008, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26936098

RESUMO

Pelvic organ prolapse (POP) is a global health problem, for which the pathophysiological mechanism remains to be fully elucidated. The loss of extracellular matrix protein has been considered to be the most important molecular basis facilitating the development of POP. Oxidative stress (OS) is a well­recognized mechanism involved in fiber metabolic disorders. The present study aimed to clarify whether OS exists in the uterosacral ligament (USL) with POP, and to investigate the precise role of OS in collagen metabolism in human USL fibroblasts (hUSLFs). In the present study, 8­hydroxyguanosine (8­OHdG) and 4 hydroxynonenal (4­HNE), as oxidative biomarkers, were examined by immunohistochemistry to evaluate oxidative injury in USL sections in POP (n=20) and non­POP (n=20) groups. The primary cultured hUSLFs were treated with exogenous H2O2 to establish an original OS cell model, in which the expression levels of collagen, type 1, α1 (COL1A1), matrix metalloproteinase (MMP)­2, tissue inhibitor of metalloproteinase (TIMP)­2 and transforming growth factor (TGF)­ß1 were evaluated by western blot and reverse transcription­quantitative polymerase chain reaction analyses. The results showed that the expression levels of 8­OHdG and 4­HNE in the POP group were significantly higher, compared with those in the control group. Collagen metabolism was regulated by H2O2 exposure in a concentration­dependent manner, in which lower concentrations of H2O2 (0.1­0.2 mM) stimulated the anabolism of COL1A1, whereas a higher concentration (0.4 mM) promoted catabolism. The expression levels of MMP­2, TIMP­2 and TGF­ß1 exhibited corresponding changes with the OS levels. These results suggested that OS may be involved in the pathophysiology of POP by contributing to collagen metabolic disorder in a severity­dependent manner in hUSLFs, possibly through the regulation of MMPs, TIMPs and TGF­ß1 indirectly.


Assuntos
Colágeno/metabolismo , Fibroblastos/metabolismo , Ligamentos/citologia , Estresse Oxidativo , Prolapso de Órgão Pélvico/metabolismo , Apoptose/efeitos dos fármacos , Biomarcadores , Estudos de Casos e Controles , Sobrevivência Celular/efeitos dos fármacos , Feminino , Fibroblastos/efeitos dos fármacos , Guanosina/análogos & derivados , Guanosina/biossíntese , Humanos , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Imuno-Histoquímica , Pessoa de Meia-Idade , Prolapso de Órgão Pélvico/fisiopatologia , Espécies Reativas de Oxigênio/metabolismo
19.
Mol Med Rep ; 12(4): 5342-8, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26238938

RESUMO

The aim of the present study was to explore the underlying mechanisms of the roles of mechanical factors in the pathogenesis of pelvic organ prolapse (POP). The experiments were performed on fibroblasts derived from uterosacral ligaments and cardinal ligaments of patients who received total hysterectomy due to benign disease excluding POP. Fibroblasts were cultured after collagenase digestion and identified by morphological observation and immunocytochemical methods. A four­point bending device was used to subject fibroblasts at passage 4­6 to strains of 0, 1,333 µ (1 mm), 2,666 µ (2 mm) or 5,333 µ (4 mm) at a frequency of 0.1 Hz for 4 h. Intracellular reactive oxygen species (ROS) were quantified using the fluorescent probe 2',7'­dichlorodihydrofluorescein diacetate. Changes in the mitochondrial membrane potential were verified using the fluorescent dye JC­1, and apoptosis was detected using Annexin V/propidium iodide staining and flow cytometric analysis. Mechanical strain changed the morphology and adherence ability of parametrial ligament fibroblasts. Furthermore, the production of ROS was significantly increased and the mitochondrial membrane potential obviously declined with the enhancement of mechanical stress loading. In addition, the apoptotic rate of fibroblasts subjected to high mechanical strain was significantly increased compared with that in fibroblast under low­intensity strain. In conclusion, the present study showed that mechanical strain enhanced intracellular ROS levels, decreased the mitochondrial membrane potential and increased the apoptotic rate in human parametrial ligament fibroblasts, which may contribute to POP.


Assuntos
Fibroblastos/metabolismo , Fibroblastos/patologia , Ligamentos/citologia , Estresse Oxidativo , Estresse Mecânico , Apoptose , Células Cultivadas , Feminino , Humanos , Espaço Intracelular/metabolismo , Potencial da Membrana Mitocondrial , Prolapso de Órgão Pélvico , Cultura Primária de Células
20.
J Obstet Gynaecol Res ; 41(7): 1049-55, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25773925

RESUMO

AIM: Genital fistula is one of the most devastating injuries in women. Despite advances in medical care, it continues to be a distressing problem, and the success rate of repair surgery is still limited. We herein describe our experience with the surgical approach using Foley catheter to repair genital fistula after gynecological surgery. METHODS: We retrospectively reviewed 29 patients who had received genital fistula repair surgery with Foley catheter between October 2011 and December 2013. Based on traditional transvaginal genital fistula repair surgery, we inserted a Foley catheter into the bladder or intestine through the fistula opening. As a result, the fistula opening could be tracked, which allows for a clear view to improve fistula repair. All 29 patients were followed up at 1, 4, and 12 weeks postoperatively. RESULTS: Of the 29 patients, 28 had successful surgical outcome (96.55% success rate). The mean operative time was 85 ± 8.1 min. The mean blood loss was 109 ± 23.4 mL. No intraoperative complications were observed. The mean postoperative hospitalization time was 10 ± 2.8 days. The follow-up rate was 100%. CONCLUSIONS: Repair of transvaginal genital fistula using Foley catheter had a high success rate, short operative time, minimal blood loss, low morbidity and short hospital stay. Therefore, this approach is minimally invasive and effective.


Assuntos
Genitália Feminina/lesões , Complicações Pós-Operatórias/prevenção & controle , Cateterismo Urinário/métodos , Fístula Vaginal/cirurgia , Adulto , Idoso , China/epidemiologia , Feminino , Seguimentos , Genitália Feminina/cirurgia , Hospitais Universitários , Humanos , Tempo de Internação , Pessoa de Meia-Idade , Duração da Cirurgia , Estudos Retrospectivos
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