Jie Min

Therapeutic Effects of Punicalagin Against Ovarian Carcinoma Cells in Association With β-Catenin Signaling Inhibition.

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, β-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 β-catenin pathway was suppressed as shown by the down-regulations of β-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 β-catenin signaling pathway.

Therapeutic Effect and Mechanism of Electrical Stimulation in Female Stress Urinary Incontinence.

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 Massons trichrome and Van Gieson staining, and the expressions of collagen, transforming growth factor (TGF)-β1-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-β1, 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 TGFβ1-Smad2/3 pathway. T-type calcium channels might be involved in these processes.

Role of mechanical strain-activated PI3K/Akt signaling pathway in pelvic organ prolapse

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 four-point bending device, human uterosacral ligament fibroblasts (hUSLF) were exposed to mechanical tensile strain at a frequency of 0.3 Hz and intensity of 5333 µε, 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 Mn-superoxide 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.

The Anti-HSV-2 Effect of Alumen: In Vitro and In Vivo Experimental Studies

This study investigated the anti-HSV-2 effect of alumen through in vitro and in vivo experiments. Viable cell counting was employed to assess the toxicity of alumen on Vero cells. The inhibition rate of HSV-2 was defined as the cytopathic effect (CPE) of the cells infected with the virus. Alumen suppositories of different concentrations were vaginally applied to the guinea pigs which were then infected with HSV-2 via a vaginal route. The clinical symptoms were observed and the local virus titer calculated. The results showed that alumen had an in vitro anti-HSV-2 effect by means of antiviral duplication, direct killing of the virus, and antiviral adsorption. Alumen suppositories of different concentrations could reduce or completely inhibit HSV-2 infection in guinea pigs. It was concluded that alumen had an in vitro anti-HSV-2 effect through multiple approaches and it could suppress in vivo vaginal HSV-2 infection of guinea pig to some extent.SummaryThis study investigated the anti-HSV-2 effect of alumen through in vitro and in vivo experiments. Viable cell counting was employed to assess the toxicity of alumen on Vero cells. The inhibition rate of HSV-2 was defined as the cytopathic effect (CPE) of the cells infected with the virus. Alumen suppositories of different concentrations were vaginally applied to the guinea pigs which were then infected with HSV-2 via a vaginal route. The clinical symptoms were observed and the local virus titer calculated. The results showed that alumen had an in vitro anti-HSV-2 effect by means of antiviral duplication, direct killing of the virus, and antiviral adsorption. Alumen suppositories of different concentrations could reduce or completely inhibit HSV-2 infection in guinea pigs. It was concluded that alumen had an in vitro anti-HSV-2 effect through multiple approaches and it could suppress in vivo vaginal HSV-2 infection of guinea pig to some extent.

Astragalus polysaccharides increase the sensitivity of SKOV3 cells to cisplatin

BackgroundMultidrug resistance in malignant tumours hinders the treatment of tumours. Studies showed that astragalus polysaccharides (APS), one major active ingredient of astragalus, enhanced the sensitivity of non-small cell lung cancer and liver cancer cells to chemotherapeutic drug. However, the effect of APS on ovarian cancer is still unclear. In this study, we will examine the sensitizing effect of APS on SKOV3 cells to cisplatin and explore the possible mechanism.MethodsMTT assay was employed to examine the viability of SKOV3 after treatment with APS and cisplatin. The cell apoptosis rate was determined by flow cytometry. The expression of Bax, Bcl-2, Caspase-3, and c-Jun N-terminal kinases 1/2 (JNK1/2) was measured using Western blotting and RT-PCR.ResultsAPS synergistically promoted the inhibitory effect of cisplatin on SKOV3 cell viability. Flow cytometry showed that APS promoted cisplatin-induced apoptosis of SKOV3 cell lines. Further studies showed that APS down-regulated the expression of Bcl2, increased the expression of Bax and caspase 3 and activated JNK1/2 signalling pathway. The JNK inhibitors significantly rescued the proliferation inhibition induced by the drugs.ConclusionsAstragalus polysaccharides increased the sensitivity of SKOV3 cells to cisplatin potentially by activating the JNK pathway. The apoptosis-related genes may contribute to the process. Thus, APS may be useful for the treatment of ovarian cancer as an enhancer of chemosensitivity.

The role of GPX1 in the pathogenesis of female pelvic organ prolapse

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.

Mechanism of Mechanical Trauma-Induced Extracellular Matrix Remodeling of Fibroblasts in Association with Nrf2/ARE Signaling Suppression Mediating TGF-β1/Smad3 Signaling Inhibition

Stress urinary incontinence (SUI) is a common hygienic problem affecting the quality of womens life worldwide. In this research, we revealed the involvement and regulation of extracellular matrix (ECM) remodeling, oxidative damage, and TGF-β1 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 TGFβ1, 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 μstrains treatment. Both mTGF-β1 pretreatment and Nrf2 overexpression could reverse mechanical injury-induced TGFβ1/Smad3 signaling inhibition and ECM remodeling, whereas mTGF-β1 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-β1/Smad3 suppression in mice. Therefore, we deduce that mechanical injury-induced ECM remodeling might be associated with Nrf2/ARE signaling suppression mediating TGF-β1/Smad3 signaling inhibition. This might reflect a new molecular target for SUI researches.

Role of transforming growth factor β‑1 in the pathogenesis of pelvic organ prolapse: A potential therapeutic target

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 factor-β1 (TGF-β1) 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 TGF-β1 were examined by Massons trichrome staining, immunohistochemistry and RT-qPCR using USL biopsies. In vitro, human USL fibroblasts (hUSLFs) were primary cultured, pre-treated with recombinant TGF-β1 (0, 5, or 10 ng/ml) and then subjected to cyclic mechanical stretching (CMS; 0 or 5,333 με strain). Changes in the expression levels of collagen type I/III, elastin, TIMP-2, MMP-2/9 and Smad were detected. Our results revealed that at the tissue level, the expression of collagen fibers, elastin, TIMP-2 and TGF-β1 was significantly reduced in the POP group, while the activities of MMP-2/9 were significantly upregulated, compared with the control group. Statistical analysis indicated that the mRNA expression of TGF-β1 inversely correlated with the severity of POP partially. Our in vitro experimental data demonstrated that a CMS of 5333 με strain promoted the degradation of ECM proteins, inhibited the synthesis of TIMP-2, and upregulated the proteolytic activities of MMP-2/9. Pre-treatment with TGF-β1 attenuated the loss of ECM by stimulating the synthesis of TIMP-2 and inhibiting the activities of MMP-2/9 through the TGF-β1/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. TGF-β1 not only has a specific value in predicting the severity of POP, but should also be considered as a novel therapeutic target for POP.