The bifunctional role of hydrazine as a potent nucleophile and antioxidant was investigated for the rapid aminolysis of RAFT polymers within minutes in air with effective suppression of the formation of disulfides. Using both dithioesters and trithiocarbonates as model compounds, we showed that hydrazine exhibited a significantly improved aminolysis rate when compared with a commonly used primary alkyl amine. On the basis of the exellent results with CTAs, we further studied the aminolysis of RAFT polymers prepared with either dithioesters or trithiocarbonates. In accord with the aminolysis results on CTAs, hydrazine aminolyzed RAFT polymers in an impressively short time and, more importantly, it significantly suppressed the formation of disulfides as comfirmed with GPC.
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 four‑point bending device was used to subject fibroblasts at passage 4‑6 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 JC‑1, 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 low‑intensity 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.
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 well-recognized 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, 8-hydroxyguanosine (8-OHdG) and 4 hydroxynonenal (4-HNE), as oxidative biomarkers, were examined by immunohistochemistry to evaluate oxidative injury in USL sections in POP (n=20) and non-POP (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)-β1 were evaluated by western blot and reverse transcription-quantitative polymerase chain reaction analyses. The results showed that the expression levels of 8-OHdG and 4-HNE in the POP group were significantly higher, compared with those in the control group. Collagen metabolism was regulated by H2O2 exposure in a concentration-dependent manner, in which lower concentrations of H2O2 (0.1–0.2 mM) stimulated the anabolism of COL1A1, whereas a higher concentration (0.4 mM) promoted catabolism. The expression levels of MMP-2, TIMP-2 and TGF-β1 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 severity-dependent manner in hUSLFs, possibly through the regulation of MMPs, TIMPs and TGF-β1 indirectly.
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-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 μstrain's 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.
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 Masson's 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.
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 Kit‑8 assay. In addition, the cell cycle distribution, protein expression levels of B‑cell lymphoma 2 (Bcl‑2)‑associated X protein (Bax), Bcl‑2, tissue inhibitor of metalloproteinase (TIMP)-2, TIMP‑3 and the β‑catenin pathway, and the activities of matrix metalloproteinase (MMP)‑2 and MMP‑9 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 wound‑healing 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 MMP‑2 and MMP‑9, upregulation of TIMP‑2 and TIMP‑3, cell‑cycle arrest in the G1 phase, induction of apoptosis via alterations of Bcl‑2 and Bax, and downregulation of β‑catenin 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 β-catenin signaling pathway.
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.
Jagged1, the essential ligand of the Notch signalling pathway, is highly expressed in metastatic prostate cancer, and its high expression in breast cancer is linked to poor survival rates. However, the mechanism of Jagged1′s involvement in platinum‐resistant ovarian cancer has not been thoroughly elucidated to date. The purpose of the present study was to investigate the roles of Jagged1 in the platinum resistance of ovarian cancer and its possible mechanisms. Compared with a platinum responsive group of ovarian epithelial cell carcinomas, we found the positive staining intensity of Notch1, Notch2, Jagged1, STAT 3 and Epithelial‐mesenchymal transition ( EMT ) proteins were lower in a platinum‐resistant group. The DDP ‐resistant ovarian cancer cell line (C13K) had a higher IC 50 of DDP than its parental cell line ( OV 2008) ( P < 0.05) and acquired an EMT phenotype and invasive characteristics. Inhibiting or knockdown of Jagged1 expression could not only reduce its capacity of migration and invasion but also reverse EMT and down‐regulate the expression of serine 727‐phosphorylated STAT 3 ( pS 727) at the protein level but not total STAT 3 or tyrosine 705‐phosphorylated STAT 3 ( pY 705) in C13K cells. Furthermore, it was found that crosstalk between the Jagged1/Notch and JAK / STAT 3 signalling pathways were involved in Jagged1‐promoting EMT in C13K cells. Experiments in vivo showed a reduced micrometastatic tumour burden in the lung, liver and spleen of mice implanted with C13K cells with knocked‐down Jagged1 compared with mice implanted with control cells. All of these results demonstrate that Jagged1 can crosstalk with the JAK / STAT 3 pathway, and they all cooperate to promote the aberrant occurrence of EMT , further reinforcing the abilities of invasion and migration of platinum‐resistant ovarian cancer in vivo and in vitro.
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