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Background: It has been proved that PDGF-BB could protect the chondrocytes via multiple mechanisms. The present study aims to further investigating the anti-inflammatory and chondroprotective effects of platelet-derived growth factor (PDGF)-BB on osteoarthritis (OA).Methods: We established an osteoarthritis model in Sprague-Dawley rats through intraarticular injection of 3 mg/50 µL monosodium iodoacetate (MIA). The effects of PDGF-BB on MIA-induced OA were assessed by histopathological staining, real-time PCR, and western blotting. Chondrocytes were transfected with or without SOX-9 siRNA, induced by MIA and treated with PDGF-BB, and cell viability, proinflammatory cytokines, and specific markers were analyzed. For mechanistic analysis, the expression of p-SOX-9/SOX-9, p-RunX-2/RunX-2, and p-PKA/PKA and activation of the JAK2/STAT3, PI3K/AKT, and p38 pathways were analyzed.Results: PDGF-BB significantly inhibited the degeneration of cartilage in MIA-induced OA with reductions in proinflammatory cytokines (IL-1, IL-6, collagen Ⅹ, MMPs and ADATMS) and increases in collagen II, aggrecan, and integrin α5β1, and these effects could be reversed by a PDGFR-β antagonist. Consistently, the in vitro results showed similar effects. PDGF-BB treatment suppressed the activation of the JAK2/STAT3, PI3K/AKT, and p38 signaling pathways after MIA induction in both cartilage and chondrocytes. In addition, activation of PKA by PDGF-BB enhanced SOX-9 phosphorylation, which increased anabolic activity, and attenuated RunX-2 phosphorylation, which decreased catabolic activity. The PKA inhibitor and SOX-9 siRNA suppressed PDGF-BB-mediated chondroprotection.Conclusions: PDGF-BB could attenuate OA development by regulating the JAK2/STAT3 PI3K/AKT and p38 signaling pathways by inhibiting inflammation and enhancing cell proliferation via PKA-mediated regulation of SOX-9/RunX-2.

The increase of oxidative stress is one of the important characteristics of mammalian luteal regression. Previous investigations have revealed the essential role of reactive oxygen species (ROS) in luteal cell death during luteolysis, while it is unknown how ROS is regulated in this process. Considering the decrease of blood flow and increase of PGF2α during luteolysis, we hypothesized that the HIF-1α pathway may be involved in the regulation of ROS in the luteal cell of the late corpus luteum (CL). Here, by using a pseudopregnant rat model, we showed that the level of both HIF-1α and its downstream BNIP3 was increased during luteal regression. Consistently, we observed the increase of autophagy level during luteolysis, which is regulated in a Beclin1-independent manner. Comparing with early (Day 7 of pseudopregnancy) and middle CL (Day 14), the level of ROS was significantly increased in late CL, indicating the contribution of oxidative stress in luteolysis. Inhibition of HIF-1α by echinomycin (Ech), a potent HIF-1α inhibitor, ameliorated the upregulation of BNIP3 and NIX, as well as the induction of autophagy and the accumulation of ROS in luteal cells on Day 21 of pseudopregnancy. Morphologically, Ech treatment delayed the atrophy of the luteal structure at the late-luteal stage. An in vitro study indicated that inhibition of HIF-1α can also attenuate PGF2α-induced ROS and luteal cell apoptosis. Furthermore, the decrease of cell apoptosis can also be observed by ROS inhibition under PGF2α treatment. Taken together, our results indicated that HIF-1α signaling is involved in the regression of CL by modulating ROS production via orchestrating autophagy. Inhibition of HIF-1α could obviously hamper the apoptosis of luteal cells and the process of luteal regression.

Osteoarthritis (OA) is a common joint disease that mainly affects the diarthrodial joints. Treatments for OA include non‐pharmacological interventions, topical and oral therapies, intra‐articular therapies and joint surgery. However, all the treatments mentioned above mainly aim to control the symptoms instead of improving or reversing the joint condition. In this research, we observed the effect of recombinant platelet‐derived growth factor (PDGF)‐BB on OA in a monosodium iodoacetate (MIA)–induced rat model and revealed the possible mechanisms. In vitro, the level of inflammation in the chondrocytes was gradually alleviated, and the apoptosis rate was gradually decreased by PDGF‐BB at increasing concentrations. The levels of p‐p38, Bax and caspase‐3 decreased, and the level of p‐Erk increased with increasing PDGF‐BB concentration. In vivo, PDGF‐BB could significantly reverse chondrocyte and matrix loss. Furthermore, high concentrations of PDGF‐BB could alleviate cartilage hyperplasia to remodel the tissue. The level of collagen II was up‐regulated, and the levels of collagen X and apoptosis were down‐regulated by increasing concentrations of PDGF‐BB. In conclusion, recombinant PDGF‐BB alleviated OA by down‐regulating caspase‐3‐dependent apoptosis. The effects of PDGF‐BB on OA mainly include inhibiting chondrocyte loss, reducing cartilage hyperplasia and osteophyte formation, and regulating collagen anabolism.

The mechanical properties of cells could serve as an indicator for disease progression and early cancer diagnosis. This study utilized atomic force microscopy (AFM) to measure the viscoelastic properties of ovarian cancer cells and then examined the association with the invasion of ovarian cancer at the level of living single cells. Elasticity and viscosity of the ovarian cancer cells OVCAR-3 and HO-8910 are significantly lower than those of the human ovarian surface epithelial cell (HOSEpiC) control. Further examination found a dramatic increase of migration/invasion and an obvious decease of microfilament density in OVCAR-3 and HO-8910 cells. Also, there was a significant relationship between viscoelastic and biological properties among these cells. In addition, the elasticity was significantly increased in OVCAR-3 and HO-8910 cells after the treatment with the anticancer compound echinomycin (Ech), while no obvious change was found in HOSEpiC cells after Ech treatment. Interestingly, Ech seemed to have no effect on the viscosity of the cells. Ech significantly inhibited the migration/invasion and significantly increased the microfilament density in OVCAR-3 and HO-8910 cells, which was significantly related with the elasticity of the cells. An increase of elasticity and a decrease of invasion were found in OVCAR-3 and HO-8910 cells after Ech treatment. Together, this study clearly demonstrated the association of viscoelastic properties with the invasion of ovarian cancer cells and shed a light on the biomechanical changes for early diagnosis of tumor transformation and progression at single-cell level.

The development of tumor is closely related to extracellular matrix, which changes the biomechanical behavior of cells.Research have prepared polyacrylamide hydrogel substrates of differing stiffness according to the hardness values of breast tissue under normal and tumor physiological conditions. Then AFM was used to measure the mechanical properties of breast cells with different degrees of malignancy grown on different stiffness substrates. To explore the reasons for the changes in the young’s modulus of three breast cells, the distribution of cellular actin filaments were observed with a confocal microscope. These results showed that when the substrate hardened, the viscoelasticity of benign breast cells increased significantly, and the other two cancer cells also changed to some extent. We also found that the harder the substrate, the more conducive to the spreading behavior of cells, and the weaker response of malignant cells to substrates.

Objectives: To evaluate the effect of in-hospital pulmonary rehabilitation (PR) on short-term pulmonary functional recovery in patients with COVID-19.Methods: Patients with COVID-19 (n = 123) were divided into two groups (PR group or Control group) according to recipient of pulmonary rehabilitation. Six-min walk distance (6MW), heart rate (HR), forced vital capacity (FVC), forced expiratory volume in 1 s (FEV1), diffusing capacity of the lung for carbon monoxide (DLCO), and CT scanning were measured at the time of discharge, 1, 4, 12, and 24 weeks.Results: At week one, both PR group and Control group showed no significant changes in pulmonary function. At 4 and 12 weeks, 6MW, HR, FVC, FEV1, and DLCO improved significantly in both groups. However, the improvement in the PR group was greater than the Control group. Pulmonary function in the PR group returned to normal at 4 weeks [FVC (% predicted, PR vs. Control): 86.27 ± 9.14 vs. 78.87 ± 7.55; FEV1 (% predicted, PR vs. Control) 88.76 ± 6.22 vs. 78.96 ± 6.91; DLCO (% predicted, PR vs. Control): 87.27 ± 6.20 vs. 77.78 ± 5.85] compared to 12 weeks in the control group [FVC (% predicted, PR vs. Control): 90.61 ± 6.05 vs. 89.96 ± 4.05; FEV1 (% predicted, PR vs. Control) 94.06 ± 0.43 vs. 93.85 ± 5.61; DLCO (% predicted, PR vs. Control): 91.99 ± 8.73 vs. 88.57 ± 5.37]. Residual lesions on CT disappeared at week 4 in 49 patients in PR group and in 28 patients in control group (p = 0.0004).Conclusion: Pulmonary rehabilitation could accelerate the recovery of pulmonary function in patients with COVID-19.

Background We have previously shown that high salt stimulates the expression of miR-429 in the renal medulla, which induces mRNA decay of HIF prolyl-hydroxylase 2 (PHD2), an enzyme to promote the degradation of hypoxia-inducible factor (HIF)-1α, and increases the HIF-1α-mediated activation of antihypertensive genes in the renal medulla, consequently promoting extra sodium excretion. Our preliminary results showed that high salt-induced increase of miR-429 was not observed in Dahl S rats. This present study determined whether correction of this impairment in miR-429 would reduce PHD2 levels, increase antihypertensive gene expression in the renal medulla and attenuate salt-sensitive hypertension in Dahl S rats. Methods Lentiviruses encoding rat miR-429 were transfected into the renal medulla in uninephrectomized Dahl S rats. Sodium excretion and blood pressure were then measured. Results Transduction of lentiviruses expressing miR-429 into the renal medulla increased miR-429 levels, decreased PHD2 levels, and upregulated HIF-1α target gene NOS-2, which restored the adaptive mechanism to increase the antihypertensive gene after high-salt intake in Dahl S rats. Functionally, overexpression of miR-429 transgene in the renal medulla significantly improved pressure natriuretic response, enhanced urinary sodium excretion, and reduced sodium retention upon extra sodium loading, and consequently, attenuated the salt-sensitive hypertension in Dahl S rats. Conclusions Our results suggest that the impaired miR-429-mediated PHD2 inhibition in response to high salt in the renal medulla may represent a novel mechanism for salt-sensitive hypertension in Dahl S rats and that correction of this impairment in miR-429 pathway could be a therapeutic approach for salt-sensitive hypertension.

Vascular endothelial growth factor (VEGF) plays an essential role in luteal angiogenesis, the present study therefore utilized luteal cells cultured in vitro to further investigate the activation and contribution of nuclear factor (NF)-κB to VEGF expression induced by human chorionic gonadotrophin (HCG). The present results showed HCG induced VEGF expression as well as hypoxia-inducible factor (HIF)-1α mRNA and protein expressions, which was blocked by NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC). Further analysis found that these increases of VEGF and HIF-1α mRNA induced by HCG were also blocked by NF-κB siRNA transfection, which was consistent with PDTC treatment. However, HIF-1α siRNA treatment significantly decreased HCG induced-VEGF expression with no effect on NF-κB mRNA expression. Furthermore, combination of HIF-1α siRNA and PDTC treatment did not further decrease VEGF mRNA expression, and the result of chromatin immunoprecipitation indicated NF-κB may regulate HIF-1α transcription through binding with its promoter. Taken together, the present results clearly demonstrated that NF-κB was activated to regulate VEGF expression by increasing HIF-1α transcription in luteal cells treated with HCG. Therefore, the present study provided a new and important mechanism of luteal angiogenesis during the formation of corpus luteum in mammals.

Extracellular vesicles (EVs) are a heterogeneous group of endogenous nanoscale vesicles that are secreted by various cell types. Based on their biogenesis and size distribution, EVs can be broadly classified as exosomes and microvesicles. Exosomes are enveloped by lipid bilayers with a size of 30–150 nm in diameter, which contain diverse biomolecules, including lipids, proteins and nucleic acids. Exosomes transport their bioactive cargoes from original cells to recipient cells, thus play crucial roles in mediating intercellular communication. Breast cancer is the most common malignancy among women and remains a major health problem worldwide, diagnostic strategies and therapies aimed at breast cancer are still limited. Growing evidence shows that exosomes are involved in the pathogenesis of breast cancer, including tumorigenesis, invasion and metastasis. Here, we provide a straightforward overview of exosomes and highlight the role of exosomes in the pathogenesis of breast cancer, moreover, we discuss the potential application of exosomes as biomarkers and therapeutic tools in breast cancer diagnostics and therapeutics.

FoxO1 is a member of the forkhead transcription factor family subgroup O (FoxO), which is expressed in many cell types, and participates in various pathophysiological processes, including cell proliferation, apoptosis, autophagy, metabolism, inflammatory response, cytokine expression, immune differentiation, and oxidative stress resistance. Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in the women of childbearing age, which is regulated via a variety of signaling pathways. Currently, the specific mechanism underlying the pathogenesis of PCOS is still unclear. As an important transcription factor, FoxO1 activity might be involved in the pathophysiology of PCOS. PCOS has been associated with insulin resistance and low-grade inflammatory response. Therefore, the studies regarding the role of FoxO1 in the incidence and associated complications of PCOS will help provide novel ideas for establishing the treatment strategy of PCOS.

Owing to the avascular structure of the ovarian follicle, proliferation of granulosa cells (GCs) and development of follicles occur under hypoxia, which is obviously different from the cell survival requirements of most mammalian cells. We hypothesized that autophagy may exert an inhibitory effect on GC apoptosis. To decipher the underlying mechanism, we constructed a rat follicular development model using pregnant mare serum gonadotropin and a cell culture experiment in hypoxic conditions (3% O2). The present results showed that the autophagy level was obviously increased and was accompanied by the concomitant elevation of hypoxia inducible factor (HIF)-1α and BNIP3 (Bcl-2/adenovirus E1B 19kDa-interacting protein 3) in GCs during follicular development. The levels of Bax (Bcl2-associated X) and Bcl-2 (B-cell lymphoma-2) were increased, while the activation of caspase-3 exhibited no obvious changes during follicular development. However, inhibition of HIF-1α attenuated the increase in Bcl-2 and promoted the increase in Bax and cleaved caspase-3. Furthermore, we observed the downregulation of BNIP3 and the decrease in autophagy after treatment with a specific HIF-1α activity inhibitor (echinomycin), indicating that HIF-1α/BNIP3 was involved in autophagy regulation in GCs in vivo. In an in vitro study, we also found that hypoxia did not obviously promote GC apoptosis, while it significantly enhanced the activation of HIF-1α/BNIP3 and the induction of autophagy. Expectedly, this effect could be reversed by 3-methyladenine (3-MA) treatment. Taken together, these findings demonstrated that hypoxia drives the activation of HIF-1α/BNIP3 signaling, which induces an increase in autophagy, protecting GC from apoptosis during follicular development.

During the luteinization after ovulation in mammalian ovary, the containing cells undergo an energy consuming function re-determination process to differentiate into luteal cells under avascular environment. Previous evidences have delineated the contribution of autophagy to the cell differentiation and the catabolic homeostasis in various types of mammalian cells, whereas few interest had been focused on the involvement of autophagy in the luteinization of granulosa cells during the formation of early corpus luteum. Herein, the present study investigated that expression and contribution of autophagy during granulosa cell luteinization and early luteal development through in vivo and in vitro experiments. The results clearly demonstrated that HIF-1α/BNIP3-mediated autophagy plays a vital role in the luteinization of granulosa cells during the early luteal formation in vivo and in vitro. In the neonatal corpus luteum, HIF-1α up-regulated BNIP3 expressions, which contributed to the autophagic initiation by disrupting beclin1 from Bcl-2/beclin1 complex and protected cells from apoptosis by curbing the skew of mitochondria balance under avascular niche. Notably, Inhibition of HIF-1α activity by echinomycin enhanced the levels of cytoplasmic cytochrome c and cell apoptosis in the nascent corpus luteum. These findings revealed that HIF-1α/BNIP3-mediated autophagy enabled the process of granulosa cell luteinization and protected the granulosa-lutein cells from further apoptosis under hypoxia niche. To our knowledge, the present study firstly clarified that HIF-1α/BNIP3-mediated autophagy contributes to the luteinization of granulosa cells during the formation of pregnant corpus luteum, which will help us further understanding the luteal biology and provide us new clues for the treatment of luteal insufficiency.

Hypoxia and oxidative stress are the common causes of various types of kidney injury. During recent years, the studies on hypoxia inducible factor- (HIF-) 1 attract more and more attention, which can not only mediate hypoxia adaptation but also contribute to profibrotic changes. Through analyzing related literatures, we found that oxidative stress can regulate the expression and activity of HIF-1α through some signaling molecules, such as prolyl hydroxylase domain-containing protein (PHD), PI-3K, and microRNA. And oxidative stress can take part in inflammation, epithelial-mesenchymal transition, and extracellular matrix deposition mediated by HIF-1 via interacting with classical NF-κB and TGF-β signaling pathways. Therefore, based on previous literatures, this review summarizes the contribution of oxidative stress to HIF-1-mediated profibrotic changes during the kidney damage, in order to further understand the role of oxidative stress in renal fibrosis.

In mammals, developing ovarian follicles transform from primordial follicles to primary follicles, secondary follicles, and mature follicles, accompanied by changes in follicular secretory functions. FoxO3a is a member of the forkhead transcription factor family (FoxO), which plays an important role in the cell cycle, DNA damage repair, apoptosis, oxidative stress, and energy metabolism. Recent studies have shown that FOXO3a is involved in the physiological regulation of follicular development and pathological progression of related ovarian diseases, which will provide useful concepts and strategies for retarding ovarian aging, prolonging the ovarian life span, and treating ovarian diseases. Therefore, the regulation of FOXO3a expression, as well as the physiological contribution during ovarian follicular development are detailed in this paper, presenting an important reference for the further study of ovarian biology.

Objective To investigate the effect of progressive resistance training using resistance (elastic) bands on subacromial bursitis following triamcinolone acetonide injection. Design Randomized clinical trial. Patients A total of 68 patients with subacromial bursitis were randomized to a triamcinolone acetonide group or a triamcinolone acetonide plus resistance band training group. Methods Visual analogue scale, Constant scores, range of motion, proprioception, and muscle strength were evaluated at pretreatment and at 3, 12 and 24 weeks' follow-up. Re-treatment ratio was calculated at one-year follow-up. Results At 3 and 12 weeks, both the triamcinolone acetonide group and triamcinolone acetonide plus resistance band training group showed a significant improvement in Visual analogue scale score, Constant score, range of motion, proprioception and muscle strength. Although the scores in the triamcinolone acetonide group had not increased at 24 weeks compared with baseline, the scores in the triamcinolone acetonide plus resistance band training group showed continued improvement at 24 weeks. A lower proportion of patients in the triamcinolone acetonide plus resistance band training bands group than in the triamcinolone acetonide group had received re-treatment at one-year follow-up (12.1% vs 82.9%). Conclusion Progressive resistance training with resistance (elastic) bands has the advantages of extending the benefits of corticosteroid injection and maintaining long-term effects on shoulder function in patients with subacromial bursitis.

Renal fibrosis is a common pathological process where certain primary or secondary kidney diseases can continue to progress to the end-stage of the kidney disease; however, the molecular mechanisms underlying renal fibrosis remain unclear. Recently, research focusing on examining the function of inflammasomes has attracted a great deal of attention, and data derived from these research projects have increased our understanding of the effects and regulation of inflammasomes during renal fibrosis. Based on this, the present review summarizes recent findings in regard to NLRP3 inflammasome functions during various kidney diseases, and these findings indicate that the NLRP3 inflammasome not only mediates the inflammatory response but is also associated with pyroptosis, mitochondrial regulation, and myofibroblast differentiation during renal fibrosis. These novel findings provide us with a more in-depth understanding of the pathogenesis of renal fibrosis and will aid in the identification of new targets that can be used for the prevention and treatment of this disease.

Lipids and proteins in the plasma membrane are laterally heterogeneous and formalised as lipid rafts featuring unique biophysical properties. However, the self-assembly mechanism of lipid raft cannot be revealed even its physical properties and components were determined in specific physiological processes. In this study, two-photon generalised polarisation imaging and fluorescence correlation spectroscopy were used to study the fusion of lipid rafts through the membrane phase and the lateral diffusion of lipids in living breast cancer cells. A self-assembly model of lipid rafts associated with lipid diffusion and membrane phase was proposed to demonstrate the lipid sorting ability of lipid rafts in the plasma membrane. The results showed that the increased proportion of slow subdiffusion of GM1 -binding cholera toxin B-subunit (CT-B) was accompanied with an increased liquid-ordered domain during the β-estradiol-induced fusion of lipid rafts. And slow subdiffusion of CT-B was vanished with the depletion of lipid rafts. Whereas the dialkylindocarbocyanine (DiIC18 ) diffusion was not specifically regulated by lipid rafts. This study will open up a new insight for uncovering the self-assembly of lipid rafts in specific pathophysiological processes.

Simple Summary The present study demonstrates that induction of autophagy-related proteins in corpus luteum is regulated by Akt/mTOR signaling and autophagy may exert influences on progesterone production by controlling the pool of lipid droplets in luteal cells during the luteal development of pregnant rats. Furthermore, mitophagy-related proteins were also induced during the initiation of luteal regression in pregnant rats, which may play an essential role in the maintenance of mitochondrial homeostasis. These findings will shed light on the role of autophagy during the luteal development of pregnant ovaries in vivo in mammals. Abstract The contribution of autophagy to catabolic balance has been well-established in various types of cells, whereas the involvement of autophagy in progesterone synthesis during rat pregnancy still remains unknown. Therefore, the present study was designed to evaluate the role of autophagy in progesterone production during the luteal development of pregnant rats. The results showed autophagy-related proteins was maintained at a low level on day 10 after pregnancy, significantly induced on day 16 and subsided to a relative low level on day 21, which was consistent with the changes of serum progesterone levels. The findings further indicated the contribution of autophagy to progesterone production was regulated by inactivation of Akt/mTOR signaling during the luteal development of pregnant rats in in vivo and in vitro experiments. Further investigations revealed autophagy may be involved in the surge of progesterone production in pregnant rats, as inhibition of autophagy by 3-MA compromised serum progesterone levels. Furthermore, 3-MA treatment also leveled down the number of lipid droplets in luteal cells, implying that autophagy may affect the production of progesterone by manipulating the formation of lipid droplets in luteal cells. In addition, the results suggested that mitophagy was mobilized during the primary stage of luteolysis and inhibition of autophagy promoted the increase of redundant mitochondrial and cytoplasmic cytochrome C in luteal cells of pregnant rats. Taken together, the present study indicated that autophagy-related proteins were induced by the inactivation of Akt/mTOR signaling and then contributed to the progesterone production possibly by affecting the formation of intracellular lipid droplets during the luteal development of pregnant rats. To our knowledge, this will provide a new insight into the important mechanism of autophagy regulating progesterone production in ovaries of pregnant mammals.

Quick hemostats for non-lethal massive traumatic bleeding in battlefield and civilian accidents are important for reducing mortality and medical costs. Chitosan (CS) has been widely used as a clinic hemostat. To enhance its hemostatic efficiency, Zn2+ in the form of zinc alginate (ZnAlg) was introduced to CS to make porous CS@ZnAlg microspheres with ZnAlg component on the surface. Such microspheres were prepared by successive steps of micro-emulsion, polyelectrolyte adhesion, and thermally induced phase separation. Their structure and hemostatic performance were analyzed by SEM, FT-IR, XPS and a series of in vitro hemostatic experiments including thromboelastography analysis. The composite microspheres had an outer and internal interconnected porous structure. Their size, surface area, and water absorption ratio were ca. 70μm, 48m2/g, and 1850%, respectively. Compared to the neat chitosan microspheres, the CS@ZnAlg microspheres showed shorter onset of clot formation, much faster in vitro and in vivo whole blood clotting, bigger clot, less blood loss, and shorter hemostatic time in the rat liver laceration and tail amputation models. The synergetic hemostatic effects from (1) the electrostatic attraction between chitosan component and red blood cells, (2) the activation of coagulation factor XII by Zn2+ of zinc alginate component, and (3) physical blocking by microsphere matrix, contributed to the enhanced hemostatic performance of CS@ZnAlg microspheres.

It is established that the physiological effects of insulin are primarily mediated by the insulin signaling pathway. However, a defective insulin signaling is closely associated with the clinical manifestations of polycystic ovary syndrome (PCOS), which include excess androgen levels, insulin resistance and anovulation, and is involved in the pathophysiology of PCOS at the molecular level. Dimethyldiguanide (DMBG) has been widely employed to alleviate reproduction dysfunction in women with PCOS, however, the exact mechanism of this effect remains unclear. The objective of the present study was to investigate the effects of DMBG on the expression of the insulin signaling pathway in the ovaries of rats with PCOS, and to identify the potential underlying molecular mechanisms of these effects in PCOS. In the present study, a PCOS rat model was induced by letrozole, and successful establishment of the model was confirmed by examining ovarian histology and determining serum testosterone levels, by hematoxylin and eosin staining and ELISA, respectively. Subsequently, the expression of two key elements of insulin signaling, insulin receptor substrate (IRS)‑2 and phosphatidylinositol 3‑kinase (PI3K), was determined by immunohistochemistry and western blot analysis. The results demonstrated that IRS‑2 and PI3K expression was markedly decreased in PCOS ovaries, which was rescued by DMBG treatment. These results indicate that IRS‑2/PI3K signaling may be involved in the development of PCOS and the therapeutic effects of DMBG on PCOS. To further confirm the effects of DMBG on insulin signaling expression during this process, the expression of an additional two downstream proteins, phosphoinositide‑dependent kinase‑1 (PDK‑1) and the mammalian target of rapamycin (mTOR), was also investigated in the present study, and the results demonstrated that the expression of PDK‑1 and mTOR was significantly reduced in PCOS ovaries and increased following DMBG treatment, further indicating that altered insulin signaling may have an important role in the development and treatment of PCOS. In conclusion, the results of the present study indicate that the reduced expression of proteins involved in insulin signaling may contribute to the development of the clinical features of PCOS, and DMBG reverses reduced expression of insulin signaling components, by a mechanism that is yet to be determined, to attenuate certain symptoms of PCOS, such as obesity. To the best of our knowledge, the present study is the first to provide data regarding the detailed changes of insulin signaling during the development and treatment of PCOS, and may provide an important reference for clinical PCOS treatment.

Diabetes triggers abnormal ovarian follicular development and consequently leads to infertility. Here, we established a type 2 diabetes mouse model by feeding with high fat diet (HFD) for 15/20 weeks and assessed the effect of diabetes on follicular development and ovarian angiogenesis. After fed with HFD for 15 weeks, mice had the characteristics of type 2 diabetes, which was much more serious after 20 weeks on HFD. After 20 weeks on HFD, the mice had shown abnormal ovarian morphology with hyaline appearance, much less blood vessel, follicular development arrest and less of granulosa cells (GCs) in mature follicles, but not in ovaries from 15 weeks on HFD. Elevated makers of DNA damage, ER stress and apoptosis of GCs were observed in ovaries from HFD for 20 weeks. Additionally, diabetes significantly suppressed ovarian angiogenesis with the evidence of down-regulation of CD31 via inhibiting HIF1α-VEGF signaling pathway in time-dependent. We concluded that diabetes triggers abnormal ovarian function via inducing GCs apoptosis and suppressing ovarian angiogenesis.

Cellular mechanical properties could serve as a prominent indicator for disease progression and early cancer diagnosis. This study utilized atomic force microscopy (AFM) to measure the viscoelastic properties and then examined their association with the invasion of ovarian cancer at living single cell level. The results demonstrated the elasticity and viscosity of ovarian cancer cell OVCAR-3 and HO-8910 significantly decreased than those of HOSEpiC, the ovarian cancer control cell. Further examination found the dramatic increase of migration/invasion and the obvious decease of microfilament density in OVCAR-3 and HO-8910 cells compared with those of HOSEpiC cells. And there was a significant relationship between viscoelastic and biological properties among these cells. In addition, the elasticity was significantly increased in OVCAR-3 and HO-8910 cells after the treatment of anticancer compound echinomycin (Ech), while no obvious change was found in HOSEpiC cells after Ech treatment. Interestingly, Ech seemed no effects on the viscosity of these cells. Furthermore, Ech significantly inhibited the migration/invasion and significantly increased the microfilament density in OVCAR-3 and HO-8910 cells compared with those of HOSEpiC cells, which was significantly related with the elasticity among these cells. Notably, an increase of elasticity and a decrease of invasion were found in OVCAR-3 and HO-8910 cells with Ech treatment. Together, this study clearly demonstrated the association of viscoelastic properties with the invasion of ovarian cancer cells and shed a light on the biomechanical changes for early diagnosis of tumor transformation and progression at single cell level.

Obesity has a negative effect on ovarian functions, which is reported to increase the risk of infertility. The mechanism underlying obesity‑induced infertility is not yet clear. The present study established a high‑fat diet (HFD)‑induced obesity mouse model to elucidate the mechanisms underlying the effect of HFD‑induced obesity on follicular development in the mouse ovary. The 4‑week‑old female mice were fed with HFD or normal control (NC) diet for 15 or 20 weeks. Body mass index was used to demonstrate that the mice were obese following HFD treatment. The follicular development of the ovaries from the HFD group mice was retarded in a time‑dependent manner, as demonstrated by morphological and histological examination of the ovaries. Further investigation via western blot analysis demonstrated that the activity of the transcription factor, forkhead box O3a (FoxO3a), was increased by HFD through downregulated FoxO3a phosphorylation, which may contribute to the inhibited development of ovarian follicles. To determine the regulatory mechanism of FoxO3 on the follicular development, the expression levels of FoxO3a target protein, Smad1/5/8, were also determined and there was significant decrease in phosphorylated Smad1/5/8 in the ovaries from the HFD group compared with the NC group, indicating that FoxO3a/Smad1/5/8 may be important in the regulation of follicular development. The expression levels of the upstream regulator of FoxO3a, Akt, were also examined and it was demonstrated that Akt phosphorylation was significantly reduced in the HFD group compared with the NC group, indicating that Akt/FoxO3a may be also involved in follicular development. Together, the experiments demonstrated that HFD‑induced obesity affected the activity of the Akt/FoxO3a/Smad1/5/8 signaling pathway in a time‑dependent manner during the follicular development of the mouse ovary, leading to abnormal follicular development. These findings may provide part of a theoretical basis for the clinical prevention and treatment of obesity-associated female infertility.

The phosphatase and tensin homolog on chromosome 10 (PTEN) is one of important tumor suppressor proteins in ovarian cancer via negatively regulating the phosphatidylinositol 3-kinase–AKT signaling pathway and controlling genomic stability. Recent studies showed the physiological function of PTEN was closely related with its subcellular compartments. But only a few technologies could quantitatively measure the concentration of PTEN at different subcellular compartments in living cells. In this study, we used fluorescence correlation spectroscopy to measure the concentrations and dynamics of EGFP-PTEN in ovarian cancer cells HO-8910. Our results showed the increasing concentration of PTEN in the cytoplasm had an opposite trends with the nucleus after the oxidative stress stimulation which was induced by H2O2. Furthermore, the altered diffusion of PTEN at different subcellular compartments also illustrated the PTEN was trafficked from the cytoplasm to nucleus.

MicroRNA (miR) 429 has been shown to inhibit epithelial-to-mesenchymal transition (EMT) in cancer cells. However, the role of miR429 in EMT in non-cancer cells has not been defined, especially in the kidneys. The present study determined whether miR429 participated in angiotensin (ANG) II-induced EMT and fibrogenesis in renal cells. In NRK-52E cells, a rat proximal tubular cell line, incubation of ANG II (10−9 M) for 24 h significantly reduced the level of miR429 by 60% and increased the protein levels of mesenchymal markers α-smooth muscle actin and fibroblast-specific protein-1 by threefold and decreased epithelial marker E-cadherin by 60%, which was blocked by losartan, an AT1 receptor antagonist. Treatment of cells with miR429 inhibitor produced similar changes in the above EMT markers to that induced by ANG II. In cells overexpressed with miR429 transgene, ANG II-induced increases in collagen were abolished. Male Sprague-Dawley rats were infused with ANG II (200 ng·kg−1·min−1) for 12 days, and the levels of miR429 in the kidneys were reduced by 75%. Intrarenal transfection of lentivirus expressing miR429 also reversed the ANG II-induced changes in the EMT markers and collagen in the kidneys. The ANG II-induced increase in urinary albumin was significantly inhibited by miR429 transgene. There was no difference in the increases of blood pressure between ANG II- and ANG II+miR429 transgene-treated rats. These data suggest that ANG II-induced inhibition of miR429 contributes to ANG II-induced transdifferentiation and fibrogenesis in renal cells and that miR429 protects against ANG II-induced kidney damages.

Our previous study has demonstrated aerobic exercise contributed to the improvement of endothelial vasodilation functions by nitric oxide (NO) signaling activation during pre-diabetes mellitus. The present study was designed to examine the contribution of hypoxia inducible factor (HIF)-1alpha to the regulation of endothelial nitric oxide synthase (eNOS) expressions in the vascular endothelium of prediabetic rats with HIF-1alpha specific inhibitor echinomycin (Ech) and/or aerobic exercise. The results showed the expressions of eNOS mRNA and protein dramatically decreased after Ech treatment (p

The regulatory mechanism of granulosa cells (GCs) proliferation during the follicular development is complicated and multifactorial, which is essential for the oocyte growth and normal ovarian functions. To investigate the role of high fat diet (HFD) on the proliferation of GCs, 4-week old female mice were fed with HFD or normal control diet (NC) for 15 weeks or 20 weeks and then detected the expression level of some regulatory molecules of cell cycle and apoptosis. The abnormal ovarian morphology was observed at 20 weeks. Further mechanistic studies indicated that HFD induced-obesity caused elevated apoptotic levels in GCs of the ovaries in a time-dependent manner. Moreover, cell cycle progress was also impacted after HFD fed. The cell cycle inhibitors, p27(Kip1) and p21(Cip1), were significantly induced in the ovaries from the mice in HFD group when compared with that in the ovaries from the mice in NC group. Subsequently, the expression levels of Cyclin D1, D3 and CDK4 were also significantly influenced in the ovaries from the mice fed with HFD in a time-dependent manner. The present results suggested that HFD induced-obesity may trigger cell cycle arrest and excessive apoptosis of GCs, causing the abnormal follicular development and ovarian function failure.

Vascular endothelial growth factor (VEGF)-dependent angiogenesis has a crucial role in the corpus luteum formation and their functional maintenances in mammalian ovaries. A previous study by our group reported that activation of hypoxia‑inducible factor (HIF)‑1α signaling contributes to the regulation of VEGF expression in the luteal cells (LCs) in response to hypoxia and human chorionic gonadotropin. The present study was designed to test the hypothesis that HIF prolyl‑hydroxylases (PHDs) are expressed in LCs and overexpression of PHD2 attenuates the expression of VEGF induced by hypoxia in LCs. PHD2-overexpressing plasmid was transfected into LC2 cells, and successful plasmid transfection and expression was confirmed by reverse transcription quantitative polymerase chain reaction and western blot analysis. In addition, the present study investigated changes of HIF‑1α and VEGF expression after incubation under hypoxic conditions and PHD2 transfection. PHD2 expression was significantly higher expressed than the other two PHD isoforms, indicating its major role in LCs. Moreover, a significant increase of VEGF mRNA expression was identified after incubation under hypoxic conditions, which was, however, attenuated by PHD2 overexpression in LCs. Further analysis also indicated that this hypoxia‑induced increase in the mRNA expression of VEGF was consistent with increases in the protein levels of HIF‑1α, which is regulated by PHD-mediated degradation. In conclusion, the results of the present study indicated that PHD2 is the main PHD expressed in LCs and hypoxia‑induced VEGF expression can be attenuated by PHD2 overexpression through HIF‑1α‑mediated mechanisms in LCs. This PHD2-mediated transcriptional activation may be one of the mechanisms regulating VEGF expression in LCs during mammalian corpus luteum development.

The corpus luteum is a temporary endocrine structure in mammals that plays an important role in the female reproductive cycle and is formed from a ruptured and ovulated follicle with rapid angiogenesis. Vascular endothelial growth factor (VEGF) is thought to be vital in normal and abnormal angiogenesis in the ovary, but the molecular regulation of luteal VEGF expression during corpus luteum development in vivo is still poorly understood at present. Therefore, we examined whether hypoxia-inducible factor-1a (HIF-1a) is induced and regulates VEGF expression and luteal function in vivo using a pseudopregnant rat model treated with a small-molecule inhibitor of HIF-1a, echinomycin. Corpus luteum development in the pseudopregnant rat ovary was determined after measuring plasma progesterone concentration and ovarian prostaglandin F2a content to reflect changes in HIF-1a and VEGF on different days of this developmental process. At day 7, the corpus luteum was formed and the expression of HIF- 1a/VEGF reached a maximum, while a significant decrease in HIF-1a/ VEGF expression was observed when luteolysis occurred at day 13. Additionally, echinomycin blocked luteal development by inhibiting VEGF expression mediated by HIF-1a and following luteal function by detecting the progesterone changes at day 7. These results demonstrated that HIF-1a-mediated VEGF expression might be an important mechanism regulating ovarian luteal development in mammals in vivo, which may provide new strategies for fertility control and for treating some types of ovarian dysfunction, such as polycystic ovarian syndrome, ovarian hyperstimulation syndrome, and ovarian neoplasia.

Dimethyl carbonate (DMC) is a widely used industrial chemical, which may be increasingly used in the future. However, its toxicity profile remains largely unknown. The present study was designed to investigate the effects of DMC exposure on the ovaries and the effect of autophagy activation on follicular development. Rats were randomly divided into a control group and low, medium and high dose DMC groups (all n=10). Histological analyses identified no marked differences in the rate of apoptosis between the control and low dose groups; however, marked apoptosis occurred in the medium and high dose groups. The expression of cleaved caspase-3 was significantly increased in the medium and high dose groups, which was consistent with changes observed in the expression of Bcl-2 and Bax. These results indicated that DMC exposure induces toxicity on ovarian function via the induction of apoptosis. The increased expression of the autophagy-related proteins light chain 3II, beclin-1 and p62 following exposure to DMC further indicated that autophagy was activated primarily in the granulosa cells of ovarian follicles in a dose-dependent manner. In addition, the changes in the expression of hypoxia inducible factor 1 α subunit (HIF-1α) and its target protein BCL2 interacting protein 3 (BNIP3) indicated that they may serve a role in the follicular development process induced by DMC. The results of the current study demonstrated that DMC exposure activated autophagy in the ovarian tissue. Furthermore, exposure to low doses of DMC may protect follicular development by activating the HIF-1α/BNIP3 signaling pathway. Taken together, these results indicate that exposure to medium and high doses of DMC induced follicular atresia by activating the apoptotic signaling pathway. This may be an important mechanism of regulating follicular development and ovarian function in mammals.

Background We have previously shown that high salt intake suppresses the expression of prolyl hydroxylase domain-containing protein 2 (PHD2), an enzyme promoting the degradation of hypoxia-inducible factor (HIF)-1α, and increases HIF-1α along with its target genes in the renal medulla, which promotes sodium excretion and regulates salt sensitivity of blood pressure. However, it remains unknown how high salt inhibits the expression of PHD2. Method and results The current study first revealed that high-salt-induced PHD2 inhibition was due to the enhanced decay of mRNA. We then found that high salt significantly increased the expression of miR-429, which was subsequently proven to target the 3'-untranslated region of PHD2 and reduce PHD2 levels, in the renal medulla. To define the functional role of renal medullary miR-429 in the regulation of PHD2/HIF-1α-mediated renal adaptation to high salt intake and salt sensitivity of blood pressure, we locally inhibited miR-429 in the renal medulla by locked nucleic acid anti-miR-429 in uninephrectomized rats. Our results demonstrated that inhibition of miR-429 remarkably increased the levels of PHD2, which disrupted PHD2-associated adaptive activation of HIF-1α-mediated gene expression in response to high salt in the renal medulla and consequently inhibited urinary sodium excretion, enhanced sodium retention in response to chronic sodium overloading, and as a result, produced a salt-sensitive hypertension. Conclusion It is concluded that miR-429 is an important upstream mediator in PHD2/HIF-1α-associated renal adaptation to high salt intake and that deficiency in miR-429-mediated PHD2 inhibition in response to high salt in the renal medulla may represent a pathogenic mechanism for salt-sensitive hypertension.

Background: Our previous study has detected a stem cell deficiency in the renal medulla in Dahl salt-sensitive (S) rats. This study determined whether infusion of valproic acid (VA), an agent known to stimulate the stem cell function, attenuated salt-sensitive hypertension in Dahl S rats. Methods: Uninephrectomized Dahl S rats were infused with vehicle or VA (50mg/kg/d) into the renal medulla and fed with a low (LS) or high salt diet (HS). Stem cell marker and number were analyzed by immunohistochemistry, Real-time RT-PCR and Western blot. Sodium excretion and blood pressure were measured. Results: VA significantly increased the mRNA and protein levels of FGF2, a stem cell niche factor, and CD133, a stem cell marker. The number of CD133+ cells was significantly increased in the renal medulla in VA-treated rats. Meanwhile, high salt-induced increases in the mRNA level of proinflammatory factors interleukin-1β and interleukin-6 were blocked in VA-treated rats. Functionally, sodium excretion in response to the blood pressure increase and acute sodium loading was significantly enhanced, sodium retention attenuated, high salt-induced increase of blood pressure reduced in VA-treated rats. Conclusion: Activation of stem cell function by VA inhibits the activation of proinflammatory factors and attenuates salt-sensitive hypertension in Dahl S rats.

Prediabetes is considered to be an at a risk state, with an annualized 5-10% conversion rate of diabetes and the similiar proportion converting back to normoglycaemia. The global incidence of prediabetes is increasing and will reach to 471 million patients in 2030. The elevation of blood sugar is a continuum and hence prediabetes cannot be considered an entirely benign status and present insulin resistance (IR) and β-cell dysfunction, so that the development of prediabetic complications and vascular endothelial dysfunction. Therefore, it is crucial to early recognition and treatment of prediabetic individuals. For prediabetic individuals, lifestyle modification is the cornerstone of diabetes prevention with evidence of a 40-70% relative risk reduction. In the review, we include the possible mechanism of IR-induced vascular endothelial dysfunction, and exercise intervention improving the vascular endothelial dysfunction. These results showed clearly positive effects of physical exercise intervention and provided a direct clinical evidence of the effectiveness of this approach for preventing early diabetes

Adult stem cell deficiency has been implicated in the pathogenic mechanism for various diseases. Renal medullary dysfunction is one of the major mechanisms for the development of hypertension in Dahl salt-sensitive (S) rats. The present study first detected a stem cell deficiency in the renal medulla in Dahl S rats and then tested the hypothesis that transplantation of mesenchymal stem cells (MSCs) into the renal medulla improves salt-sensitive hypertension in Dahl S rats. Immunohistochemistry and flowcytometry analyses showed a significantly reduced number of stem cell marker CD133+ cells in the renal medulla from Dahl S rats compared with controls, suggesting a stem cell deficiency. Rat MSCs or control cells were transplanted into the renal medulla in uninephrectomized Dahl S rats, which were then treated with a low- or high-salt diet for 20 days. High-salt-induced sodium retention and hypertension was significantly attenuated in MSC-treated rats compared with control cell-treated rats. Meanwhile, high-salt-induced increases of proinflammatory factors, monocyte chemoattractant protein-1, and interleukin-1β, in the renal medulla were blocked by MSC treatment. Furthermore, immunostaining showed that high-salt-induced immune cell infiltration into the renal medulla was substantially inhibited by MSC treatment. These results suggested that stem cell defect in the renal medulla may contribute to the hypertension in Dahl S rats and that correction of this stem cell defect by MSCs attenuated hypertension in Dahl S rats through anti-inflammation.Stem cell defect in the renal medulla may contribute to salt-sensitive hypertension Stem cell therapy is a potential therapeutic strategy for salt-sensitive hypertension Normal stem cell inhibits the inflammatory response to high salt in the renal medulla.

Impaired glucose tolerance (IGT) is a pre-diabetic metabolic state involving heterogeneous and dynamic changes between the normal and diabetic state. The present study aimed to investigate the endocrine regulation of endothelium-dependent dysfunction in middle-aged patients with IGT and in patients with a normal glucose tolerance (NGT). An oral glucose tolerance test was performed to determine the NGT and IGT states. Physiological and biochemical analyses were performed. The carotid artery structure and function were investigated with Doppler supersonic diagnostic equipment. The functioning of the vascular endothelium was analyzed with physiological and biochemical indices in the IGT group. The results showed a significant reduction in endothelium-dependent vasodilation, but not in endothelium-independent vasodilation in the IGT group compared with those of the NGT group. It was identified that the intima-media thickness of the carotid artery and expression levels of endothelin-1 were significantly higher, whereas the endothelium-derived factor C-type natriuretic peptide levels were significantly lower in the IGT group compared with those in the NGT group. Notably, significant correlations were identified between endocrinological changes and body composition, including fat and glucose metabolism, in the IGT group. Our data indicate that vascular endothelial functions may be impaired by fat and glucose metabolism and body composition in IGT patients during prediabetes mellitusare.

Cryptocaryon irritans is a parasitic ciliate and responsible for cryptocaryosis of ocean teleostean. In this paper, one gene homologous to 14-3-3 was isolated from cDNA library of C. irritans trophont/protomont stage and designated as Ci14-3-3. The full-length cDNA of the gene was 892bp with an open reading frame of 744bp, which encoded a polypeptide of 247 amino acids with a predicted molecular weight of 28.4kDa. After modification of the non-universal genetic codes, the open reading frame of Ci14-3-3 was inserted into plasmid pGEX-4T-1, transformed into Escherichia coli DH5α strain and then expressed as a glutathione S transferase fusion protein (rCi14-3-3). The result of western blot analysis showed that the rCi14-3-3 had antigenicity and the Ci14-3-3 gene in C. irritans was expressed at all stages of life cycle. The endogenous Ci14-3-3 not only distributed in cytoplasm, but also presented on the plasma membrane and the front end of cytostome in newly hatched theronts. However, when theronts were dying the protein appeared as dot-like aggregates around the nucleuses. The murine anti-rCi14-3-3 sera were capable of causing agglutination/immobilization of theronts, suggesting its potential for vaccine development.

Endothelial dysfunction (ED) is an early pathophysiological change in patients with impaired glucose tolerance (IGT) during prediabetes mellitus. This study was designed to test the hypothesis that exercise intervention contributes to the reversal of vascular endothelium-dependent dysfunction in middle-aged patients with IGT. Following exercise intervention, significant changes in endothelin (ET)-1, C-type natriuretic peptide (CNP), ΔDia-P, oral glucose tolerance test (OGTT)2h, fasting insulin, homeostasis model of assessment-insulin resistance (HOMA-IR), body fat percentage, waist circumference and waist to hip ratio were measured. However, no marked changes in carotid artery intima-media thickness (IMT), fasting blood glucose and BMI were observed following exercise intervention. Validity analysis of index changes in the two exercise intervention groups further confirmed there was no change. Exercise intervention increased CNP levels, decreased ET-1 levels and increased ΔDia-P, indicating improved vascular endothelium function. Decreased HOMA-IR following exercise suggests enhanced insulin sensitivity. Exercise intervention also improved glucose metabolism via decreased OGTT2h and fasting insulin. In addition, decreased waist circumference, ratio of waist to hip and body fat percentage following exercise intervention improved changes of body composition, including BMI, body fat and waist circumference. These results indicate that exercise intervention may reverse vascular endothelium-dependent dysfunction in middle-aged patients with IGT. This study also provided direct clinical data supporting the use of exercise intervention to prevent diabetes mellitus (DM) during the early stage.

Previous studies by our group have indicated that exercise intervention can ameliorate endothelial dysfunction, which is an early pathophysiological change of prediabetes mellitus. The present study aimed to test the hypothesis that nitric oxide synthases (NOSs), which are expressed in blood vessel endothelium, contribute to the mitigation of vascular endothelium-dependent dysfunction by aerobic exercise in prediabetes mellitus. A prediabetic rat model was established by feeding the rats an additional high-energy diet, and was confirmed by testing blood glucose levels, the area-under-the-curve for the blood glucose tests (P

Frizzled 3 is an important receptor in the Wnt/β-catenin pathway, a conserved signaling pathway that regulates gene expression and controls diverse developmental processes. However, the role of this protein during follicular development in the adult ovary is not known. The present study was designed to investigate the expression and localization of Frizzled 3 mRNA and protein during the estrous cycle in the mouse ovary through in situ hybridization (ISH), real-time quantitative polymerase chain reaction, immunohistochemistry and western blot. ISH results showed that in proestrus, high expression of Frizzled 3 was found in the granulosa and stroma with weak levels in the corpus luteum. In estrus and diestrus, the stroma had high Frizzled 3 expression, but levels were low in granulosa cells and corpus luteum. In the metestrus, moderate expression of Frizzled 3 was found in the stroma but low to no expression was found in luteal cells and follicles. The mRNA and protein levels of Frizzled 3 were found to be the highest in proestrus and diestrus compared to estrus and metestrus (P < 0.05), confirming the ISH results. During estrus and diestrus, high Frizzled 3 expression was observed in the stroma and moderate levels in granulosa cells, and during estrus and proestrus, low expression was seen in the oocyte cell membrane. The western blot results further confirmed this change during the estrous cycle. Together, these results indicate that Frizzled 3 is involved in regulating follicular development and oocyte maturation during the estrous cycle.

Renal medullary hypoxia-inducible factor (HIF)-1α and its target genes, such as haem oxygenase and nitric oxide synthase, have been indicated to play an important role in the regulation of sodium excretion and blood pressure. HIF prolyl hydroxylase domain-containing proteins (PHDs) are major enzymes to promote the degradation of HIF-1α. We recently reported that high salt intake suppressed the renal medullary PHD2 expression and thereby activated HIF-1α-mediated gene regulation in the renal medulla in response to high salt. To further define the functional role of renal medullary PHD2 in the regulation of renal adaptation to high salt intake and the longer term control of blood pressure, we transfected PHD2 expression plasmids into the renal medulla in uninephrectomized rats and determined its effects on pressure natriuresis, sodium excretion after salt overloading and the long-term control of arterial pressure after high salt challenge. It was shown that overexpression of PHD2 transgene increased PHD2 levels and decreased HIF-1α levels in the renal medulla, which blunted pressure natriuresis, attenuated sodium excretion, promoted sodium retention and produced salt sensitive hypertension after high salt challenge compared with rats treated with control plasmids. There was no blood pressure change in PHD2-treated rats that were maintained in low salt diet. These results suggested that renal medullary PHD2 is an important regulator in renal adaptation to high salt intake and a deficiency in PHD2-mediated molecular adaptation in response to high salt intake in the renal medulla may represent a pathogenic mechanism producing salt sensitive hypertension.

Hypoxia inducible factor (HIF)-1α-mediated gene activation in the renal medulla in response to high salt intake plays an important role in the control of salt sensitivity of blood pressure. High salt-induced activation of HIF-1α in the renal medulla is blunted in Dahl S rats. The present study determined whether the impairment of the renal medullary HIF-1α pathway was responsible for salt sensitive hypertension in Dahl S rats. Renal medullary HIF-1α levels were induced by either transfection of HIF-1α expression plasmid or chronic infusion of CoCl2 into the renal medulla, which was accompanied by increased expressions of anti-hypertensive genes, cyclooxygenase-2 and heme oxygenase-1. Overexpression of HIF-1α transgenes in the renal medulla enhanced the pressure natriuresis, promoted the sodium excretion and reduced sodium retention after salt overload. As a result, hypertension induced by 2-week high salt was significantly attenuated in rats treated with HIF-1α plasmid or CoCl2. These results suggest that an abnormal HIF-1α in the renal medulla may represent a novel mechanism mediating salt-sensitive hypertension in Dahl S rats and that induction of HIF-1α levels in the renal medulla could be a therapeutic approach for the treatment of salt-sensitive hypertension.

Vascular endothelial growth factor (VEGF)-dependent angiogenesis plays a crucial role in the corpus leteum formation and their functional maintenances in mammalian ovaries. We recently reported that the activation of hypoxia-inducible factor (HIF)-1α signaling contributes to the regulation of VEGF expression in the luteal cells (LCs) in response to hypoxia and human chorionic gonadotropin (HCG). This study was designed to test the hypothesis that, HIF prolyl-dydoxylases (PHDs) express in LCs and overexpression of PHD attenuates the expression of VEGF induced by HCG in LCs. By real-time PCR and western blot analysis, we examined the expression of PHDs, confirmed the plasmid transfection and their expression and also investigated the changes of HIF-1α and VEGF expression after treatment with HCG and PHD2 transgenes. PHD2 expression was significantly higher than the others, indicating its main roles. Moreover, a significant increase of VEGF mRNA was found after HCG treatment, while this increased VEGF mRNA was also blocked by PHD2 overexpression in LCs. Further analysis also found that, this HCG-induced increase of VEGF mRNA was consistent with the level of HIF-1α protein, which is regulated by HIF prolyl-dydoxylase -mediated degradation. Taken together, our results indicated that, PHD2 mainly expressed in LCs and HCG-induced VEGF expression can be blocked by PHD2 overexpression through HIF-1α -mediated mechanism in LCs. This PHD2-mediated transcriptional activation may be one of the important mechanisms regulating VEGF expression in LCs during mammalian corpus leteum development. Key words : Hypoxia-inducible factor-1α, HIF prolyl-dydoxylase, vascular endothelial growth factor, human chorionic gonadotropin, luteal cells.

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite formed by phosphorylation of sphingosine. S1P has been indicated to play a significant role in the cardiovascular system. It has been shown that the enzymes for S1P metabolism are expressed in the kidneys. The present study characterized the expression of S1P receptors in the kidneys and determined the role of S1P in the control of renal hemodynamics and sodium excretion. Real-time RT-PCR analyses showed that S1P receptors S1P1, S1P2, and S1P3 were most abundantly expressed in the renal medulla. Immunohistochemistry revealed that all three types of S1P receptors were mainly located in collecting ducts. Intramedullary infusion of FTY720, an S1P agonist, produced a dramatic increase in sodium excretion by twofold and a small but significant increase in medullary blood flow (16%). Administration of W146, an S1P1 antagonist, into the renal medulla blocked the effect of FTY720 and decreased the sodium excretion by 37% when infused alone. The antagonists of S1P2 and S1P3 had no effect. FTY720 produced additive natriuretic effects in combination with different sodium transporter inhibitors except amiloride, an epithelial sodium channel blocker. In the presence of nitric oxide synthase inhibitor l-NAME, FTY720 still increased sodium excretion. These data suggest that S1P produces natriuretic effects via activation of S1P1 in the renal medulla and this natriuretic effect may be through inhibition of epithelial sodium channel, which is nitric oxide independent. It is concluded that S1P is a novel diuretic factor in the renal medulla and may be an important regulator of sodium homeostasis.

We recently reported that HIF-1α plays a critical role in the regulation of vascular endothelial growth factor (VEGF) expression in the developing letual cells (LCs) and VEGF-dependent angiogenesis is essential for normal luteal development. Although it is believed that hypoxia is the primary inducer of VEGF, recent reports have also shown that human chorionic gonadotrophin (hCG) up-regulates VEGF expression in developing corpus luteum (CL). Therefore the present study was designed to test the induced effects of hCG on the expression of VEGF and HIF-1α in LCs under normoxic and hypoxic conditions. In addition, we also investigated whether the signaling pathways such as phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) and mitogen-activated protein kinase (MAPK) are involved in hCG-induced VEGF in LCs. A significant increase of VEGF mRNA was found in LCs treated with hCG, which was consistent with the changes of HIF-1α protein, even under hypoxic conditions. However, there was no obvious changes of HIF-1α mRNA in hCG-treated LCs between normoxic and hypoxic conditions, indicating hCG induces VEGF expression by increasing transcription of HIF-1α, while hypoxia mainly increases HIF-1α protein stability. When LCs were pretreated with inhibitors, we found that the PI3K/mTOR signaling pathway is required for HIF-1α and VEGF expression induced by hCG, while the MAPK pathway is not required. Together, these results suggest that activation of IP3K/mTOR signaling pathway contributes to the induction of VEGF and HIF-1α in hCG-treated LCs. To our knowledge this will provide a new insight into the important mechanism of hCG/LH-induced VEGF-dependent angiogenesis in the bovine ovary.

The present study was designed to test the hypothesis that hypoxia inducible factor (HIF)-1α mediates profibrotic effects of angiotensin (ANG) II and to determine whether HIF prolyl-hydroxylase, the enzyme that promotes the degradation of HIF-1α, is involved in the profibrotic action of ANG II. In cultured renal medullary interstitial cells, ANG II (10−6 M) treatment for 20 hours remarkably increased HIF-1α levels, which was accompanied by the significant upregulation of collagen I/III and tissue inhibitor of metalloproteinases (TIMP)-1. HIF-1α siRNA decreased HIF-1α levels and completely blocked the effects of ANG II on collagen I/III and TIMP-1. HIF-1α siRNA also abolished ANG II-induced elevation of proliferating cell nuclear antigen, a marker of cell proliferation, and vimentin, a marker of cell transdifferentiation. HIF-2α siRNA did not affect the action of ANG II on collagen I/III and TIMP-1. Overexpression of PHD2 transgene, the predominant renal HIF prolyl-hydroxylase, attenuated ANG II-induced profibrotic action and silencing of PHD2 gene enhanced ANG II-induced profibrotic action. Removal of H2O2 eliminated ANG II-induced profibrotic effects. Two week ANG II infusion (150 ng/Kg/min) increased the expression of HIF-1α and α-smooth muscle actin in the renal medullary interstitial cells in vivo. Our data suggest that HIF-1α mediates ANG II-induced profibrotic effects through activation of cell transdifferentiation and that redox regulation of PHD plays a critical role in ANG II-induced activation of HIF-1α and consequent cell proliferation, transdifferentiation and abnormal extracellular matrix metabolism in renal cells.

High salt induces the expression of transcription factor hypoxia-inducible factor (HIF) 1α and its target genes in the renal medulla, which is an important renal adaptive mechanism to high-salt intake. HIF prolyl-hydroxylase domain-containing proteins (PHDs) have been identified as major enzymes to promote the degradation of HIF-1α. PHD2 is the predominant isoform of PHDs in the kidney and is primarily expressed in the renal medulla. The present study tested the hypothesis that PHD2 responds to high salt and mediates high-salt–induced increase in HIF-1α levels in the renal medulla. In normotensive rats, high-salt intake (4% NaCl, 10 days) significantly inhibited PHD2 expressions and enzyme activities in the renal medulla. Renal medullary overexpression of the PHD2 transgene significantly decreased HIF-1α levels. PHD2 transgene also blocked high-salt–induced activation of HIF-1α target genes heme oxygenase 1 and NO synthase 2 in the renal medulla. In Dahl salt-sensitive hypertensive rats, however, high-salt intake did not inhibit the expression and activities of PHD2 in the renal medulla. Correspondingly, renal medullary HIF-1α levels were not upregulated by high-salt intake in these rats. After transfection of PHD2 small hairpin RNA, HIF-1α and its target genes were significantly upregulated by high-salt intake in Dahl salt-sensitive rats. Overexpression of PHD2 transgene in the renal medulla impaired renal sodium excretion after salt loading. These data suggest that high-salt intake inhibits PHD2 in the renal medulla, thereby upregulating the HIF-1α expression. The lack of PHD-mediated response to high salt may represent a pathogenic mechanism producing salt-sensitive hypertension.