1α,25-Dihydroxyvitamin D3 Improves Follicular Development and Steroid Hormone Biosynthesis by Regulating Vitamin D Receptor in the Layers Model.

1α,25-Dihydroxyvitamin D3 (VitD3) is the active form of vitamin D, and it regulates gene expression and protein synthesis in mammalian follicle development. However, the function of VitD3 in the follicular development of layers remains unclear. This study investigated, through in vivo and in vitro experiments, the effects of VitD3 on follicle development and steroid hormone biosynthesis in young layers. In vivo, ninety 18-week-old Hy-Line Brown laying hens were randomly divided into three groups for different treatments of VitD3 (0, 10, and 100 µg/kg). VitD3 supplementation promoted follicle development, increasing the number of small yellow follicles (SYFs) and large yellow follicles (LYFs) and the thickness of the granulosa layer (GL) of SYFs. Transcriptome analysis revealed that VitD3 supplementation altered gene expression in the ovarian steroidogenesis, cholesterol metabolism, and glycerolipid metabolism signaling pathways. Steroid hormone-targeted metabolomics profiling identified 20 steroid hormones altered by VitD3 treatment, with 5 being significantly different among the groups. In vitro, it was found that VitD3 increased cell proliferation, promoted cell-cycle progression, regulated the expression of cell-cycle-related genes, and inhibited the apoptosis of granulosa cells from pre-hierarchical follicles (phGCs) and theca cells from prehierarchical follicles (phTCs). In addition, the steroid hormone biosynthesis-related genes, estradiol (E2) and progesterone (P4) concentrations, and vitamin D receptor (VDR) expression level was significantly altered by VitD3. Our findings identified that VitD3 altered the gene expression related to steroid metabolism and the production of testosterone, estradiol, and progesterone in the pre-hierarchical follicles (PHFs), resulting in positive effects on poultry follicular development.

Key parameters of physiological responses to acute heat stress in two commercial layers determined by Fisher discriminant analyses.

Given the escalating global warming and the intense nature of modern poultry production, layers are becoming increasingly susceptible to heat stress. This stress disrupts the physiological processes of layers, which leads to reduced productivity and welfare. To address this issue, it is crucial to first evaluate the stress response systematically. However, such evaluations are still lacking in this field. The objective of this study was to accurately monitor the impact of thermal stress and identify common and key indicators that would support decision-making to maintain layer welfare and productivity under stress. We constructed two heat stress models to reflect moderate (32 °C) to severe (36 °C) stress effects and obtained a comprehensive profile of blood physiological parameters associated with the layers' responses to heat stress. We found that genetic differences had limited influence on their physiological responses to heat stress after 32 °C heat challenges. Using 8 selected and significantly changed parameters, layers' physiological status under heat stress could be accurately determined (judgmental accuracy of 98%). As ambient temperature increased to 36 °C, birds suffered more severe challenges that parameters changed in larger percentages. Additionally, breed variations of the physiological responses became apparent, a Fisher discriminant function based on 5 selected parameters could distinguish heat stress effects at 32 °C or 36 °C with 80% accuracy. The results obtained from this study provide two discriminant models for assessing heat stress and shed lights on developing effective and widely applicable heat stress mitigation strategies targeting these indicators.

Reversion of malignant phenotypes of human glioblastoma cells by ß-elemene through ß-catenin-mediated regulation of stemness-, differentiation- and epithelial-to-mesenchymal transition-related molecules.

BACKGROUND: Glioblastoma is the most common and lethal type of primary brain tumor. ß-Elemene, a natural plant drug extracted from Curcuma wenyujin, has shown strong anti-tumor effects in various tumors with low toxicity. However, the effects of ß-elemene on malignant phenotypes of human glioblastoma cells remain to be elucidated. Here we evaluated the effects of ß-elemene on cell proliferation, survival, stemness, differentiation and the epithelial-to-mesenchymal transition (EMT) in vitro and in vivo, and investigated the mechanisms underlying these effects. METHODS: Human primary and U87 glioblastoma cells were treated with ß-elemene, cell viability was measured using a cell counting kit-8 assay, and treated cells were evaluated by flow cytometry. Western blot analysis was carried out to determine the expression levels of stemness markers, differentiation-related molecules and EMT-related effectors. Transwell assays were performed to further determine EMT of glioblastoma cells. To evaluate the effect of ß-elemene on glioblastoma in vivo, we subcutaneously injected glioblastoma cells into the flank of nude mice and then intraperitoneally injected NaCl or ß-elemene. The tumor xenograft volumes were measured every 3 days and the expression of stemness-, differentiation- and EMT-related effectors was determined by Western blot assays in xenografts. RESULTS: ß-Elemene inhibited proliferation, promoted apoptosis, impaired invasiveness in glioblastoma cells and suppressed the growth of animal xenografts. The expression levels of the stemness markers CD133 and ATP-binding cassette subfamily G member 2 as well as the mesenchymal markers N-cadherin and ß-catenin were significantly downregulated, whereas the expression levels of the differentiation-related effectors glial fibrillary acidic protein, Notch1, and sonic hedgehog as well as the epithelial marker E-cadherin were upregulated by ß-elemene in vitro and in vivo. Interestingly, the expression of vimentin was increased by ß-elemene in vitro; this result was opposite that for the in vivo procedure. Inhibiting ß-catenin enhanced the anti-proliferative, EMT-inhibitory and specific marker expression-regulatory effects of ß-elemene. CONCLUSIONS: ß-Elemene reversed malignant phenotypes of human glioblastoma cells through ß-catenin-involved regulation of stemness-, differentiation- and EMT-related molecules. ß-Elemene represents a potentially valuable agent for glioblastoma therapy.

The role of 8-OH-DPAT on the rat neuronal apoptosis after diffuse brain injury coupled with secondary brain injury.

The potential role of 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) on rat neuronal apoptosis after diffuse brain injury (DBI) coupled with secondary brain injury (SBI) was investigated. One hundred and twelve adult male Wister rats weighing 305-355 g were randomly divided into four groups and received an intraperitoneal injection of 8-OH-DPAT (0.5 mg/kg) or an equal volume of normal saline. Neurological severity score (NSS) was recorded and the injured extent was observed after hematoxylin-eosin (HE) staining. The neuronal cell apoptosis index and the expression of Bax and Bcl-2 were detected by TUNEL method and immunohistochemistry respectively. We found a higher NSS value for rats in the DBI + SBI groups compared with those in normal control and sham-operated control groups (P < 0.01). HE staining showed that 8-OH-DPAT treatment could alleviate the occurrence of injury in rats CA3 hippocampus and PFC. The neuronal apoptosis index decreased in the 8-OH-DPAT treatment group compared with the NS group (P< 0.05) and gradually increased at 6 h, reached the peak level at 72 h and still had a high performance at 168 h in not only CA3 hippocampus but also PFC. Expression of Bax and Bcl-2 increased after DBI + SBI, however, with 8-OH-DPAT treatment Bcl-2 expression increased while Bax expression decreased. 8-OH-DPAT had an inhibitory effect on the rat neuronal apoptosis in CA3 hippocampus and PFC after DBI coupled with SBI.

Monoterpenoid indole alkaloids from Alstonia yunnanensis and their cytotoxic and anti-inflammatory activities.

The 80% ethanol extract of Alstonia yunnanensis afforded five new monoterpenoid indole alkaloids: 11-hydroxy-6,7-epoxy-8-oxo-vincadifformine (1), 14-chloro-15-hydroxy- vincadifformine (2), perakine N(4)-oxide (3), raucaffrinoline N(4)-oxide (4), and vinorine N(1),N(4)-dioxide (5), together with three known compounds: 11-methoxy-6,7-epoxy-8-oxo- vincadifformine (6), vinorine N(4)-oxide (7) and vinorine (8). The structures of the isolated compounds were established based on 1D and 2D (1H-1H-COSY, HMQC, HMBC, and ROESY) NMR spectroscopy, in addition to high resolution mass spectrometry. The isolated compounds were tested in vitro for cytotoxic potential against seven tumor cell lines and anti-inflammatory activities. Compounds 3, 4 and 7 exhibited weak cytotoxicity against the tested cell lines and selective inhibition of Cox-2 (> 85%).

The protective mechanism for the blood-brain barrier induced by aminoguanidine in surgical brain injury in rats.

This study was performed to investigate the mechanism of blood-brain barrier (BBB) permeability change, which was induced by aminoguanidine (AG) after surgical brain injury (SBI) in rats. Compared to control group, AG (150 mg/kg, i.p.) significantly reduced Evans blue extravasation into brain tissue at 24 h after surgical resection, it also induced a 32% decrease of malondialdehyde (MDA) values and a 1.1-fold increase of the glutathione (GSH) levels at 12 h after injury. The expression of inducible nitric oxide synthase (iNOS) reached the peak value at 24 h after SBI, which was significantly attenuated after AG treatment. In addition, ZO-1 protein was up-regulated by AG (150 mg/kg) treatment at 24 h after SBI. Our results indicated that AG could protect the BBB after SBI, which could be correlated with antioxidative property, the down-regulation of iNOS and up-regulation of tight junction protein expression.

Gene delivery by a cationic and thermosensitive nanogel promoted established tumor growth inhibition.

AIM: In vivo stability and consequent high tumor accumulation is highly desired for nonviral gene therapy. MATERIALS & METHODS: Here, a well-defined cationic nanogel system (NPS) was facilely prepared for gastric tumor therapy. RESULTS: The physical chemical properties of NPS were finely regulated and investigated. In vitro transfer efficiency of NPS was obviously promoted due to stable polyplex structure, small size, narrow size distribution and weak surface potential. Interestingly, the transfection was further enhanced by its passive targeting function. Intratumor accumulation was significantly promoted post intravenous administrated to Balb/c nude mice. Thus, the established gastric tumor (N87) growth was significantly inhibited by p53 as delivered by NPS. CONCLUSION: Such noncytotoxic cationic thermosensitive NPS can be effective for practicable gene therapy.

ß-elemene inhibits stemness, promotes differentiation and impairs chemoresistance to temozolomide in glioblastoma stem-like cells.

Accumulating evidence indicates that glioblastoma stem-like cells (GSCs) are key factors in tumour development, recurrence and chemoresistance. The impairment of stemness and the enhancement of differentiation contributes to the weakening of radiation and chemotherapy resistance of GSCs. We previously found that ß-elemene was an effective anti-glioblastoma agent and chemosensitizer. In this study, we examined the distribution of CD133(+) cells in human glioblastoma tissues by immunohistochemistry. Following treatment with ß-elemene, the formation of GSC spheres was investigated by manual counting, the proliferation of GSCs was measured with a Cell Counting Kit-8 (CCK-8) assay, and the dispersion of GSC spheres was observed with an inverted microscope. GSC spheres were treated with ß-elemene, and the expression levels of CD133, ATP-binding cassette subfamily G member 2 (ABCG2) and glial fibrillary acidic protein (GFAP) were examined by western blotting. After treatment with ß-elemene, the volumes and weights of GSC xenografts were measured, and the expression of CD133, ABCG2 and GFAP was evaluated through immunohistochemistry analysis. After treatment with ß-elemene and temozolomide (TMZ), GSC viability was examined by the CCK-8 assay, and the volumes and weights of xenografts were measured. We found that CD133(+) cells were assembled in some vascular walls and also sparsely distributed in other parts of glioblastoma tissues. ß-elemene decreased the formation of GSC spheres, dispersed GSC spheres and inhibited the proliferation of GSCs in vitro and in vivo. In the GSC spheres and xenografts treated with ß-elemene, the expression of CD133 and ABCG2 was significantly downregulated, and the expression of GFAP increased. Furthermore, the sensitivity of GSCs to TMZ was enhanced in vitro and in vivo. These results suggest that ß-elemene impaired the stemness of GSC spheres, promoted their differentiation and sensitized GSCs to TMZ. ß-elemene will hopefully become a valuable agent to enhance the effects of radiotherapy and chemotherapy.

Expression of pro-inflammatory cytokines and the risk of intracranial aneurysm.

Intracranial aneurysm (IA) lingers as a potentially devastating clinical problem, in which inflammation acts as a critical contributor to the pathogenesis of this disease. Cytokines play a major role in regulating inflammation. The aim of this study was to gain insight in the inflammatory response in IA by assessing plasma cytokine profiles. Plasma levels of 10 cytokines were quantified by multiplex protein arrays in 66 patients with IA and 78 healthy controls. Results showed that plasma level of interleukin 1 beta (IL-1ß) was 2.4-fold higher in patients than in controls (p < 0.05). The level of monocyte chemoattractant protein-1 (MCP-1) was 2.8-fold higher in patient than in controls (p < 0.01). The level of tumor necrosis factor-alpha (TNF-α) was 2.1-fold higher in cases than in controls (p < 0.001). When comparing the expression of cytokines in IA patients with different characteristics, cases with ruptured aneurysm revealed increased level of MCP-1 than those with unruptured aneurysm (p < 0.05), whereas cases with multiple numbers of aneurysms demonstrated higher levels of MCP-1 and TNF-α than those with single aneurysm (p < 0.05 and p < 0.05, respectively). These data indicated that IL-1ß, MCP-1, and TNF-α were associated with increased risk of IA and may affect the development of this disease.

An efficient and non-enzymatic method for isolation and culture of endothelial cells from the nidus of human cerebral arteriovenous malformations.

In this report, we describe an efficient and non-enzymatic method for isolating and culturing endothelial cells (ECs) from the nidus of surgically resected arteriovenous malformation (AVM) specimens. These cultured cells possessed typical phenotypic markers (i.e. von Willebrand factor and CD34), as well as morphological and ultrastructural characteristics of ECs. However, they had activated Notch-1 signaling, which plays a critical role in the development of AVM. The present study suggests that hypoxic endothelial cells from the nidus of human cerebral arteriovenous malformation (CAVMECs) have angiogenic potentials, as our data showed that VEGF gene expression and cell proliferation were more evident with prolonged hypoxia. In our study, we successfully used the vascular tissue explants adherent method to isolate and culture CAVMECs with high purity. This may prove to be a useful tool for studying the molecular mechanisms that mediate abnormal vessel development and maintenance in AVM.

8-hydroxy-2-(di-n-propylamino)tetralin intervenes with neural cell apoptosis following diffuse axonal injury.

Previous studies have reported a neuroprotective effect of 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) against traumatic brain injury. In accordance with the Marmarou method, rat models of diffuse axonal injury were established. 8-OH-DPAT was intraperitoneally injected into model rats. 8-OH-DPAT treated rats maintained at constant temperature served as normal temperature controls. TUNEL results revealed that neural cell swelling, brain tissue necrosis and cell apoptosis occurred around the injured tissue. Moreover, the number of Bax-, Bcl-2- and caspase-3-positive cells increased at 6 hours after diffuse axonal injury, and peaked at 24 hours. However, brain injury was attenuated, the number of apoptotic cells reduced, Bax and caspase-3 expression decreased, and Bcl-2 expression increased at 6, 12, 24, 72 and 168 hours after diffuse axonal injury in normal temperature control and in 8-OH-DPAT-intervention rats. The difference was most significant at 24 hours. All indices in 8-OH-DPAT-intervention rats were better than those in the constant temperature group. These results suggest that 8-OH-DPAT inhibits Bax and caspase-3 expression, increases Bcl-2 expression, and reduces neural cell apoptosis, resulting in neuroprotection against diffuse axonal injury. This effect is associated with a decrease in brain temperature.