Dendrotoxin-�� suppresses tumor growth induced by human lung adenocarcinoma A549 cells in nude mice

Voltage-gated K⁺ (Kv) channels have been considered to be a regulator of membrane potential and neuronal excitability. Recently, accumulated evidence has indicated that several Kv channel subtypes contribute to the control of cell proliferation in various types of cells and are worth noting as potential emerging molecular targets of cancer therapy. In the present study, we investigated the effects of the Kv1.1-specific blocker, dendrotoxin-κ (DTX-κ), on tumor formation induced by the human lung adenocarcinoma cell line A549 in a xenograft model. Kv1.1 mRNA and protein was expressed in A549 cells and the blockade of Kv1.1 by DTX-κ, reduced tumor formation in nude mice. Furthermore, treatment with DTX-κ significantly increased protein expression of p21^Waf1/Cip1, p27^Kip1, and p15^INK4B and significantly decreased protein expression of cyclin D3 in tumor tissues compared to the control. These results suggest that DTX-κ has anti-tumor effects in A549 cells through the pathway governing G1-S transition.     

Regulation of matrix metalloproteinase-9 protein expression by 1α,25-(OH)2D3 during osteoclast differentiation

To investigate 1α,25-(OH)₂D₃ regulation of matrix metalloproteinase-9 (MMP-9) protein expression during osteoclast formation and differentiation, receptor activator of nuclear factor κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) were administered to induce the differentiation of RAW264.7 cells into osteoclasts. The cells were incubated with different concentrations of 1α,25-(OH)₂D₃ during culturing, and cell proliferation was measured using the methylthiazol tetrazolium method. Osteoclast formation was confirmed using tartrate-resistant acid phosphatase (TRAP) staining and assessing bone lacunar resorption. MMP-9 protein expression levels were measured with Western blotting. We showed that 1α,25-(OH)₂D₃ inhibited RAW264.7 cell proliferation induced by RANKL and M-CSF, increased the numbers of TRAP-positive osteoclasts and their nuclei, enhanced osteoclast bone resorption, and promoted MMP-9 protein expression in a concentration-dependent manner. These findings indicate that 1α,25-(OH)₂D₃ administered at a physiological relevant concentration promoted osteoclast formation and could regulate osteoclast bone metabolism by increasing MMP-9 protein expression during osteoclast differentiation.     

Dietary taurine attenuates hydrogen peroxide-impaired growth performance and meat quality of broilers via modulating redox status and cell death signaling

Oxidative stress seriously affects poultry production. Nutritional manipulations have been effectively used to alleviate the negative effects caused by oxidative stress. This study investigated the attenuating effects and potential mechanisms of dietary taurine on the growth performance and meat quality of broiler chickens challenged with hydrogen peroxide (H₂O₂). Briefly, a total of 192 male Arbor Acres broilers (28 d old) were randomly categorized into three groups: non-injection of birds on basal diets (control), 10.0% H₂O₂ injection of birds on basal diets (H₂O₂), and 10.0% H₂O₂ injection of birds on basal diets supplemented with 5 g/kg taurine (H₂O₂ + taurine). Each group consisted of eight cages of eight birds per cage. Results indicated that H₂O₂ administration significantly reduced growth performance and impaired breast meat quality by decreasing ultimate pH and increasing shear force value (P < 0.05). Dietary taurine improved the body weight gain and feed intake and decreased feed/gain ratio of H₂O₂-challenged broilers. Meanwhile, oxidative stress induced by intraperitoneal injection of H₂O₂ suppressed the nuclear factor-κB (NF-κB) signaling and initiated autophagy and apoptosis. Compared with the H₂O₂ group, taurine supplementation restored the redox status in the breast muscle by decreasing levels of reactive oxygen species and contents of oxidative products and increasing antioxidant capacity (P < 0.05). Moreover, upregulated mRNA expression of NF-κB signaling-related genes, including NF-κB subunit 1 (p50) and B-cell CLL/lymphoma 2 (Bcl-2), and enhanced protein expression of NF-κB were observed in the H₂O₂ + taurine group (P < 0.05). Additionally, dietary taurine decreased the expression of caspase family, beclin1, and microtubule-associated protein 1light chain 3 beta (LC3-II; P < 0.05), thereby rescuing autophagy and apoptosis in breast muscle induced by H₂O₂. Collectively, dietary supplementation with taurine effectively improves growth performance and breast meat quality of broilers challenged with H₂O₂, possibly by protecting against oxidative injury and modulating cell death signaling.     

Involvement of the Ca2+ signaling pathway in osteoprotegerin inhibition of osteoclast differentiation and maturation

The purpose of this study was to determine whether the Ca²⁺ signaling pathway is involved in the ability of osteoprotegerin (OPG) to inhibit osteoclast differentiation and maturation. RAW264.7 cells were incubated with macrophage colony-stimulating factor (M-CSF) + receptor activator of nuclear factor-κB ligand (RANKL) to stimulate osteoclastogenesis and then treated with different concentrations of OPG, an inhibitor of osteoclast differentiation. The intracellular Ca²⁺ concentration [Ca²⁺]_i and phosphorylation of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) in the different treatment groups were measured by flow cytometry and Western blotting, respectively. The results confirmed that M-CSF + RANKL significantly increased [Ca²⁺]_i and CaMKII phosphorylation in osteoclasts (p < 0.01), and that these effects were subsequently decreased by OPG treatment. Exposure to specific inhibitors of the Ca²⁺ signaling pathway revealed that these changes varied between the different OPG treatment groups. Findings from the present study indicated that the Ca²⁺ signaling pathway is involved in both the regulation of osteoclastogenesis as well as inhibition of osteoclast differentiation and activation by OPG.     

Signaling pathways involved in liver injury and regeneration in rabbit hemorrhagic disease, an animal model of virally-induced fulminant hepatic failure

Management of fulminant hepatic failure (FHF) continues to be one challenging problem, and experimental animal models resembling its clinical conditions are still needed. Rabbit hemorrhagic disease (RHD) fullfils many requirements of an animal model of FHF. This work investigated changes in MAPK, NF-κB, AP-1 and STAT pathways during RHD-induced liver injury. Rabbits were infected with 2 × 10⁴ hemagglutination units of an RHD virus isolate. Apoptosis was documented by the presence of caspase-3 activity and substantial PARP proteolysis at 36 and 48 h postinfection (pi). Infection induced a marked and maintained expression of TNF-α from 12 h pi, while there was only a transitory increase in IL-6 expression. Expression of phosphorylated (p)-JNK, p-p38 and p-ERK1/2 was significantly elevated at 12 h pi. At 48 h pi p-JNK expression was maintained at a maximum level, while that of p-p38 returned to normality and there was no p-ERK1/2 expression. Activation of NF-κB and AP-1 and increased expression of VCAM-1 and COX-2 were observed. No significant changes were detected in activation of STAT1 and STAT3, while SOCS3 expression increased significantly. The current findings suggest that activation of JNK is an essential component in liver injury mediated by the RHD virus and that lack of activation of STAT3, probably mediated by SOCS3 over-expression, would contribute to the inhibition of the regenerative response. Data show the presence of molecular mechanisms contributing to liver damage and the lack of regeneration and they support the usefulness of this model to investigate novel therapeutical modalities in FHF.     

Protective effect of butylated hydroxylanisole against hydrogen peroxide-induced apoptosis in primary cultured mouse hepatocytes

Butylated hydroxyanisole (BHA) is a synthetic phenolic compound consisting of a mixture of two isomeric organic compounds: 2-tert-butyl-4-hydroxyanisole and 3-tert-butyl-4-hydroxyanisole. We examined the effect of BHA against hydrogen peroxide (H₂O₂)-induced apoptosis in primary cultured mouse hepatocytes. Cell viability was significantly decreased by H₂O₂ in a dose-dependent manner. Additionally, H₂O₂ treatment increased Bax, decreased Bcl-2, and promoted PARP-1 cleavage in a dose-dependent manner. Pretreatment with BHA before exposure to H₂O₂ significantly attenuated the H₂O₂-induced decrease of cell viability. H₂O₂ exposure resulted in an increase of intracellular reactive oxygen species (ROS) generation that was significantly inhibited by pretreatment with BHA or N-acetyl-cysteine (NAC, an ROS scavenger). H₂O₂-induced decrease of cell viability was also attenuated by pretreatment with BHA and NAC. Furthermore, H₂O₂-induced increase of Bax, decrease of Bcl-2, and PARP-1 cleavage was also inhibited by BHA. Taken together, results of this investigation demonstrated that BHA protects primary cultured mouse hepatocytes against H₂O₂-induced apoptosis by inhibiting ROS generation.     

The Control of Reactive Oxygen Species Influences Porcine Oocyte In Vitro Maturation

The aim of this study was to examine the effect of varying intracellular reactive oxygen species (ROS) levels during oocyte in vitro maturation with enzymatic ROS production systems (xanthine + xanthine oxidase or xanthine + xanthine oxidase + catalase), scavenger systems (catalase or superoxide dismutase + catalase) or cysteine on porcine oocyte maturation. Oocyte ROS levels showed an increase when H₂O₂ or O₂?^‐ production systems were added to the culture medium (p < 0.05). On the other hand, the presence of ROS scavengers in the maturation medium did not modify oocyte ROS levels compared with the control after 48 h of maturation, but the addition of cysteine induced a decrease in oocyte ROS levels (p < 0.05). The ROS production systems used in this work did not modified the percentage of oocyte nuclear maturation, but increased the decondensation of sperm head (p < 0.05) and decreased the pronuclear formation (p < 0.05). In turn, the addition of O₂?^‐ and H₂O₂ scavenging systems during in vitro maturation did not modify the percentage of oocytes reaching metaphase II nor the oocytes with decondensed sperm head or pronuclei after fertilization. However, both parameters increased in the presence of cysteine (p < 0.05). The exogenous generation of O₂?^‐ and H₂O₂ during oocyte in vitro maturation would not affect nuclear maturation or later sperm penetration, but most of the spermatozoa cannot progress to form the pronuclei after fusion with the oocyte. The decrease in endogenous ROS levels by the addition of cysteine would improve pronuclear formation after sperm penetration.     

The role of Bcl-xL and nuclear factor-��B in the effect of taxol on the viability of dendritic cells

Taxol has been used effectively in cancer therapies. Our previous study demonstrated that taxol induced altered maturation and improved viability of dendritic cells (DCs). However, the effects of taxol on DC viability have not been fully elucidated. In the present study, flow cytometric analyses revealed that taxol treatment significantly increased the number of viable DCs and the expression levels of a representative anti-apoptotic protein Bcl-xL. Furthermore, mobilization of the p65 subunit of nuclear factor-κB (NF-κB) from the cytosol to the nucleus in DCs was observed by confocal microscopy. An inhibition assay using N-p-tosyl-_L-phenylalanine chloromethyl ketone confirmed that NF-κB was intimately involved in the effects of taxol on DC viability. In addition, we investigated the mechanisms of taxol enhancement of DC viability. Since taxol is a popular anticancer agent used in clinic, this study may provide a rationale for the use of taxol in DC immunotherapy to treat cancer patients. Taken together, these results confirm that taxol increases DC viability, and this information may provide new insights for new clinical applications of both taxol and DCs.     

Dopamine Release Suppression Dependent on an Increase of Intracellular Ca2+ Contributed to Rotenone-induced Neurotoxicity in PC12 Cells

Rotenone is an inhibitor of mitochondrial complex I that produces a model of Parkinson’s disease (PD), in which neurons undergo dopamine release dysfunction and other features. In neurons, exocytosis is one of the processes associated with dopamine release and is dependent on Ca²⁺ dynamic changes of the cell. In the present study, we have investigated the exocytosis of dopamine and the involvement of Ca²⁺ in dopamine release in PC12 cells administrated with rotenone. Results demonstrated that rotenone led to an elevation of intracellular Ca²⁺ through Ca²⁺ influx by opening of the voltage-gated Ca²⁺ channel and influenced the soluble N-ethylmaleimide attachment protein receptor (SNARE) proteins expression (including syntaxin, vesicle-associated membrane protein 2 (VAMP₂) and synaptosome-associated protein 25 (SNAP-25)); pretreatment with a blocker of L-type voltage-activated Ca²⁺ channels (nifedipine) decreased the intracellular dopamine levels and ROS formation, increased the cell viability and enhanced the neurite outgrowth and exocytosis of synaptic vesicles. These results indicated that the involvement of intracellular Ca²⁺ was one of the factors resulting in suppression of dopamine release suppression in PC12 cells intoxicated with rotenone, which was associated with the rotenone-induced dopamine neurotoxicity.     

蒙古马母体与胎儿胃肠道NF-κB信号通路相关基因mRNA相对表达水平研究

[目的] 研究蒙古马母体与胎儿胃肠道不同区段NF-κB信号通路相关基因的mRNA相对表达水平。[方法] 选取3匹体况良好的妊娠蒙古马进行屠宰,屠宰过程中取出胎儿,分别采集母体及胎儿的胃、十二指肠、空肠、回肠、盲肠、大结肠、小结肠和直肠组织;采用实时荧光定量PCR方法（qPCR）,对NF-κB信号通路上6个相关基因（NF-κB p50、NF-κB p65、NFKBIA、IL-1β、TNF-α和IL-8）的mRNA相对表达量进行分析,比较母体和胎儿胃肠道不同区段组织中各基因的表达差异。[结果] NF-κB信号通路上6个基因在蒙古马母体和胎儿胃肠道不同区段8个组织中均有表达;总体来看,NF-κB p50、NF-κB p65和NFKBIA基因的mRNA相对表达量在母体和胎儿上均较高,IL-1β、TNF-α和IL-8基因的mRNA相对表达量均较低。在胃肠道区段8个组织中,6个基因的mRNA相对表达量基本呈现母体高于胎儿的特征;在回肠和盲肠组织中,母体NF-κB p50基因的mRNA相对表达量显著（P<0.05）高于胎儿;在大结肠组织中,母体NF-κB p65基因和NFKBIA基因的mRNA相对表达量显著（P<0.05）高于胎儿。[结论] NF-κB信号通路上6个基因在蒙古马母体和胎儿胃肠道8个组织中均有表达,且存在一定差异;母体部分肠道区段组织中的NF-κB p50、NF-κB p65、NFKBIA基因mRNA相对表达量显著高于胎儿。     

Periplaneta americana peptide decreases apoptosis of pig-ovary granulosa cells induced by H2O2 through FoxO1

Oxidative stress can induce apoptosis of granulosa cells and lead to follicular atresia, thereby reducing the number of pigs giving birth. The aim of this study was to investigate the protective effect of Periplaneta americana peptide (PAP) on the apoptosis of the granulosa cells of pig ovaries (PGCs) induced by hydrogen peroxide (H₂O₂) via FoxO1. PGCs were treated with H₂O₂ to establish a cell apoptosis model. Cell viability was measured using the cell counting kit-8 (CCK-8) assay, and cell apoptosis was detected using flow cytometry. The malondialdehyde (MDA) level and nitric oxide (NO) content were detected to reflect the oxidative stress. Western blotting, qRT-PCR and overexpression were undertaken to determine the expression of FoxO1 and caspase-3, and immunofluorescence was used to detect FoxO1 in the nucleus and cytoplasm. PGCs were treated with 100 μM H₂O₂ for 6 hr, which resulted in oxidative damage and apoptosis and an apoptosis rate for PGCs of 32.95%. Next, PGCs were treated with 400 μg/ml PAP for 24 hr to repair the apoptosis induced by H₂O₂. PAP improved cell viability in H₂O₂-stimulated PGCs, the increased MDA level and NO content caused by H₂O₂ stimulation were reversed and the apoptotic rate of PGCs was reduced. The qRT-PCR and Western blotting results indicated that PAP decreased the H₂O₂-induced apoptosis and the expression of FoxO1 and caspase-3 in PGCs. The effect of PAP was the same following FoxO1 overexpression. FoxO1 was expressed in the nucleus when stimulated by H₂O₂ or overexpression; however, it migrated to the cytoplasm following PAP treatment. PAP decreased the apoptosis of PGCs induced by H₂O₂ by regulating FoxO1 expression and nuclear translocation.     

Modulation of the immunogenicity of the Trypanosoma congolense cysteine protease, congopain, through complexation with ��2-macroglobulin

The protozoan parasite Trypanosoma congolense is the main causative agent of livestock trypanosomosis. Congopain, the major lysosomal cysteine proteinase of T. congolense, contributes to disease pathogenesis, and antibody-mediated inhibition of this enzyme may contribute to mechanisms of trypanotolerance. The potential of different adjuvants to facilitate the production of antibodies that would inhibit congopain activity was evaluated in the present study. Rabbits were immunised with the recombinant catalytic domain of congopain (C2), either without adjuvant, with Freund’s adjuvant or complexed with bovine or rabbit α₂-macroglobulin (α₂M). The antibodies were assessed for inhibition of congopain activity. Rabbits immunised with C2 alone produced barely detectable anti-C2 antibody levels and these antibodies had no effect on recombinant C2 or native congopain activity. Rabbits immunised with C2 and Freund’s adjuvant produced the highest levels of anti-C2 antibodies. These antibodies either inhibited C2 and native congopain activity to a small degree, or enhanced their activity, depending on time of production after initial immunisation. Rabbits receiving C2-α₂M complexes produced moderate levels of anti-C2 antibodies and these antibodies consistently showed the best inhibition of C2 and native congopain activity of all the antibodies, with maximum inhibition of 65%. Results of this study suggest that antibodies inhibiting congopain activity could be raised in livestock with a congopain catalytic domain-α₂M complex. This approach improves the effectiveness of the antigen as an anti-disease vaccine candidate for African trypanosomosis.     

Vitamin E regulates bovine granulosa cell apoptosis via NRF2-mediated defence mechanism by activating PI3K/AKT and ERK1/2 signalling pathways

High-yield dairy cows are usually subject to high-intensive cell metabolism and produce excessive reactive oxygen species (ROS). Once ROS is beyond the threshold of scavenging ability, it can induce oxidative stress, imperilling the reproductive performance of cows. The study was to investigate the effects of vitamin E (VE) on H₂O₂-induced proliferation and apoptosis of bovine granulosa cells and the underlying molecular mechanism. Granulosa cells were pretreated with VE for 24 hr and then treated with H₂O₂ for 6 hr. The results showed that VE treatment decreased the intracellular ROS levels, increased the MDA content, and improved the antioxidant enzyme activity in a dose-dependent manner. Furthermore, VE treatment promoted the proliferation and inhibited apoptosis in granulosa cells by up-regulation of CCND1 and BCL2 levels and down-regulation of P21, BAX, and CASP3 levels. The cytoprotective effects of VE were attributed to the activation of the NRF2 signalling pathway. Knockdown of the NRF2 impaired the cytoprotective effects of VE on granulosa cells. Besides, the PI3K/AKT and ERK1/2, but not the p38 signalling pathway is involved in the regulation of VE-mediated cell proliferation and apoptosis. The PI3K/AKT inhibitor LY294002 and ERK1/2 inhibitor SCH772984 inhibited the VE-induced granulosa cell proliferation and promoted apoptosis, whereas the p38 inhibitor SB203580 had the opposite effects. These results were confirmed by proliferation and apoptosis-related gene expression at mRNA and protein levels. The results also showed that the PI3K/AKT inhibitor LY294002 and ERK1/2 inhibitor SCH772984 inhibited VE-induced NRF2, GCLC, GCLM, and HO-1 expression, whereas the p38 inhibitor SB203580 not. Overall, the results demonstrated that VE-regulated granulosa cell proliferation and apoptosis via NRF2-mediated defence system by activating the PI3K/AKT and ERK1/2 signalling pathway.     

Oxidative Stress Produced by Xanthine Oxidase Induces Apoptosis in Human Extravillous Trophoblast Cells

Oxidative stress has been recognized as an important factor in the pathophysiology of preeclampsia. It has been reported that the expression of xanthine oxidase (XO) in the cytotrophoblast and plasma hydrogen peroxide (H₂O₂) level are significantly higher in preeclamptics than in control women. The aim of this study was to clarify the biological influence of reactive oxygen species (ROS) produced by XO on extravillous trophoblast (EVT) cells. TCL1 cells, a human immortalized EVT cell line, were incubated with xanthine and XO (X/XO). We then measured the cell number, urate level of the culture media and the apoptotic cell ratio. Similar experiments were performed with additional administration of allopurinol, catalase, L-NAME or D-NAME, and with administration of H₂O₂ in substitution for X/XO. We assessed the effects of H₂O₂ on invasion ability, tube-like formation and protein expression of HIF1A and ITGAV of TCL1. Finally, the apoptotic cell ratio using primary cultured trophoblasts was measured following exposure to H₂O₂. X/XO decreased the relative cell number and increased the urate level and apoptotic cell ratio significantly. Elevation of the urate level and apoptotic cell ratio was attenuated by allopurinol and catalase, respectively. L-NAME and D-NAME had no influence on these effects. H₂O₂ also decreased the relative cell number. Pretreatment with H₂O₂ significantly inhibited the invasion ability, tube-like formation and HIF1A and ITGAV of TCL1. H₂O₂ also induced apoptosis in primary cultured trophoblasts. In conclusion, ROS produced by XO induced apoptosis and affected EVT function including invasion and differentiation.     

Estrogen receptor independent neurotoxic mechanism of bisphenol A, an environmental estrogen

Bisphenol A (BPA), a ubiquitous environmental contaminant, has been shown to cause developmental toxicity and carcinogenic effects. BPA may have physiological activity through estrogen receptor (ER) -α and -β, which are expressed in the central nervous system. We previously found that exposure of BPA to immature mice resulted in behavioral alternation, suggesting that overexposure of BPA could be neurotoxic. In this study, we further investigated the molecular neurotoxic mechanisms of BPA. BPA increased vulnerability (decrease of cell viability and differentiation, and increase of apoptotic cell death) of undifferentiated PC12 cells and cortical neuronal cells isolated from gestation 18 day rat embryos in a concentration-dependent manner (more than 50 µM). The ER antagonists, ICI 182,780, and tamoxifen, did not block these effects. The cell vulnerability against BPA was not significantly different in the PC12 cells overexpressing ER-α and ER-β compared with PC12 cells expressing vector alone. In addition, there was no difference observed between BPA and 17-β estradiol, a well-known agonist of ER receptor in the induction of neurotoxic responses. Further study of the mechanism showed that BPA significantly activated extracellular signal-regulated kinase (ERK) but inhibited anti-apoptotic nuclear factor kappa B (NF-κB) activation. In addition, ERK-specific inhibitor, PD 98,059, reversed BPA-induced cell death and restored NF-κB activity. This study demonstrated that exposure to BPA can cause neuronal cell death which may eventually be related with behavioral alternation in vivo. However, this neurotoxic effect may not be directly mediated through an ER receptor, as an ERK/NF-κB pathway may be more closely involved in BPA-induced neuronal toxicity.     

Feline adipose tissue-derived mesenchymal stem cells pretreated with IFN-γ enhance immunomodulatory effects through the PGE2 pathway

BackgroundPreconditioning with inflammatory stimuli is used to improve the secretion of anti-inflammatory agents in stem cells from variant species such as mouse, human, and dog. However, there are only few studies on feline stem cells.ObjectivesThis study aimed to evaluate the immune regulatory capacity of feline adipose tissue-derived (fAT) mesenchymal stem cells (MSCs) pretreated with interferon-gamma (IFN-γ).MethodsTo assess the interaction of lymphocytes and macrophages with IFN-γ-pretreated fAT-MSCs, mouse splenocytes and RAW 264.7 cells were cultured with the conditioned media from IFN-γ-pretreated MSCs.ResultsPretreatment with IFN-γ increased the gene expression levels of cyclooxygenase-2, indoleamine 2,3-dioxygenase, hepatocyte growth factor, and transforming growth factor-beta 1 in the MSCs. The conditioned media from IFN-γ-pretreated MSCs increased the expression levels of M2 macrophage markers and regulatory T-cell markers compared to those in the conditioned media from naive MSCs. Further, prostaglandin E₂ (PGE₂) inhibitor NS-398 attenuated the immunoregulatory potential of MSCs, suggesting that the increased PGE₂ levels induced by IFN-γ stimulation is a crucial factor in the immune regulatory capacity of MSCs pretreated with IFN-γ.ConclusionsIFN-γ pretreatment improves the immune regulatory profile of fAT-MSCs mainly via the secretion of PGE₂, which induces macrophage polarization and increases regulatory T-cell numbers.     

β-irradiation (166Ho patch)-induced skin injury in mini-pigs: effects on NF-κB and COX-2 expression in the skin

In the present study, the detrimental effect of β-emission on pig skin was evaluated. Skin injury was modeled in mini-pigs by exposing the animals to 50 and 100 Gy of β-emission delivered by ¹⁶⁶Ho patches. Clinicopathological and immunohistochemical changes in exposed skin were monitored for 18 weeks after β-irradiation. Radiation induced desquamation at 2~4 weeks and gradual repair of this damage was evident 6 weeks after irradiation. Changes in basal cell density and skin depth corresponded to clinically relevant changes. Skin thickness began to decrease 1 week after irradiation, and the skin was thinnest 4 weeks after irradiation. Skin thickness increased transiently during recovery from irradiation-induced skin injury, which was evident 6~8 weeks after irradiation. Epidermal expression of nuclear factor-kappa B (NF-κB) differed significantly between the untreated and irradiated areas. One week after irradiation, cyclooxygenase-2 (COX-2) expression was mostly limited to the basal cell layer and scattered among these cells. High levels of COX-2 expression were detected throughout the full depth of the skin 4 weeks after irradiation. These findings suggest that NF-κB and COX-2 play roles in epidermal cell regeneration following β-irradiation of mini-pig skin.     

Inhibition of lipopolysaccharide-induced suppression of luteal function in isolated perfused bovine ovaries

Recently, we observed that lipopolysaccharide (LPS) suppresses corpus luteum (CL) function in isolated perfused ovaries. It remained unclear if this suppression was due to increased luteal PGF_2α secretion or LPS-induced apoptosis. Therefore, possible impacts of PGF_2α and LPS were inhibited by a non-steroidal anti-inflammatory drug (flunixin) and an endotoxin-binding agent (polymyxin B), respectively. Bovine ovaries with a mid-cycle CL were collected immediately after slaughter and perfused for 240 min. After 50 min of equilibration, either flunixin or polymyxin B (5 μg/ml of each) were added to the perfusion medium of six ovaries, respectively. All ovaries (n = 12) were treated with E. coli LPS (0.5 μg/ml) 60 min after the onset of perfusion, and received 500 I.U. of hCG after 210 min of perfusion. Progesterone and PGF_2α were measured in the effluent perfusate every 10 and 30 min, respectively. Biopsies of the CL were collected every 60 min to determine the mRNA expression of the cytokine TNFA and factors of apoptosis (CASP3, -8). Flunixin-treatment inhibited the increase of PGF_2α after LPS-challenge that was observed in the polymyxin B-treated (PX-LPS) ovaries. After hCG-stimulation, progesterone secretion increased (P < 0.05) in group PX-LPS but not in the flunixin-treated (F-LPS) ovaries. Compared to initial values before LPS-challenge, luteal mRNA expression of TNFA and CASP3 was increased (P < 0.05) in group F-LPS at 120 and 180 min, respectively, and those of CASP8 was decreased (P < 0.05) in PX-LPS at 60 and 120 min after LPS-treatment. In conclusion, although flunixin managed to inhibit PGF_2α, it did not suffice to successfully prevent LPS-induced apoptosis. However, endotoxin-binding polymyxin B resulted in luteal responsiveness to hCG after LPS-challenge.