Berberine ameliorates iron levels and ferroptosis in the brain of 3 × Tg-AD mice.

BACKGROUND: Berberine (BBR) is a natural alkaloid extracted from the herb Coptis chinensis. This compound has the ability to penetrate the blood-brain barrier (BBB) and exhibit neuroprotective value in the treatment of Alzheimer's disease (AD). AD is a neurodegenerative disease characterized by ß-amyloid (Aß) deposition, hyperphosphorylated tau and other characters. Iron accumulation and ferroptosis were also detected in AD brain, which can result in neuronal damage. However, it is still unclear whether BBR can suppress ferroptosis in AD and alleviate its underlying pathology. PURPOSE: This study investigated whether BBR may affect ferroptosis and related signaling pathways in triple transgenic AD (3 × Tg-AD) mice. METHODS: Four-month-old 3 × Tg-AD mice received oral administration of BBR at a dose of 50 mg/kg for 7.5 months. Cognitive function and anxiety levels in mice were assessed using the morris water maze test, open field test, and novel object recognition test. Western blot, immunohistochemistry, and ICP-MS were employed to assess the pathology of AD, brain iron metabolism, and ferroptosis signaling pathways. Transmission electron microscopy was used to detect mitochondrial changes. The synergistic effects of BBR combined with Nrf2 were investigated using molecular docking programs and surface plasmon resonance technology. Co-inmunoprecipitation assay was used to examine the effect of BBR on the binding ability of Nrf2 and Keap1. RESULTS: The results indicated that chronic treatment of BBR mitigated cognitive disorders in 3 × Tg-AD model mice. Reductions in Aß plaque, hyperphosphorylated tau protein, neuronal loss, and ferroptosis in the brains of 3 × Tg-AD mice suggested that BBR could alleviate brain injury. In addition, BBR treatment attenuated ferroptosis, as evidenced by decreased levels of iron, MDA, and ROS, while enhancing SOD, GSH, GPX4, and SLC7A11. Consistent with the in vivo assay, BBR inhibited RSL3-induced ferroptosis in N2a-sw cells. BBR increased the expression levels of GPX4, FPN1 and SLC7A11 by regulating Nrf2 transcription levels, thereby inhibiting ferroptosis. Molecular docking programs and surface plasmon resonance technology demonstrated the direct combination of BBR with Nrf2. Co-inmunoprecipitation analysis showed that BBR inhibited the interaction between Keap1 and Nrf2. CONCLUSION: For the first time, these results showed that BBR could inhibit iron levels and ferroptosis in the brains of 3 × Tg-AD model mice and partially protect against RSL3-induced ferroptosis via the activation of Nrf2 signaling.

Alteration of faecal microbiota balance related to long-term deep meditation.

Background: Advancements in research have confirmed that gut microbiota can influence health through the microbiota-gut-brain axis. Meditation, as an inner mental exercise, can positively impact the regulation of an individual's physical and mental health. However, few studies have comprehensively investigated faecal microbiota following long-term (several years) deep meditation. Therefore, we propose that long-term meditation may regulate gut microbiota homeostasis and, in turn, affect physical and mental health. Aims: To investigate the effects of long-term deep meditation on the gut microbiome structure. Methods: To examine the intestinal flora, 16S rRNA gene sequencing was performed on faecal samples of 56 Tibetan Buddhist monks and neighbouring residents. Based on the sequencing data, linear discriminant analysis effect size (LEfSe) was employed to identify differential intestinal microbial communities between the two groups. Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) analysis was used to predict the function of faecal microbiota. In addition, we evaluated biochemical indices in the plasma. Results: The α-diversity indices of the meditation and control groups differed significantly. At the genus level, Prevotella and Bacteroides were significantly enriched in the meditation group. According to the LEfSe analysis, two beneficial bacterial genera (Megamonas and Faecalibacterium) were significantly enriched in the meditation group. Functional predictive analysis further showed that several pathways-including glycan biosynthesis, metabolism and lipopolysaccharide biosynthesis-were significantly enriched in the meditation group. Moreover, plasma levels of clinical risk factors were significantly decreased in the meditation group, including total cholesterol and apolipoprotein B. Conclusions: Long-term traditional Tibetan Buddhist meditation may positively impact physical and mental health. We confirmed that the gut microbiota composition differed between the monks and control subjects. The microbiota enriched in monks was associated with a reduced risk of anxiety, depression and cardiovascular disease and could enhance immune function. Overall, these results suggest that meditation plays a positive role in psychosomatic conditions and well-being.

Effects of Selenoprotein S Knockdown on Endoplasmic Reticulum Stress in ATDC5 Cells and Gene Expression Profiles in Hypertrophic Chondrocytes.

Selenoprotein S (SelS), a member of the selenoprotein family, is mainly located on the endoplasmic reticulum (ER) membrane. SelS is involved in a variety of biological processes, including oxidative stress, inflammation, glucose metabolism regulation, and ER-associated protein degradation (ERAD). This study was designed to explore the role of SelS in chondrocytes. It was confirmed that SelS is a Se-sensitive selenoprotein in low-selenium rat and cell models. ER stress was not induced in SelS knockdown ATDC5 cells. However, treatment of ATDC5 cells with tunicamycin (Tm), an ER stress inducer, increased the expression of SelS, and knockdown of SelS aggravated ER stress induced by Tm, suggesting that SelS is a regulatory molecule involved in ER stress in chondrocytes. Both osteoarthritis and Kashin-Beck disease are osteochondral diseases associated with hypertrophic chondrocyte abnormalities. Therefore, ATDC5 cells were induced to hypertrophic chondrocytes. SelS was knocked down and RNA sequencing was performed. Bioinformatics analysis of the differentially expressed genes (DEGs) revealed that SelS knockdown affected a variety of biological processes, including cell adhesion, osteoclast differentiation, and extracellular matrix homeostasis. Collectively, this study verified that SelS is sensitive to selenium levels and is an ER stress-responsive molecule. Knocking down SelS can cause abnormal expression of adhesion molecules and matrix homeostasis disorder in hypertrophic chondrocytes.

MMP-10 Deficiency Effects Differentiation and Death of Chondrocytes Associated with Endochondral Osteogenesis in an Endemic Osteoarthritis.

OBJECTIVE: The objective of this study was to determine the matrix metalloproteinase-10 (MMP-10) expression pattern and to assess how it contributes to endochondral osteogenesis in Kashin-Beck disease (KBD). DESIGN: The cartilages of KBD patients, Sprague-Dawley rats fed with selenium (Se)-deficient diet and/or T-2 toxin, and ATDC5 cells were used in this study. ATDC5 cells were induced into hypertrophic chondrocytes using a 1% insulin-transferrin-selenium (ITS) culture medium for 21 days. The expressions of MMP-10 in the cartilages were visualized by immunohistochemistry. The messenger RNA (mRNA) and protein expression levels were determined by real-time polymerase chain reaction (RT-PCR) and Western blotting. MMP-10 short hairpin RNA (shRNA) was transfected into hypertrophic chondrocytes to knock down the gene expression of MMP-10. Meanwhile, the cell death of MMP-10-knockdown chondrocyte was detected using flow cytometry. RESULTS: The expression of MMP-10 was decreased in the growth plates of children with KBD. A decreased expression of MMP-10 also was observed in the growth plates of rats fed with an Se-deficient diet and/or T-2 toxin exposure. The mRNA and protein expression levels of MMP-10 increased during the chondrogenic differentiation of ATDC5 cells. MMP-10 knockdown in hypertrophic chondrocytes significantly decreased the gene and protein expression of collagen type II (Col II), Col X, Runx2, and MMP-13. Besides, the percentage of cell apoptosis was significantly increased after MMP-10 knockdown in hypertrophic chondrocytes. CONCLUSION: MMP-10 deficiency disrupts chondrocyte terminal differentiation and induces the chondrocyte's death, which impairs endochondral osteogenesis in the pathogenesis of KBD.

Downregulation of Insulin-Like Growth Factor-1 Receptor Mediates Chondrocyte Death and Matrix Degradation in Kashin-Beck Disease.

PURPOSE: To explore the relationship between insulin-like growth factor (IGF)-1R expression and the pathological progression of Kashin-Beck disease (KBD). DESIGN: KBD cartilage samples were collected from 5 patients. Additionally, T-2 toxin was administered to rats fed a selenium (Se)-deficient diet, and their knee joints were collected. Human C28/I2 chondrocytes and mouse hypertrophic ATDC5 chondrocytes were cultured in vitro and treated with T-2 toxin and Se supplementation. Subsequently, the cultured human and mouse chondrocytes were treated with the IGF-1R inhibitor, picropodophyllin. Chondrocyte death and caspase-3 activity were analyzed using flow cytometry and a specific kit, respectively. Protein and mRNA expression levels of IGF-1R and matrix molecules were measured using immunohistochemistry, western blotting, and quantitative real-time reverse transcription-polymerase chain reaction analyses. RESULTS: The cartilages from patients with KBD and T-2 toxin-treated rats on a Se-deficient diet showed significantly decreased expression of IGF-1R compared to cartilages from controls. T-2 toxin decreased IGF-1R mRNA and protein levels in both C28/I2 and hypertrophic ATDC5 chondrocytes in a dose-dependent manner; however, Se supplementation reduced the decrease of IGF-1R induced by T-2 toxin. Furthermore, inhibition of IGF-1R resulted in chondrocyte death of C28/I2 and hypertrophic ATDC5 chondrocytes, as well as decreased type II collagen expression and increased MMP-13 expression at the mRNA and protein levels. CONCLUSION: Downregulation of IGF-1R was associated with KBD cartilage destruction. Therefore, inhibition of IGF-1R may mediate chondrocyte death and extracellular matrix degeneration related to the pathological progression of KBD.

Reduction of Smad2 caused by oxidative stress leads to necrotic death of hypertrophic chondrocytes associated with an endemic osteoarthritis.

OBJECTIVE: The occurrence and development of an endemic OA, Kashin-Beck disease (KBD), is closely related to oxidative stress induced by free radicals. The aim of the study was to find the key signalling molecules or pathogenic factors as a potential treatment strategy for KBD. METHODS: Real-time PCR and western blotting were performed to detect the mRNA and protein expression levels in cells and tissues. Immunohistochemical staining was assayed in rat models and human samples obtained from children. The type of cell death was identified by annexin V and propidium iodide staining with flow cytometry. RESULTS: Oxidative stress decreased levels of Smad2 and Smad3 in hypertrophic chondrocytes both in vitro and in vivo. In the cartilage of KBD patients, the expression of Smad2 and Smad3 proteins in the middle and deep zone was significantly decreased with an observed full deletion in the deep zone of some samples. Reduction of Smad2 protein induced necrotic death of hypertrophic chondrocytes, while reduction of Smad3 protein induced apoptosis. The reduction of Smad2 protein was not accompanied by Smad3 protein reduction in hypertrophic chondrocyte necrosis. Furthermore, the reduction of Smad2 also impaired the construction of tissue-engineered cartilage in vitro. CONCLUSION: These studies reveal that oxidative stress causes necrosis of hypertrophic chondrocytes by downregulating Smad2 protein, which increases the pathogenesis of KBD cartilage. The importance of Smad2 in the development of KBD provides a new potential target for the treatment of KBD.

LncRNA MIAT regulated by selenium and T-2 toxin increases NF-κB-p65 activation, promoting the progress of Kashin-Beck Disease.

LncRNA myocardial infarction associated transcript (MIAT) has been shown to be involved in osteoarthritis (OA), but its role in Kashin-Beck Disease (KBD) has rarely been reported. In this study, rats were administered with low selenium and/or T-2 toxin for 4 weeks to establish a KBD animal model. The serum selenium level, TNF-α and IL-1ß contents, phosphorylated p65 (p-p65) and MIAT expression were increased in each intervention group. Next, we isolated the primary epiphyseal chondrocytes, and found that selenium treatment reversed the effects of T-2 toxin on chondrocyte injury, p-p65 and MIAT expression. In addition, MIAT overexpression or T-2 toxin treatment led to increased cell death, apoptosis, inflammation, NF-κB-p65 pathway activation and MIAT expression, which was rescued by selenium treatment or MIAT siRNA transfection. Our results suggested that lncRNA MIAT regulated by selenium and T-2 toxin increased the activation of NF-κB-p65, thus being involved in the progress of KBD.

Decreased Expression of Heat Shock Protein 47 Is Associated with T-2 Toxin and Low Selenium-Induced Matrix Degradation in Cartilages of Kashin-Beck Disease.

Recent evidence suggests a role of type II collagen in Kashin-Beck disease (KBD) degeneration. We aimed to assess the abnormal expression of heat shock protein 47 (HSP47) which is associated with a decrease in type II collagen and an increase in cartilage degradation in KBD. Hand phalange cartilages were collected from KBD and healthy children. Rats were administered with T-2 toxin under the selenium (Se)-deficient diet. ATDC5 cells were seeded on bone matrix gelatin to construct engineered cartilaginous tissue. C28/I2 and ATDC5 cells and engineered tissue were exposed to different concentrations of T-2 toxin with or without Se. Cartilage degeneration was determined through histological evaluation. The distribution and expression of type II collagen and HSP47 were investigated through immunohistochemistry, western blotting, and real-time PCR. KBD cartilages showed increased chondronecrosis and extracellular matrix degradation in deep zone with decreased type II collagen and HSP47 expression. The low-Se + T-2 toxin animal group showed a significantly lower type II collagen expression along with decreased HSP47 expression. Decreased type II collagen and HSP47 in C28/I2 and ATDC5 cells induced by T-2 toxin showed a dose-dependent manner. Hyaline-like cartilage with zonal layers was developed in engineered cartilaginous tissues, with decreased type II collagen and HSP47 expression found in T-2 toxin-treated group. Se-supplementation partially antagonized the inhibitory effects of T-2 toxin in chondrocytes and cartilages. HSP47 plays a role in the degenerative changes of KBD and associated with T-2 toxin-induced decreased type II collagen expression, further promoting matrix degradation.

Expression and localization of the small proteoglycans decorin and biglycan in articular cartilage of Kashin-Beck disease and rats induced by T-2 toxin and selenium deficiency.

Kashin-Beck disease (KBD) is an endemic degenerative osteoarthropathy of uncertain etiology. Our study sought to identify a correlation between small proteoglycans decorin and biglycan expression and Kashin-Beck Disease. Immunohistochemistry was used to assess the decorin and biglycan levels in cartilage specimens from both child KBD patients, and rats fed with T-2 toxin under a selenium-deficient condition. Real-time PCR and Western blot were used to assess mRNA and protein levels of decorin and biglycan in rat cartilages, as well as in C28/I2 chondrocytes stimulated by T-2 toxin and selenium in vitro. The result showed that decorin was reduced in all zones of KBD articular cartilage, while the expression of biglycan was prominently increased in KBD cartilage samples. Increased expression of biglycan and reduced expression of decorin were observed at mRNA and protein levels in the cartilage of rats fed with T-2 toxin and selenium- deficiency plus T-2 toxin diet, when compared with the normal diet group. Moreover, In vitro stimulation of C28/I2 cells with T-2 toxin resulted in an upregulation of biglycan and downregulation of decorin, T-2 toxin induction of biglycan and decorin levels were partly rescued by selenium supplement. This study highlights the focal nature of the degenerative changes that occur in KBD cartilage and may suggest that the altered expression pattern of decorin and biglycan have an important role in the onset and pathogenesis of KBD.

Gene Expression Profile of Hypertrophic Chondrocytes Treated with H2O2: A Preliminary Investigation.

Objective To identify the osteogenesis genes whose expression is altered in hypertrophic chondrocytes treated with H2O2. Methods Murine chondrogenitor cells (ATDC5) were differentiated into hypertrophic chondrocytes by Insulin-Transferrin-Selenium (ITS) treatment, and then treated with H2O2. Suitable conditions (concentration, time) were determined by using the MTT assay. After total RNA isolation and cDNA synthesis, the levels of 84 genes were determined using the PCR array, whereas quantitative RT-PCR was carried out to validate the PCR array data. Result We identified 9 up-regulated genes and 12 down-regulated genes, encoding proteins with various functions, such as collagen proteins, transcription factors, proteins involved in skeletal development and bone mineral metabolism, as well as cell adhesion molecules. Quantitative RT-PCR confirmed the altered expression of 5 down-regulated genes (Smad2, Smad4, transforming growth factor $\beta$ receptor 1, transforming growth factor $\beta$ receptor 3, and matrix metalloproteinase 10). Conclusions H2O2 significantly changed the expression of several genes involved in a variety of biological functions. Because of the link between oxidative damage and Kashin-Beck disease, these genes may also be involved in the deep-zone necrosis of the cartilage observed in Kashin-Beck disease.

T-2 Toxin Alters the Levels of Collagen II and Its Regulatory Enzymes MMPs/TIMP-1 in a Low-Selenium Rat Model of Kashin-Beck Disease.

The objectives of this study are to assess T-2 toxin's involvement in low selenium (Se)-induced Kashin-Beck disease (KBD) in rats and unveil the mechanisms underlying this disease. Two hundred thirty rats were randomly divided into two groups after weaning and fed normal or low-Se diets (n = 115), respectively, for a month. After low-Se model confirmation, rats in each group were subdivided into five: two subgroups (n = 20) were fed their current diets (normal or low-Se diets, respectively) for 30 and 90 days, respectively; two other subgroups (n = 25) received their current diets + low T-2 toxin (100 ng/g BW/day) for 30 and 90 days, respectively; and 25 rats were fed their current diets + high T-2 toxin (200 ng/g BW/day) for 30 days. Articular cartilage samples were extracted for hematoxylin and eosin (H&E) staining and immunohistochemistry. Western blot and reverse transcription-polymerase chain reaction (RT-PCR) were used to assess protein and mRNA levels, respectively, of collagen II, matrix metalloproteinase (MMP-1), MMP -3, MMP-13, and tissue inhibitor of metalloproteinase-1 (TIMP-1). Low Se and T-2 toxin synergistically affected animal fitness. Interestingly, low Se + T-2 toxin groups showed KBD characteristics. MMP-1, -3, and -13 mRNA and protein levels generally increased in low-Se groups, while collagen II and TIMP-1 levels showed a downward trend, compared with normal diet fed animals for the same treatment (P < 0.05). T-2 toxin's effect was dose but not time dependent. Low Se and T-2 toxin synergistically alter the expression levels of collagen II as well as its regulatory enzymes MMP-1, MMP-3, MMP-13, and TIMP-1, inducing cartilage damage. Therefore, T-2 toxin may cause KBD in low-Se conditions.

Increased levels of IL-6, IL-1ß, and TNF-α in Kashin-Beck disease and rats induced by T-2 toxin and selenium deficiency.

The objective of this study is to investigate the possible role of inflammatory mediators such as IL-6, IL-1ß, and TNF-α in Kashin-Beck disease (KBD) children and rats fed with T-2 toxin under a selenium-deficient nutrition status in order to determine possible mechanism underlying KBD. Sprague-Dawley rats were administered a selenium-deficient diet for 4 weeks prior to their exposure to T-2 toxin for 4 weeks. The morphology of joint cartilages of KBD children and rats was examined by light microscopy, and the expression of proteoglycans was determined by histochemical staining. The serum levels of IL-6, IL-1ß, and TNF-α were determined by enzyme-linked immunosorbent assay. IL-6, IL-1ß and TNF-α were localized by immunohistochemistry, and their mRNA levels were detected by real-time RT-PCR. The serum levels of IL-6 were significantly elevated in rats fed with selenium-deficient, T-2 toxin, and T-2 toxin plus selenium-deficient diets compared to those in the normal diet, while the serum levels of IL-1ß and TNF-α were significantly increased only in the T-2 toxin plus selenium-deficient diet group. IL-6, IL-1ß and TNF-α protein and mRNA levels in cartilage were significantly higher in rats with diets of T-2 toxin and T-2 toxin plus selenium deficiency than in rats fed normal or selenium-deficient diet. While staining for the cytokines in cartilages of KBD children was significantly higher than that in controls. T-2 toxin under a selenium-deficient nutritional status induces increased levels of IL-6, IL-1ß, and TNF-α in serum and cartilages, which may account for the pathological mechanism underlying the cartilage damage in KBD.

Protective effects of selenium on oxidative damage and oxidative stress related gene expression in rat liver under chronic poisoning of arsenic.

Arsenic (As) is a toxic metalloid existing widely in the environment, and chronic exposure to it through contaminated drinking water has become a global problem of public health. The present study focused on the protective effects of selenium on oxidative damage of chronic arsenic poisoning in rat liver. Rats were divided into four groups at random and given designed treatments for 20 weeks. The oxidative damage of liver tissue was evaluated by lipid peroxidation and antioxidant enzymes. Oxidative stress related genes were detected to reflect the liver stress state at the molecular level. Compared to the control and Na2SeO3 groups, the MDA content in liver tissue was decreased and the activities of antioxidant enzymes were increased in the Na2SeO3 intervention group. The mRNA levels of SOD1, CAT, GPx and Txnrd1 were increased significantly (P<0.05) in the combined Na2SeO3+NaAsO2 treatment group. The expressions of HSP70 and HO-1 were significantly (P<0.05) increased in the NaAsO2 group and reduced in the combined treatment group. The results indicate that long-term intake of NaAsO2 causes oxidative damage in the rat liver, and Na2SeO3 protects liver cells by adjusting the expression of oxidative stress related genes to improve the activities of antioxidant enzymes.

Histopathology of chondronecrosis development in knee articular cartilage in a rat model of Kashin-Beck disease using T-2 toxin and selenium deficiency conditions.

The objective of this study is to observe pathogenic lesions of joint cartilages in rats fed with T-2 toxin under a selenium deficiency nutrition status in order to determine possible etiological factors causing Kashin-Beck disease (KBD). Sprague-Dawley rats were fed selenium-deficient or control diets for 4 weeks prior to their being exposed to T-2 toxin. Six dietary groups were formed and studied 4 weeks later, i.e., controls, selenium-deficient, low T-2 toxin, high T-2 toxin, selenium-deficient diet plus low T-2 toxin, and selenium-deficient diet plus high T-2 toxin. Selenium deficiencies were confirmed by the determination of glutathione peroxidase activity and selenium levels in serum. The morphology and pathology (chondronecrosis) of knee joint cartilage of experimental rats were observed using light microscopy and the expression of proteoglycans was determined by histochemical staining. Chondronecrosis in deep zone of articular cartilage of knee joints was seen in both the low and high T-2 toxin plus selenium-deficient diet groups, these chondronecrotic lesions being very similar to chondronecrosis observed in human KBD. However, the chondronecrosis observed in the rat epiphyseal growth plates of animals treated with T-2 toxin alone or T-2 toxin plus selenium-deficient diets were not similar to that found in human KBD. Our results indicate that the rat can be used as a suitable animal model for studying etiological factors contributing to the pathogenesis (chondronecrosis) observed in human KBD. However, those changes seen in epiphyseal growth plate differ from those seen in human KBD probably because of the absence of growth plate closure in the rat.

The effects of mycotoxins and selenium deficiency on tissue-engineered cartilage.

OBJECTIVE: To investigate the effects of 3 mycotoxins, deoxynivalenol (DON), nivalenol (NIV) and T-2 toxin, in the presence and absence of selenium (Se) on the metabolism of tissue-engineered cartilage to mimic conditions found in Kashin-Beck disease (KBD) environments. MATERIALS AND METHODS: Chondrocytes were seeded onto bone matrix gelatin (BMG) to construct engineered cartilage. The 3 toxins were added to the culture media for 3 weeks followed by immunhistochemical analyses of collagens type II and X, aggrecan, matrix metalloproteinases 1 and 3 (MMP-1 and MMP-3), MMP inhibitors 1 and 3 (TIMP-1 and TIMP-3) and α(2) macroglobulin (α2M). RESULTS: Type II collagen was decreased while type X collagen was increased in response to DON, NIV and T-2 toxin. Aggrecan was reduced by all 3 mycotoxins. Compared with the control, the 3 toxins decreased the expression of α2M, TIMP-1 and TIMP-3, and increased the expression of MMP-1 and MMP-3. Se could partially inhibit the effects of DON, NIV and T-2 toxins. CONCLUSION: Under the low Se condition, the 3 mycotoxins produced procatabolic changes in cartilage resulting in the loss of aggrecan and type II collagen and promoted a hypertrophic phenotype of chondrocytes characterized by increasing type-X-collagen expression, enhancing the expression of MMPs, while weakening the TIMPs. Se could partially block the effects mentioned above. These results support the hypothesis that the combination of mycotoxin stress and Se deficiency would be the causative factors for KBD.

Luteolin induces mitochondria-dependent apoptosis in human lung adenocarcinoma cell.

The effects of luteolin on the proliferation of A549 cells were evaluated by MTT and clone formation assays. DNA ploidy and apoptotic cell percentage were calculated by flow cytometry. The expression of Bax, Bcl-xl, Bcl-2, Mcl-1, caspase-9, caspase-3, and PARP was analyzed by Western blotting. The membrane potential of mitochondria was detected by JC-1 fluorescence microscopy assay. Our results demonstrated that luteolin could inhibit the proliferation of A549 cells via induction of apoptosis, with the evidence of characteristic morphological changes of apoptosis in the nucleus. Furthermore, DNA flow cytometric analysis indicated that luteolin induced a S phase arrest of the cell cycle. The membrane potential of mitochondria was decreased. The protein levels of Bax, Bcl-xl, Bcl-2, Mcl-1, caspase-9, caspase-3, and PARP were activated after treatment with luteolin. Luteolin can inhibit the proliferation of A549 cells and trigger mitochondria- dependent apoptosis in them.

Oxidant damage in Kashin-Beck disease and a rat Kashin-Beck disease model by employing T-2 toxin treatment under selenium deficient conditions.

Kashin-Beck disease (KBD) is an endemic degenerative osteoarthropathy, but the mechanisms underlying its pathogenesis remains unclear. This study compares antioxidant capacity and lipid peroxidation using a novel model, in which rats were administered a selenium-deficient diet for 4 weeks prior to their exposure to T-2 toxin for 4 weeks. Changes in cell morphology and empty chondrocyte lacunae indicative of cell death, as well as cartilage proteoglycan loss in the deep zone of articular cartilage of knee joints were observed in rats with selenium-deficient diet plus T-2 toxin treatment. These changes were similar to those observed previously in KBD. The levels of thiobarbituric acid reactive substances (TBARS), indicative of lipid peroxidation in serum and cartilage, were significantly increased in all experimental groups compared to the normal diet group, while the levels of antioxidants, measured as total antioxidant capacity (T-AOC), catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidases (GPX), in serum and cartilage were significantly lower than that in the normal diet group. The mRNA expression of those antioxidants in cartilage tissue was significantly reduced by T-2 toxin alone or by selenium-deficient diet plus T-2 toxin treatment. These results indicate that increasing TBARS and decreasing antioxidants in serum and cartilage by T-2 toxin treatment with a selenium-deficient nutritional status may alter oxidative stress in joint tissues and contribute to the pathological process of cartilage damage in KBD.

Effects of T-2 toxin and selenium on chondrocyte expression of matrix metalloproteinases (MMP-1, MMP-13), α2-macroglobulin (α2M) and TIMPs.

T-2 toxin is regarded as an important etiological factor of Kashin-Beck disease, and supplementation of selenium-salt partly prevents Kashin-Beck disease. The present study investigated the effects of T-2 toxin on the degradation of type II collagen in human chondrocytes in vitro. Human chondrocytes were isolated and cultured on bone matrix gelatin to form an artificial cartilage model in vitro with or without T-2 toxin and selenium. Immunohistochemistry analyses showed that T-2 toxin decreased type II collagen staining and selenium appeared to prevent the decrease in type II collagen induced by T-2 toxin in engineered cartilage. Then, Western blot and RT-PCR analyses showed that an increase in MMP-13 and MMP-1 expressions, and a decrease in the expression of the general endoproteinase inhibitor (α(2)M) were induced by T-2 toxin. Gelatin reverse zymography showed that TIMP-1 and TIMP-2 levels were decreased in a dose-dependent manner after exposure of T-2 toxin. Selenium had a protective role by increasing the level of type II collagen protein through down-regulation of MMP-13 protein and mRNA expression and up-regulation of TIMP-1 and TIMP-2 expressions. These data suggest T-2 toxin induces cartilage matrix degradation by the up-regulation of MMP-13 and TIMP-1, and down-regulation of TIMP-2 and α(2)M expressions.

High selenium status in individuals exposed to arsenic through coal-burning in Shaanxi (PR of China) modulates antioxidant enzymes, heme oxygenase-1 and DNA damage.

BACKGROUND: Although interactions of arsenite and selenite in mammalian organisms have been suggested by literature data, the antioxidant effects and biochemical mechanisms of dietary selenium on human populations exposed to inorganic arsenic are not fully understood. METHODS: Total blood, urine and hair concentrations of arsenic and selenium were determined in all individuals by hydride generation atomic fluorescence spectrometry. The individuals with skin lesions were subsequently classified as "High As group" and "High Se/As group" and controls were classified as "High Se group" and "Control group" according to their blood selenium concentrations. RESULTS: High selenium status was correlated with elevated activities of serum superoxide dismutase, glutathione peroxidase, catalase, and reduced levels of malondialdehyde, and increased RNA and protein expression of heme oxygenase-1 in peripheral blood mononuclear cells (PBMC) of individuals in the high arsenic group. Urinary 8-hydroxy-2'-deoxyguanosine levels were positively associated with blood arsenic, but inversely with blood and hair selenium among individuals with skin lesions, whereas mRNA are protein levels of 8-oxoganine DNA glycosylase 1 in PBMC increased in the "High Se/As group" compared to the "High As group". CONCLUSIONS: Inorganic arsenic exposure is associated with oxidative stress, which may be prevented by high selenium status via its antioxidative activity and detoxification effect possibly through the formation of an arsenic and selenium containing metabolite, the seleno-bis(S-glutathionyl) arsinium ion.

Effects of moniliformin and selenium on human articular cartilage metabolism and their potential relationships to the pathogenesis of Kashin-Beck disease.

OBJECTIVE: To investigate the effects of mycotoxin moniliformin (MON) on the metabolism of aggrecan and type II collagen in human chondrocytes in vitro and the relationship between MON and Kashin-Beck disease (KBD). METHODS: Human chondrocytes were isolated and cultured on bone matrix gelatin to form an artificial cartilage model in vitro with or without MON toxin. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The expression of aggrecan and type II collagen in the cartilage was determined using immunocytochemical staining. RESULTS: MON toxin inhibited chondrocyte viability in dose-dependent and time-dependent manners. MON reduced aggrecan and type II collagen syntheses in the tissue-engineered cartilage. MON also increased the expression of matrix metalloproteinase-1 (MMP-1), MMP-13, BC4 epitopes, and CD44 in cartilages. However, the expression of 3B3(-) epitopes in cartilages was inhibited by MON. Selenium partially alleviated the damage of aggrecan induced by MON toxin. CONCLUSION: MON toxin promoted the catabolism of aggrecan and type II collagen in human chondrocytes.