Wedelolactone suppresses breast cancer growth and metastasis via regulating TGF-ß1/Smad signaling pathway.

OBJECTIVE: Breast cancer is a malignant tumor with high invasion and metastasis. TGF-ß1-induced epithelial-mesenchymal transition (EMT) is crucially involved in the growth and metastasis of breast cancer. Wedelolactone (Wed) is extracted from herbal medicine Ecliptae Herba, which is reported to have antineoplastic activity. Here, we aimed to elucidate the efficacy and mechanism of Wed against breast cancer. METHODS: The effects of Wed on migration and invasion of 4T1 were detected. The expression of EMT-related markers was detected by Western blot and qPCR. The 4T1 orthotopic murine breast cancer model was established to evaluate the therapeutic effect of Wed on the growth and metastasis of breast cancer through TGF-ß1/Smad pathway. RESULTS: Wed inhibited the proliferation, migration and invasion of 4T1. It exhibited concentration-dependent inhibition of p-Smad2/3. Wed also reversed the expression of EMT-markers induced by TGF-ß1. In addition, Wed suppressed the growth and metastasis of breast cancer in mice. It also affected p-Smad3 expression as well as EMT-related genes, suggesting that its anti-breast cancer effect may be related to the TGF-ß1/Smad pathway. CONCLUSION: Wed reverses EMT by regulating TGF-ß1/Smad pathway, potentially serving as a therapeutic agent for breast cancer. Wed is expected to be a potential drug to inhibit TGF-ß1/Smad pathway-related diseases.

Flavonoid extracted from Epimedium attenuate cGAS-STING-mediated diseases by targeting the formation of functional STING signalosome.

Hyperactivation of the cyclic-GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signalling pathway has been shown to be associated with the development of a variety of inflammatory diseases, and the discovery of an inhibitor of the cGAS-STING signalling pathway holds great promise in the therapeutic interventions. Epimedium flavonoid (EF), a major active ingredient isolated from the medicinal plant Epimedium, has been reported to have good anti-inflammatory activity, but its exact mechanism of action remains unclear. In the present study, we found that EF in mouse bone marrow-derived macrophages (BMDMs), THP-1 (Tohoku Hospital Pediatrics-1) as well as in human peripheral blood mononuclear cells (hPBMC) inhibited the activation of the cGAS-STING signalling pathway, which subsequently led to a decrease in the expression of type I interferon (IFN-ß, CXCL10 and ISG15) and pro-inflammatory cytokines (IL-6 and TNF-α). Mechanistically, EF does not affect STING oligomerization, but inhibits the formation of functional STING signalosome by attenuating the interaction of interferon regulatory factor 3 (IRF3) with STING and TANK-binding kinase 1 (TBK1). Importantly, in vivo experiments, EF has shown promising therapeutic effects on inflammatory diseases mediated by the cGAS-STING pathway, which include the agonist model induced by DMXAA stimulation, the autoimmune inflammatory disease model induced by three prime repair exonuclease 1 (Trex1) deficiency, and the non-alcoholic steatohepatitis (NASH) model induced by a pathogenic amino acid and choline deficiency diet (MCD). To summarize, our study suggests that EF is a potent potential inhibitor component of the cGAS-STING signalling pathway for the treatment of inflammatory diseases mediated by the cGAS-STING signalling pathway.

Network pharmacology-based strategy for predicting therapy targets of Ecliptae Herba on breast cancer.

Breast cancer is a prevalent malignancy affecting women globally, characterized by significant morbidity and mortality rates. Ecliptae Herba is a traditional herbal medicine commonly used in clinical practice, has recently been found to possess antitumor properties. In order to explore the underlying material basis and molecular mechanisms responsible for the anti-breast cancer effects of Ecliptae Herba, we used network pharmacology and experimental verification. UPLC-MS/MS was utilized to identify compounds present in Ecliptae Herba. The active components of Ecliptae Herba and its breast cancer targets were screened using public databases. Hub genes were identified using the STRING and Metascape database. The R software was utilized for visual analysis of GO and KEGG pathways. The affinity of the hub targets for the active ingredients was assessed by molecular docking analysis, which was verified by experimental assessment. A total of 178 targets were obtained from the 10 active components of Ecliptae Herba, while 3431 targets associated with breast cancer were screened. There were 144 intersecting targets between the components and the disease. Targets with a higher degree, namely EGFR and TGFB1, were identified through the hub subnetwork of PPI. GO and KEGG analyses revealed that Ecliptae Herba plays an important role in multiple cancer therapeutic mechanisms. Moreover, molecular docking results showed that the core components had good binding affinity with key targets. Finally, it was confirmed that TGF-ß1 might be a potential crucial target of Ecliptae Herba in the treatment of breast cancer by cytological experiments, and the TGF-ß1/Smad signaling pathway might be an important pathway for Ecliptae Herba to exert its therapeutic effects. This study elucidated the active ingredients, key targets, and molecular mechanisms of Ecliptae Herba in the treatment of breast cancer, providing a scientific foundation and therapeutic mechanism for the prevention and treatment of breast cancer with Traditional Chinese medicine.

Polysaccharide extract from Isatidis Radix inhibits multiple inflammasomes activation and alleviate gouty arthritis.

The polysaccharide extract from Isatidis Radix exhibits potent antiinflammatory and antiviral activities, but the mechanism of Isatidis Radix polysaccharide (IRP) remains obscure. Herein, we reported that IRP blocked the activation of nod-like receptor pyrin domain-containing 3 (NLRP3) inflammasome, leading to the inhibiting of caspase-1 cleavage and IL-1ß secretion. Mechanistically, IRP did not inhibit NLRP3 inflammasome through suppressing mitochondrial reactive oxygen species (mtROS) production. However, IRP can significantly suppress the oligomerization of apoptosis-associated speck-like protein (ASC) and subsequently block the formation of inflammasome. Next, we evaluate the role of IRP in monosodium urate (MSU)-induced gout in vivo which is a NLRP3-associated disease. We also observed that oral administration of IRP can reduce the increased ankle thickness and the secretion of IL-1ß, IL-18, IL-6, TNF-α and MPO of the mouse ankle joints caused by MSU crystals. Furthermore, flow cytometry analysis highlighted a significant modulation of T helper 17 cells (Th17)/regulatory T cells (Treg) following IRP treatment in MSU induced gout. Overall, our findings suggest that IRP has comprehensive and potent antiinflammatory effects and provide a reasonable therapeutic strategy in preventing inflammasome-associated diseases, such as inflammatory gouty arthritis.

[Molecular mechanism of luteolin regulating lipoxygenase pathway against oxygen-glucose deprivation/reperfusion injury in H9c2 cardiomyocytes based on molecular docking].

The aim of this study was to investigate the mechanism of luteolin regulating lipoxygenase pathway against oxygen-glucose deprivation/reperfusion(OGD/R) injury in H9 c2 cardiomyocytes. First, Discovery Studio 2019 was used for the molecular docking of luteolin with three key enzymes including lipoxygenase 5(ALOX5), lipoxygenase 12(ALOX12), and lipoxygenase 15(ALOX15) in lipoxygenase pathway. The docking results showed that luteolin had high docking score and similar functional groups with the original ligand. From this, H9 c2 cardiomyocytes were cultured in vitro, and then the injury model of H9 c2 cardiomyocytes was induced by deprivation of oxygen-glucose for 8 h, and rehabilitation of oxygen-glucose for 12 h. Cell viability was detected by tetrazolium(MTT) colorimetry. H9 c2 cardiomyocytes were observed with a fluorescence inverted microscope, and colorimetry was used to detect the level of lactate dehydrogenase(LDH) in cell supernatant. The results showed that luteolin could significantly protect the morphology of H9 c2 cells, significantly improve the survival rate of H9 c2 cardiomyocytes in OGD/R injury model, reduce the level of LDH in cell supernatant, inhibit cytotoxicity, and maintain the integrity of cell membrane. The inflammatory cytokines interleukin-6(IL-6) and tumor necrosis factor-α(TNF-α) were detected by enzyme-linked immunosorbent assay. Compared with the model group, luteolin can significantly reduce the release of IL-6 and TNF-α. Western blot was employed to detect the protein levels of ALOX5, ALOX12, and ALOX15 in lipoxygenase pathway. After luteolin intervention, the protein levels of ALOX5, ALOX12, and ALOX15 were significantly down-regulated compared with those in model group. These results indicate that luteolin can inhibit the release of IL-6 and TNF-α by restraining the activation of lipoxygenase pathway, thereby playing a protective role in the cardiomyocyte injury model induced by OGD/R.

[Analysis of epidemiological characteristics of drug induced liver injury associated with Baixianpi Preparations].

A retrospective study was performed in drug-induced liver injury(DILI) cases associated with Dictamni Cortex(Baixianpi,BXP) Preparations,which were treated at grade Ⅲ class A liver disease hospitals from 2008 to 2016 and spontaneously reported for adverse reactions between 2012 and 2016 at HILI Cloud(hilicloud.net). The results showed 25 DLII cases associated with BXP Preparations treated at grade Ⅲ class A liver disease hospitals during the 9 years,including only 14 cases in line with the clinical diagnostic criteria of Guidelines for the Diagnosis and Treatment of Herb-Induced Liver Injury. And 74 DILI cases associated with BXP Preparations spontaneously reports adverse reactions,and 18. 92% of them had unreasonable medication,including polypharmacy(21. 43%),overdose(28. 57%) and repeated dosage(50%). And 47 DILI cases used BXP Preparations to treat psoriasis and vitiligo(a total of59. 57%). The time range of taking BXP Preparations until liver injury occurred was 1-366 d,with the median of 18 d. The dose of BXP Preparations was estimated to be 0. 09-12 g·d-1. And the cumulative dosage of taking drugs until liver injury occurred was 1. 1-336 g. Obvious associations with time-toxicity as well as quantity-toxicity could not be found based on the wide range of time-toxicity relations and quantity-toxicity relations. On the basis of the study,we found that DILI cases associated with BXP Preparations commonly occurred in patients with immune diseases,such as psoriasis and vitiligo,indicating specific individual differences. The results suggested that DILI cases associated with BXP Preparations would be correlated with the property of idiosyncratic drug-induced liver injury. In conclusion,the risk of liver injury clinically caused by BXP Preparations should be paid more attention,and the studies on the mechanism of idiosyncratic drug-induced liver injury must be enhanced,and those on risk factors,like irrational drug use,should be strengthened. Moreover,the evaluation of the risk-to-benefit ratio is supposed to be performed for the sake of improving the risk prevention and control standards for BXP preparations,and ensuring safe and rational clinical application of BXP Preparations.

The study of GPX3 methylation in patients with Kashin-Beck Disease and its mechanism in chondrocyte apoptosis.

OBJECTIVE: Selenium deficiency is a risk factor for Kashin-Beck Disease (KBD), an endemic osteoarthropathy. Although promoter hypermethylation of glutathione peroxidase 3 (GPX3) (a selenoprotein) has been identified in several cancers, little is known about promoter methylation and expression of GPX3 and their relation to selenium in KBD. The present study was thus conducted to investigate this research question. METHODS: Methylation and expressions of GPX3 in whole blood drawn from 288 KBD patients and 362 healthy controls and in chondrocyte cell line were evaluated using methylation-specific PCR and qRT-PCR, respectively. The protein levels of PI3K/Akt/c-fos signaling in the whole blood and chondrocyte cell line were determined with Western blotting. Chondrocytes apoptosis were detected by Hoechst 33342 and Annexin V-FITC/PI staining. RESULTS: GPX3 methylation was increased, GPX3 mRNA was decreased, and protein levels in the PI3K/Akt/c-fos signaling pathway were up-regulated in the whole blood collected from KBD patients as compared with healthy controls. Similar results were obtained for chondrocytes injured by oxidative stress. There was a significant, decreasing trend in GPX3 expression across groups of unmethylation, partial methylation, and complete methylation for GPX3, in sequence. Compared with unmethylation group, protein levels in PI3K/Akt/c-fos pathway were enhanced in partial and complete methylation groups. Treatment of chondrocytes with sodium selenite resulted in reduced methylation and increased expression of GPX3 as well as down-regulated level of PI3K/Akt/c-fos proteins. CONCLUSIONS: The methylation and expression of GPX3 and expression of PI3K/Akt/c-fos pathway are altered in KBD and these changes are reversible by selenium supplementation.

Assessment on the Efficacy and Safety of Aidi Injection Combined with Vinorelbine and Cisplatin for Treatment of Advanced Nonsmall Cell Lung Cancer.

BACKGROUND: The aim of this study was to assess the efficacy and safety of vinorelbine and cisplatin (NP chemotherapy) alone or in combination with Aidi injection for the treatment of advanced nonsmall cell lung cancer (NSCLC). METHODS: Pertinent publications were identified in PubMed, EMBASE, Cochrane Library, CNKI, CQVIP, and Wanfang databases, up to December 8, 2015. After quality assessment of all included randomized controlled trials evaluating Aidi injection combined with NP chemotherapy for the treatment of advanced NSCLC, a meta-analysis was performed by Review Manager 5.2 and STATA 12.0 for statistical analyses. RESULTS: Twelve studies including 509 and 503 cases in the experimental and control groups, respectively, were finally analyzed. The meta-analysis revealed that when cisplatin dose ranging from 20 to 40 mg/m 2 , combination of Aidi injection and NP chemotherapy was statistically different compared with NP chemotherapy alone in enhancing efficiency (relative risk [RR] = 1.24, 95% confidence interval [CI] [1.05-1.47], P = 0.010) and reducing the incidence of Grade II or above nausea and vomiting (RR = 0.49, 95% CI [0.30-0.80], P = 0.005). Meanwhile, with cisplatin ranging from 80 to 120 mg/m 2 , no significant differences in efficiency (RR = 1.11, 95% CI [0.87-1.42], P = 0.390) and Grade II or above nausea and vomiting (RR = 0.88, 95% CI [0.71-1.10], P = 0.260) were obtained. In addition, Aidi injection combined with NP chemotherapy was superior to NP chemotherapy alone in improving the quality of life, alleviating Grade II or above leukopenia and thrombocytopenia. CONCLUSIONS: Aidi injection combined with NP chemotherapy can enhance efficiency, improve the quality of life, and decrease adverse effects in patients with advanced NSCLC.

Effects of sodium selenite on c-Jun N-terminal kinase signalling pathway induced by oxidative stress in human chondrocytes and c-Jun N-terminal kinase expression in patients with Kashin-Beck disease, an endemic osteoarthritis.

The c-Jun N-terminal kinases (JNK) are members of the mitogen-activated protein kinase family and are activated by environmental stress. Se plays an important role in the biological pathways by forming selenoprotein. Selenoproteins have been shown to exhibit a variety of biological functions including antioxidant functions and maintaining cellular redox balance, and compromise of such important proteins would lead to oxidative stress and apoptosis. We examined the expression levels of JNK in Kashin-Beck disease (KBD) patients, tested the potential protective effects of sodium selenite on tert-butyl hydroperoxide (tBHP)-induced oxidative injury and apoptosis in human chondrocytes as well as its underlying mechanism in this study. We produced an oxidative damage model induced by tBHP in C28/I2 human chondrocytes to test the essential anti-apoptosis effects of Se in vitro. The results indicated that the expression level of phosphorylated JNK was significantly increased in KBD patients. Cell apoptosis was increased and molecule expressions of the JNK signalling pathway were activated in the tBHP-injured chondrocytes. Na2SeO3 protected against tBHP-induced oxidative stress and apoptosis in cells by increasing cell viability, reducing reactive oxygen species generation, increasing Glutathione peroxidase (GPx) activity and down-regulating the JNK pathway. These results demonstrate that apoptosis induced by tBHP in chondrocytes might be mediated via up-regulation of the JNK pathway; Na2SeO3 has an effect of anti-apoptosis by down-regulating the JNK signalling pathway.

[Effect of Nivalenol and selenium on IL-1beta and TNF-alpha secretion in cultured chondrocytes].

AIM: To investigate the effect of Nivalenol(NIV) and Selenium(Se) on the levels of IL-1beta and TNF-alpha in the cultured chondrocytes. METHODS: Human chondrocytes cultured in vitro were treated with or without NIV and Se. The morphology of chondrocytes was observed by optic microscope. The DNA content was determined by UV Spectrophotometry. The levels of IL-1beta and TNF-alpha in cultured medium were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Hematoxylin & eosin staining indicated there was cell necrosis in the cartilage reconstructed in vitro from both NIV group and NIV+Se group. Compared with the group of NIV toxin, the damage of chondrocytes was less severe when Se was added. NIV could inhibit chondrocyte DNA synthesis. The content of DNA with NIV was lowest than that in other groups. The levels of IL-1beta and TNF-alpha with NIV were significantly higher than control group (P<0.05). After Se was added, the levels did not change significantly compared with the groups without Se. CONCLUSION: NIV toxin could superinduce IL-1beta and TNF-alpha secretion in chondrocytes, which may be the key mechanism of chondrocyte injury by NIV. Se can partially alleviate the effects of NIV on chondrocytes cultured in vitro.

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.

Butenolide induced cytotoxicity by disturbing the prooxidant-antioxidant balance, and antioxidants partly quench in human chondrocytes.

Butenolide (BUT), a mycotoxin produced by Fusarium species, was detected often in corns or grains from endemic Kashin-Beck disease (KBD) areas in China. In this study, we evaluated the cytotoxicity of BUT on chondrocytes and the possible toxic mechanism with the aim of understanding the pathogenesis and of directing future therapeutic interventions for KBD. Exposure of human chondrocytes and engineered cartilage to high concentration of BUT (> 1 microg/ml) resulted in significant cytotoxicity, manifested by losses in cell viability and changes in cell morphology. BUT with high concentration (> 1 microg/ml) also induced significant oxidative damage to chondrocytes in vitro evidenced by increasing both lipid peroxidation and endogenous antioxidants. Furthermore, free radical scavenging agents, such as selenium (Se), vitamin C (VC) and vitamin E (VE), partly blocked BUT-induced oxidative damage. In conclusion, this finding indicates that BUT induces cytotoxicity to human chondrocytes, and the disturbance of prooxidant-antioxidant balance may play a pivotal role in BUT-induced injuries in chondrocytes. Moreover, Se, VC or VE can quench the toxic effects of BUT to a certain extent, which will possibly direct future therapeutic interventions against KBD.

T-2 toxin-induced apoptosis involving Fas, p53, Bcl-xL, Bcl-2, Bax and caspase-3 signaling pathways in human chondrocytes.

OBJECTIVE: To investigate the effects of T-2 toxin on expressions of Fas, p53, Bcl-xL, Bcl-2, Bax and caspase-3 on human chondrocytes. METHODS: Human chondrocytes were treated with T-2 toxin (1-20 ng/ml) for 5 d. Fas, p53 and other apoptosis-related proteins such as Bax, Bcl-2, Bcl-xL, caspase-3 were determined by Western blot analysis and their mRNA expressions were determined by reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: Increases in Fas, p53 and the pro-apoptotic factor Bax protein and mRNA expressions and a decrease of the anti-apoptotic factor Bcl-xL were observed in a dose-dependent manner after exposures to 1-20 ng/ml T-2 toxin, while the expression of the anti-apoptotic factor Bcl-2 was unchanged. Meanwhile, T-2 toxin could also up-regulate the expressions of both pro-caspase-3 and caspase-3 in a dose-dependent manner. CONCLUSION: These data suggest a possible underlying molecular mechanism for T-2 toxin to induce the apoptosis signaling pathway in human chondrocytes by regulation of apoptosis-related proteins.

Promotion of the articular cartilage proteoglycan degradation by T-2 toxin and selenium protective effect.

OBJECTIVE: To identify the relationship between T-2 toxin and Kashin-Beck disease (KBD), the effects of T-2 toxin on aggrecan metabolism in human chondrocytes and cartilage were investigated in vitro. METHODS: Chondrocytes were isolated from human articular cartilage and cultured in vitro. Hyaluronic acid (HA), soluble CD44 (sCD44), IL-1beta and TNF-alpha levels in supernatants were measured by enzyme-linked immunosorbent assay (ELISA). CD44 content in chondrocyte membrane was determined by flow cytometry (FCM). CD44, hyaluronic acid synthetase-2 (HAS-2) and aggrecanases mRNA levels in chondrocytes were determined using reverse transcription polymerase chain reaction (RT-PCR). Immunocytochemical method was used to investigate expressions of BC-13, 3-B-3(-) and 2-B-6 epitopes in the cartilage reconstructed in vitro. RESULTS: T-2 toxin inhibited CD44, HAS-2, and aggrecan mRNA expressions, but promoted aggrecanase-2 mRNA expression. Meanwhile, CD44 expression was found to be the lowest in the chondrocytes cultured with T-2 toxin and the highest in control plus selenium group. In addition, ELISA results indicated that there were higher sCD44, IL-1beta and TNF-alpha levels in T-2 toxin group. Similarly, higher HA levels were also observed in T-2 toxin group using radioimmunoprecipitation assay (RIPA). Furthermore, using monoclonal antibodies BC-13, 3-B-3 and 2-B-6, strong positive immunostaining was found in the reconstructed cartilage cultured with T-2 toxin, whereas no positive staining or very weak staining was observed in the cartilage cultured without T-2 toxin. Selenium could partly inhibit the effects of T-2 toxin above. CONCLUSION: T-2 toxin could inhibit aggrecan synthesis, promote aggrecanases and pro-inflammatory cytokines production, and consequently induce aggrecan degradation in chondrocytes. These will perturb metabolism balance between aggrecan synthesis and degradation in cartilage, inducing aggrecan loss in the end, which may be the initiation of the cartilage degradation.

[Butenolide induces apoptosis of cultured chondrocytes: study of its mechanism].

OBJECTIVE: To observe cell apoptosis and Bcl-2 and Bax expression changes of chondrocytes induced by butenolide (BUT) and the inhibitory effect of selenium against BUT-induced chondrcyte apoptosis, to gain insights into the mechanism by which BUT induces chondrcyte apoptosis. METHODS: Cartilage tissue reestablished from human fetal articular chondrocytes in vitro were treated with BUT at the concentrations of 0.1, 1.0 and 5.0 microg/ml and with the protective factor selenium. TUNEL method was used to detect chondrocyte apoptosis, which was quantified by flow cytometry. Immunohitochemistry was performed to analyze the expression of Bcl-2 and Bax in the reestablished cartilage tissue. RESULTS: BUT exposure induced chondrocyte apoptosis, and the apoptosis rate increased with the concentration increment of BUT from 0 to 1.0 mg/ml, resulting also increased positive expression rate of Bcl-2 and Bax(P<0.05). The apoptosis rate of chondrocytes in BUT+ selenium group was significantly lower than that of BUT groups (P<0.05), as was the positivity rate of Bcl-2 and Bax expression (P<0.05). CONCLUSION: BUT induces chondrocyte apoptosis in positive relation with BUT concentration (from 0 to 1.0 mg/ml) and causes increased expressions of Bcl-2 and Bax. Selenium can inhibit the chondrocyte apoptosis induced by BUT.

[Effect of NO and Fas pathway on T-2 induced apoptosis in chondrocytes].

OBJECTIVE: To investigate the relationship of T-2 toxin-induced chondrocytes apoptosis with nitric oxide(NO) and Fas apoptosis pathway. METHODS: Human chondrocytes cultured in vitro were treated with different concentrations of T-2 toxin at different time (1-5 d). Cell viability of the treated cells was measured by MTT assay. Apoptotic ultrostructural changes of the treated cells were observed with electron microscopy. Biological changes of apoptosis were detected by annexin V/PI Flow cytometer (FCM). The levels of NO in culture media were detected by colorimetric method of Griess assay. Nitric oxide synthase (iNOS) and Fas protein were measured by Western blot. RESULTS: In this study the results shown the dose-dependent and time-dependent effects of T-2 toxin, within a range of concentration (1-2000 ng/mL) and a period of time (1-5 d), on the T-2 toxin-treated chondrocytes. Apoptotic body was found in T-2 toxin-treated chondrocytes by electron microscopy. Early-stage apoptosis rate and late-stage apoptosis rate were both increased in T-2 toxin-treated cells when compared with non-treated cells in a dose-dependent manner. The levels of NO in T-2 toxin-treated culture media were higher than that of normal control. Over-expressions of iNOS and Fas protein were detected in T-2 toxin-treated cells. T-2 toxin-induced apoptosis was noted to be significtnly correlated with the level of NO production and the levels of iNOS and Fas protein expression. CONCLUSION: T-2 toxin can enhance NO production and upregulate the expression of iNOS and Fas protein. Both NO and Fas signaling pathway are involved in T-2 toxin-induced apoptosis.

T-2 toxin induces apoptosis, and selenium partly blocks, T-2 toxin induced apoptosis in chondrocytes through modulation of the Bax/Bcl-2 ratio.

T-2 toxin is one of the mycotoxins, a group of type A trichothecenes produced by several fungal genera including Fusarium species. In the present study, we have investigated the apoptotic effects of T-2 toxin on chondrocytes and the relationship between T-2 toxin induced chondrocyte apoptosis and its influence on Bcl-2/Bax protein and mRNA expression. We have also examined the inhibitory effects of selenium on chondrocyte apoptosis induced by T-2 toxin. We have combined morphological and biological techniques to establish the relevance of apoptosis in human chondrocyte death induced by T-2 toxin. Treatment with T-2 toxin caused accelerated apoptosis in a concentration dependent manner. The apoptosis induced by T-2 toxin involved an increased Bax/Bcl-2 ratio. Bcl-2 mRNA expression remained unchanged in chondrocyte apoptosis induced by T-2 toxin treatment, while Bax mRNA expression increased following treatment with T-2 toxin. Selenium could partly block the apoptosis of chondrocytes induced by T-2 toxin through decreasing the Bax/Bcl-2 ratio. These results suggest that, under our experimental conditions, apoptosis of chondrocytes can be induced by T-2 toxin (1-20ng/mL) via the Bcl-2 and Bax proteins, and the Bax/Bcl-2 ratio may play a critical role in governing the susceptibility to apoptosis induced by T-2 toxin in human chondrocytes.

[The effect of retinoic acid on induction of osteoporotic model rats and the possible mechanism].

OBJECTIVE: To observe the effect of retinoic acid on induction of osteoporotic model in rats and analyze the mechanism therein involved. METHODS: SD rats were treated with retinoic acid 80 mg/(kg x d) by gastrogavage for 15 days to induce osteoporosis and were killed in batches at 0, 30 and 60 days after drug withdrawal. The levels of Ca, P, BGP, E2, IGF-1, AKP and TRAP in serum were assayed, the collagen and proteoglycan contents of bone and bone mineral density (BMD) were determined, and the morphological changes in cancellous and cortical bone and growth plate cartilage (GPC) of femurs from the experimental rats were observed. RESULTS: After 15 days of induction by retinoic acid, the serum E2 and BGP contents of rats were obviously decreased, the activities of AKP and TRAP were significantly increased, and the levels of BMD were lowered. The masses of spongy bone and cortical bone of femurs from the rats were decreased, and the number of chondrocytes in GPC was reduced. At 30 days, after drug withdrawal, the masses of spongy bone and cortical bone of femurs from the osteoporotic model rats still showed reduction; the activities of AKP in serum were lower than those at 15 days after drug redrawal, but were still higher than those of normal group rats; the chondrocytes in GPC were increased, the serum BGP and Ca contents were increased. At 60 days, after drug withdrawal, only the masses of femoral spongy bone of the osteoporotic model rats continuously showed obvious reduction, the other indices, including BGP, E2, AKP, TRAP and the masses of cortical bone, showed no significant difference between the two groups. CONCLUSION: The short-term effect of retinoic acid on induction of rat's osteoporotic model was noticeable, but the long-term effect was not so good, and the bone loss of spongy bone existed longer and was more obvious than that of cortical bone.