Ammonia induce lung tissue injury in broilers by activating NLRP3 inflammasome via Escherichia/Shigella.

Respiratory tract diseases are closely related to atmosphere pollution. Ammonia is one of the harmful pollutants in the atmosphere environment, which has a great threat to human and animal respiratory tract health, but the mechanism of causing diseases is not clear. In this study, broiler lung tissue was used as a model to study the effect of high ammonia on respiratory tract diseases through the relationship between respiratory microflora, NLRP3 inflammasome, and inflammatory factors. For this, we validated the occurrence of lung tissue inflammation under ammonia exposure and detected the lung tissue microbial constituent by 16S rDNA sequencing. Moreover, the relative expression levels of NLRP3 and caspase-1 mRNA and the content of IL-1ß and IL-6 were measured. After 7-D ammonia exposure, the proportion of the phylum Proteobacteria and the genus Escherichia/Shigella in lung tissue was significantly increased, the expression levels of NLRP3 and caspase-1 mRNA were significantly increased, and the content of IL-1ß in lung tissue and serum was higher than that in the control group. In conclusion, high ammonia induced lung tissue inflammation via increasing the proportion of Escherichia/Shigella, activating NLRP3 inflammasome, and promoting IL-1ß release. These findings provided a reference for the prevention and control of respiratory tract diseases in humans and animals caused by ammonia pollution.

The role of real-time shear wave elastography in the diagnosis of idiopathic nephrotic syndrome and evaluation of the curative effect.

PURPOSE: To evaluate the use of real-time shear wave elastography (SWE) in the assessment of renal elasticity and the efficacy of steroid treatment in adult idiopathic nephrotic syndrome (INS). METHODS: This study included 120 patients with INS. Patients were divided into steroid-sensitive and steroid-resistant groups. Renal biopsy was performed. Thirty healthy subjects were recruited as controls. Young's modulus (YM) of the renal parenchyma was measured by SWE. The YM values in each group were compared using glomerular sclerosis index (GI) and renal interstitial fibrosis (RIF). RESULTS: The YM values were significantly different between the INS and control groups, as well as between the steroid-sensitive and steroid-resistant groups (P < 0.05). Higher YM values were associated with steroid sensitivity. The area under the receiver operating characteristic curve for the YM value in the INS group vs. control group was 0.871 (95% CI 0.815-0.927) and in the steroid-resistant group vs. control, and steroid-sensitive groups was 0.836 (95% CI 0.765-0.908). The corresponding cut-off values were 7.96 and 10.73 m/s, with 81.7% and 86.0% sensitivities, 93.3% and 77.9% specificities, and Youden index 0.750 and 0.639, respectively. Spearman correlation analysis showed that the YM value in the renal parenchyma was positively correlated with GI (r = 0.631, P < 0.05) and RIF (r = 0.606, P < 0.05). CONCLUSION: SWE technology is a potential method for non-invasive quantitative measurement of renal parenchyma stiffness to determine the pathological changes of INS renal parenchyma and evaluate the effectiveness of steroid therapy.

Induction of apoptosis in human cervical carcinoma HeLa cells by active compounds from Hypericum ascyron L.

Hypericum ascyron L. (Great St. Johnswort), which belongs to the Hypericaceae family, has been used for the treatment of hematemesis, metrorrhagia, rheumatism, swelling, stomach ache, abscesses, dysentery and irregular menstruation for >2,000 years in China. The aim of the present study was to clarify the anticancer activity compounds from H. ascyron L. and the underlying molecular mechanism. Anticancer activity of H. ascyron L. extract was evaluated using an MTT assay. To confirm the anticancer mechanism of activity compounds, Hoechst 33258, Annexin V-fluorescein isothiocyanate/propidium iodide, 2',7'-dichlorodihydrofluorescein diacetate, rhodamine 123 staining and caspase-3 activity analysis were performed. The results demonstrated that the anti-proliferative action of the mixture of kaempferol 3-O-ß-(2″-acetyl) galactopyranoside (K) and quercetin (Q) (molar ratio, 1:1) was significantly increased compared with either of these two compounds separately, and the active fraction of the H. ascyron L. extract |(HALE). HALE, indicating that the anti-proliferative function of H. ascyron L. may be a synergic effect of K and Q. Furthermore, the inhibitory effect of KQ on the growth of HeLa cells was mediated by the induction of apoptosis. To the best of our knowledge, the present study is the first to identify that KQ exhibits significant anti-proliferation activity on HeLa cells via the apoptotic pathway, and is also the first to evaluate the anticancer potential of H. ascyron L. The results of the present study may provide a rational base for the use of H. ascyron L. in the clinic, and shed light on the development of novel anticancer drugs.

Quercetin and aconitine synergistically induces the human cervical carcinoma HeLa cell apoptosis via endoplasmic reticulum (ER) stress pathway.

Up till now, studies have not been conducted on how the combination of Quercetin (Q), Aconitine (A) and apoptosis induction affects human cervical carcinoma HeLa cells. The result of our findings shows that the combination of Q and A (QA) is capable of synergistically inhibiting the proliferation of HeLa cells in a number of concentrations. QA synergistically inhibits the proliferation of MDR1 gene in the HeLa cells. It is concluded based on our result that QA induces apoptosis and ER stress just as QA-induced ER stress pathway may mediate apoptosis by upregulating mRNA expression levels of eIF2α, ATF4, IRE1, XBP1, ATF6, PERK and CHOP in the HeLa cells. The up-regulating of mRNA expression level of GRP78 and activation of UPR are a molecular basis of QA-induced ER stress.

PFP alleviates nonalcoholic steatohepatitis fatty liver in both Apo E-/- mice and Changliver cell[S].

High-calorie food leads to nonalcoholic fatty liver disease (NAFLD) through the dysregulation of genes involved in lipid metabolism, but the precise mechanism is still unknown. Pomegranate flowers are used to treat diabetes mellitus in traditional Uighur medicine. Here we sought to investigate the effect and mechanism of pomegranate flower polyphenols (PFP) on NAFLD Apo E-/- mice induced by a high-fat diet (HFD) and whether PFP improves NAFLD through decreasing oxidative stress. PFP supplementation in mice significantly reduced the HFD-induced gains in body weight compared with the mice fed only with HFD. It also significantly reduced HFD-induced increases in serum lipids, including cholesterol and triglyceride. Consistent with the reduced liver weight, hepatic lipid accumulation, and the size of lipid droplets in the epididymal fat pads were also reduced by PFP supplementation. To further investigate how PFP may reduce obesity, we analyzed lipid metabolism-related genes in the liver. PFP supplementation altered expression profiles of several lipid metabolism-related genes, including ACC, AMPK, CPT-1α, FAS, LDLR, Leptin, LXR, PON1, PPAR, SirT3, and SREBP, relative to those in HFD control mice. The expression patterns of these genes observed by quantitative reverse transcriptase-polymerase chain reaction and AMPK, SirT3, ACC2, and CPT-1A expression were confirmed by immunohistochemical assays. Collectively, our results indicate that PFP prevents HFD-induced obesity in Apo E-/- mice, and its anti-obesity effects may be related to the regulation of lipogenesis at the level of transcription.

[Clinical and Pathologic Features of Myeloid Sarcoma].

OBJECTIVE: To explore the clinicopathologic features, differential diagnosis and therapy of myeloid sarcoma. METHODS: The clinical data including clinical manifestations, laboratorial tests, histopathologicical examination, immunohistochemistry and clinical prognosis of 10 patients with myeloid sarcoma were analyzed retrospectively. Among 10 patients, 5 male and 5 female, aged 23 to 71 years old (median = 36 years). RESULTS: 2 cases of myeloid sarcoma were secondary from chronic myeloid leukemia, and 1 cases of myeloid sarcoma occurred after the allogeneic hematopoietic stem cell transplantation due to acute myeloid leukemia, and the others lacked the anamnesis of malignancies. The neoplasms occurred at bone, brain, skin, breast, epididymis, uterine cervix, small intestine, ovary and lymph nodes. Microscopically, the tumor cells were round or oval, which infiltrated diffusely or arranged in single-file. The cytoplasm was scarce and immature eosinophils were scattered. The nuclei were round, oval or focally irregular, and the mitosis was visible. The neoplasms were positive for MPO, CD34, CD43, CD45, CD99 and CD117 by immunohistochemical staining. 4 patients progressed into acute myeloid leukemia from 2 to 10 months after the diagnosis of myeloid sarcoma. All of them achieved complete remission after inductive chemotherapy, but 3 patients relapsed from 3 to 12 months after remission and only survived for 14 to 23 months. 4 patients were treated by using chemotherapy before bone marrow abnormality, and with the disease-free survival for 1 to 48 months. CONCLUSION: Myeloid sarcoma needs to be distinguished from lymphoblastic lymphoma, Burkitt's lymphoma, blastic plasmacytoid dendritic cell neoplasms and so on. The diagnosis and differential diagnosis of myeloid sarcoma are dependent on the pathological and immunohisto-chemical features. The chemotherapy and allogeneic hematopoietic stem cell transplantation of acute myeloid leukemia are the main methods for treatment of myeloid sarcoma.

Efficacy of pEgr-1-endostatin combined with ionizing radiation on hypoxic conditions in nude mice bearing SKOV3 ovarian carcinoma.

Hypoxia occurs in a wide range of solid tumors, and is strongly associated with radio-resistance of malignant tumors. The aim of the present study was to investigate the effect of endostatin combined with ionizing radiation (IR) on hypoxic conditions. A total of 24 mice bearing SKOV3 ovarian carcinoma were divided into three groups. Following injection with pEgr-1-endostatin plasmid for 12 h, the mice in the endostatin-IR-treated group were exposed to 300 cGy/min X-ray for 48 h, and the IR-treated group was exposed to the same condition. Then, the expression of endostatin, hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF) was detected by reverse transcription-polymerase chain reaction, ELISA, immunohistochemistry and western blotting. In addition, the tumor microvessel density (MVD) was examined by immunohistochemistry analysis of cluster of differentiation 31-positive cells. The results revealed that pEgr-1-endostatin was successfully induced by IR. The level of endostatin messenger RNA in the endostatin-IR-treated group was significantly higher than that in the control and IR-treated groups (F=380.078, P<0.001). Statistical differences were also examined at the protein level by western blotting and ELISA. An obvious increase in MVD was observed in the IR-treated group compared with that in the control group (t=7.040, P<0.001), and a significant decrease in MVD was observed in the endostatin-IR-treated group compared with that in the control group (t=18.153, P<0.001). By comparing the morphology of the tumor vasculature in the three groups, it was noticed that the microvessels in the endostatin-IR-treated group were more regularly distributed and had fewer giant branches than those in the IR-treated group. Further investigation revealed that the expression levels of HIF-1α and VEGF in the endostatin-IR-treated group were lower compared with those in the control (t=5.339, P=0.001; and t=13.880, P<0.001, respectively) and the IR-treated groups (t=12.930, P<0.001; and t=14.050, P<0.001, respectively). Our findings suggested that endostatin decreased the number of microvessels via the HIF-1/VEGF signaling pathway, and that pEgr-1-endostatin combined with IR may improve hypoxic conditions and may be a novel approach for treating solid tumors.

Effectiveness of ultrasound-guided percutaneous microwave ablation for symptomatic uterine fibroids: a multicentre study in China.

PURPOSE: To evaluate the clinical efficacy of ultrasound-guided percutaneous microwave ablation (PMWA) therapy for symptomatic uterine fibroids in a multicentre study. MATERIALS AND METHODS: Patients with symptomatic uterine fibroids who underwent PMWA at multiple treatment centres in China between January 2013 and August 2015 were prospectively studied to compare the reduction rate of uterine fibroids, haemoglobin level and uterine fibroid symptom and health-related quality of life questionnaire (UFS-QOL) scores before and at 3, 6 and 12 months after ablation. RESULTS: A total of 311 patients (405 leiomyomas) from eight treatment centres underwent the treatment (age, 29-55 years; mean ± SD, 41 ± 5.11 years). The mean diameter of the myomas ranged from 2.03 to 12.50 cm (mean, 5.10 ± 1.28 cm) and the volume ranged from 4.40 to 1022.14 cm3 (mean, 95.01 ± 70.29 cm3). Forty-eight myomas were identified as FIGO type 1/2 fibroids, 256 as type 3/4 fibroids and 101 as type 5/6 fibroids. The mean ablation rate was 86.6% (54.0-100%). The mean reduction rate was 63.5%, 78.5% and 86.7% at 3, 6 and 12 months posttreatment, respectively. The haemoglobin level increased significantly from 88.84 ± 9.31 g/L before treatment to 107.14 ± 13.32, 116.05 ± 7.66 and 117.79 ± 6.51 g/L at 3, 6 and 12 months posttreatment, respectively (p = .000). The symptom severity score (SSS) and health-related quality of life (HRQL) scores were also significantly improved posttreatment compared with before treatment (p = .000). CONCLUSION: PMWA is an effective, minimally invasive treatment for symptomatic leiomyomas that can significantly improve the quality of life of patients.

Variables associated with vaginal discharge after ultrasound-guided percutaneous microwave ablation for adenomyosis.

Objective The aim of this study was to analyse the significant variables for vaginal discharge after ultrasound-guided percutaneous microwave ablation (PMWA) therapy. Materials and methods PMWA was performed on 117 patients with adenomyosis from October 2012 to July 2014. The presence or absence, colour, quantity and duration of vaginal discharge, which was different from pre-ablation, were recorded within 1 year after PMWA. Patients were categorised into G1 (n = 26, without vaginal discharge), G2 (n = 40, vaginal discharge lasting 1 to 19 days), and G3 (n = 51, vaginal discharge lasting ≥20 days) groups. The potentially correlative variables were analysed. Variables with significant correlations with vaginal discharge post-ablation were identified via binary logistic regression analysis. Results The differences in adenomyosis type, pre-ablation uterine volume, total microwave ablation energy, total non-perfused volume (NPV) and minimum distance from the non-perfused lesion (NPL) margin to the endomyometrial junction (EMJ) among groups were statistically significant (p = 0.005, p = 0.000, p = 0.000, p = 0.005 and p = 0.000, respectively). Minimum distance from the NPL margin to the EMJ was the strongest predictor of vaginal discharge post-ablation with odds ratio (OR) 0.632, p = 0.018, 95% CI 0.432-0.923. Patients with diffuse adenomyosis were more likely to have prolonged vaginal discharge (≥20 days) post-ablation (OR 3.461, p = 0.000, 95% CI 1.759-7.536). Conclusion The minimum distance from the NPL margin to the EMJ and adenomyosis type were significantly associated with vaginal discharge post-ablation.

Fusion with pep-1, a cell-penetrating peptide, enhances the transmembrane ability of human epidermal growth factor.

Administration of macromolecule compositions in medicine and cosmetics always exhibited low bioavailability due to the limitation of transmembrane transport. Here, human epidermal growth factor (hEGF) was fused with glutathione S-transferase (GST) and Pep-1, the first commercial cell-penetrating peptide, in Escherichia coli. The fusion protein was firstly purified with the affinity chromatography, and then the GST tag was released by TEV protease. Final purification was achieved by the ion exchange chromatography. The biological activities and the transmembrane ability of the obtained products were determined using scratch wound-healing assay, MTT analysis, and immunofluorescence assay. The results showed that both rhEGF and Pep-1-fused hEGF were soluble expressed in E. coli. The fusion of Pep-1 could markedly increase the transmembrane ability of EGF, whereas it did not interfere with the growth-stimulating and migration-promoting functions of hEGF on fibroblasts. This research provided a novel strategy for the transmembrane transport of protein-derived cosmetics or drugs.

Quality and antitumour activity evaluation of extract of Hypericum ascyron.

Similarity assessment of complex chromatographic profiles is a potential tool for the identification and quality control of herbal medicinal products to guarantee the expected biological activity. In this paper, a high-performance liquid chromatography method was established for controlling the quality of extract of Hypericum ascyron for the first time. With this method, the correlation coefficients of similarity of 10 batches extract of H. ascyron were >0.97. The extract of H. ascyron displayed steadily inhibitorty activities on the growth of human cervical cancer Hela cell lines. Therefore, the present study successfully set up a sensitive efficient method which might confirm stable biological activity of the extract of H. ascyron.

Downregulation of both EGFR and ErbB3 improves the cellular response to pemetrexed in an established pemetrexed­resistant lung adenocarcinoma A549 cell line.

Epidermal growth factor receptor (EGFR) and ErbB3 (HER3) play important roles in the regulation of cell proliferation, differentiation, anti-apoptosis and chemoresistance; however, their dysregulation in pemetrexed (PEM) resistance remains unclear. The aim of the present study was to clarify the relationship between PEM resistance and gene expression of EGFR and ErbB3, by establishing the PEM-resistant lung adenocarcinoma A549 cell line, A549/PEM. Compared with A549 cells, the A549/PEM cells were significantly more resistant to PEM (P=0.0024). The downregulation of S phase and arrest at G1 stage were detected in the A549/PEM cell line when compared to the A549 cells (P<0.05). The apoptosis rate of A549/PEM cells was much lower than that of the A549 cells after a 24 h continuous exposure to PEM (P<0.001). Real-time PCR and western blotting demonstrated the overexpression of EGFR and ErbB3 in A549/PEM cells. However, downregulation of EGFR or ErbB3 by lentiviral delivered shRNAs in A549/PEM cells showed no significant correlation with PEM sensitivity while silencing both EGFR and ErbB3 increased the cellular response to PEM in the A549/PEM cells and significantly decreased phosphorylation of STAT3, AKT and ERK. Together, these data suggest that either high expression of EGFR or ErbB3 plays a critical role in the cellular response to PEM in human lung adenocarcinoma cells though EGFR/ErbB3-dependent pathways.

[Clinical significance of tissue factor and vascular endothelial growth factor expressions on CD14+ monocytes in patients with non Hodgkin lymphoma].

OBJECTIVE: To explore the values of tissue factor (TF) and vascular endothelial growth factor (VEGF) expressions on peripheral CD14+ monocytes in disease assessment, prognosis, and short-term efficacy evaluation of non-Hodgkin lymphoma (NHL) patients. METHODS: TF and VEGF expressions on CD14+monocytes in 47 NHL patients (disease group) before chemotherapy and after 4 chemotherapy cycles and in 30 healthy subjects (control group) were detected by flow cytometry, and the potential relationship among TF, VEGF, International Prognostic Index (IPI), and short-term efficacy were analyzed. RESULTS: TF and VEGF expressions on CD14 + monocytes in disease group were significantly higher than those in control group ( all P <0. 01) and positive correlation was showed between them (r = 0. 708, P = 0.00). TF and VEGF expressions in Ann Arbor stage III and IV (n = 22 and 19) , symptomatic (n = 22) , lactate dehydrogenase (LDH) increased (n = 21) , Eastern Cooperative Oncology Group (ECOG) score 2-4 (n = 12) and extranodal lesions >1 (n = 16) groups were significantly higher than those in Ann Arbor stage II (an = 6) , asymptomatic (an =25) , LDH normal (n = 26) , ECOG score 0-1 ( n = 35) and extranodal lesions ~1 ( na = 31) groups, respectively (all P <0.05). The expressions of TF and VEGF on CD14 + monocytes in high-risk (n = 7) or high-middle-risk (n = 11) groups were significantly increased compared with low-risk (n = 15) or low-middle-risk(n = 14) groups, respectively (all P <0. 01). TF and VEGF expressions in non-remission group before chemotherapy (n = 11) were both obviously higher than those in remission group (an = 36, all P <0. 01) , and after chemotherapy their expressions in remission group were significantly lower than those before chemotherapy (all P <0. 01) , while such significant changes were not observed in the non-remission group ( all P > 0. 05). CONCLUSION: The high expressions of TF and VEGF on peripheral CD14 + monocytes can be useful markers in dis-ease assessment, prognosis evaluation and short-term efficacy observation of NHL patients.

[Relationship between coagulation function and international prognostic index in lymphoma patients].

The study was aimed to investigate the clinical significance of coagulation function changes in lymphoma patients and to analyze the relationship between their changes and international prognostic index (IPI). The prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT) and fibrinogen (FIB) were detected by magnetic bead method in 75 lymphoma patients and 20 healthy persons. The dehydrogenase (LDH) level was detected by rate method in all lymphoma patients and healthy persons. The results showed that (1) the APTT and FIB more obviously increased in lymphoma patients which displayed as hyperfibrinogenemia, as compared with control group (p < 0.05, p < 0.01); no obvious changes of coagulation indexes presented in patients with different ages and extranodal lesions (p > 0.05, p < 0.01). (2) APTT and FIB levels in stage III and IV patients were much higher than those in the stage II (p < 0.05 and < 0.01), and FIB level in stage IV group was significantly higher than those in the stage III (p < 0.05). FIB level in symptomatic group was significantly higher than that in asymptomatic group (p < 0.01). (3) APTT and FIB in increased LDH group were obviously higher than those in control group (p < 0.05, p < 0.01). Furthermore, FIB in increased LDH group was higher than that in normal LDH group (p < 0.05). FIB in performance status (PS) 2 - 4 groups increased significantly as compared with those in PS 0-1 group (p < 0.01). (4)FIB levels in the low-middle-risk, high-middle-risk and high-risk groups were significantly higher than those in control group (p < 0.01), while FIB levels in high-middle-risk and high-risk groups were higher than those in low-risk group (p < 0.05). (5) the number of FIB increased patients in symptomatic group, increased LDH group, PS 2 - 4 group and Ann Arbor stage III-IV group were much higher than those in counterparts (p < 0.05 or 0.01).There were positive correlations between FIB and LDH level, PS grades, Ann Arbor stages as well as risk grades respectively (p < 0.05 or 0.01). It is concluded that lymphoma patients usually accompany with hyperfibrinogenemia which may be influenced by Ann Arbor stage, systemic symptom, LDH level and PS grade. FIB is supposed to be an effective indication of prognosis in lymphoma patients.

[Influence of dipyridamole on expression of PAC-1 and CD62p in patients with malignant lymphoma].

This study was purposed to explore the expressions of platelet-activated markers PAC-1 and CD62p in peripheral blood of malignant lymphoma patients and the influence of dipyridamole on their expression. 32 lymphoma patients were divided into simple chemotherapy group (simple group) and chemotherapy plus dipyridamole group (combined group) randomly, and 15 healthy peoples were selected as control group. The dipyridamole of 100 mg/day was given to the patients in combined group. The expression levels of PAC-1, CD62p and fibrinogen (Fib) were detected by flow cytometry and magnetic bead method on day 0, 3, 7 and 14 of chemotherapy respectively. The results showed that the levels of PAC-1, CD62p and Fib in lymphoma patients were significantly higher than those in control group (p < 0.01, 0.05), moreover there was positive correlation between levels of PAC-1 and Fib (r = 0.549, p < 0.01). PAC-1 expression on day 0 and 3 of chemotherapy in simple group was higher than that on day 14 (p < 0.05, 0.01) and CD62p expression on day 3 of chemotherapy was higher than that on day 0, 7 and 14 (p < 0.05, 0.01). PAC-1 expression in combined group on day 14 of chemotherapy was lower than than on day 0 and 3 (p < 0.05, 0.01), and CD62p on day 14 was lower than that on day 3 of chemotherapy (p < 0.05); PAC-1 and CD62p expressions in combined group on day 3, 7 and 14 of chemotherapy were decreased than those in simple group, but Fib level was not changed significantly. It is concluded that the patients with malignant lymphoma usually accompany with platelet activation and hyperfibrinogenemia in peripheral blood. Applying dipyridamole routine dosage in chemotherapy can efficiently restrain platelet activation.

Inducible nitric oxide synthase is involved in the oxidation stress induced by HIV-1 gp120 in human retina pigment epithelial cells.

BACKGROUND: The human immunodeficiency virus-1 (HIV-1) envelope glycoprotein gp120 has been implicated in the development of AIDS-associated retinopathy. The present study tested the hypothesis that gp120 may induce oxidative stress including up regulation of inducible nitric oxide synthase (iNOS) and production of malondialdehyde (MDA) and nitric oxide (NO) to mediate retinopathy in retinal pigment epithelial (RPE) cells. METHODS: Human RPE cell line D407 was cultured and treated with gp120. HIV-1 gp120 protein induced lipid peroxidation product MDA. NO production and iNOS expression were examined in vitro by spectrophomtometry, real-time PCR, Western blotting, and confocal microscope. RESULTS: Addition of gp120 was able to induce RPE cells to produce NO and MDA in time- and dose-dependent manners (P < 0.05). Similarly, gp120 was also capable of up-regulating iNOS mRNA and protein in D407 cells in time- and dose-dependent manners. CONCLUSIONS: Gp120 induces oxidative stress in D407 cell by stimulating MDA and NO production, which is mediated by up-regulating iNOS expression. Gp120 may mediate oxidation stress in AIDS-associated retinopathy.

Reactive oxygen species stimulates receptor activator of NF-kappaB ligand expression in osteoblast.

It has been established that reactive oxygen species (ROS) such as H2O2 or superoxide anion is involved in bone loss-related diseases by stimulating osteoclast differentiation and bone resorption and that receptor activator of NF-kappaB ligand (RANKL) is a critical osteoclastogenic factor expressed on stromal/osteoblastic cells. However, the roles of ROS in RANKL expression and signaling mechanisms through which ROS regulates RANKL genes are not known. Here we report that increased intracellular ROS levels by H2O2 or xanthine/xanthine oxidase-generated superoxide anion stimulated RANKL mRNA and protein expression in human osteoblast-like MG63 cell line and primary mouse bone marrow stromal cells and calvarial osteoblasts. Further analysis revealed that ROS promoted phosphorylation of cAMP response element-binding protein (CREB)/ATF2 and its binding to CRE-domain in the murine RANKL promoter region. Moreover, the results of protein kinase A (PKA) inhibitor KT5720 and CREB1 RNA interference transfection clearly showed that PKA-CREB signaling pathway was necessary for ROS stimulation of RANKL in mouse osteoblasts. In human MG63 cells, however, we found that ROS promoted heat shock factor 2 (HSF2) binding to heat shock element in human RANKL promoter region and that HSF2, but not PKA, was required for ROS up-regulation of RANKL as revealed by KT5720 and HSF2 RNA interference transfection. We also found that ROS stimulated phosphorylation of extracellular signal-regulated kinases (ERKs) and that PD98059, the inhibitor for ERKs suppressed ROS-induced RANKL expression either in mouse osteoblasts or in MG63 cells. These results demonstrate that ROS stimulates RANKL expression via ERKs and PKA-CREB pathway in mouse osteoblasts and via ERKs and HSF2 in human MG63 cells.

Construction and identification of eukaryotic expression vector of human full-length PLCgamma1 gene.

OBJECTIVE: To construct the eukaryotic expression vector of human full-length PLCgamma1 gene for further study of the role of PLCgamma1 in cancer invasion. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) technique was used to amplify human full-length PLCgamma1 gene from MG63 cells with a pair of specific primers containing the restriction sites for HindIII and NotI. After purification, the product of RT-PCR was digested with HindIII and NotI before insertion into the corresponding sites of eukaryotic expression vector pLNCX2, yielding the recombinant plasmid pLNCX2/PLCgamma1. PCR, restriction endonuclease analysis and DNA sequencing were performed to identify the recombinant eukaryotic expression vector pLNCX2/PLCgamma1. RT-PCR and Western blotting were used to detect the expression of the PLCgamma1 gene in LoVo cells after transient transfection via Lipofectamine TM 2000. RESULTS: A 3 878-bp full-length PLCgamma1 gene fragment was successfully amplified by RT-PCR and inserted into eukaryotic expression vector pLNCX2. After digestion by HindIII and NotI, the recombinant eukaryotic expression vector pLNCX2/PLCgamma1 yielded a 3 878-bp fragment (PLCgamma1 gene) and a 6 100 bp fragment (vector). HindIII-BglII digestion was also done to verify the correctness of the recombinant plasmid, resulting in the identification of the fragments as expected. Sequencing analysis further confirmed the results. In addition, RT-PCR and Western blotting verified that the PLCgamma1 could overexpress in LoVo cells after transfection with recombinant eukaryotic expression vector pLNCX2/PLCgamma1. CONCLUSION: The recombinant eukaryotic expression vector pLNCX2/PLCgamma1 has been constructed successfully.

Hydrogen peroxide in the Burkitt's lymphoma cell line Raji provides protection against arsenic trioxide-induced apoptosis via the phosphoinositide-3 kinase signalling pathway.

Many anticarcinogenic drugs kill tumour cells by inducing apoptosis. We examined the effects of hydrogen peroxide (H(2)O(2)) on arsenic trioxide (As(2)O(3))-induced cell killing. Low concentrations of H(2)O(2) (200 micromol/l) inhibited the ability of As(2)O(3) to induce apoptosis in the Burkitt's lymphoma cell line Raji. H(2)O(2) altered the form of cell death from apoptosis to pyknosis/necrosis and also lowered the degree of cell killing by As(2)O(3). H(2)O(2) was capable of preventing caspase-3 activation induced by As(2)O(3) in Raji cells. Incubation of cells with a phosphoinositide-3 kinase (PI-3K) inhibitor, wortmannin (100 nmol/l), blocked the effects of H(2)O(2) on As(2)O(3)-induced caspase-3 activation. In addition, the PI-3K inhibitor partially blocked the effects of H(2)O(2) on up-regulation of Bcl-2 and Bcl-X(L) protein expression, down-regulation of Bax protein expression, and phosphorylation of Bcl-2 and IkappaBalpha. This investigation demonstrated for the first time that low concentrations of H(2)O(2) provide protection against the in vivo of As(2)O(3)-induced apoptosis. PI-3K plays a crucial role in enhancing cell survival during H(2)O(2), inhibiting As(2)O(3)-induced apoptosis in the Burkitt's lymphoma cells. As(2)O(3)-induced cancer cell apoptosis may be enhanced by certain antioxidants in the treatment protocol.

[Hydrogen peroxide inhibits arsenic trioxide-induced apoptosis of Burkitt lymphoma cells].

OBJECTIVE: To examine the effects of hydrogen peroxide (H2O2) on arsenic trioxide (As2O3)-induced apoptosis of human Burkitt lymphoma cells and evaluate the value of fluorescence microscope in analyzing cellular apoptosis. METHODS: As2O3 was used to induce apoptosis in human Burkitt lymphoma cells BJAB, and the cellular changes were analyzed quantitatively using transmission electron microscope and fluorescence microscope after staining with Hoechst 33342/ propidium iodide (PI). RESULTS: Under fluorescence microscope and electron microscope, BJAB cells displayed chromatin aggregation, nuclear margination and fragmentation in response to As2O3 treatment. In the presence of relatively low levels of H2O2 (200 micromol/L), the cell death resulting from apoptosis was inhibited with pyknosis and necrosis becoming the major pathway, while As2O3 failed to induce cell apoptosis. The cells showed none of the typical markers of apoptosis nor any characteristic necrotic changes, and the nuclei exhibited condensation instead of swelling. In addition, the rate of cell death induced by As2O3 was decreased in the presence of H2O2. CONCLUSIONS: Low levels of H2O2 (200 micromol/L) inhibits As2O3-induced (at clinically achievable concentrations) apoptosis of the human malignant lymphoma cells. Fluorescence microscopy makes possible quantitative analysis of apoptosis, capable of distinguishing not only cell apoptosis from necrosis, but also membrane-intact from membrane-permeable apoptotic cells.