Uptake of bacterial lipopolysaccharide and expression of tumor necrosis factor α mRNA in isolated rat intrahepatic bile duct epithelial cells *

AIM To study the uptake of bacterial lipopolysaccharides (LPS) and expression of tumor necrosis factor α mRNA (TNF α mRNA) with cultured rat intrahepatic bile duct epithelial cells.

Influence of neoadjuvant chemotherapy on proliferation, apoptosis and multi-drug resistance in osteosarcoma cells

Objective: To study the influence of neoadjuvant chemotherapy on the expression of cyclin D1, Bcl-2, PCNA and P- gp in osteosarcoma cells and the relationship between the expression and tumor cell necrosis rate (TCNR) after chemotherapy. Methods: By using immunohistochemistry, the expression of cyclin D1, Bcl-2, PCNA and P-gp was detected in 23 cases of osteosarcoma and TCNR were calculated. Results: The pre-chemotherapy positive expression rate of cyclin D1, Bcl-2, PCNA and P-gp was 73.9%, 69.6%, 91.3% and 21.7%, respectively, and that post-chemotherapy positive expression rate was 52.1%, 34.8%, 43.5% and 56.5%, respectively. The positive expression rate of Bcl-2 and PCNA after chemotherapy was much lower than that before chemotherapy (P=0.039, 0.034). After chemotherapy, the expression rate of P-gp was higher (P=0.021) and the expression of cyclin D1 had no statistically significant difference (P=0.180) comparing with that before chemotherapy. No correla- tion existed between the expression of cyclin D1, Bcl-2, PCNA, P-gp and TCNR before chemotherapy (P=0.155, 0.371, 1.000 and 0.640). There was a negative correlation between the expression of Bcl-2, PCNA, P-gp and TCNR (P=0.009, 0.012 and 0.015), but no relationship existed between the cyclin D1 and TCNR (P=0.100) after chemotherapy. Conclusion: Chemotherapy could inhibit proliferation and induce apoptosis of osteosarcoma cells. At the same time, due to the overexpression of the P-gp, the drug resistance of the osteosarcoma cells was increased. The detection of cyclin D1, Bcl-2, PCNA and P-gp in osteosarcoma samples before chemotherapy might not be used to predict the curative effect of the chemotherapy.

Quinacrine pretreatment reduces microwave-induced neuronal damage by stabilizing the cell membrane

Quinacrine, widely used to treat parasitic diseases, binds to cell membranes. We previously found that quinacrine pretreatment reduced microwave radiation damage in rat hippocampal neurons, but the molecular mechanism remains poorly understood. Considering the thermal effects of microwave radiation and the protective effects of quinacrine on heat damage in cells, we hypothesized that quinacrine would prevent microwave radiation damage to cells in a mechanism associated with cell membrane stability. To test this, we used retinoic acid to induce PC12 cells to differentiate into neuron-like cells. We then pretreated the neurons with quinacrine （20 and 40 mM） and irradiated them with 50 mW/cm^2 microwaves for 3 or 6 hours. Flow cytometry, atomic force microscopy and western blot assays revealed that irradiated cells pretreated with quinacrine showed markedly less apoptosis, necrosis, and membrane damage, and greater expression of heat shock protein 70, than cells exposed to microwave irradiation alone. These results suggest that quinacrine stabilizes the neuronal membrane structure by upregulating the expression of heat shock protein 70, thus reducing neuronal injury caused by microwave radiation.

MESANGIAL CELL PROLIFERATION INDUCED BY TNF-α IS DETERMINED BY LEVELS OF CYCLIN-KINASE INHIBITOR P27

Objective To investigate the role of cyclin-kinase inhibitor p27 on proliferation of mesangial cell(MC) induced by tumor necrosis factor α( TNF-α ). Methods The p27 protein of MC lysate was detected with western blotting analysis. The degree of MC proliferation was estimated through [^3H] thymidine incorporation. The effect of reducing p27 expression on MC proliferation was analysed with p27 antisense oligodeoxynucleotide (ODN). Results TNF-α(200000U/L) decreased p27 level to (0.6±0.1 ) from (1. 1±0.1 ) of MC lysate cultured in serum free DMEM for 24h ( P<0.01 ) and increased [^3H] thymidine incorporation to ( 2060±112 ) from (685±53) cpm/well( P<0.01 ). p27 antisense ODN transfection decreased p27 level of MC stimulated by TNF-α for 24h [(0.3±0.1 ) vs (0.6±0.1), P <0.01 ] and increased [^3H] thymidine incorporation [(2420±130) vs (2060±112) cpm/well, P <0.05]. Conclusion The decline of p27 protein maybe play an important role in MC proliferation induced by TNF-α.

Necrostatin-1 protection of dopaminergic neurons

Necroptosis is characterized by programmed necrotic cell death and autophagic activation and might be involved in the death process of dopaminergic neurons in Parkinson＇s disease. We hypothesized that necrostatin-1 could block necroptosis and give protection to dopaminergic neurons. There is likely to be crosstalk between necroptosis and other cell death pathways, such as apoptosis and autophagy. PC12 cells were pretreated with necroststin-1 1 hour before exposure to 6-hydroxydopamine. We examined cell viability, mitochondrial membrane potential and expression patterns of apoptotic and necroptotic death signaling proteins. The results showed that the autophagy/lysosomal pathway is involved in the 6-hydroxydopamine-induced death process of PC12 cells. Mitochondrial disability induced overactive autophagy, increased cathepsin B expression, and diminished Bcl-2 expression. Necrostatin-1 within a certain concentration range（5–30 μM） elevated the viability of PC12 cells, stabilized mitochondrial membrane potential, inhibited excessive autophagy, reduced the expression of LC3-II and cathepsin B, and increased Bcl-2 expression. These findings suggest that necrostatin-1 exerted a protective effect against injury on dopaminergic neurons. Necrostatin-1 interacts with the apoptosis signaling pathway during this process. This pathway could be a new neuroprotective and therapeutic target in Parkinson＇s disease.

Polymorphisms in tumor necrosis factor genes and susceptibility to esophageal squamous cell carcinoma and gastric cardiac adenocarcinoma in a population of high incidence region of North China

Background We investigated the possible association of the functional polymorphisms in the tumor necrosis factor （TNF） genes with susceptibility to esophageal squamous cell carcinoma （ESCC） and gastric cardiac adenocarcinoma （GCA）.Methods The TNF-α-308G/A and TNF-β ＋ 252G/A single nucleotide polymorphisms （SNPs） were genotyped using polymerase-chain reaction-restriction fragment length polymorphism （PCR-RFLP） analysis, in 555 cancer patients （291 ESCC and 264 GCA） and 437 healthy controls in a high incidence region of North China. Results Among healthy controls, frequencies of the TNF-α 1/1, 1/2 and 2/2 genotypes were 89.4% ,9.2% and 1.4% respectively, while frequencies of the TNF-β B1/B1, B1/B2 and B2/B2 genotypes were 12.6% , 32.3% and 55.1%, respectively. No significant difference was found in the overall genotype and allelotype distribution of the TNF-α-308G/A and TNF-β ＋ 252G/A SNPs among cancer patients and controls. However, both the B1/B1 genotype and B1/B2 genotype significantly increased the risk of developing ESCC [ the age and gender adjusted odds ratio （OR） =2.04 and 1.91, 95% confidence interval （CI） = 1.04 -4.43 and 1.14 - 2.60, respectively] and GCA （the age and gender adjusted OR =2. 68 and 2. 64, 95% CI = 1.14 -6.29 and 1.47 -4.72, respectively） in individuals with negative family history of UGIC, in comparison with the B2/B2 genotype. When the two TNF polymorphisms were combined and analyzed, individuals with the TNF-β B1/B2 and TNF-α1/2 or 2/2 genotypes significantly reduced the risk of developing ESCC and GCA, in comparison with those harboring the TNF-β B2/B2 and TNF-α 1/1 genotypes （ the age and gender adjusted OR = 0.37 and 0. 34, 95% CI =0. 15 -0.92 and 0. 13 -0.90, respectively）. Conclusions Therefore, the TNF-α -308G/A and TNF-β ＋ 252G/A genotyping may be used as a stratification markers to predicate the risk of ESCC and GCA development in North China.

Multi-endpoint assays reveal more severe toxicity induced by chloraminated effluent organic matter than chloraminated natural organic matter

Disinfection by chloramination produces toxic byproducts and the difference in toxicity of reclaimed and drinking water treated by chloramination remains unclear.This study investigated cytotoxic effects at the same concentrations of dissolved organic matter and showed that chloraminated effluent organic matter(EfOM)induced 1.7 times higher cytotoxicity than chloraminated natural organic matter(NOM)applied to simulate drinking water.Chloraminated EfOM induced more reactive nitrogen species than chloraminated NOM,and chloraminated EfOM and NOM induced similar and higher levels of reactive oxygen species than the negative control,respectively.Consequently,intracellular macromolecule damage indicated by DNA/RNA damage marker 8–hydroxy-(deoxy)guanosine and the intracellular protein carbonyl concentration induced by chloraminated EfOM was higher and slightly more than that induced by chloraminated NOM,respectively.These data were consistent with the effects on cell physiological processes.Cell cycle arrest mainly occurred in G2 phase by chloraminated EfOM and NOM.Early apoptotic cells,which could return to normal,increased upon exposure to high concentrations of chloraminated EfOM and NOM.Moreover,necrotic cells were significantly increased from 0.5%to 2.5%when the concentration increased from 20-to 60-fold chloraminated EfOM,but were not obviously changed by chloraminated NOM.These results indicated that the comprehensive intracellular changes induced by toxic substances in chloraminated EfOM were more irreversible and induced more cell death than chloraminated NOM.

Intravenous transplantation of mesenchymal stem cells attenuates oleic acid induced acute lung injury in rats

Background Acute lung injury （ALl） and end-stage acute respiratory distress syndrome （ARDS） were among the most common causes of death in intensive care units. The activation of an inflammatory response and the damage of pulmonary epithelium and endotheliumwerethe hallmark of ALI/ARDS. Recent studies had demonstrated the importance of mesenchymal stem cells （MSCs） in maintaining the normal pulmonary endothelial and epithelial function as well as participating in modulating the inflammatory response and they are involved in epithelial and endothelial repair after injury. Here, our study demonstrates MSCs therapeutic potential in a rat model of ALI/ARDS. Methods Bone marrow derived MSCs were obtained from Sprague-Dawley （SD） rats and their differential potential was verified. ALl was induced in rats byoleic acid （OA）, and MSCs were transplanted intravenously. The lung injury and the concentration of cytokines in plasma and lung tissue extracts were assessed at 8 hours, 24 hours and 48 hours after OA-injection. Results The histological appearance and water content in rat lung tissue were significantly improved at different time points in rats treated with MSCs. The concentration of tumor necrosis factor-a and intercellular adhesion molecular-1 in rats plasma and lung tissue extracts were significantly inhibited after intravenous transplantation of MSCs, whereas interleukin-10 was significantly higher after MSCs transplantation at 8 hours, 24 hours and 48 hours after OA-challenge. Conclusions Intravenous transplantation of MSCs could maintain the integrity of the pulmonary alveolar-capillary barrier and modulate the inflammatory response to attenuate the experimental ALI/ARDS. Transplantation of MSCs could be a novel cell-based therapeutic strategy for prevention and treatment of ALI/ARDS.

Effects of Silver Nanoparticles on Soil Microbial Communities and Bacterial Nitrification in Suburban Vegetable Soils

Silver nanoparticles （AgNPs） are widely used antimicrobial compounds; however, they may pose a threat to non-targeted bacteria in the environment. In this study high-throughput sequencing was used to investigate the effects of different concentrations of AgNPs （10, 50, and 100 mg kg-1） on soil microbial community structure during short-term （7 d） exposure. The amounts of Acidobacteria, Actinobacteria, Cyanobacteria, and Nitrospirae significantly decreased with increasing AgNP concentration; meanwhile, several other phyla （e.g., Proteobacteria and Planctomycetes） increased and dominated. Nitrosomonas europaea, a well-characterized ammonia- oxidizing bacterium, was used to study the sensitivity of bacteria to AgNPs and ionic silver （Ag＋）. Flow cytometry was used to monitor the toxicity of low （1 mg L-l）, middle （10 mg L-l）, and high concentrations （20 mg L-1） of AgNPs, as well as Ag＋ （1 mg L-1） released into the medium from 20 mg L-1 concentration of AgNPs, towards N. europaea. After 12 h of exposure, the survival rate of N. europaea treated with 1 mg L-1 Ag＋ was significantly lower than those treated with low （1 mg L-1） and middle concentrations （10 mg L-1） of AgNPs, but the survival rate in the treatment with high concentration （20 mg L-1） of AgNPs was much lower. Additionally, necrosis rates were higher in the treatment with 20 mg L-1 AgNPs. Electron microscopy showed that Ag＋ caused serious damage to the cell wall of N. europaea, disintegrated the nucleoids, and condensed next to the cell membrane; however, dissolved Ag＋ is only one of the antibacterial mechanisms of AgNPs.