Effects of a GSK-3ß inhibitor on the renal expression levels of RANK, RANKL and NF-κB in a rat model of diabetic nephropathy.

The present study aimed to investigate the effects of glycogen synthase kinase-3ß (GSK-3ß) on the expression levels of receptor activator of nuclear factor (NF)-κB (RANK), RANK ligand (RANKL) and NF-κB in the renal tissues of rats modeling diabetic nephropathy (DN). The rats were allocated at random into three groups, as follows: Normal control group (NC), the DN model group (DNM group) and the DN model lithium chloride (LiCl) intervention group (DNI group). Urinary proteins were examined by staining with the Coomassie Brilliant Blue dye for 24 h. Histochemical analyses of kidney tissue sections were conducted using hematoxylin and eosin staining, after which the kidney pathology of the rats was observed. In addition, the mRNA and protein expression levels of GSK-3ß, RANK, RANKL and NF-κB in the renal tissues were detected using reverse transcription-quantitative polymerase chain reaction and immunohistochemistry, respectively. As compared with the NC group, the level of urinary protein was significantly increased in the DNM group (P<0.05); however, as compared with the DNM Group, the level of urinary protein at 12 weeks was significantly decreased in the DNI group (P<0.05). As compared with the NC group, marked pathological changes were detected, and the mRNA and protein expression levels of GSK-3ß, RANK, RANKL and NF-κB were significantly increased, in the renal tissues of the DNM group. Conversely, pathological alterations in the renal tissues were attenuated, and the mRNA and protein expression levels of GSK-3ß, RANK, RANKL and NF-κB were significantly decreased (P<0.05), in the DNI group, as compared with the DNM group. The results of the present study suggested that GSK-3ß, RANK, RANKL and NF-κB may be crucially involved in the development of DN, and that LiCl may effectively attenuate DN by reducing the expression levels of GSK-3ß, RANK, RANKL and NF-κB.

[Detection of regional lymph node micrometastasis and its impact on long-term survival of non-small cell lung cancer (NSCLC) patients].

BACKGROUND & OBJECTIVE: Regional lymph node micrometastasis, which can not be found by routine pathologic examination, may be detected by immunohistochemistry after radical operation of tumors. This study was to investigate regional lymph node micrometastasis in non-small cell lung cancer (NSCLC) and its impact on prognosis. METHODS: The expressions of cytokeratin (CK) in 684 specimens from 78 stage pT1-2N0M0 NSCLC patients, treated from Jan. 1996 to Dec. 2003, were detected by EnVision method using anti-CK antibody AE1/AE3. The correlation of CK expression to prognosis was analyzed. RESULTS: The overall detection rate of CK was 26.9%û the detection rates were 5.9% in stage p-IA patients and 32.8% in stage p-IB patients. The 1-, 3-, 5-, and 8-year survival rates were significantly higher in non-micrometastasis group than in micrometastasis group (90.80% vs. 60.00%, 69.70% vs. 48.48%, 55.76% vs. 29.09%, and 37.17% vs. 0, P=0.008)û the median survival time were 87 months and 23 months, respectively. CK and KPS score were independent prognostic factors of NSCLC (P=0.014, P<0.001). Compared with surgery alone, surgery plus postoperative radiochemotherapy prolonged survival, but the difference was not significant (P=0.063). CONCLUSIONS: The detection rate of lymph node micrometastases may be increased with the progression of NSCLC. Micrometastasis is an independent prognostic factor of NSCLC.

[Fluorosis on expression of nicotinic acetylcholine receptors in protein and gene levels in human SH-SY5Y neuroblastoma cells].

OBJECTIVE: To investigate the influence of fluorosis on nicotinic acetylcholine receptors (nAChRs) in protein and gene levels in SH-SY5Y cells and the mechanism of the receptor modification. METHODS: SH-SY5Y cells, a human neuroblastoma cell line, were incubated with different concentrations of fluoride or with antioxidant for 48 hours. The functions of cells were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) method, and protein oxidation detected by carbonyl content; the alpha3 and alpha7 nAChR subunits in protein level were measured by Western blotting and in mRNA level by RT-polymerase chain reaction (RT-PCR). RESULTS: In high-dose group as compared to the control, the decreased MTT (49%), increased protein oxidation (72%), and lower expression of alpha3 (51%) and alpha7 (47%) nAChR subunit proteins were obviously observed in SH-SY5Y cells. There were no changes in expression of nAChR subunit mRNAs between the cells treated with fluoride and those un-treated in controls. Prior treatment with antioxidant resulted in preventing the decrease of nAChR protein in cells exposed to the high doses of fluoride. CONCLUSION: Fluorosis should result in damage of cells and the declined expression of nAChRs in protein levels, but no influences on gene expression of the receptors in human neuroblastoma neurons. The decreased nAChR proteins might be involved in the mechanism of oxidative stress induced by fluorosis.

Decreased nicotinic receptors in PC12 cells and rat brains influenced by fluoride toxicity--a mechanism relating to a damage at the level in post-transcription of the receptor genes.

In order to reveal mechanisms of the decreased nicotinic acetylcholine receptors (nAChRs) resulted from fluoride toxicity, we treated PC12 cells by different concentrations of fluoride (0.1-100 ppm) for 48 h, and exposed rats to high doses of fluoride (30 and 100 ppm) in their drinking water for 7 months. The expression of nAChRs at mRNA and protein levels, neurotoxicity and oxidative stress were analyzed in the study. The results indicated that there were no significant changes at mRNA level of the nAChR alpha3, alpha7, beta2 subunits in PC12 cells, and alpha4, alpha7, beta2 subunits in rat brains between the groups with fluorosis and controls. A significant decline in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction, and increased levels of protein oxidation and lipid peroxidation were observe in PC12 cells treated with high doses of fluoride or rat brains with chronic fluorosis. The decreases of nAChR alpha3 and alpha7 subunit proteins in PC12 cells resulted from fluoride toxicity were mostly prevented by a pretreatment with antioxidant. The results suggest that the deficit of nAChRs induced by fluoride toxicity occurs at the level of post-transcription of the receptor gene, in which a mechanism might be involved in the damage by oxidative stress.

Selective decreases of nicotinic acetylcholine receptors in PC12 cells exposed to fluoride.

In an attempt to elucidate the mechanism by which excessive fluoride damages the central nervous system, the effects of exposure of PC12 cells to different concentrations of fluoride for 48 h on nicotinic acetylcholine receptors (nAChRs) were characterized here. Significant reductions in the number of binding sites for both [3H]epibatidine and [125I]alpha-bungarotoxin, as well as a significant decrease in the B(max) value for the high-affinity of epibatidine binding site were observed in PC12 cells subjected to high levels of fluoride. On the protein level, the alpha 3 and alpha 7 subunits of nAChRs were also significantly decreased in the cells exposed to high concentrations of fluoride. In contrast, such exposure had no significant effect on the level of the beta 2 subunit. These findings suggest that selective decreases in the number of nAChRs may play an important role in the mechanism(s) by which fluoride causes dysfunction of the central nervous system.

Chronic fluoride toxicity decreases the number of nicotinic acetylcholine receptors in rat brain.

In order to investigate the molecular mechanism(s) underlying brain dysfunction caused by chronic fluorosis, neuronal nicotinic acetylcholine receptors (nAChRs) in the brain of rats receiving either 30 or 100 ppm fluoride in their drinking water for 7 months were analyzed in the present study employing ligand binding and Western blotting. There was a significant reduction in the number of [3H]epibatidine binding sites in the brain of rats exposed 100 ppm of fluoride, but no alteration after exposed to 30 ppm. On the other hand, the number of [125I]alpha-BTX binding sites was significantly decreased in the brains of rats exposed to both levels of fluoride. Western blotting revealed that the level of the nAChR alpha4 subunit protein in the brains of rats was significantly lowered by exposure to 100 ppm, but not 30 ppm fluoride; whereas the expression of the alpha7 subunit protein was significantly decreased by both levels of exposure. In contrast, there was no significant change in the level of the beta2 subunit protein in the brains of rats administered fluoride. Since nAChRs play major roles in cognitive processes such as learning and memory, the decrease in the number of nAChRs caused by fluoride toxicity may be an important factor in the mechanism of brain dysfunction in the disorder.