Effect of nuclear factor-κB signaling pathway on the expression of hypoxia-inducible factor-1 and vascular endothelial growth factor in nasal polyp cells / 新乡医学院学报

Objective To observe the effect of nuclear factor-κB (NF-κB) signaling pathway on the expression of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) in nasal polyp cells under hypoxic cultivation,and to investigate the relationship between NF-κB signaling pathway and the development of nasal polyp.Methods The nasal polyp and inferior turbinate tissue specimens were collected in the First Affiliated Hospital of Jiamusi University from January 2012 to December 2014.The nasal polyp and inferior turbinate tissues were taken to obtain nasal polyp cells and inferior turbinate cells,then the cells were cultured in primary culture,and the cells were cultured under hypoxia when they grew to 90％.When the cells were cultured in vitro to 90％,the NF-κB inhibitor BAY11-7082 was added (inhibitor intervention group),the other cells without inhibitor were used as controls (no inhibitor group),then the cells in the two groups were cultured under hypoxia.The cells were collected when they were cultured for 0,3,6 and 9 hours,respectively;and the expression of HIF-1α,VEGF and NF-κB p65 protein in the cells were detected by Western blot.Results Compared with 0 hour,the expression of HIF-1α,VEGF and NF-κB p65 protein in nasal polyp cells increased significantly after 3,6 and 9 hours of hypoxic cultivation (P ＜ 0.05);however,the expression of HIF-1α,VEGF and NF-κB p65 protein in inferior turbinate cells was not statistically significant (P ＞ 0.05).The expression of HIF-1α,VEGF and NF-κB p65 protein in nasal polyposis cells after 6 hours of hypoxic cultivation was significantly higher than that after 3 and 9 hours of hypoxic cultivation (P ＜ 0.05);but there was no significant difference in the expression of HIF-1α,VEGF and NF-κB p65 protein in nasal polyp cells between 3 and 9 hours of hypoxic cultivation (P ＞ 0.05).Compared with 0 hour,the expression of HIF-1α and VEGF protein in nasal polyp cells of no inhibitor group increased significantly after 3,6 and 9 hours of hypoxic cultivation (P ＜ 0.05);and the expression of HIF-1α and VEGF protein in nasal polyp cells after 6 hours of hypoxic cultivation was significantly higher than that after 3 and 9 hours of hypoxic cultivation in no inhibitor group (P ＜ 0.05).But there was no significant difference in the expression of HIF-1α and VEGF protein in nasal polyp cells of no inhibitor group between 3 and 9 hours of hypoxic cultivation (P ＞ 0.05).There was no significant difference in the expression of HIF-1 α and VEGF protein in nasal polyp cells of the inhibitor intervention group among 0,3,6 and 9 hours of hypoxic cultivation (P ＞ 0.05).There was no significant difference in the expression of HIF-1α and VEGF protein in nasal polyp cells between no inhibitor group and inhibitor intervention group at 0 hour of hypoxic cultivation (P ＞0.05).The expression of HIF-1α and VEGF protein in nasal polyp cells of inhibitor intervention group was significantly lower than that of no inhibitor group after 3,6 and 9 hours of hypoxic cultivation (P ＜ 0.05).Conclusion The expression of HIF-1α,VEGF and NF-κB p65 protein increased in nasal polyp cells under hypoxia condition.NF-κB signaling pathway may mediate hypoxia-induced HIF-1α and VEGF protein expression,and participate in the occurrence and development of nasal polyp.

[Case-control study on the relationship of chronic low back pain of facet joint origin with the distribution of nerve endings and neuropeptide: a quantitative histological analysis].

OBJECTIVE: To investigate the density and distribution of nerve endings and neuropeptide Y (NPY) in lumbar facet joints of patients with low back pain. METHODS: Fifteen patients without low back pain were selected as control group (group A). Facet joint samples in group A were obtained during the operation or lumbar spinal canal tumor they suffered from. Those patients with low back pain were divided into three groups according to their different origins of pain, such as not from facet joint (group B, 15 patients) ,from facet joint only (group C, 20 patients), or from facet joint partially (group D, 20 patients). Different origins were determined by VAS after facet joint block. The density and distribution of nerve ending and neuropeptide in the capsular tissues were analyzed by a modified gold chloride staining and immunochemistry respectively. RESULTS: Compared with the ones in group A and B, the fact joints in group C and D were more inclined to be degenerated and got more nerve endings. NPY was expressed mainly in the facet joint of patients with low back pain in group C and D. In addition, there was a significant relationship between the distribution of nerve endings and NPY expression,while none of them were related with MRI Fujiwara grade of facet joint. CONCLUSION: These results suggest that the number of mechanoreceptors, neural sprouting and secreted peptides in the facet joint capsules vary with the change of mechanical or nociceptive stimulation, which may promote the development of low back pain in return.

Uniaxial cyclic stretch promotes osteogenic differentiation and synthesis of BMP2 in the C3H10T1/2 cells with BMP2 gene variant of rs2273073 (T/G).

Ossification of the posterior longitudinal ligament of the cervical spine (OPLL) is characterized by the replacement of ligament tissues with ectopic bone formation, and this result is strongly affected by genetic and local factors. Two single nucleotide polymorphisms (SNPs) of rs2273073 (T/G) and rs235768 (A/T) of bone morphogenetic protein 2 (BMP2) gene which are associated with OPLL have been reported in our previous report. In this study, we confirmed the connection in 18 case samples analysis of BMP2 gene in OPLL patients; additionally, it was also shown from the OPLL patients with ligament tissues that enchondral ossification and expression of BMP2 were significantly higher compared with the non-OPLL patients by histological examination, immunohistochemistry and Western blotting analysis. To investigate the underlying mechanism, we studied the effect of SNPs in cell model. The C3H10T1/2 cells with different BMP2 gene variants were constructed and then subjected to uniaxial cyclic stretch (0.5 Hz, 10% stretch). In the presence of mechanical stress, the expression of BMP2 protein in C3H10T1/2 cells transfected by BMP2 (rs2273073 (T/G)) and BMP2 (rs2273073 (T/G), rs235768 (A/T)) were significantly higher than the corresponding static groups (P<0.05). In conclusion, these results suggested that BMP2 gene variant of rs2273073 (T/G) could not only increase cell susceptibility to bone transformation similar to pre-OPLL change, but also increase the sensibility to mechanical stress which might play an important role during the progression of OPLL.

LIPUS enhance elongation of neurites in rat cortical neurons through inhibition of GSK-3beta.

OBJECTIVE: Low-intensity pulsed ultrasound (LIPUS) has been reported to enhance proliferation and to alter protein production in various kinds of cells. In the present study, we measured the neurites length after LIPUS treatment to define the effectiveness of LIPUS stimulation on neurons, and then we examined the acticity of GSK-3beta to study the intracellular mechanism of neurite's outgrowth. METHODS: LIPUS was applied to cultured primary rat cortical neurons for 5 minutes every day with spatial- and temporal average intensities (SATA) of 10 mW/cm(2), a pulse width of 200 microseconds, a repetition rate of 1.5 KHz, and an operation frequency of 1 MHz. Neurons were photographed on the third day after LIPUS treatment and harvested at third, seventh, and tenth days for immunoblot and semi-quantitative RT-PCR analysis. RESULTS: Morphology change showed that neurite extension was enhanced by LIPUS. There was also a remarkable decrease of proteins, including p-Akt, p-GSK-3beta, and p-CRMP-2, observed on the seventh and tenth days, and of GSK-3beta mRNA expression, observed on the seventh day, in neurons treated with LIPUS. CONCLUSION: LIPUS can enhance elongation of neurites and it is possible through the decreased expression of GSK-3beta.

Effect of low-intensity pulsed ultrasound on the expression of neurotrophin-3 and brain-derived neurotrophic factor in cultured Schwann cells.

It is generally known that low-intensity pulsed ultrasound (LIPUS) accelerates peripheral nerve tissue regeneration. However, the precise cellular mechanism involved is still unclear. The purpose of this study was to determine how the Schwann cells respond directly to LIPUS stimuli. Thus, we investigated the effect of LIPUS on cell proliferation, neurotrophin-3 (NT-3), and brain-derived neurotrophic factor (BDNF) mRNA expression in rat Schwann cells. Schwann cells were enzymatically isolated from postnatal 1-3 day rat sciatic nerve tissue and cultured in the six-well plate. The ultrasound was applied at a frequency of 1 MHz and an intensity of 100 mW/cm(2) spatial average temporal average for 5 minutes/day. The control group was cultured in the same way but without the administration of ultrasound. Immunohistochemistry demonstrated that more than 98% of the experimental and control cells were positive for S-100, NT-3, and BDNF. With 5-bromo-2'-deoxyuridine (BrdU) assay, the stimulated cells also exhibited an increase in the rate of cell proliferation on days 4, 7, 10, and 14. Further investigation found that mRNA expression of NT-3 was significantly upregulated in experimental groups compared with the control 14 days after the LIPUS stimulation (the ratio of NT-3/beta-actin was 0.56 +/- 0.13 vs. 0.41 +/- 0.09, P < 0.01), whereas the mRNA expression of BDNF was significantly downregulated in experimental groups compared with the control (the ratio of BDNF/beta-actin was 0.51 +/- 0.05 vs. 0.60 +/- 0.08, P < 0.05). These results demonstrated that the application of LIPUS promotes cell proliferation and NT-3 gene expression in Schwann cells, and involved in the alteration of BDNF gene expression.