Halofuginone attenuates intervertebral discs degeneration by suppressing collagen I production and inactivating TGFß and NF-кB pathway.

Most low back pain is caused by intervertebral discs (IVD) degeneration, a disease that prevalence is increasing with age. Halofuginone, an analog of ferbrifugine isolated from plant Dichroa febrifuga, has drawn much attention in recent years for the wide range of bioactivities in malaria, cancer, fibrotic and autoimmune diseases. In this study, we evaluated the benefit effects of halofuginone in IVD degeneration treatment in a validated rabbit puncture model. Halofuginone treatment could attenuate disc degeneration by suppressing the decrease of discs height and nucleus pulposus signal strength. Besides, halofuginone treatment could suppress mRNA and protein expression of collagen I in nucleus pulposus. This might possibly due to the inactivation of transform growth factor-ß (TGFß) signal pathway by down-regulating p-Samd3 and up-regulating inhibitory Smad7. Then, we evaluated the effects of halofuginone treatment on nuclear factor of kappa B (NF-κB) signal pathway and its downstream pro-inflammatory cytokines. The level of p-p65 and p-IκBα was down-regulated in halofuginone treated group, indicating the inactivation of NF-κB signal pathway. The mRNA expression of interleukin 1ß (IL-1ß), tumor necrosis factor α (TNF-α), interleukin 6 (IL-6) and interleukin 8 (IL-8) was decreased in nucleus pulposus too, indicating the down-regulation of pro-inflammatory cytokines. In conclusion, halofuginone treatment could attenuate IVD degeneration and this was possibly due to suppressing of collagen I production and inactivation of TGFß and NF-κB signal pathway in nucleus pulposus of degenerated discs. These results suggest that halofuginone has the potential for IVD degeneration treatment, but more research is needed to validate this.

Gene expression of NMDA and AMPA receptors in different facial motor neurons.

OBJECTIVES/HYPOTHESIS: Facial motor neurons (FMNs) are involved in the remodeling of the facial nucleus in response to peripheral injury. This study aimed to examine the gene expression of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR) and N-methyl-D-aspartate subtype of ionotropic glutamate receptor (NMDAR) in reinnervating dormant FMNs after facial nerve axotomy. STUDY DESIGN: Animal study. METHODS: Rat models of facial-facial anastomosis were set up and raised until the 90th day. By laser capture microdissection (LCM), the reinnervating neurons labeled by Fluoro-Ruby (FR) were first captured, and the remaining (dormant) neurons identified by Nissl staining were captured in the facial nucleus of the operated side. The total RNA of two types of neurons were extracted, and the gene expressions of AMPAR and NMDAR were studied by real-time quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). RESULTS: Messenger RNA (mRNA) of AMPAR subunits (GluR1, GluR2, GluR3, and GluR4) and NMDAR subunits (NR1, NR2a, NR2b, NR2c, and NR2d) was detected in reinnervating and dormant neurons. The relative ratios exhibited that the expressions of GluR1, GluR4, NR2a, NR2b, NR2c, and NR2d mRNA were lower, whereas the expressions of GluR2, GluR3, and NR1 mRNA were higher in dormant FMNs than in reinnervating counterparts. CONCLUSIONS: LCM in combination with real-time qRT-PCR can be employed for the examination of gene expression of different FMNs in a heterogeneous nucleus. The adaptive changes in AMPAR and NMDAR subunit mRNA might dictate the regenerative fate of FMNs in response to the peripheral axotomy and thereby play a unique role in the pathogenesis of facial nerve injury and regeneration.

[Growth inhibition effection of perlecan anti-sense cDNA on human laryngeal carcinoma xnograft in nude mice].

OBJECTIVE: To observe growth inhibition effect of perlecan anti-sense cDNA (pAP) on human laryngeal carcinoma xnografted in nude mice. To vertify its antitumor effect and mechanism in vivo, and it may be useful as a biomarker in carcinoma of larynx cancer. METHOD: Created the model of human laryngeal carcinoma xnograft in nude mice. To observe growth of those xnografts in nude mice and draw growth curve of xnografted. The expression of perlecan mRNA and portein in xnografts were examined by RT-PCR and immunohistochemistry. RESULT: Volume of xnografts in the group transfected by the plasmids of pAP were significant small as compared with other two groups made by the wild type cells and phpApr-neol cells (P < 0.05). It was showed that the expression of perlecan mRNA and protein were significantly reduced in the tumor of pAP transfected Hep-2 cells as compared with the tumors transfected by the wild type cells and phßApr-neol cells (P < 0.01). CONCLUSION: These data raise the possibility that pAP many play key roles in the growth of those xnografts in nude mice.

miR-17 inhibitor suppressed osteosarcoma tumor growth and metastasis via increasing PTEN expression.

MicroRNAs (miRNAs) play essential roles in cancer development and progression. Here, we investigated the role of miR-17 in the progression and metastasis of osteosarcoma (OS). miR-17 was frequently increased in OS tissues and cell lines. Inhibition of miR-17 in OS cell lines substantially suppressed cell proliferation, migration, and invasion. Phosphatase and tensin homolog (PTEN) was identified as a target of miR-17, and ectopic expression of miR-17 inhibited PTEN by direct binding to its 3'-untranslated region (3'-UTR). Expression of miR-17 was negatively correlated with PTEN in OS tissues. Together, these findings indicate that miR-17 acts as an oncogenic miRNA and may contribute to the progression and metastasis of OS, suggesting miR-17 as a potential novel diagnostic and therapeutic target of OS.

[Comparison of lentivirus and adenovirus vector mediated gene transfer into cultured spiral ganglion cells].

OBJECTIVE: We aimed to compare the characteristics between lentivirus and adenovirus vector mediated gene transfer into cultured spiral ganglion cells (SGCs). METHOD: SGCs from newborn rats were cultured and exposed to lentivirus-GFP and adenovirus-GFP vectors. GFP expression and the cell morphology were evaluated under epi-fluorescence microscope at 3 days and 7 days after exposure. Survival number of SGCs was counted, and the average percentage of SGCs with GFP expression was calculated, and axon length was measured by ImageJ software. RESULT: Cultured SGCs were transfected by either adenovirus or lentivirus vector successfully. The adenovirus vector presented an instant and efficient transfection. However, the expression of GFP went down after 7 days. In lentivirus-GFP group, GFP expression was detected at 7 days after exposure, and the number of cells with GFP expression increased gradually in the following days. Statistical analysis revealed that there were no differences in survival number of SGCs and average axon length among lentivirus-GFP group, adenovirus-GFP group and control group. CONCLUSION: Cultured SGCs can be transfected by either lentivirus vector or adenovirus vector safely and efficiently. SGCs are more susceptible to adenovirus vector, but GFP persists for a longer period after the lentivirus-mediated gene transfer.

Isolating RNAs from rat facial motor neurons with laser capture microdissection after facial-facial anastomosis.

Both reinnervated and dormant neurons can be observed in facial nucleus following facial-facial anastomosis. Although they may play different roles in the remodeling mechanism of facial nucleus during facial nerve injury and regeneration, comprehensive gene profiling analysis of these two neuron types has never been performed due to the difficulty in isolating specific neuron populations and extracting sufficient amount of RNAs from the heterogeneous facial nucleus. In this study, we developed a method to isolate the Fluoro-Ruby (FR) retrogradely labeled reinnervated facial motor neurons and the Nissl stained dormant neurons in two steps with laser capture microdissection (LCM) technology. The quality and yield of RNAs extracted from these isolated neurons were confirmed with Agilent 2100 Bioanalyzer. This modified LCM based neuron isolation method will facilitate the studies of the roles and interactions of different neurons that co-exist in the same facial nucleus after peripheral injury.

[A study of spiral ganglion cells death pathway induced by hydrogen peroxide].

OBJECTIVE: To make clear the molecular pathways involved in hydrogen peroxide-induced spiral ganglion cells death. METHOD: The spiral ganglion cells of the newly born rats were primary cultured. Then the SGCs were exposed to hydrogen peroxide for different concentrations (0, 100, 200, 500 micromol/L) and for different hours (2, 4, 6 h). Cell nucleic were stained simultaneously with the DNA binding dyes Hoechst 33258 and propidium iodide. RESULT: At lower concentrations of hydrogen peroxide, apoptosis was the main reason for cell death. At higher concentrations of hydrogen peroxide, the cells died mainly by necrosis. CONCLUSION: The effects of hydrogen peroxide are dose and time dependency. Reactive oxygen species may play a role as an early molecule signal in the mechanism of SGCs death.

[Adenovirus-mediated delivery of bcl-2 gene attenuates cisplatin-induced degeneration of cultured spiral ganglion cells.].

OBJECTIVE: To assess the protection against cisplatin-induced ototoxicity by adenovirus-mediated overexpression of the bcl-2 gene in cultured spiral ganglion cells (SGC). METHODS: SGC from P3 rats were cultured in vitro and exposed to adenovirus vector carrying green fluorescent protein gene (Ad-GFP), followed by immunocytochemical analysis for expression of the neuron-specific marker Neurofilament 200 (NF200) and detection under laser scanning confocal fluorescence microscope. Then, SGC were transduced by Ad-bcl-2 and the expression of human bcl-2 protein was evaluated by Western Blot. Finally, the cultures of SGC were divided into 4 groups: the group of Ad-bcl-2 transfection followed by cisplatin treatment, the group of Ad-GFP transfection followed by cisplatin treatment, the group of cisplatin treatment only and the untreated group. Cisplatin worked for 48 hours at a concentration of 2 microg/ml. Outcome measures included survival number of SGC and longest neurite length by using ImageJ software. RESULTS: SGC were cultured successfully in vitro and transfected by adenovirus vector safely and efficiently. By Western Blot, human bcl-2 protein was expressed in the group after exposure to Ad-bcl-2, but not in the Ad-GFP transfected SGC. Cisplatin exposure resulted in shrinking of neuritis and pyknosis of cell body, even cell death. Expression of bcl-2 in the SGC provided a significant level of protection against cisplatin-induced SGC degeneration. CONCLUSIONS: Our results suggest that SGC can be transduced by adenovirus vector safely and efficiently in vitro. Adenovirus-mediated delivery of the bcl-2 gene attenuates cisplatin-induced SGC degeneration.

Plastic changes of synapses and excitatory neurotransmitter receptors in facial nucleus following facial-facial anastomosis.

The remodeling process of synapses and neurotransmitter receptors of facial nucleus were observed. Models were set up by facial-facial anastomosis in rat. At post-surgery day (PSD) 0, 7, 21 and 60, synaptophysin (p38), NMDA receptor subunit 2A and AMPA receptor subunit 2 (GluR2) were observed by immunohistochemical method and semi-quantitative RT-PCR, respectively. Meanwhile, the synaptic structure of the facial motorneurons was observed under a transmission electron microscope (TEM). The intensity of p38 immunoreactivity was decreased, reaching the lowest value at PSD day 7, and then increased slightly at PSD 21. Ultrastructurally, the number of synapses in nucleus of the operational side decreased, which was consistent with the change in P38 immunoreactivity. NMDAR2A mRNA was down-regulated significantly in facial nucleus after the operation (P<0.05), whereas AMPAR2 mRNA levels remained unchanged (P>0.05). The synapses innervation and the expression of NMDAR2A and AMPAR2 mRNA in facial nucleus might be modified to suit for the new motor tasks following facial-facial anastomosis, and influenced facial nerve regeneration and recovery.

Plastic Changes of Synapses and Excitatory Neurotransmitter Receptors in Facial Nucleus Following Facial-facial Anastomosis / 华中科技大学学报（医学）（英德文版）

The remodeling process of synapses and eurotransmitter receptors of facial nucleus were observed. Models were set up by facial-facial anastomosis in rat. At post-surgery day (PSD) 0, 7, 21 and 60, synaptophysin (p38), NMDA receptor subunit 2A and AMPA receptor subunit 2 (GIuR2) were observed by immunohistochemical method and emi-quantitative RT-PCR, respectively. Meanwhile, the synaptic structure of the facial motorneurons was observed under a transmission electron microscope (TEM). The intensity of p38 immunoreactivity was decreased, reaching the lowest value at PSD day 7, and then increased slightly at PSD 21. Ultrastructurally, the number of synapses in nucleus of the operational side decreased, which was consistent with the change in P38 immhnoreactivity. NMDAR2A mRNA was down-regulated significantly in facial nucleus after the operation (P00<0.05), whereas AMPAR2 mRNA levels remained unchanged (P>0.05). The synapses innervation and the expression of NMDAR2A and AMPAR2 mRNA in facial nucleus might be modified to suit for the new motor tasks following facial-facial anastomosis, and influenced facial nerve regeneration and recovery.

[Excitotoxic effects of glutamate on the in vitro spiral ganglion cells].

OBJECTIVE: To explore the excitotoxic effects of glutamate on the in vitro primarily cultured spiral ganglion cell (SGC) of rat. METHOD: Spiral ganglion cells (SGCs) were cultured in vitro for 4 days, and exposed to 1 mmol/L glutamate for 24 hours. Damaged cells double-labeling with Hoechest33258 and PI were observed by fluorescence microscope. Ffluo-3 and CLSM for measurement of intracellular calcium levels were also utilized. RESULT: Most cells were damaged and the intracellular calcium increased and after exposure to 1 mmol/L glutamate (P < 0.05). CONCLUSION: Glutamate excitotoxicity was associated with free intracellular calcium ion concentration elevation in SGC.

[Application of rat tail collagen in the primary culture of rats marginal cells of stria vascularis].

OBJECTIVE: To study the feasibility of application of rat-tail collagen in the primary culture of rats marginal cells of stria vascularis. METHOD: The effect of self-made rat-tail collagen on the primary culture of rats marginal cells of stria vascularis was observed and estimated. RESULT: When cochlear stria vascularis fragment was isolated and cultured for 24 h, a few culture cells appeared around the fragments. About 48 to approximately 72 h later, clusters of culture cells could be seen,these cells showed the cobblestone shape under the microscope. The immunohistochemistry and the transmission electron microscopy showed the epithelial origination of these cells. CONCLUSION: It is feasible to the primarily culture the rats marginal cells of stria vascularis with self-made rat-tail collagen.

Expression of vascular endothelial growth factor and cyclooxygenase-2 in laryngeal squamous cell carcinoma and its significance.

In order to study the expressions of vascular endothelial growth factor (VEGF) and cyclooxygenase-2 (COX-2) in human laryngeal squamous cell carcinoma (LSCC) and its significance, the expression of VEGF mRNA and COX-2 mRNA in 62 cases of LSCC and 54 adjacent noncancerous laryngeal tissues and 9 normal human laryngeal mucous tissues was detected by using techniques of semi-quantitative RT-PCR. It was found that the expression level of VEGF and COX-2 mRNA was significantly increased in LSCC as compared with that in the normal human laryngeal mucous tissues (both P < 0.01), and the expression level of VEGF and COX-2 mRNA were significantly increased in stage Ill + IV tissues of LSCC as compared with the stage I + II tissues of LSCC (P < 0.01). There was a high positive correlation between VEGF and COX-2 expression in LSCC (r = 0.756, P < 0.01). These data raise the possibility that VEGF and COX-2 may play key roles in the growth, invasion and metastasis of LSCC.

[Experimental study of primary culture methods for spiral ganglion cells of newborn rats].

OBJECTIVE: To optimize culture condition of spiral ganglion cells (SGCs) in vitro and to obtain highly purified SGCs. METHOD: The spiral ganglions from newborn rats were digested in 0.25% trypsin and 0.001% DNase. The SGCs suspension was plated at a density of 10(9) cells/L. The cells were kept in serum free medium (DMEM/F12+B27 supplement). And 5 micromol/L cytosine arabinoside (Ara-C) was added to the medium on the second day and maintained for 48 hours. Serum medium with or without Ara-C was set as a control. The morphology and the purity of SGCs were observed under microscope. RESULT: Highly purified SGCs can be harvested in serum free medium (DMEM/F12+B27 supplement) with Ara-C. The SGCs were identified with mouse anti neurofilament protein antibody by immunohistochemistry methods. CONCLUSION: The method used in this study is an optimal means to culture and purify SGCs that can meet the needs of further study.

[Expression of perlecan in laryngeal carcinoma cell and its significance].

OBJECTIVE: To study the expression of perlecan in human laryngeal carcinoma cells and its significance. METHOD: The expression of perlecan mRNA in human laryngeal carcinoma cells, Hep-2 cells were investigated by using the techniques of semi-quantify RT-PCR. The expression of perlecan in the Hep-2 cells was investigated by using the techniques of immunohistochemistry. RESULT: It showed that the expression level of perlecan and perlecan mRNA significantly increased in Hep-2 cells as compared with the normal cells, Hacat cells (P < 0.01). CONCLUSION: These data raise the possibility that perlecan many play key roles in the growth,invasion and metastasis of human laryngeal carcinoma cells through either a paracrine or an autocrine mechanism.

[Suppression of proliferation of Hep-2 cells by stable expression of anti-sense perlecan cDNA].

OBJECTIVE: To study the suppression of proliferation of Hep-2 cells by stable expression of anti-sense perlecan cDNA. METHOD: In this study, the plasmids of recombination eukaryotic expression vector perlecan anti-sense cDNA (pAP) were transfected into Hep-2 cells by using cationic liposome (lipofectamine 2000) and divided into three groups: non-transfected group, WT group; transfection with no load carrier, neo group; and transfection with the pAP plasmid, pAP group. Semi quantify RT-PCR, western blot assay and MTT assay were used to detected the expression of perlecan mRNA and protein in the three groups; the level of cell proliferation; and the responsivity of basic fibroblast growth factor (bFGF). RESULT: It was showed that the expression of perlecan mRNA and protein were significantly reduced in the pAP group compared with WT group and ph beta Apr-neol transfected group ( P < 0.01). In the presence of 1 microg/L of bFGF in low serum (0.1% FCS), the pAP transfected cells showed a reduced proliferation rate (MTT assay) while the wild type cells and ph beta Apr-neol transfected cells grew rapidly. CONCLUSION: The growth of Hep-2 cells could be inhibited significantly by perlecan anti-sense cDNA plasmids transfection.

[Expression of vascular endothelial growth factor and its receptors in laryngeal carcinoma cell and its significance].

OBJECTIVE: To study the expression of vascular endothelial growth factor (VEGF) and both the Flt-1 and KDR high affinity VEGF receptors in human laryngeal carcinoma cells and its significance. METHOD: In this study, we investigated the expression of VEGF mRNA and both the Fit-1 mRNA and KDR mRNA high affinity VEGF receptors in human laryngeal carcinoma cells using techniques of semi-quantify RT-PCR. RESULT: It was showed that the expression level of VEGF mRNA was significantly increased in human laryngeal carcinoma cells as compared with the normal cells, Hacat cells (P <0.05), and there was the high expression level of Flt-1 mRNA in the Hep-2 cells, but not KDR mRNA. CONCLUSION: These data raise the possibility that VEGF and its receptors many play key roles in the growth, invasion and metastasis of human laryngeal carcinoma cells through either a paracrine or an autocrine mechanism.

[Construction of recombinant adenovirus containing human Bcl-2 and its expression in the spiral ganglion cells].

OBJECTIVE: To construct the adenoviral vector containing human Bcl-2 gene and to study the expression of the gene in the spiral ganglion cells (SGC) in vitro. METHODS: Human Bcl-2 cDNA obtained from the plasmid pUC-CAGGS/Bcl-2 was cloned into the plasmid pAdTrack-CMV. Then, pAdTrack/Bcl-2 was cotransferred with adenoviral backbone vector into E. coli strain BJ5183. The recombinant adenoviral plasmid was identified by restriction analysis with Pac I and transfected into HEK293 cells to package and amplify recombinant adenovirus particles which would be identified by Electron microscope. After the adenovirus infected the rat spiral ganglion cells, the expression of Bcl-2 gene was detected by Western Blot and RT-PCR. RESULTS: The recombinant AdEGFP/Bcl-2 plasmid was correctly constructed and confirmed by restriction endonuclease analysis. The viral particles in HEK293 cells were identified by Electron microscope. RT-PCR and Western blot showed that Bcl-2 gene was exactly transcripted and expressed in transgene SGC. CONCLUSIONS: The method based on homologous recombination in bacteria is simple and high efficient. The recombinant adenoviral vector containing human Bcl-2 cDNA was constructed and the transgene SGC expressed human Bcl-2 gene in vitro successfully. It provided foundation for the further study of protection for the impaired SGC by hBcl-2 gene.

[Alteration of the auditory function and auditory nerve pathology in Guinea pigs with peripheral nerve demyelination].

OBJECTIVE: To study the auditory function and auditory nerve pathological changes of the experimental allergic neuritis (EAN) animal model in Guinea pigs. METHOD: To establish the animal model of EAN, heterologous myelin basic protein (MBP) of peripheral nerve was used to the immunize Guinea pigs. Serum anti-MBP-IgG levels, sciatic nerve conduction velocity (NCV), hearing thresholds and wave latencies, and pathological changes in auditory nerve, sciatic nerve and cochlea were observed. The immune lesions in inner ear were examined with immunohistochemistry. Auditory brainstem response (ABR) and compound action potential (CAP) were assayed weekly before and after immunization for a period of seven weeks. In the control group, antigen was replaced by the 0.9% saline. RESULT: Serum anti-MBP-IgG levels increased significantly in experimental group comparing with the control group (P < 0.01). Seiatic nerve conduction velocity decreased after immunization, as comparing with the control group (P < 0.01). Sciatic nerve and auditory nerve showed the existence of demyelination. There was significant increase of ABR and CAP thresholds and the latencies of wave I, III, V and N1 in experimental group when compared to the controls (P < 0.01), but no significant changes in inter-peak latencies of I-III, III-V were found( P > 0.05). In addition, there were 4 Guinea pigs (8 ears) in which only appeared lengthened wave latencies with normal hearing thresholds. Immunohistochemistry indicated that antibodies to inner ear from the MBP-sensitized animals distributed in cochlear nerve, neurofiber of inner ear and spiral ganglion cells. Cochlear scanning electron microscopy showed the pathology of inner hair cell with stereociliary disorder and cytoplasmic protrusions. CONCLUSION: The results suggest that auditory nerve demyelination are involved in this experimental allergic neuritis animal model and sciatic nerve demyelination as well, and the auditory abnormalities are elicited. This animal model seems to be a promising one of auditory dysfunction due to demyelination disorders.