The Protective effect of the endoplasmic reticulum stress-related factors BiP/GRP78 and CHOP/Gadd153 on noise-induced hearing loss in guinea pigs.

CONTEXT: The audiological features and cochlear morphology of individuals with noise-induced hearing loss (NIHL) are well characterized. However, the molecular processes in the cochlea are not well understood. AIMS: To explore the role of the endoplasmic reticulum stress (ERS) response in the guinea pig model of cochlear damage induced by exposure to intense noise. SETTINGS AND DESIGN: A pilot case-control study. SUBJECTS AND METHODS: Forty-eight guinea pigs were divided into four equal groups. At 1, 4, or 14 days (d) post-exposure, the auditory brainstem responses (ABRs) were tested before sacrificing the subjects. The expression levels of the binding immunoglobulin protein/glucose-regulated protein 78 (BiP/GRP78) and C/EBP-homologous protein/growth arrest and DNA damage-inducible gene 153 (CHOP/Gadd153) proteins were evaluated using immunohistochemistry and Western blotting. The number of cochlear hair cells with altered nuclei was counted using confocal fluorescence microscopy. STATISTICAL ANALYSIS USED: One-way analysis of variance (ANOVA) and the least squares difference (LSD) test. RESULTS: The outer hair cells (OHCs) showed changes of apoptosis, necrosis, and loss after noise exposure. In the 1- and 4-d groups, more apoptotic cells were found than necrotic cells (P < 0.01). The level of BiP/GRP78 was significantly higher in all three experimental groups compared to the control group (P < 0.01). The level of CHOP/Gadd153 was increased at 1 d post-exposure, achieving a peak that was maintained until 4 d, after which it returned to baseline levels by 14 d post-exposure. CONCLUSIONS: ERS response was activated by inducing the expression of BiP/GRP78 to lessen the extent of the resulting cellular damage and activating the CHOP/Gadd153 pathway to eliminate the most severely damaged cells.

[Determination of hexabromocyclododecane in coatings by gas chromatography-mass spectrometry].

A method has been developed for the determination of hexabromocyclododecane (HBCD) in fire proof coatings by gas chromatography-mass spectrometry (GC-MS). The sample was extracted with dichloromethane and purified through an organic membrane before analysis with GC-MS. The characteristic fragments (m/z 157, 239, 319, 401) and the quantitative ion (m/z 239) were selected. With the optimized conditions, the good linear relationship was obtained between the peak area and the mass concentration of HBCD in the range of 5 to 100 mg/L with the correlation coefficient more than 0. 999. The spiked recoveries in the coatings of acrylic and epoxy resins were 92.9% - 116.3% with the RSDs not more than 8%. The LOD (S/N > or = 3) of HBCD was 30 microg/g, and the LOQ (S/N > or = 10) was 100 microg/g, which were much lower than the international maximum residue limit. The method is simple, quick, accurate and precise, which can meet the requirements of the European Commission Regulation (EC) No. 1907/2006 and Norway PoHS instruction (Prohibition on Certain Hazardous Substances in Consumer Products) for the determination of HBCD. It is suitable for the analysis of HBCD in fire proof coatings.

[Endoplasmic reticulum molecular chaperone involved in the impairment of inner ear consistent with the mimetic aging rats].

OBJECTIVE: To explore the involvement of endoplasmic reticulum molecular chaperone GRP78 in the impairment of inner ear consistent with the mimetic aging model. METHOD: Twenty-four Wistar rats were randomly divided into two groups. Model group was induced by daily hypodermic injection of 10% D-galactose (800 mg x kg(-1) x d(-1)) for 8 weeks and the control group was given saline accordingly. Spatial learning and memory was measured by Morris-Water-Maze. Colorimetry was used to analyze superoxide dismutase (SOD) and malondialdehyde (MDA) extracted from inner ear tissue. Hearing threshold of rats were detected with Auditory brainstem response (ABR). In addition, expression of GRP78 in the inner ear was detected by immunohistochemistry, RT-PCR and Western blot. The control group was studied parallel. RESULT: The escape latency in the model group injected with D-galactose was markedly longer than that in the control group. Accordingly, the changes of SOD and MDA were more significant in the model group, the difference between two groups was significant (t-test, P<0.01). the variation of ABR in two groups was observed, There was no statistically difference of the hearing in the model group compared with the control group (P>0.05). The expression of GRP78 was significantly different between two groups, which is increased in the inner ear tissue of model group (P<0.01). CONCLUSION: The impairment of inner ear tissue partly dued to the oxidative stress in the model, which was induced by D-galactose and endoplasmic reticulum molecular chaperone was thought to contribute to the impairment mechanism of inner ear in mimetic aging model.

[Role of caspase 12 in apoptosis of cochlea induced by intense noise in guinea pigs].

OBJECTIVE: To investigate the relationship between caspase 12 activation and endoplasmic reticulum stress mediated apoptosis of guinea pig cochlea cells induced by intense noise. METHODS: Thirty-two guinea pigs were randomly divided into 4 groups. The guinea pigs in the experiment groups were exposed to 4 kHz narrow band noise at 120 dB SPL for 4 h. After the noise expose for 1, 4, 14 days of the experiment guinea pigs, auditory brainstem response (ABR) of the guinea pigs on experiment and control groups were tested before decapitated. Four guinea pig's cochleae of every group were taken to paraffin section, and the rest was extracted the total protein. Apoptosis was tested by terminal deoxynucleotidyl transferase (TDT)-mediated deoxyuridine triphosphate (d-UTP) nick and labeling method (TUNEL) and transmission electron microscopy. Expression of caspase 12, Bip/GRP78 was tested by immunohistochemistry and Western blot methods. RESULTS: The observation by transmission electron microscopy showed the features characteristic of apoptotic cells in the Corti and SGC of 1d after the noise expose, but no in the control. There were higher expressions of Tunel-Positive cells in the OHC, SGC and SV of experiment groups, and there was significant differences compared with the control group (P < 0.01). Protein levels of Bip/GRP78 and caspase 12 were risen up after noise exposed, and there all were significant differences compared with the control group (P < 0.01). CONCLUSION: Intense noise causes cochlea cell lesion by inducing apoptosis to result in and caspase 12 induced endoplasmic reticulum stress-related apoptosis plays an important role in the procedure of apoptosis.

[Study of the effect of JNK signal transduction pathway in intense noise-induced apoptosis in cochlea of guinea pig].

OBJECTIVE: To investigate the mechanism of intense noise-induced cochlea cells death in guinea pig, and the effect of JNK signal transduction pathway in the procedure of cochlea cells apoptosis by intense noise-induced. METHOD: Thirty-two guinea pigs were randomly divided into 4 groups. The guinea pigs in the experiment groups were exposed to 4 kHz narrow band noise at 120 dB SPL for 4 h. After the noise expose for 1, 4, 14 days of the experiment guinea pigs, ABR of the guinea pigs on experiment and control groups were tested before put them to death. Four guinea pig's cochleas of every group were taken to paraffin section, and the rest was extracted the total cochlear's protein. Apoptosis was tested by terminal deoxynucleotidyl Transferase (TdT)-mediated deoxyuridine triphosphate (d-UTP) nick and labeling method (TUNEL). The phosphorylation of JNK and c-Jun were tested by immunohistochemistry and western blot methods. RESULT: Tunel-Positive cells in the Corti's, SGC and SV of experiment groups, and there have significant differences compared with the control group (P<0.01) and Tunel-Positive cells are most in 1 d experiment group. The positive cells of P-JNK and P-c-Jun could be detected in guinea pig's cochleas after noise exposed, but no positive cells were found in the control. Protein levels of P-JNK and P-c-Jun were risen up and activated quickly after noise exposed, and achieved peak in 1 d, 4 d and then fallen-offs, but still maintained higher levels within 14 d. CONCLUSION: Intense noise causes cochlea cell lesion by inducing apoptosis to result in and JNK signal transduction pathway plays an important role in the procedure of apoptosis.

[Effect of the intense noise on hearing function and cochlea morphology in rat].

OBJECTIVE: To observe the changes of auditory electrophysiology and inner ear pathology in rat cochlea after the noise exposure, and to offer the experimental data for exploring the mechanism of noise-damaged cochlea. METHOD: The rats in the study group were exposed to a intense narrow band noise centered at 4 kHz at the leave of 120 dB (SPL) for 4 h. The exposed cochleae were collected at various intervals (1 or 21 days) after the noise exposure. Auditory function was monitored by measuring thresholds of auditory brain stem responses (ABR). The morphological changes in rat cochlear hair cell (HC) were examined by HC nuclei stained with Propidium iodide (PI), a fluorescent dye specifically labelling the nuclear DNA and scanning electron microscopy (SEM). The number of spiral ganglion cells was calculated using pathologic technique. RESULT: The thresholds of ABR in the study group were significantly greater than that in the normal control group (P < 0.01). Examined at 1 day after the noise exposure, normal, apoptosis, necrotic and missing out hair cell (OHC) could be distinguished with PI staining, whereas the apoptosis OHC were not found at 21 days. Significant OHC loss was found in as compared to the normal control group (P < 0.01). There was not significant difference in the calculation of spiral ganglion cells (P > 0.05). SEM revealed the injured stereocilia of OHC (disarrangement, collapse) and OHC loss in the study group, which was more severe in OHC3 than the other two rows of OHC. CONCLUSION: The intense noise used in our study could injure the rat cochlea and bring permanent threshold shift (PTS). Under this condition, the death modes of HC in the cochlea include apoptosis and necrosis in the fore part, whereas necrotic is the major mode in the evening of exposure. The injured stereocilia of OHC and OHC loss could remain the most consistent correlate of PTS.

[Culture, identification and label of embryonic rat neural stem cells].

OBJECTIVE: To explore the methods of culture, identification and label of embryonic rat neural stem cells. METHOD: The cells isolated from fetal rat hippocampus were identified with nestin immunocytochemical fluorescent staining. The cellular multiplication was observed by immunocytochemical fluorescence co-label after accession of BrDU. The neural stem cells (NSCs) were marked with fluorescent dye, bisbenzimide (Hoechest33342) and induced to differentiate. The differentiated cells were detected with Neuron Specific Enolase (NSE) and Glial Fibrillary Acidic Protein (GFAP) immunocytochemical fluorescent staining respectively. RESULT: Nest-like clusters of neural stem cells were obtained in suspension and the cells could be differentiated into neurons and astrocytes which maintaining the main characteristics of NSCs after 8 passages of culture. The label efficiency of cells with Hoechest33342 was 97% and no attenuation of fluorescent brightness was observed after 8 passages of culture. The cellular fluorescence was observed in the NSCs and the differentiated cells. CONCLUSION: The cells from embryonic rat hippocampus possessed the abilities of division, multiplication and self-renew, which were believed to be the main characteristics of NSCs of the central nervous system. The cells could be efficiently labeled with fluorescent dye and could be used as donor cells in experimental research on NSCs transplantation.

Effect of DRB on the biological characteristics of human laryngeal carcinoma Hep-2 cell line.

In order to study the effect of 5, 6-Dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB) on the biological characteristics of human laryngeal carcinoma Hep-2 cell line in vitro, Hep-2 cells cultured in vitro were treated with different concentrations of DRB. Changes in cell proliferation, apoptotic rate and invasiveness were detected by MTT assay, flow cytometry (FCM) and matrigel in vitro invasion assay, respectively. It was found that DRB inhibited the proliferation of Hep-2 cells in a dose-and time-dependent manner. After being treated with 0, 10, 20, 40, 80 microm mol/L DRB for 24 h, the apoptotic rate in Hep-2 cells was (0.68+/-0.19)%, (1.95+/-0.12)%, (8.51+/-0.26)%, (11.26+/-0.17)% and (14.99+/-0.32)%, respectively. The matrigel in vitro invasion assay revealed that DRB began to inhibit the invasion of Hep-2 cells at the concentration of 5 microm mol/L, and with the increase of DRB concentration, the inhibitory effect was enhanced. It was suggested that DRB could influence the essential biological characteristics of Hep-2 cells, inhibit Hep-2 cells proliferation, reduce invasive ability and induce apoptosis of Hep-2 cells.

[Determination of phthalic acid esters in textiles by solid phase extraction-gas chromatography].

A method was established for the simultaneous determination of some phthalic acid esters, namely, dimethyl phthalate (DMP), diethyl phthalate (DEP), dipropyl phthalate (DPrP), dibutyl phthalate (DBP), diamyl phthalate (DAP), dihexyl phthalate (DHP), benzyln-butyl phthalate (BBP), di-(2-ethylhexyl) phthalate (DEHP), dicyclohexyl phthalate (DCHP), di-n-octyl phthalate (DNOP), diisononyl phthalate (DINP) and diisodecyl phthalate (DIDP) in textiles by solid phase extraction (SPE) coupled with gas chromatography (GC). The phthalic acid esters in textiles were extracted by Soxhlet extraction with hexane, the extracts were then cleaned up and enriched by a strong anion exchange (SAX) SPE cartridge. The parameters affecting the purification efficiency of SPE cartridge, such as solvent conditioning, rinsing, and elution, were studied. Conditioning with 5 mL hexane and rinsing with 3 mL isooctane were proved to be the optimal conditions. Of the several solvent ratios (ethylacetate in hexane) used for selective elution of phthalic acid esters from the SAX SPE cartridge, the 15% (v/v) content for ethylacetate in hexane gave the best result. Under the optimized conditions, the recoveries of phthalic acid esters for spiked standards (n=7) were 86.3%-102.7%, and the relative standard deviations (RSDs) were less than 5%. In this method the detection limits for DMP, DEP, DPrP, DBP, DAP, BBP, DCHP, DEHP, DNOP were all below 1 mg/kg, and the detection limits for DINP and DIDP were 1.74 mg/kg and 1.55 mg/kg respectively. This SPE-GC method is sensitive, accurate and suitable for the analysis of phthalate environmental hormones in textiles.

[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.