Otoprotective effects of mouse nerve growth factor in DBA/2J mice with early-onset progressive hearing loss.

As it displays progressive hair-cell loss and degeneration of spiral ganglion neurons (SGNs) characterized by early-onset progressive hearing loss (ePHL), DBA/2J is an inbred mouse strain widely used in hearing research. Mouse nerve growth factor (mNGF), as a common exogenous nerve growth factor (NGF), has been studied extensively for its ability to promote neuronal survival and growth. To determine whether mNGF can ameliorate progressive hearing loss (PHL) in DBA/2J mice, saline or mNGF was given to DBA/2J mice of either sex by daily intramuscular injection from the 1st to the 9th week after birth. At 5, 7, and 9 weeks of age, in comparison with vehicle groups, mNGF groups experienced decreased auditory-evoked brainstem response (ABR) thresholds and increased distortion product otoacoustic emission (DPOAE) amplitudes, the prevention of hair cell loss, and the inhibition of apoptosis of SGNs. Downregulation of Bak/Bax and Caspase genes and proteins in cochleae of mice receiving the mNGF treatment was detected by real-time PCR, Western blot, and immunohistochemistry. This suggests that the Bak-dependent mitochondrial apoptosis pathway may be involved in the otoprotective mechanism of mNGF in progressive hearing loss of DBA/2J mice. Our results demonstrate that mNGF can act as an otoprotectant in the DBA/2J mice for the early intervention of PHL and, thus, could become of great value in clinical applications. © 2017 Wiley Periodicals, Inc.

Caffeine Ameliorates Age-Related Hearing Loss by Downregulating the Inflammatory Pathway in Mice.

OBJECTIVE: Age-related hearing loss (ARHL), also known as presbycusis, is a debilitating sensory impairment that affects the elderly population. There is currently no ideal treatment for ARHL. Long-term caffeine intake was reported to have anti-aging effects in many diseases. This study is to identify whether caffeine could ameliorate ARHL in mice and analyze its mechanism. METHODS: Caffeine was administered in drinking water to C57BL/6J mice from the age of 3 months to 12 months. The body weight, food intake and water intake of the mice were monitored during the experiment. The metabolic indicators of serum were detected by ELISA. The function of the hearing system was evaluated by ABR and hematoxylin and eosin staining of the cochlea. Genes' expression were detected by Q-PCR, immunofluorescencee and Western blot. RESULTS: The results showed that the ARHL mice exhibited impaired hearing and cochlear tissue compared with the young mice. However, the caffeine-treated ARHL mice showed improved hearing and cochlear tissue morphology. The expression of inflammation-related genes, such as TLR4, Myd88, NF-κB, and IL-1ß, was significantly increased in the cochleae of ARHL mice compared with young mice but was down-regulated in the caffeine-treated cochleae. CONCLUSIONS: Inflammation is involved in ARHL of mice, and long-term caffeine supplementation could ameliorate ARHL through the down-regulation of the TLR4/NF-κB inflammation pathway. Our findings provide a new idea for preventing ARHL and suggest new drug targets for ARHL treatment.

Endolymphatic Hydrop Phenotype in Familial Norrie Disease Caused by Large Fragment Deletion of NDP.

Norrie disease (ND; OMIM 310600), a rare X-linked recessive genetic disorder, is characterized by congenital blindness and occasionally, sensorineural hearing loss, and developmental delay. The congenital blindness of ND patients is almost untreatable; thus, hearing is particularly important for them. However, the mechanism of hearing loss of ND patients is unclear, and no good treatment is available except wearing hearing-aid. Therefore, revealing the mechanism of hearing loss in ND patients and exploring effective treatment methods are greatly important. In addition, as a serious monogenic genetic disease, convenient gene identification method is important for ND patients and their family members, as well as prenatal diagnosis and preimplantation genetic diagnosis to block intergenerational transmission of pathogenic genes. In this study, a Norrie family with two male patients was reported. This pedigree was ND caused by large fragment deletion of NDP (norrin cystine knot growth factor NDP) gene. In addition to typical severe ophthalmologic and audiologic defects, the patients showed new pathological features of endolymphatic hydrops (EH), and they also showed acoustic nerves abnormal as described in a very recent report. PCR methods were developed to analyze and diagnose the variation of the family members. This study expands the understanding of the clinical manifestation and pathogenesis of ND and provides a new idea for the treatment of patients in this family and a convenient method for the genetic screen for this ND family.

Role of Endoplasmic Reticulum Stress in Otitis Media.

Endoplasmic reticulum (ER) stress occurs in many inflammatory responses. Here, we investigated the role of ER stress and its associated apoptosis in otitis media (OM) to elucidate the mechanisms of OM and the signaling crosstalk between ER stress and other cell damage pathways, including inflammatory cytokines and apoptosis. We examined the expression of inflammatory cytokine- and ER stress-related genes by qRT-PCR, Western blotting, and immunohistochemistry (IHC) in the middle ear of C57BL/6J mice after challenge with peptidoglycan polysaccharide (PGPS), an agent inducing OM. We also evaluated the effect of the suppression of ER stress with tauroursodeoxycholic acid (TUDCA), an ER stress inhibitor. The study revealed the upregulation of ER stress- and apoptosis-related gene expression after the PGPS treatment, specifically ATF6, CHOP, BIP, caspase-12, and caspase-3. TUDCA treatment of PGPS-treated mice decreased OM; reduced the expression of CHOP, BIP, and caspase 3; and significantly decreased the proinflammatory gene expression of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). These results suggest that PGPS triggers ER stress and downstream proinflammatory gene expression in OM and that inhibition of ER stress alleviates OM. We propose that ER stress plays a critical role in inflammation and cell death, leading to the development of OM and points to ER stress inhibition as a potential therapeutic approach for the prevention of OM.

Otitis media induced by peptidoglycan-polysaccharide (PGPS) in TLR2-deficient (Tlr2(-/-)) mice for developing drug therapy.

BACKGROUND: Toll like receptor 2 (TLR2) signaling can regulate the pathogenesis of otitis media (OM). However, the precise role of TLR2 signaling in OM has not been clarified due to the lack of an optimal animal model. Peptidoglycan-polysaccharide (PGPS) of the bacterial cell wall can induce inflammation by activating the TLR2 signaling. This study aimed at examining the pathogenic characteristics of OM induced by PGPS in Tlr2(-/-) mice, and the potential therapeutic effect of sodium aescinate (SA) in this model. METHODS: Wild-type (WT) and Tlr2(-/-) mice were inoculated with streptococcal PGPS into their middle ears (MEs) and treated intravenously with vehicle or SA daily beginning at 3days prior to PGPS for 6 consecutive days. The pathologic changes of individual mice were evaluated longitudinally. RESULTS: In comparison with WT mice, Tlr2(-/-) mice were susceptible to PGPS-induced OM. Tlr2(-/-) mice displayed greater hearing loss, tympanic membrane damage, ME mucosal thickening, longer inflammation state, cilia and goblet cell loss. SA-treatment decreased neutrophil infiltration, modulated TLR2-related gene expression and improved ciliary organization. CONCLUSIONS: PGPS induced a relatively stable OM in Tlr2(-/-) mice, providing a new model for OM research. Treatment with SA mitigated the pathogenic damage in the ME and may be valuable for intervention of OM.

[Correlation of osteopontin expression and laryngeal squamous cell carcinoma infiltration and metastasis].

OBJECTIVE: To investigate osteopontin (OPN) expression in plasma and tissue of patients with layngeal squamous cell carcinoma and analyze its role in invasion, metastasis, and clinical significance in laryngeal quamous cell carcinoma. METHOD: Enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry were used to detect expression of OPN in plasma and tissue of 60 cases of laryngeal squamous cell carcinoma, 20 cases of adjacent normal laryngeal tissue and 20 cases of plasma from healthy subjects. RESULT: The expression of plasma OPN was closely correlated with clinical stage and cervical lymphatic metastasis in laryngeal squamous cell carcinoma (P < 0.05), but no significant correlation with the tumor location, pathological grade, gender and age (P > 0.05). The expression of OPN increased in plasma during cancer development: laryngeal squamous cell carcinoma (38.089 ± 9.225) ng/ml, healthy subjects (18.563 ± 9.308) ng/ml. There was a significant difference between the groups (P < 0.05). The expression of OPN in tissue was closely correlated with clinical stage (P < 0.05), pathological grade (P < 0.05) and cervical lymphatic metastasis (P < 0.05) in laryngeal squamous cell carcinoma adjacent atypical hyperplastic epithelium and carcinoma. The expression of OPN increased in tissue during cancer development: laryngeal squamous cell carcinoma (56.67%), adjacent normal laryngeal tissue (15.00%). There was a significant difference between the groups (P < 0.05). Elevated expression of plasma OPN is positively correlated with the expression of OPN in tissue in laryngeal squamous cell carcinoma patients (r = 0. 871, P < 0.05). CONCLUSION: OPN plays an important role in the infiltration, metastasis and carcinogenesis in laryngeal squamous cell carcinoma. Combination of serum OPN, tissue OPN detection can be used as diagnostic and surveillance indicators for laryngeal squamous cell carcinoma infiltration and metastasis.

[Expression of p-STAT3 in laryngeal squamous carcinoma and its correlation with PTEN].

OBJECTIVE: To study the expression of p-STAT3 and PTEN in human laryngeal squamous carcinoma, to explore their relations and clinical significance. METHOD: Formalin-fixed and paraffin-embedded tissues from 67 cases of laryngeal squamous carcinoma, 25 cases of normal mucosa over 2.0 cm away from tumor margin in 25 patients with total or subtotal laryngectomy were evaluated for the expression of p-STAT3, PTEN by SP immunohistochemistry, the levels of these proteins in tissues and their correlation with clinicopathological parameters of laryngeal squamous carcinoma were analyzed. The prognostic analysis was performed by Kaplan-Merier. RESULT: The expression rates of p-STAT3 protein in laryngeal squamous carcinoma and normal control laryngeal mucous tissues were 71.64%, 16% respectively. There was significant difference between them (Zc = 4.7052, P < 0.01); The expression rates of PTEN protein in laryngeal squamous carcinoma and normal control laryngeal mucous tissues were 41.79%, 96% respectively. There was significant difference between them (Zc = 5.7037, P < 0.01); The expressions of p-STAT3 and PTEN in laryngeal squamous carcinoma were associated with clinical stage, differentiation grade, lymph nodal metastases and prognosis (P < 0.01). There was a negative correlation between the expression of p-STAT3 and PTEN,and their correlation coefficient was r = -0.5148 (P < 0.01). p-STAT3 positive expression rate in patients survived over a 5 years follow up was 56.25% (18/32), which was obviously lower than the rate 82.35% (14/17) in those dead. CONCLUSION: The expression of p-STAT3 and PTEN may take important roles in the tumorigenesis, aggressiveness, metastases and prognosis of laryngeal squamous carcinoma. The high expression of p-STAT3 was negatively correlated with the lower PTEN in laryngeal squamous carcinoma, which suggested that PTEN may be a downstream target gene of p-STAT3.

[Relationship and expression of hepatocyte growth factor and vascular endothelial growth factor in serum preoperative and postoperative patients with laryngeal squamous cell cancer].

OBJECTIVE: To investigate the role and clinical value of the serum level of hepatocyte growth factor (HGF) and vascular endothelial growth factor(VEGF) in laryngeal squamous cell cancer and the relationship between HGF, VEGF and clinicopathological factors. METHOD: We measured serum HGF and VEGF level in 54 laryngeal squamous cell cancer patients using ELISA to demonstrate the variation of HGF, VEGF level before operation and at day 1, 3, 7, 14 post-surgery. The concentration of the serum HGF, VEGF content in 35 normal healthy people and in 30 vocal cords polyps patients were measured by the quantitative sandwich enzyme linked immunoassay technique. RESULT: (1) The level of serum HGF and VEGF in the patients with laryngeal squamous cell cancer was significantly higher than that of normal healthy people and vocal cord polyps patients (P < 0.01). (2) The level of serum HGF, VEGF in the patients with laryngeal squamous cell cancer was related to clinical stage, differentiation situation, lymph nodes metastasis (P < 0.01), but it was independent of age at diagnosis, gender and tumor location (P > 0.05). (3) There was a significantly positive correlation between preoperative serum VEGF and HGF levels (r = 0.7667, P < 0.01). (4) Post-operational serum HGF levels in 54 laryngeal squamous cell cancer patients who underwent surgical intervention increased significantly, peaked at day 3 after operation. Serum HGF levels of survivors during follow-up period gradually decreased at day 7 and day 14 after operation. The postoperative serum levels of VEGF were decreased significantly than that of preoperation. CONCLUSION: HGF and VEGF may play an important role in the development and progression of human laryngeal cancer. Elevated serum HGF and VEGF levels predict a more aggressive biological behavior in laryngeal squamous cell cancer.

[Expression of Skp2 and PTEN and its correlation in glottic carcinoma].

OBJECTIVE: To investigate the expression and clinical significance of Skp2 and PTEN in glottic carcinoma and the relationship between the two genes. METHOD: Formalin-fixed and paraffin-embedded tissues, which came from 42 cases of glottic carcinoma and 16 cases of atypical hyperplasia of vocal fold and 27 cases of vocal cord polyp, were detected for the expression of Skp2, PTEN by SP immunohistochemistry, then we analyzed the result statistically. RESULT: The expression rates of Skp2 protein in vocal cord polyp, atypical hyperplasia of vocal cord and glottic carcinoma were 11.11%, 37.50%, 40.48% respectively. There was significant difference among them (P < 0.01); the expression rates of PTEN protein in vocal cord polyp, atypical hyperplasia of vocal cord and glottic carcinoma were 100.00%, 75.00%, 52.38% respectively. There was significant difference among them (P < 0.05), the expressions of Skp2 and PTEN in glottic carcinoma were associated with clinical stage, lymph nodal metastases and prognosis (P < 0.05); there was a negative correlation between the expression of Skp2 and PTEN, and their correlation coefficient was r= -0.4301 (P < 0.01). CONCLUSION: The expressions of Skp2 and PTEN may play an important roles in the tumorigenesis, metastases and poor prognosis of glottic carcinoma. These changes may be the early molecular event of the carcinogenesis. The high expression of Skp2 was negative correlation with the lower PTEN in glottic carcinoma.

[Expression of two kinds of tumor correlation protein in laryngeal carcinoma and precancerous lesions].

OBJECTIVE: To study the expression of S phase kinase associated protein 2 (Skp2), phosphatase and tensin homolog deleted on chromosome ten (PTEN) in human laryngeal carcinoma and precancerous lesions, to explore their relations and clinical significance. METHODS: Formalin-fixed and paraffin-embedded tissues from 79 cases of laryngeal carcinoma, 16 cases of atypical hyperplasia of vocal fold,14 cases of adult laryngeal papillomas and 27 cases of vocal cord polyps were evaluated for the expression of Skp2, PTEN by SP immunohistochemistry, the levels of these proteins in tissues with the different types of lesion and their correlation with clinicopathological parameters of laryngeal carcinoma were analyzed. RESULTS: The expression rates of Skp2 in vocal cord polyps, adult laryngeal papillomas, atypical hyperplasia of vocal cord and laryngeal carcinoma were 11. 11% ,14. 29% ,37. 50% ,39. 24% respectively. There was significant difference among them( Hc = 11. 57, P <0. 01). The expression rates of PTEN protein in vocal cord polyps,adult laryngeal papillomas, atypical hyperplasia of vocal cord and laryngeal carcinoma were 100% ,92. 86% ,75. 00%, 56. 96% respectively . There was significant difference among them (Hc = 62. 86, P<0. 05). There was a negative correlation between the expression of Skp2 and PTEN,and their correlation coefficient was r = -0. 4512(P <0. 01). Patients with Skp2 expression in laryngeal carcinoma revealed poorer five yeas survival rate than patients with negative expression of Skp2 (x2 = 21. 46, P = 0. 000). CONCLUSIONS: The expression of Skp2 and PTEN took important roles in the tumorigenesis, aggressiveness, metastases of laryngeal carcinoma. The high expression of Skp2 was negative correlation with the lower PTEN in laryngeal carcinoma, which suggested that PTEN may regulate the expression of Skp2.