Considering the protective effect of exendin-4 against oxidative stress in spiral ganglion neurons.

Objectives: The protection of spiral ganglion neurons (SGNs) is crucial for hearing loss. Exendin-4 has been shown to have neuroprotective effects in several neurological disorders. Therefore, this study aimed to investigate the effect of the glucagon-like protein-1 receptor (GLP-1R) agonist exendin-4 on kanamycin-induced injury in mouse SGNs in vitro. Materials and Methods: In this study, GLP-1R expression in SGNs was verified by immunofluorescence and immunohistochemical staining. In vitro-cultured SGNs and the organ of Corti were exposed to kanamycin with or without exendin-4 treatment. The cell survival rate was measured using the cell counting kit-8 assay, and the damage to auditory nerve fibers (ANF) projecting radially from the SGNs was evaluated using immunofluorescence staining. Reactive oxygen species (ROS) content was determined by flow cytometry, and glutathione peroxidase (GSH-Px) content, superoxide dismutase (SOD) activity, and malondialdehyde (MDA) content were determined by spectrophotometry. Protein expression of nuclear factor erythroid-2-related factor 2/heme oxygenase-1 (Nrf2/HO-1) was detected using western blotting. Results: GLP-1R was expressed in SGNs. Treatment with 1 mM kanamycin for 24 hr induced SGN damage. Exendin-4 (100 nM) had a protective effect against kanamycin-induced SGN cell injury, improved cell survival rate, reduced nerve fiber injury, increased SOD activity and GSH-Px level, and reduced MDA and ROS contents. The Nrf2/HO-1 pathway was activated. Conclusion: Exendin-4 alleviates oxidative damage and exerts neuroprotective effects in kanamycin-induced SGN injury through the Nrf2/HO-1 signaling pathway. Exendin-4 has the potential to prevent or treat hearing loss due to SGN damage.

Mastoid obliteration and external auditory canal reconstruction using 3D printed bioactive glass S53P4 /polycaprolactone scaffold loaded with bone morphogenetic protein-2: A simulation clinical study in rabbits.

Introduction: The lack of good prosthetic materials and objective standards has limited the promotion of mastoid obliteration and external auditory canal reconstruction, and the quality of the surgery varies. In this study, bioactive glass S53P4 (S53P4), the most popular artificial prosthetic material, was modified and combined with polycaprolactone (PCL) and bone morphogenetic protein-2 (BMP-2) to produce an individualized biological scaffold using 3D printing technology to explore a better material and method for mastoid obliteration and external auditory canal reconstruction. Methods: 3D-printed S53P4/PCL scaffolds were fabricated from 3D reconstruction data of bone defect areas in New Zealand rabbits simulating "Canal Wall Down Mastoidectomy". The water absorption, swelling rate, porosity, and Young's modulus of the scaffold were measured, and the morphology and pore size of the scaffold were observed using scanning electron microscopy. The cytotoxicity of the S53P4/PCL scaffolds was detected using the CCK8 assay, and the in vitro antibacterial activity of the S53P4/PCL scaffolds was detected using the inhibition circle method. The BMP-2-loaded S53P4/PCL scaffolds were prepared using the drop-in lyophilization method and implanted into animal models. The biocompatibility, osteogenic activity, and external auditory canal repair of the scaffolds were observed using endoscopy, micro-CT, and histological examination. Results: The S53P4/PCL scaffold was highly compatible with the defective area of the animal model, and its physicochemical properties met the requirements of bone tissue engineering. In vitro experiments showed that the S53P4/PCL scaffold was non-cytotoxic and exhibited better antibacterial activity than the same volume of the S53P4 powder. In vivo experiments showed that the S53P4/PCL scaffold had good biocompatibility and osteogenic activity, and could effectively repair bone defects and reconstruct the normal morphology of the external auditory canal in animal models. Furthermore, its osteogenic activity and repair ability were significantly improved after loading with BMP-2. Conclusions: The 3D printed S53P4/PCL scaffold has great potential for clinical mastoid obliteration and external auditory canal reconstruction.

Association Between Sigmoid Sinusoidal Tinnitus and Low-Frequency Sensorineural Hearing Loss: A Retrospective Study at a Single Center.

BACKGROUND We aimed to explore the correlation between patients' sigmoid sinusoidal tinnitus (SST) and low-frequency sensorineural hearing loss (LFSHL) and illustrate the underlying mechanism. MATERIAL AND METHODS Seven healthy volunteers with normal hearing were subjected to 125-, 250-, and 500-Hz pure sound and different white noise-masking intensities. A retrospective analysis was made on the clinical data and postoperative follow-up data of 59 patients with SST in the First Affiliated Hospital of Chongqing Medical University. The patients' sex, age, chief complaints, affected site, concomitant symptoms, course of disease, pure-tone audiometry (PTA) results, tinnitus discomfort loudness scale results, imaging examination, and complications were collected. RESULTS The results of the simulation experiment showed that the threshold of each frequency segment was higher after noise masking than before masking; the intensity of noise masking was positively correlated with hearing loss, and the changes of the hearing threshold of the 3 frequencies before and after masking were statistically significant (P<0.05). Fifty-nine patients with SST were documented between January 2015 and January 2020. After the operation, their low-frequency hearing was recovered to normal; 11 cases had significantly alleviated tinnitus and 9 cases were cured. CONCLUSIONS SST often causes corresponding pseudo-low-frequency hearing loss due to the noise-masking effect. The center frequency of tinnitus appears not to be 250-Hz or 500-Hz octave frequency of PTA, barring the detection of the pseudo-hearing loss in the audiometry chart of most patients. Surgery positively affects patients with SST, and the pseudo-LFSHL can be completely recovered after the operation as a result of tinnitus elimination.

MiR-1204 sensitizes nasopharyngeal carcinoma cells to paclitaxel both in vitro and in vivo.

Nasopharyngeal carcinoma (NPC) is an endemic tumor with a relatively high incidence in Southern China and Southeast Asia. Paclitaxel combination chemotherapy has been used for treatment of advanced NPC. However, treatment failure often occurs due to development of acquired paclitaxel resistance. In this study, we first established a paclitaxel-resistant CNE-1/Taxol, HNE-2/Taxol and 5-8F/Taxol cell sublines by treating the parental CNE-1, HNE-2 and 5-8F cells with increasing doses of paclitaxel for about 5 months, respectively. Then, microRNA arrays were used to screen differentially expressed miRNAs between the CNE-1/Taxol cells and the parental CNE-1 cells. We found 13 differentially expressed miRNAs, of which miR-1204 was significantly downregulated in the paclitaxel-resistant CNE-1/Taxol cells. We restored miR-1204 expression in the CNE-1/Taxol, HNE-2/Taxol and 5-8F/Taxol cells and found that restoration of miR-1204 re-sensitized the paclitaxel-resistant CNE-1/Taxol, HNE-2/Taxol and 5-8F/Taxol cells to paclitaxel both in vitro. Finally, we demonstrated that restoration of miR-1204 in significantly inhibits tumor growth in vivo. Thus, our study provides important information for the development of targeted gene therapy for reversing paclitaxel resistance in NPC.

MiR-634 sensitizes nasopharyngeal carcinoma cells to paclitaxel and inhibits cell growth both in vitro and in vivo.

Resistance to chemotherapy is one of the key causal factors in cancer death and increasing evidence has revealed that microRNAs (miRNAs) are involved in chemoresistance in many kinds of human cancers. Paclitaxel has been used for treatment of advanced nasopharyngeal carcinoma (NPC); however, treatment failure often occurs due to development of acquired paclitaxel resistance. In this study, based on miRNA microarray screening and qRT-PCR validation, we found six differentially expressed miRNAs in our induced paclitaxel-resistant NPC CNE-1/Taxol cells. Furthermore, we clarified the role of miR-634, most significantly downregulated in the paclitaxel-resistant CNE-1/Taxol, in regulating the paclitaxel sensitivity in NPC cells. We restored miR-634 expression in the CNE-1/Taxol cells by lentivirus infection, and found restoration of miR-634 re-sensitized the CNE-1/Taxol cells to paclitaxel in vitro by MTT assay and colony formation assay. In xenograft mouse model, we found that miR-634 inhibited tumor growth and enhanced paclitaxel sensitivity. Thus, our findings provide important information for the development of targeted gene therapy for reversing paclitaxel resistance in NPC.

[Cloning of cDNAs encoding skin antimicrobial peptide precursors from Chinese brown frogs, Rana chensinensis and determination of antimicrobial, anticancer and hemolysis activity].

Amphibian skin antimicrobial peptides exhibit a broad spectrum of antimicrobial activity against Gram-positive and Gram-negative bacterium and cytotoxic activity responsible for inhibiting the growth of cancer cells. In this present study, six cDNAs encoding antimicrobial peptide precursors were cloned from the skin of Chinese brown frog, Rana chensinensis by RT-PCR and 3'-RACE procedure and identified as preprotemporin-1CEa, preprotemporin-1CEb, preprotemporin-1CEc, preprobrevinin-1CEa, preprobrevinin-1CEb, and preprochensinin-1, respectively. The nucleotide sequences of cDNA encoding 59-65 amino acid composed of 289-315 bp. Preprotemporin-1CEa, preprotemporin-1CEb and preprotemporin-1CEc are members of temporin family, which usually are short, hydrophobic, and C-terminally alpha-amidated antimicrobial peptides. Preprobrevinin-1CEa and preprobrevinin-1CEb were identified as the members of the brevinin-1 family of antimicrobial peptides since both peptides contain "RANA box" that it's responsible for forming Cys-bridged cyclic heptapeptides at the C-terminal region of peptide. The nucleotide acid sequence and the deduced amino acid Sequence of preprochensinin-1 were not found to be identity with any known amphibian skin defensive peptides, so, preprochensinin-1 was identified as a novel peptide precursor. Four of bioactive peptides: temporin-1CEa, temporin-1CEb, brevinin-1CEa and chensinin-1 were synthesized to investigate their antimicrobial, anticancer and haemolysis activities. The results showed that all of the synthesized antimicrobial peptides in this study inhibited the growth of the Gram-positive bacterium, and exhibited the anticancer activity against the growth of MCF-7 cells and HeLa cells. Analysis of the R. chensinensis bioactive peptides and their gene expression will be beneficial for preservation of this species.

Molecular cloning of cDNAs encoding antimicrobial peptide precursors from the skin of the Chinese brown frog, Rana chensinensis.

Skin plays a key role in the daily survival of amphibians. In the present study, six cDNAs encoding amphibian skin antimicrobial peptide precursors from the Chinese brown frog Rana chensinensis, were cloned and identified as preprobrevinin-1CEc, preprobrevinin-1CEb, preprotemporin-1CEa, preprotemporin-1CEb, preprotemporin-1CEc, and preprochensinin-1. Preprotemporin-1CEa, CEb, and CEc are members of the temporin family, which are usually short, hydrophobic, and C-terminally alpha-amidated antimicrobial peptides. Preprobrevinin-1CEa and CEb were identified as members of the brevinin-1 family of antimicrobial peptides, because both peptides contain a "Rana box" that is responsible for forming C-terminal Cys-bridged cyclic heptapeptides. The nucleotide and deduced amino acid sequences of preprochensinin-1 were not similar to any known amphibian skin defensive peptides. Four bioactive peptides were chemically synthesized according to the deduced amino acid sequences of six prepropeptides from R. chensinensis skin, and their antimicrobial, cytotoxic, and haemolytic properties were evaluated. All of the synthesized peptides inhibited the growth of Gram-positive bacteria. Brevinin-1CEa showed a broad spectrum of antimicrobial activity. The novel amphibian skin peptide chensinin-1 was active against Bacillus cereus and Streptococcus lactis at a concentration of 11.6 microM, but did not inhibit the growth of MCF-7 and HeLa cells at 200 microM, and had no haemolytic activity at a concentration of 500 microM. Temporin-1CEa exhibited the greatest ability to inhibit the growth of MCF-7 cells. Its antimicrobial and cytotoxic activities may be due to its high degree of alpha-helical confirmation and amphipathic nature.