Protective Effect Of Recombinant Adenovirus A20 On Cochlear Hair Cells In Guinea Pigs

1 Background and aimsHearing loss is the fifteenth common disease in the world. Even in developed countries, there are about 100 million people suffering from it, and 90% among them are sensorineural hearing loss. According to the data collected by a sampling survey on the handicapped in our country, auditory deformity is in a trend of rising year by year. Hearing loss due to hair cell damage is the main reason for deafness, so how to protect hair cells becomes the focus of scientific researchers. Cochlear hair cells, the terminal receptor which transforms the acoustic stimulus into chemical energy, consists two kinds of cells, inner and outer hair cells. The former are the receptor cells which transform the mechanical vibration into the action potential of the connected auditory nerves, and the later are involved in the enhancement of the auditory nerves on height-frequency selectivity, and regulation and self-protection of the cochlea. The lesion of either outer or inner hair cells can lead to irreversible sensorineural hearing loss. Although being enveloped and protected in a snail-shaped bony structure and with the help of blood-labyrinth barrier, the cochlea is relatively independent in anatomical and functional aspects. Once it is impaired, it will be difficult to communicate with outside via systemic or local circulation, and has to depend on its own metabolism to eliminate the injury factors. In this way, the key point of this issue is to improve the self-protective ability of hair cells so as to prevent the normal cells from damage. We need to find a factor which can exert consistent protection on hair cells.When there are exogenous deleterious factors, our body will generate defensive reflection to response them, while for cells, they will produce some protective proteins. Zinc finger protein A20 is one of them. This protein was firstly found as the product when TNF was used to treat human endothelial cells. Gene sequencing revealed that it encodes a new zinc protein, so was named as zinc finger protein A20. The zinc finger protein A20 exists in the cytoplasma, which negatively regulates the activity of nuclear factor-kappa B (NF-κB), and is expressed in many cells. There are four standard repeats ATTTA in the 3' untranslated region of the A20 mRNA, which indicating the unstable expression of A20. A20 exerts anti-inflammation, anti-apoptosis, and anti-necrosis effects in various cells, such as endothelial cells, and brain nerve cells. It has reported that hair cell injury results in the alteration of NF-κB and Caspase-3 signal pathways, leading to up-regulation of these two proteins. While A20 can inhibit the expressions of NF-κB and caspase-3. Thus, we infer A20 can protect hair cells through caspase-3. Since A20 is expressed rapidly and transiently, recombinant expression vector is need to obtain stable expression to perform its anti-injury effect. Adenovirus vector is well adopted in the treatment targeting inner ear genes because of its high-efficiency, low pathogenicity and high-titer.There are various causes for hair cell injury, and drug poisoning is the most common one. What's more, in this modern society and during modern war, electromagnetic radiation, especially that of high-frequency, becomes a main reason threatening human's health,. Base on this, we employed these two kinds of injury models of hearing loss. We aim to found theoretical basis for A20 improving and repairing injured auditory function by exploring its protective effect and possible molecular mechanism on inner hair cells.Based on above mentioned analyses, we established the hair cell injury models induced by gentamicin ototoxicity and high power microwave, observed A20 expression in normal and injured hair cells, then explore the protective effect of A20 by constructing recombinant adenoviral expression vector pAdEasy-1/A20 and the possible mechanism.2 Methods and results2.1 Establishment of HPM-induced hair cell injury modelGuinea pigs was placed in a microwave anechoic chamber with a reflection coefficient of approximately zero, and exposed to radiation of 30, 65 or 90 W/cm2 for 20 min. Then the animals were observed 3, 6 and 12 h after radiation. Changes of neurobehavior function and body temperature were observed in guinea pigs exposure to different power radiation, indicating that HPM leads to thermal effect injury. Significant difference was found in the evoked potentials of auditory brainstem response (ABR) in guinea pigs 6 h after being exposed to 65 W/cm2 radiation. Light and electron microscopy observed that there were more injured hair cells, especially inner hair cells, and many globular objects in the group 6 h after 65 W/cm2 radiation. These results suggested that HPM resulted in hair cell injury and 6 h after 65 W/cm2 radiation could be used as the dose for damage study. 2.2. Establishment of gentamicin -induced hair cell injury modelThis model was established by i.p. injecting 120㎎/㎏ gentamicin for 10 d. ABR, light and electron microscopy discovered that gentamicin caused hearing loss, made the damage of hair cells morphologically, especially those inner cells. The damage of first row hair cells was more severe than those at second and third rows.2.3 Expression of A20 in the cochlear hair cells of guinea pigsIn situ hybridization indicated that A20 was not expressed in normal cochlea, but begun to express 3 h after gentamicin ototoxicity or HPM radiation, declined in 6 h and almost disappeared in 12 h. These data indicated that A20 was transiently expressed only under damage condition in cochlea inner hair cells .2.4 Construction, purification and expression of adenoviral vector pAdEasy-1/20 in hair cells in vivoAfter the expression cassette of plasmid pCAGGS-FLAGmA20 was cloned into shuttle vector pAdtrack-CMV by enzyme ligation, the recombinant was amplified in E. coli Adeasy-1 and then packaged in 293 cells. After identified by single and double enzyme digestion and PCR, the virus was expanded and purified, and yield a titer of 5×109 pfu /ml with the aid of reporter gene GFP.2.5 Protective effect of recombinant adenovirus pAdEeay-1/A20 on hair cells Immunohistochemical methods indicated that the recombinant adenovirus could infect hair cells in vivo. The guinea pigs in this study were divided into normal control, pAdEeay-1 group, pAdEeay-1 and gentamicin/(or HPM) group, pAdEeay-1/A20/ gentamicin/(or HPM) group, and artificial perilymph solution/gentamicin group. There was no significant difference in hearing threshold of ABR in the animals of normal control group and of pAdEeay-1 group before and after drug administration into the cochlea, indicating that this way of administration and recombinant adenovirus had no obvious effect on the hearing threshold of ABR. Significant change was observed in animals from pAdEeay-1 and gentamicin/(or HPM) group, suggesting that there was no protective effect of pAdEeay-1 on hearing loss. The hearing threshold of ABR was significantly higher in pAdEeay-1/A20/ gentamicin/(or HPM) group and artificial perilymph solution/ gentamicin group in comparison with that in normal control group. That of pAdEeay-1/A20/ gentamicin/(or HPM) group was significantly lower than that of artificial perilymph solution/ gentamicin group. These results implied that A20 exerted protective effect on hearing loss induced by gentamicin ototoxicity or HPM radiation, but the protection was not complete. Morphological study found that there were lesser injured hair cells in pAdEeay-1/A20/ gentamicin/(or HPM) group and artificial perilymph solution/ gentamicin group than in normal control group, with the former better than the later group, indicating that A20 could protect outer and inner hair cells morphologically.2.6 Mechanism of A20 protecting hair cellsTheoretically, the pathway of A20 exerting its anti-injury effect has a crossing point with the damage mechanism of hair cells, that is, caspase-3. We aimed to investigate whether caspase-3 played a role in this protective process. Immunohistochemical method was used to detect its expression in above mentioned group. It was negatively expressed in normal control group and of pAdEeay-1 group, strongly expressed in gentamicin/(or HPM) group and pAdEeay-1/A20/ gentamicin/(or HPM) group. It was distributed in the organ of Corti, hair cells and cells of Deiters. But the caspase-3 expression was significantly milder in pAdEeay-1/A20/ gentamicin/(or HPM) group than in gentamicin/(or HPM) group.3 Conclusion3.1 HPM-induced hair cell injury model is successfully established in guinea pigs. HPM is proved to damage hair cells in a dose- and time- dependent manner, and mainly injures inner hair cells.3.2 Zinc finger protein A20 is not expressed in normal cochlear hair cells, but transiently expressed after the stimulation of external injury factors.It,s expression maintained only several hours.3.3 Zinc finger protein A20 has certain protective effect on the function and morphology of hair cells after gentamicin ototoxicity or HPM radiation via the construction of recombinant adenovirus pAdEeay-1/A20 and its transfection to hair cells.3.4 Zinc finger protein A20 may protect hair cells by inhibiting the expression of caspase-3.