[Effect of TNF-α on cisplatin-induced permeability change of blood labyrinth barrier in cochlea of C57BL/6J mice].

Abstract

Objective: To investigate the effect of tumor necrosis factor α (TNF-α) on the permeability of blood labyrinth barrier in C57BL/6J male mice and its possible mechanism. Methods: As for the design of animal experiment, twenty male C57BL/6J mice aged 6-8 weeks were randomly divided into control group and cisplatin group with 10 mice in each group by software method. The control group was intraperitoneally injected with normal saline every day, and the cisplatin group was intraperitoneally injected with 4 mg/kg cisplatin for 4 consecutive days. After administration, auditory brainstem response (ABR) was used to detect hearing changes in mice. HE staining was used to observe the morphological changes of mouse cochlea vasculature. The expression of TNF-α was detected by immunohistochemistry and ELISA. The permeability of the blood labyrinth barrier was observed by Evans blue staining. With respect to the design of cell experiment, primarily cultured cochlea pericytes (PC) and endothelial cells (EC) were divided into EC group, EC+TNF-α group, EC+PC group, EC+PC+TNF-α group, EC+PC+TNF-α+SB-3CT (MMP-9/MMP-2 secretion inhibitor) group, PC group, PC+TNF-α group, PC+TNF-α+LY294002 (PI3K/AKT pathway inhibitor) group, PC+LY294002 group. The protein expressions of ZO-1, VE-cadherin, MMP-9, MMP-2, PI3K, p-PI3K, AKT, and p-AKT were detected by Western blot. TEER and FITC-dextran penetration experiment were used to detect EC resistance and monolayer EC permeability, respectively. The data were statistically analyzed using GraphPad Prism 8 software. Results: In animal experiment, compared with control group, the ABR response threshold of mice in cisplatin group was increased (P<0.01). The vaccine ular structure of the cochlea was disordered red, wrinkled and vacuole increased. The extravasation of vascular red fluorescent dye increased (P<0.05), and also, levels of serum TNF-α and cochlear veins increased (P<0.01). In cell experiment, by treatment of EC with different concentrations of cisplatin, 20 μmol cisplatin led to the highest expression of TNF-α (P<0.01). The expression of TNF-α was the highest after 3 h intervention in EC (P<0.05). Compared with those in EC+PC group, the resistance value of EC was decreased, the permeability of monolayers EC was increased, the expression of ZO 1 and VE cadherin proteins was decreased, and however, the resistance value was increased and the permeability of EC was decreased after the intervention of SB-3CT in EC+PC+TNF-α group. The expressions of ZO-1 and VE-cadherin proteins were increased (P<0.05). The expression of MMP-9 increased after TNF-α intervention (P<0.05), the expression of MMP-2 had no significant change, and the expression of p-PI3K/PI3K and p-AKT/AKT were increased (P<0.05). The expression of MMP-9 decreased in PC+TNF-α+LY294002 group (P<0.05). Conclusion: The hearing loss of C57BL/6J male mice induced by cisplatin may be related to the increased release of TNF-α from the blood labyrinth barrier EC in the cochlear stria vascularis, and the activation of PI3K/AKT signaling pathway by TNF-α in PC, which increases the secretion of MMP-9 from PC, ultimately leads to the increased permeability of the blood labyrinth barrier.