Mechanism Of Cisplatin-induced Inflammation Affecting BLB Permeability In Stria Vascularis Of Cochlea

Objective: Cisplatin(Cis)is widely used in the treatment of tumors,but its ototoxicity mechanism is still completely clear,and there has been a lack of effective prevention and treatment measures.In this study,C57BL/6J male mice were used as the research object to explore whether cisplatin can promote the secretion of MMP-9 and destroy the permeability of stria vascularis by inducing inflammation and activating the PI3K/AKT signal pathway of pericytes(PCs)of stria vascularis in the cochlea to provide an experimental basis for the prevention and treatment of hearing loss caused by cisplatin ototoxicity.Methods: Animal experiment: control group(Control)and cisplatin group(Cis).Cell experiment:primary culture and identification of C57BL/6J mice cochlear stria vascularis PCs and endothelial cells(ECs);(1)It is divided into PC group,PC+TNF-α group,PC+IL-1β group,PC+TNF-α+ LY294002(PI3K/AKT pathway inhibitors)group,PC+IL-1β+ LY294002 group,PC+LY294002 group;(2)Divided into EC group,EC+TNF-α group,EC+IL-1β group,EC+PC group,EC+PC+TNF-α group,EC+PC+IL-1β group,EC+PC+TNF-α+SB-3CT(MMP-9/MMP-2 secretion inhibitor)group,EC+PC+IL-1β+SB-3CT group.Auditory brainstem response(ABR)was used to detect the hearing changes of mice;The morphological changes of stria vascularis in the cochlea of mice were observed by H&E staining;ELISA to detect changes in TNF-α and IL-1β expression in mouse serum and cell supernatant;Detection of TNF-α and IL-1β protein distribution expression on cochlear vascular striae by immunohistochemical techniques;Evans Blue(EB)staining was used to observe the permeability of blood labyrinth barrier(BLB);Western blot was used to detect the expression of MMP-9,inflammatory factors,tight junction protein,and PI3K/AKT-related pathway protein;Millipore Millicell ERS-2 assay for endothelial cell resistance;FITC-dextran(4 k Da)assay for endothelial barrier permeability.Results: Animal experiment:(1)Compared with the control group,the hearing threshold of Cis group mice increased(P < 0.01);(2)Compared with the control group,the stria vascularis in the cochlea of mice in the Cis group was more disordered and shrunk,and the number of vacuoles increased;(3)The red fluorescent dye extravasation of stria vascularis increased in Cis group(P < 0.05);(4)Cis group cochlear stria vascularis and serum TNF-α,IL-1β Expression increased(P < 0.05);Cell experiment:(5)The expression of TNF-α and IL-1β was the highest after 20 μmol cisplatin intervention in endothelial cells(P <0.01),and the expression of TNF-α was the highest at 3h and IL-1β was the highest at 1h(P < 0.05).(6)The results of TEER resistance showed that the resistancethe of EC+PC group was higher than the that of EC group 0.05),and EC+PC+TNF-α group and EC+PC+IL-1β compathe red with EC+PC group,the rthe esistance of endothelial cells in SB-3CT group decreased(P < 0.05),and SB-3CT can improve TNF-α and IL-1β the electrical resistance of endothelial cells decreased(P < 0.05);(7)FITC-dextran permeability results showed that monolayer endothelial cell permeability was increased in the EC+PC group compared with the EC group(P < 0.05),TNF-α and IL-1β increased endothelial cell permeability in the EC+PC group(P <0.05),and SB-3CT reversed the TNF-α and IL-1β-induced increase in endothelial cell permeability(P <0.05).(8)Western blot results show that the expression of ZO-1 and VE-cadthe herin protein in EC+PC group was higher than that in the EC group(P < 0.05),TNF-α and IL-1β decrease the expression of ZO-1 and VEcadherin protein in EC+PC group(P < 0.05),and increase the expression of ZO-1 and VE-cadherin protein in endothelial cells after SB-3CT intervention(P < 0.05);(9)Western blot results show that after TNF-α and IL-1β intervention in pericytes,MMP-9 expression increased(P < 0.05),MMP-2 expression did not change significantly,and p-PI3K/PI3 K and p-AKT/AKT expression increased(P < 0.05);Pretreatment with LY294002 reversed the increasing of MMP-9 expression by TNF-α and IL-1β induced(P < 0.05).Conclusion: Cisplatin-induced BLB permeability and resulting hearing loss may be associated with increased release of TNF-α and IL-1β from cochlear vascular striatum BLB endothelial cells,activation of the PI3K/AKT signaling pathway in pericytes,and increased secretion of MMP-9 by pericytes.