The Role Of SHH Signaling In The Regeneration Of Hair Cells In The Zebrafish Larvae Model Of Cisplatin Ototoxicity

[Background]Ototoxicity is a serious side effect of cisplatin chemotherapy,and there is no effective drug to prevent and treat cisplatin-induced ototoxicity at present.Previous studies have suggested that adult mammals lack the capacity to regenerate hair cells,but in recent years,it has been possible to regenerate the adult mammals cochlear hair cells through exogenous gene expression and promoting the differentiation of regeneration related cells.Promoting post-injury regeneration of hair cell may be another way to solve the problem of cisplatin ototoxicity.Hair cell regeneration is a complex process,Mathl,the Notch signaling pathway,bFGF and so on,have been proved to be able to regulate the regeneration of hair cells.Shh plays an important regulatory role in development of mammalian cochlea and formation of hair cell.Although numerous studies have been done to elucidate the roles of Shh signaling during cochlear development,its roles during hair cell regeneration remain largely unknown.Zebrafish has emerged as an ideal vertebrate animal model,it has about 87%genes as similar as human.Zebrafish lateral line hair cells are similar to inner ear in morphological characters and physiological function,which is located on the body surface and easy to observe,it can regenerate after injury.The model of hair cell regeneration in zebrafish is simple and effective,and it has been widely used in recently years.Previous studies showed that the mechanism of Aminoglycosides-induced ototoxicity was different from the cisplatin.Compared with neomycin,cisplatin treatment could inhibit the regeneration of hair cells,however,the underlying mechanism is still unclear.So we established the zebrafish larval model of cisplatin ototoxicity,detected the injury and regeneration of hair cells and analyzed the role of Shh signaling in regeneration of hair cells.[Objective]Established the zebrafish larval model of cisplatin ototoxicity detected the injury and regeneration of hair cells;Analyzed the role of Shh signaling in regeneration of zebrafish larvae lateral line hair cells after cisplatin treatment,and lay the foundation for Human auditory hair cells regeneration after cisplatin treatment.[Method]Cisplatin treatment commenced at 5 dpf,and the zebrafish larvae were given cisplatin at 50 umol/L for 24h,and then detected the regeneration of hair cell at Oh,6h,12h,24h,48h.To detect the role of Shh signaling involved in the regeneration of hair cells in the zebrafish larval model of cisplatin ototoxicity,then the whole-mount in situ analysis of Gill and Shh were conducted.At last,we use the Shh inhibitor Cyclompamin to inhibit the Shh signaling pathway and then detected the regeneration of zebrafish larvae lateral line hair cell.[Result](1)In the control group,the average number of hair cells in the four neuromast(SO1?SO2?O1?and OC1)was 9.675±0.64603;At 0 hour after cisplatin treatment,the average number of hair cells in the four neuromast was 0.2222±0.26352;At 6h,12h,48h after cisplatin treatment,the average number of hair cells in the four neuromast were 0.6944±0.30046?2.0750±0.55340?4.1818±0.58192?6.5250±0.50621.Compared with the control group,the percentage of the hair recovery cells in each group was 7.18%?21.45%?43.22%?67.44%.(2)In the control group,the Shh gene and Glil gene expressed in the lateral neuromast.Compared with the control group,at 6h after cisplatin treatment,the expression of Shh gene decreased significantly,then the expression of Shh gene were signficantly increased with time.The expression of the glil gene is similar to that of shh.(3)In the cyclopamine group,the average number of hair cells in the four neuromast SO1?S02?Oland OC1 in the zebrafish larval lateral is 2.2955±0.49772,the average number of hair cells in the non-inhibitor group is 3.8000 ±0.94868,the difference has statistically significant(P<0.05),The results show that cyclopamine could inhibit the regeneration of hair cells to some extent.[Conclusion](1)Cisplatin can destroy the zebrafish larval lateral hair cells,and zebrafish larval hair cells have the capacity to regenerate after injury.the zebrafish larval model of cisplatin ototoxicity and regeneration is useful.(2)Shh signaling pathway may be involved in the development and functional maintenance of the zebrafish larval lateral neuromast.(3)Shh signaling pathway is activated in the process of zebrafish larval lateral hair cells regeneration after cisplatin treatment and take part in the hair cell regeneration.Inhibit the Shh signaling pathway can decrease the number of the regenerated hair cell,the results indicated that Shh signaling plays a positive role in the regeneration of hair cells in the zebrafish larval model of cisplatin ototoxicity.