Sequential Change And Mechanism Of Autophagy And Apoptosis In Spiral Ganglion Cells And Cochlear Nucleus Neurons In Diabetic Rats

Aims and SignificanceDiabetes mellitus(DM)may cause varying degrees of damage on the hearing system,but the mechanism is not clear now.Many studies found that autophagy and apoptosis were closely associated with diabetes mellitus and its complications such as diabetic nephropathy,diabetic neuropathy and diabetic retinopathy,and played important roles on these complications.However,there are few studies on hearing impairment in diabetes mellitus.We concluded that autophagy may also play a role in diabetes-related hearing impairment.In this study,the diabetic rat models were established to observe the dynamic pathological changes of auditory system in diabetes mellitus.To investigate whether autophagy and apoptosis participate in the toxic process of high blood glucose on spiral ganglion cells(SGCs)and cochlear nucleus neurons,and their involvement in the pathogenesis of diabetes-induced hearing impairment in rats.To provide experimental basis for the study of pathogenesis of diabetes-related hearing impairment.MethodsSprague-dawley rats were induced by STZ,then the diabetic rats were divided into 4,8,12week groups according to the diabetes duration.The DPOAE and ABR were conducted in all groups.The histomorphology of SGCs in each group were observed under the optical microscope and counted the number of cells.Ultra microstructure of SGCs and cochlear nucleus were observed by transmission electron microscope.The expression of GFAP in cochlear nucleus was detected by Immunohistochemistry.Immunohistochemistry and western blotting were used to detect the expression of autophagy-related proteins(Beclin-land cleaved-LC3),apoptosis-related proteins(Bax,Bcl-2 and cleaved caspase-3)in SGCs and cochlear nucleus.To analyze the correlation between autophagy and apoptosis using Pearson correlation.Results1.The diabetic rat model was successfully established.The mission success rate is 76.50%,and the mortality rate is 23.57%.2.The amplitude of DPOAE was significantly decreased in DM12W group.The threshold of ABR was significantly elevated in DM8W and DM12W group.Compared to the control group,the latency and wave duration of each wave were prolonged significantly in DM12W group.3.There were no significantly pathological changes of SGCs and cochlear nucleus in DM4W group.The density neurons of SGCs and cochlear nucleus were decreased in DM8W and DM12W groups.The number of SGCs were significantly decreased(P<0.05).4.Ultrastructural pathology Changes.There were some pathological changes of the cochlear neurons in DM8W and DM12W groups.The myelinated nerve fibers were reduced,the myelin sheaths of nerve fibers were segmental demyelination.The axons atrophy and autophagolysosomes could be seen in cytoplasm.Some of the sciatic nerve myelin lamellar structures were presented as isolation in DM4W group.In DM8W and DM12W groups,the myelin sheaths of spiral ganglion nerves were segmental demyelination and axons atrophy,some mitochondria were swelling and vacuolization.Some mitochondria of SGCs were swelling in DM4W group.The cochlear nucleus in DM4W group,mitochondria were a slight swelling,the myelin sheaths of fibers were demyelination.In DM8 W and DM12W groups,Mitochondria were swelling and vacuolization,autophagosome and lysosomes were increased,segmental demyelination more widespread and the atrophy of axons more widespread.5.Expression of autophagy and apoptosis proteins in SGCs.Immunohistochemistry showed that expression of Beclin-1,LC3,Bax and Cleaved Caspase-3 proteins were increased in the diabetic rats groups,but expression of Bcl-2 were reduced.On the contrary,Bcl-2 was increased in the control group.Western blotting test showed that the expression level of Beclin-1 protein in the diabetic rats groups were differed significantly from that in the control group(P<0.05).The expression of Cleaved-LC3,Bax and Cleaved Caspase-3 in DM8W and DM12W groups were increased significantly compared with the control group(P<0.05).However,the expression of Bcl-2 in DM8W and DM12W groups were decreased significantly.6.Expression of proteins in cochlear nucleus.Immunohistochemical staining showed that the expression of GFAP was increased in diabetic groups.The expression of Beclin-1,LC3,Bax and Cleaved Caspase-3 proteins were up-regulated,but expression of Bcl-2 were reduced in the diabetic rats groups.On the contrary,Bcl-2 was increased in the control group.Western blotting test showed that the expression of Beclin-1 and Bax proteins were upregulated significantly in the diabetic rats groups.Compared with the control group,the expression of Cleaved-LC3 in DM8W and DM12W groups were increased significantly,while the expression of Bcl-2 protein were significantly lower in DM8 W and DM12W groups.Conclusion1.Persistent hyperglycemia may result in the chronic damage of SGCs,cochlear nerve and cochlear nucleus neurons.2.Hyperglycemia can promote the proliferation and activation of astrocytes in cochlear nucleus.3.Autophagy and apoptosis might be involved in the neurotoxicity of hyperglycemia in the SGCs and the cochlear nucleus neurons.4.With the prolongation of diabetes mellitus,the levels of autophagy and apoptosis in SGCs and cochlear nucleus neurons were gradually increased.5.Autophagy may participate in the toxic process of high blood glucose on SGCs and cochlear nucleus through Bcl-2/Beclin-1 pathway.