The Differential Expression Of Mitochondrial Dynamics In The Nervous System Of Type 1 Diabetic SD Rats

ObjectionMorphological structure,number of mitochondria and mitochondrial dynamics-related proteins in the central and peripheral nervous system of SD rats with type 1 diabetes were observed or differentiated.At the same time,rat spinal cord anterior horn motor neurons(VSC4.1)were cultured in vitro,and the changes of mitochondria in the normal group and high glucose intervention group were observed.Further explore the relationship between mitochondrial dynamics and diabetic neuropathy,the underlying mechanism of the development of diabetic neuropathy,and provide a theoretical basis and evidence for the possible mechanism of treatment of the disease.MethodThirty male Sprague-Dawley rats were randomly divided into two groups: control group(Control group)and diabetes group(DM group),control group(n=13),and diabetic group(n=12).Six rats in each group were used for transmission electron microscopy and immunofluorescence,and the remaining rats were used for immunoblotting(WB)detection.1.Transmission electron microscopy was used to observe the changes of mitochondrial morphology and quantity in neuronal cells of the cortex,hippocampus,posterior horn of the cervical cord,posterior horn of the lumbar spinal cord and dorsal root ganglia at 4 weeks.Immunofluorescence was used to observe the differential expression situation of mitochondrial dynamics-related protein 1(Drp1),mitochondrial fusion protein 1(Mfn1)and mitochondrial markers(VDAC1)in the cortex,hippocampus,posterior horn of the cervical spinal cord,and posterior horn of the lumbar spinal cord at 4 weeks.3.Western blot analysis the expression of mitochondrial dynamics,for example: mitochondrial markers(VDAC1),mitochondrial optic atrophy factor 1(Opa1),mitochondrial fusion protein 1(Mfn1),mitochondrial fusion protein 2(Mfn2),mitochondrial outer membrane small division protein1(Fis1)mitochondrial dynamics-associated protein 1(Drp1).Rat spinal cord anterior horn motor neurons(VSC4.1)were cultured in vitro.Transmission electron microscopy,Western blot and mitochondrial fluorescence staining were used to observe mitochondrial morphology,quantity and mitochondrial dynamics in normal and high glucose intervention groups.Result1.Transmission electron microscopy showed that the volume of mitochondria in the neurons of the posterior horn of the lumbar spinal cord and the dorsal root ganglion of SD rats in diabetic group increased,light and moderate swelling,and some vacuolization appeared.The mitochondria and the outer membrane are still intact,and the mitochondria tend to be slightly swollen.The intrinsic matrix particles show partial reduction or loss.The mitochondrial morphology,length,aspect ratio and quantity change are compared with the normal control group.The difference was statistically significant(p < 0.01).2.Immunofluorescence observation results showed that the perfermance of Drp1 and VDAC1 was significantly increased in the posterior horn of the lumbar spinal cord of SD rats,and the perfermance of Mfn1 was positively down.The amount was significantly reduced,and the discrepancy was statistically significant compared with the normal control group(p<0.01).3.Western blot results showed that VDAC1,Fis1 and Drp1 were found in the posterior and posterior root ganglia of SD rats in diabetic rats.The perfermance of gray value of Drp1 was significantly increased;while the perfermance of Mfn1,Mfn2 and Opa1 was significantly reduced.The discrepancy was statistically significant compared with the normal control group.(p < 0.01).4.Electron microscopy showed that the volume of mitochondria in cells increased significantly and swelled and vacuolated;a large number of swollen mitochondria accumulate each other;some mitochondrial inner membranes ruptured;mitochondria swelled and ruptured;The matrix particles were largely absent,and the mitochondrial morphological structure,length,aspect ratio and number of changes were statistically significant(p<0.01).5.The results of Western blot showed that the perfermance of the Drp1,Fis1 and VDAC1 was significantly increased in cells under high-glucose intervention;The perfermance of the Mfn1,Mfn2 and Opa1 was significantly decreased,and the difference was statistically significant compared with the normal control group(p<0.01).6.After mitochondrial Mitotracker Red fluorescent staining,the mitochondria of the normal and high-glucose group were observed statically and dynamically in cells.It was found that mitochondrial division occupied a distinct advantage in high-glucose group cells and formed a fragmented,discontinuous network.At the same time,it was found that a large number of fragments,smaller and round around the cell body.Round mitochondria,and the phenomenon of aggregation,and the number of mitochondria in the axons of neurons were showed a significant decrease,and the appearance of mitochondrial distribution loss and aggregation appeared intermittently;There are significant changes compared with normal group cells.Conclusion1.Morphological abnormalities,kinetic imbalances,and increased numbers.These changes in mitochondria are one of the potential mechanisms for the development or progression of diabetic neurological diseases;they are also the premise and basis for pathophysiological damage and dysfunction of the nervous system.2,diabetic neurological disease occurs in the early stages of diabetes,peripheral nervous system lesions begin first,and the central nervous system has not changed.3.In vitro,mitochondrial division in VSC4.1 cells stimulated by high glucose was significant and formed a discontinuous network.These changes also provide a theoretical basis for the study of the occurrence and development of diabetic neuropathy.