The Effect And Molecular Mechanism Of Congenital Kidney Deficiency On The Development Of Cerebral Cortex In Offspring Rats Based On ADAMTS-4 Splicing-Regulated Reelin

ObjectiveThis research has continued our team’s earlier research content.On the one hand,to explore the relationship between kidney and brain from the perspective of Chinese medicine,so as to provide theoretical basis for treating brain development disorder from kidney;On the other hand,following the rat model of congenital kidney deficiency,We first used A Disintegrin And Metalloproteinase with Thrombospondin motifs-4（ADAMTS-4）clipping and regulation Reelin as the breakthrough point,to study the effects of upstream and downstream factors of Reelin signal pathway on neuronal migration during brain development,and to further clarify the cause of abnormal neuronal migration and the possible mechanism of kidney regulating brain development in rats with congenital kidney deficiency,to provide experimental basis for prevention and treatment of neurological disorder.Methods（1）Theoretical Research:We collect,sort out and analyze the theoretical exposition,literature and research progress about kidney and brain by using the methods of learning original works and literature retrieval,and then,we summarize and refine on this basis.（2）Experimental Research:（1）In this experiment,40 females and 20 males were paired with3-month-old SPF rats.After conception,the first 33 females were randomly divided into three groups:control group,model group,and kidney-tonifying group,11 in each group.To make the rat model of congenital kidney deficiency,In the model group and the kidney-tonifying group,the pregnant mice were stimulated daily by the combined stress method,including the simulated earthquake and the hanging stress method of Bao Tiantong.From the first day of pregnancy,pregnant rats in the kidney-tonifying group were treated with Zuogui pill suspension,and pregnant rats in the blank control group and the model group were treated with normal saline daily.（2）The levels of free triiodothyronine（FT₃）and free thyroxine（FT₄）in the serum of pregnant mice,the serum of newborn（D0）mice and the cerebral cortex of newborn rats were detected by Enzyme Linked Immunosorbent Assay（ELISA）;The morphology of cerebral cortex in D0 rats was observed by HE stain;The expression of cortical Dicortin（DCX）and Brain Lipophilic Binding Protein（BLBP）was detected by double immunofluorescence staining in 15-day pregnant（E15）fetal rats and D0 offspring rats.（3）The protein and m RNA expression levels of ADAMTS-4 and Reelin in cerebral cortex of D0 rats with congenital kidney deficiency were detected by Western Blot（WB）and Reverse Transcription-Polymerase Chain Reaction（RT-PCR）.（4）The protein and m RNA expression levels of downstream associated proteins of Reelin signaling pathway including Dab1,Fyn and PI3K were detected by WB and RT-PCR respectively in the cerebral cortex of D0 rats with congenital kidney deficiency.（5）We observed the general condition of D21 rats with congenital kidney deficiency;measured the weight,body length and brain weight of the rats;test the learning and memory ability of D21offspring rats by Morris water maze;observe the cortical neurons of D21 offspring rats by light microscope and electron microscope;detect the protein and m RNA expression levels of ADAMTS-4,Reelin and Fyn in cerebral cortex of D21 offspring rats with congenital kidney deficiency by WB and RT-PCR respectively.Results（1）Theoretical Research:From the point of view of Chinese medicine,this paper expounds the relationship between kidney and brain,and holds that they are in harmony with Yin and Yang in attribute,the physiological characteristics are all“Hidden but not Cathartic”,the material base is connected with each other,the physiological function is coordinated with each other,and the kidney and brain affect each other pathologically,tonifying the kidney to benefit the brain.（2）Experimental Research:（1）Serum FT₃Levels in pregnant rats:There was no significant difference among different stages of pregnancy（P>0.05）.Compared with the control group,the level of FT₃in Model Group increased significantly（P<0.01）.Compared with the control group,the level of FT₃in kidney-tonifying group was significantly decreased（P<0.01）.Serum FT₄Levels in pregnant rats:There was no significant difference in control group（P>0.05）.There was a decreasing trend in model group（P<0.01）,and a decreasing trend in kidney-tonifying group（P<0.05）.Compared with the control group,the level of serum FT₄in the model group decreased significantly（P<0.01）.Compared with the model group,the level of FT₄in kidney-tonifying Group increased significantly（P<0.01）.Serum FT₃levels in D0 offspring rats:There was no statistical difference between the two groups（P>0.05）.Serum FT₄levels in D0 offspring rats:Compared with the control group,the serum FT₄content in the model group and the kidney-tonifying group decreased significantly（P<0.01,P<0.05）.Compared with model group,kidney-tonifying group increased significantly（P<0.01）.FT₃levels in cerebral cortex of D0 rats:There was no statistical difference between the two groups（P>0.05）.FT₄levels in cerebral cortex of D0 rats:Compared with the control group,the content of FT₄in model group and the kidney-tonifying group decreased significantly（P<0.01,P<0.05）.Compared with model group,kidney-tonifying group increased significantly（P<0.01）.Morphology of cerebral cortex in E15 fetal rats:In control group,the structure of cortex was clear,the level of brain cells was clear.In the model group,the structure of cortex was disordered,the molecular layer was enlarged,the cell body was distorted,the Chromatin was abnormal,and the axons of neurons were disordered.Compared with the model group,the kidney-tonifying group had clearer structure,shorter interlayer space and more neurons.Morphology of cerebral cortex in D0 offspring rats:In the control group,the cortical neurons arranged in a clear and orderly manner.In the model group,the outermost molecular layer was widened,the neurons in the outer granular layer and vertebral body cell layer were arranged in disorder,the intercellular space was enlarged,the body of neurons was shrunken,and the number of neurons was decreased.In the kidney-tonifying group,the nerve cells arranged orderly,and the structure of the molecular layer,the outer granular layer and the vertebral cell layer was clear.The number of neurons increased compared with the model group（P<0.05）.The expression of DCX and BLBP in cerebral cortex of E15 fetal rats and D0 offspring rats:Compared with the control group,the fluorescence intensity of DCX and BLBP positive expression decreased and the positive expression cells decreased in model group.Compared with the model group,the DCX and BLBP positive cells increased and the fluorescence intensity increased in the kidney-tonifying group.（2）The content of ADAMTS-4 and Reelin protein in cerebral cortex of D0 rats:Compared with the control group,the ADAMTS-4protein expression was significantly increased and the Reelin protein was significantly decreased in model group（P<0.01）.Compared with the model group,the ADAMTS-4 protein expression was significantly decreased（P<0.01）,and the Reelin protein expression was significantly increased in kidney-tonifying Group（P<0.01）.The content of ADAMTS-4 and Reelin m RNA in cerebral cortex of D0 rats:Compared with the control group,the ADAMTS-4 m RNA increased and the Reelin m RNA decreased in the model group（P<0.01,P<0.05）.Compared with the model group,ADAMTS-4 m RNA was decreased（P<0.05）,and Reelin m RNA was increased in kidney-tonifying group（P<0.05）.（3）The contents of Dab1,Fyn and PI3K in the cerebral cortex of D0 rats:Compared with the control group,the expression of Dab1,Fyn and PI3K in the model group decreased significantly（P<0.01,P<0.01,P<0.05）.Compared with the model group,the expression of Dab1,Fyn and PI3K increased in kidney-tonifying Group（P<0.05）.The contents of Dab1,Fyn and PI3K m RNA in the cerebral cortex of D0 rats:Compared with the control group,the expression of Dab1,Fyn and PI3K m RNA were significantly decreased in model group（P<0.01,P<0.01,P<0.05）.Compared with the model group,the expression of Dab1,Fyn and PI3K m RNA in kidney-tonifying group increased in different degrees（P<0.05,P<0.01,P<0.05）.（4）The general condition of D21 offspring rats:The control group was in good condition,with smooth and ruddy coat,high activity,good appetite,normal drinking water,normal urination and stool.Compared with the control group,the offspring of the model group were more easily frightened,more frequent urination,soft stool,some of them had no tail deformity or hair loss,weight loss（P<0.05）,body length shortened（P<0.05）,the wet weight of brain and brain weight index decreased（P<0.05）.Compared with the model group,the stool and urine of the rats in the kidney-tonifying group were basically normal,the hair was slightly withered and yellow without luster,there was no significant hair loss,the body weight increased（P<0.05）,the body length increased（P<0.05）,the wet brain weight and the brain weight index increased（P<0.05）.The learning and memory abillity of D21 offspring rats:Compared with the control group,the escape latency of D21 offspring in the model group was significantly prolonged（P<0.01）,the times of escape were decreased（P<0.05）,and the percentage of stay in the third target quadrant was shortened（P<0.01）.Compared with the model group,the escape latency was significantly shorter（P<0.01）,the times of crossing were increased（P<0.05）,and the percentage of goal quadrant staying was significantly increased in kidney-tonifying group（P<0.01）.Neuron morphology in the cerebral cortex of D21 offspring rats:In the model group,the molecular layer in the outermost layer of the cerebral cortex was widened,the intercellular space was enlarged,the arrangement of neurons was disordered,the cell body shrank,the number of neurons decreased,the structure of neurons was uneven,the nucleus was irregular,the nuclear membrane was damaged,the Mitochondria was broken,apoptotic bodies were found in cytoplasm.Compared with the model group,the number of neurons and the arrangement of cells in kidney-tonifying group were increased,and the structure of the molecular layer,the outer granular layer and the vertebral cell layer were clear.Compared with the model group,the kidney-tonifying group had more neurons,regular arrangement of cells,clear structure of nerve cells,smooth and clear nuclear membrane,and uniform distribution of chromatin.Content of ADAMTS-4,Reelin and Fyn protein in cerebral cortex of D21offspring rats:Compared with the control group,ADAMTS-4 protein was significantly increased in model group（P<0.01）,and the expression of Reelin and Fyn protein was significantly decreased（P<0.05,P<0.01）.Compared with the model group,the expression of ADAMTS-4 protein was decreased（P<0.05）,and the expression of Reelin and Fyn protein was increased in kidney-tonifying group（P<0.05,P<0.05）.Content of ADAMTS-4,Reelin and Fyn m RNA in cerebral cortex of D21 offspring rats:Compared with the control group,ADAMTS-4 m RNA increased（P<0.05）,and the expression of Reelin and Fyn m RNA decreased in the model group（P<0.01,P<0.05）.Compared with the model group,the expression of ADAMTS-4 m RNA decreased（P<0.05）,while the expression of Reelin and Fyn m RNA increased in kidney-tonifying group（P<0.05）.Conclusion（1）The kidney and brain are closely related.The treatment of encephalopathy from kidney has rich TCM theory support and extensive clinical practice.（2）The experimental model of congenital kidney deficiency was established by the method of“Fear of injury to pregnant rats”,and it was found that congenital kidney deficiency could reduce the level of Thyroid hormone,reducing the number of Radial glial cell（RGC）or decreasing the expression of DCX inhibits neuronal migration in the cerebral Cortex,thus affecting brain development.（3）The abnormal regulation of ADAMTS-4 on Reelin splicing,which results in the abnormal binding of Reelin to Apolipoprotein E Receptor2（APOER2）and Very Low-Density Lipoprotein Receptor（VLDLR）,and the failure to activate the downstream pathway,which cause the decreased expression of Dab1,Fyn and PI3K in the downstream of Reelin signaling pathway,is one of the important reasons for the abnormal development of cerebral cortex in rats with congenital kidney deficiency.（4）Zuogui pill was used to fill kidney essence in pregnant rats,it can increase the level of FT₄,the number of RGC and the expression of DCX,inhibit the expression of ADAMTS-4 and enhance the expression and activity of Reelin,and increase the expression of Dab1,Fyn and PI3K in the downstream of Reelin signal pathway,therefore,the abnormal migration of the neurons in the prefrontal cortex of offspring rats with kidney deficiency can be alleviated,the development of the cerebral cortex can be promoted,and the growth retardation and the low ability of learning and memory can be prevented or alleviated to some extent.