The Study Of The Brain-Renal RAS Axis Activity In Type I Diabetes Rat Model

BackgroundWith social progress and economic development,although life quality is much better,diet habit and the peace of life has been great improved,healthy problem isbecoming severely.Diabetes mellitus(DM)as a kind of metabolic disease has posed a grave threat on human health.Studies had reported that the number of worldwide diabetics would raise from 175 million in 2000 to 353 million by 2030.China and Indian diabetics are approximately account for 24%of total,and the treatment cost will grow 3 times which brought a heavy burden and pain to society and family.Recently in China,adult patients with diabetes account for about 11.6%,the number of high risk of diabetes account for 50.1%,while in DM patients,l/3 people have the opportunity to developto diabetic nephropathy(DN).DN as a chronic kidney disease,not only has the characteristics of DM,but more important is that it may lead to chronic renal failure finally,therefore DN is already an important public health problem of-worldwide.Recently,itbecomes the hot and difficult research topic in the field of nephrology,and it provides important theoretical basis for prevention and treatment to DN.It is the subject human urgently need research and development.Pathogenesis of DN relates with variety factors,including glucose metabolism,glycation,AGEs generation,hemodynamic abnormalities,oxidative stress,cytokines(transforming growth factor,vascular endothelial growth factor,inflammatory cytokines)and genetic factors.The main complications include hypertension,calcium and phosphorus metabolism disorders,cardiovascular disease,central and peripheral neuropathy,and patients with kidney disease is lethal,leading causes of disability.As is known,the renin-angiotensin system(Renin-angiotensin system,RAS)plays za key factor in the development of kidney disease1 Thc pathogenesis of DM:1.1 High glucose environment damageDiabetes mellitus(DM)results in high glucose environment,which lead to vivo glucose and lipid metabolism disorders,and glucose and lipid metabolism disorders can lead to diabetic nephropathy(DN)of the occurrence and development.Polyol metabolic disorder is one of the main DM human injury,in humans,cells with cells,and extracellular matrix are closely linked,mainly in the kidney inherent cells glomerular capillary endothelial cells,mesangial cells,podocytes,renal tubular epithelial cells,renal interstitial cells.All cells live together in the same environment there is signal link between cells.It is a dynamic equilibrium,and high glucose environment will break this balance,various types of cells within the environment of high-sugar high-fat will secrete different cytokines,and posses different function and metabolic processes.Cell within High sugar and high fat environment will directly injure renal inherent cell,high sugar and high fat will lead to cell swelling,necrosis and apoptosis.For example,high glucose environment will promote glomerular endothelial secrete more cytokines,leading cytoskeleton structure change,and increasing the inflammatory response.Glucose and lipid disorder can stimulate mesangial cells hypertrophy,proliferation,and extracellular matrix secretion.and results in the occurrence and development of renal fibrosis.High blood glucose can promote apoptosis of podocytes,and thicken the basement membrane,in addition,high fat and glucose can stimulate the activation of RAS in renal tubular epithelial cells,high glucose environment will stimulate transformation ofrenal interstitial fibroblasts to myofibroblasts,secreting large amounts of ?,? collagen which is hard to degrade,and promote kidney damage,accelerating the process of renal interstitial fibrosis.1.2 Effect of hemodynamic change on renal units perfusionOver the years,it has been noted that type ? diabetes mellitus presents high renal glomerularr filtration at the begining,and the glomerular filtration rate(GFR)can be higher 15%?40%than the normal.Type ? diabetes recently has been reported,to be the same phenomenon.As newly reported,type ? diabetes with normal blood pressure or without Proteinuria 45%has high glomerular filtration higher filtration.which suggeststhat increasing pressure within the glomerular capillary exists.For DM patients are under internal high glucose environment solong,which leads glomerular high perfusion and high filtration rate,and increases of pressure within glomerular,and damaging glomerular basement membrane,fall of podocytes,leading part of glomerular structure incompletly.With continuous high blood glucose injury aggravating,glomerular endothelial cell would be damaged and made proliferation,thrombosis,vascular fraction,vascular elasticity,and resistance increased perfusion.At different period of DM,the renal vascular high perfusion,high filtration,and glomerular high pressure persists,and increasing plasma macromolecules leaking to mesangial area.Coupled with the ability of diabetic mesangial cell depletion macromolecules reduction,it increased mesangial zone blocking,what is futher more,the accumulation of macromolecules in mesangial area can further stimulate mesangial cell proliferation,mesangial matrix production,which results in mesangial expansion,accelerate glomerular sclerosis,a sustained high pressure in glomerular can stimulate renal glomerular filtration membrane epithelial cells synthesis collagen,induced GBM thickenig;at the same time,it is possible to pass the high pressure from glomeruli to mesangial cell,and is also stimulating mesangial matrix production.In cultured mesangial,the cyclical under tensile stess increase collagen,Laminin,fibronectin productioon,TGF-?receptor mRNA of Ang-?synthesis and expression.1.3 The function of oxidative stress would promote nephron injuryIn the normal physiological conditions,there is dynamic equilibrium between the body's oxidation and antioxidant system.Oxidative stress refers that when body is stimulate by harmful substance reactive oxygen species(ROS)or nitrogen species(RNS)generate increase or clearance reduce.,and ROS or RNS are accumulating in the body which would cause molecules,cells,body damage.ROS production by oxidative stress can be rapidly cleared by body antioxidant system,but in certain pathological conditions,Body free radicals increase or antioxidant capacity decrease,oxidative capacity greatly exceeds the antioxidant capacity,oxidative stress would induced tissue damage in the early stage of diabetes.Compensatory increaseof part of antioxidant enzyme activation of kidney tissue,with the extension of the duration of diabetes,its activity gradually declined.Eventually leading to the occurrence of renal tissue ROS.SOD is one of the oxygen free radicals clear system of body,it transformperoxide into H202,and GPX and CAT solution H202 to 02 and H20.Podocytes exposed to high glucose concentrations 3-5 days,oxidase activity expression of SOD/Nox4? NAD(P)H increased,but activaty of GPx and CAT decrease.It show that Hyperglycemic broke oxidation-antioxidant balance of mouse foot cell,it may play an important role in the occurrence of diabetic nephropathy.Hyperglycemia as a start factor of DM complications,causing oxidative stress levels grow and increase ROS,non-enzymatic glycosylation protein and glucose autoxidation.Mitochondria generate excessive oxygen free radicals can activate the polyol pathway,ACEs pathway,PKC pathway,and promote the formation of free radicals.,creating a vicious cycle.Increased production of free radicals,the glomerular basement membrane peroxidation,inflammation make it thicker,while oxidative stress involved in Renal capillary endothelial cell cause by increasing of advanced glycation end products(AGEs).Under diabetic state ROS participation renal cell injury,high glucose through ROS start podocyte cell apoptosis,induced it detached from the basement membrane,and glomerular podocyte cell number decrese.1.4 Systemic inflammation resulting in kidney damageAlarge number of epidemiolog,ical,laboratory and clinical studies prove that DN is a kind disease associated with innaflammation.As early as glucose tolerance impaired,glomerular filtration rate already was significantly higher than normal in pre-diabetes and microalbuminuria increases significantly.It suggests that diabetic kidney damage may not only lead to high blood glucose.Further studies have also observed that these patients blood C-reactive protein increased,and it show that there is inflammation response in early diabetes,people did not think DN is a disease associated with inflammation at the beginning,till Bohle and other's discoveried and subsequent studies have confirmed kidney tissue in patients with DM macrophage infiltration was significantly higher than the normal kidney tissue,and the products of macrophages will further promote the development of inflammation.In recent years,numerous studies reveal that inflammation and immune responses play an important role in promoting the development of DN,same as inflammation and immunity play important role in the most acute and chronic kidney disease pathogenesis.There is studies reported that all type inflammatory factors were rise in DM patients;blood,such as TNF-a level higher than normal in DM patients with proteinuria;Monocyte chemoattractant protein(MCP-1)can promote the body monocyte macrophage accumulation within the organization,high glucose of body can stimulate glomerular mesangial cells and renal interstitial cells expressing high level MCP-1,renal pathology results showed that kidney tissue levels of MCP-1 expression was significantly increased in DM patients.2 Relationship between DM and the brain RASDM patients characterized by polydipsia,polyuria,polyphagia and gradual weight loss,have a variety of material and energy metabolic disorder in vivo due to the presence(of high glucose environment.The main complications of DM are hypertension,calcium and phosphorus metabolism disorders,cardiovascular disease,central and peripheral neuropathy,and kidney disease is the main reason of death and disability of patients.As is known,the renin-angiotensin system(Renin-angiotensin system,RAS)plays a key factor in the development of kidney disease.Studies showed that CKD rat model exist salt sensitivity regulatory mechanisms,and that is CKD rat brain paraventricular nucleus(PVN),supraoptic nucleus(SON),nuclear lower fornix(SFO)and other sympathetic regulatory mechanisms associated with the nuclei in CKD model is activated,and by sympathetic efferent,afferent nerves to regulate kidney RAS activity,in order to achieve the purpose of the regulation of kidney damage.In addition,there is a large number of research results about DM rat model show that a key part of the brain nucleus also is activated because of the activation of RAS.and the activation of the central RAS directly or indirectly lead to peripheral organs and tissues RAS activation.In the early of DM,due to the increase urination,kidney is always in high perfusion,high filtration state,hemodynamic changes stimulated kidney RAS activation.In order to adapt to this RAS kidney compensatory change,that increased pressure within the glomeruli,this condition continuous persist would damaged nephron;high glucose itself can lead directly mesangial proliferation podocytes apoptosis off,tubular epithelial cells significant inflammation and transdifferentiation.While DM will lead to systemic chronic inflammation,the expression of various types of inflammatory cytokines such as ILl,IL6,IL8,TNF-?,etc.in various tissues and organs will be increased,and the stimulation of inflammation would promoted RAS activity increased within various tissues and organs,enhanced sympathetic activation,to facilitate the process of kidney damage.In the process of DM disease,the blood and tissues and organs will accumulate a lot of glycation end products(AGEs),these substances can not only directly do harm to variety of renal parenchymal cell.but also improve the oxidative stress reaction within the tissues and organs,and oxidative stress,sympathetic activation and the activation of RAS are closely linked,this link has undoubtedly contributed directly or indirectly kidney damage process.All of the above of pathogenesis of DN seems to be has a close relationship with the RAS.Method:Part one Study of oxidative stress,inflammation indicators in circulation,brain RAS of non-diabetic rats(Non-DM)and diabetic rats(DM).1 Preparation of a animal modelThis study was intended for use classic type I diabetic rats model to model.Male Sprague Dawley rats(purchased from Experimental Animal Center of Southern Medical University)weighing 250-300g,were randomly divided into two groups,namely,Non-DM and DM groups.The DM group rats by intraperitoneal injection of streptozotocin(STZ,dissolved in pH4.2 sodium citrate solution)at a dose of 60mg/kg.Non-DM intraperitoneal injection pH4.2 sodium citrate solution at a dose of 1ml/100g.after injection of STZ 3d,5d,7d,collect DM rat tail vein blood to test blood glucose,if three times the mean random blood glucose levels>16.8mol/ml can be considered successful model,otherwise abandoned them.Randomized:group Samples Non-DM n=15 DM n=15All rats were measured blood glucose level and blood pressure values before randomization.After modeling,nonnal feeding 6 weeks in SPF level conditions,then execute them and collect material.2.Experimental data and specimen collectionI)Measurements of urine samples,weight and blood pressure,All rats caged alone in metabolic cage,for three consecutive days to collect 24h urine 3 days before execution,and after recording the total amount of urine reserve urine specimens 4ml,1600g,centrifugation 20min in 4 C surrounding,and packing and save in-80 0C environment.Test rat blood pressure through tail artery by Non-invasive blood pressure monitor(softron BP-98A,Japan)2)Collectionof rat blood fresh tissue perfused tissue.Time to reach the end of the experiment,inject 3%sodium pentobarbital by intraperitoneal injection to anesthetized rats(0.15ml/100g),followed laparotomy,collected blood through puncture aorta and quickly open the chest,use a 16#gavage needle puncture from the left ventricle to the aortic arch,and cut the right atrial appendage,use 200ml saline(include heparin 20U/ml)which was Pre-cooling in 4 0C perfusion,you can see the liver,skin,lungs and muscles quickly becomes pale,determining perfusion ends when liquid outflow from the right atrial appendage clear and colorless.Then take some brain nuclei,brain,heart,kidney,liver and other tissues thrown directly into liquid nitrogen.This step is to take fresh tissue to do prepare for subsequent RT-PCR and WB do to prepare3)Collection of the fixed tissuesUse 200ml saline(include heparin 20U/ml)which was Pre-cooling in 4 0C quickly perfusion,and use 4%paraformaldehyde(PH 7.2)slow perfusion 1.5h,ten carefully cut the skull and take brain,cut brain along coronal into 2mm thick slices in the brain mold of rats,put into 4%paraformaldehyde solution and fixed 6h in 4 ?state.After the water rinse 2h,gradient alcohol dehydration,xylene transparency,wax dipped embedded.Kidney,heart and liver are equally cut 2mm thick slices,fixed 24h in 4 C state,pure water rinse 2h,gradient alcohol dehydration,xylene transparency,wax dipped embedded.after processing cut 4um of all fixed tissue to make film and dye.4)Plasma angiotensin ?:radioactive immunoassay5)Determination of plasma norepinephrine(NE)levels:ELISA assay The blood urea nitrogen and serum creatinine testing:uses Clinical automatic biochemical analyzer6)Urinary 8-iso-prostaglandin-2 Determination:ELISA assay Urine creatinine and urea nitrogen testing:uses Clinical automatic biochemical analyzer7)The detection of blood brain barrier permeability:inject Evans blue through tail vein,measure per gram of brain tissue Evans blue content 0.5h later,namely,the concentration of brain tissue the permeability higher of rat blood brain barrmer.4h urinary protein:use Coomassie brilliant blue-G250 to assay8)The detection of SON,PVN,SFO and other key brain nucleus,RAS and oxidative stress indicators:fresh brain tissue and fixed brain nuclei were collected by 2,3portion respectively use the way WB,RT-PCR and immunohistochemistry to detect the expression of RAS indicators AGT,AT1 and oxidative stress indicators NOX2,NOX49)Detect kidney RAS,oxidative stress,inflammatory index:kidneys were collected by 2,3portion respectively use the way WB,RT-PCR and immunohistochemistry to detect the expression of RAS indictors indicators AGT,AT1 and oxidative stress indicators NOX2.?NOX4 and inflammatory indicators MCP-1.Statistical analysisUse the Image-Pro Plus 7.0 software to analyse immunohistochemical Image,and use Image J software to analyse of protein expression.Data analyzed by SPSS 13.0 statistical package,and data is expressed with a mean±sd.Mean differences between the two groups of rats are using two independent sample t-test,with P<0.05 difference is statistically significant.Part tow Study of oxidative stress,inflammation indicators in circulation,brain RAS in different groups1 Preparation of animal modelThis research use classic type I diabetic rats model to model.Male Sprague Dawley rats(purchased from Experimental Animal Center of Southern Medical University)weighing 250-300g.All rats were injectedwith streptozotocin(STZ,dissolved in pH4.2 sodium citrate solution)at a dose of 60mg/kg through intraperitoneal,after injection of STZ 3d,5d,7d,collect DM rat tail vein blood to test blood glucose,if three times the mean random blood glucose levels>16.8mol/ml can be considered successful model,otherwise abandoned them.The part of the experiment group was as follows:group Sample size IG 0 mg/kg/d Losartan n=10 IG 1 mg/kg/d Losartan n=10 IG 50mg/kg/d Losartan n=10 IG 500mg/kg/d Losartan n=10 ICV Omg/kg/d Losartan n=10 ICY 1 mg/kg/d Losartan n=10 ICV 5.76?g/kg/d Clonidine n=10 RDX(Removal of the renal nerves n=10 around)ICV 4.5?g/kg/d Tempol n=10 IG 30mg/kg/d Tempol n=10IG 15mg/kg/d Hydralazine,n=10All rats are measured in blood glucose level and blood pressure values before randomization.Conducted variety of interventions modeling a week after modeling,reared six weeks in SPF levelEnvironment,then execute them and collect maternal.Implant injection pump to Intracerebroventricular:intraventricular injection using ALZET micro-osmotic pump(Model:2006)with ALZET Brain Infusion Kit 2 packages;use Digital Lab Standard TM Stereotaxic brain stereotaxic instrument,referring to"GEORGE PAXINOS&CHARLES WATSON" rat brain atlas,Bregma=0mm,midline side 1.5mm,under the skull 4.5mm.All the intervention group continued to be administered 42 days.2.Experimental data and specimen collection2.Experimental data and specimen collection1)Measurements of urine samples,weight and blood pressure,All rats caged alone in metabolic cage,for three consecutive days to collect 24h urine 3 days before execution,and after recording the total amount of urine reserve urine specimens 4ml,1600g,centrifugation 20min in 4? surrounding,and packing and save in-80? environment.Test rat blood pressure through tail artery by Non-invasive blood pressure monitor(softron BP-98A,Japan)2)Collection of rat blood fresh tissue perfused tissue.Time to reach the end of the experiment,inject 3%sodium pentobarbital by intraperitoneal injection to anesthetized rats(0.15ml/100g),followed laparotomy,collected blood through puncture aorta and quickly open the chest,use a 16#gavage needle puncture from the left ventricle to the aortic arch,and cut the right atrial appendage,use 200ml saline(include heparin 20U/ml)which was Pre-cooling in 4 ? perfusion,you can see the liver,skin,lungs and muscles quickly becomes pale,determining perfusion ends when liquid outflow from the right atrial appendage clear and colorless.Then take some brain nuclei,brain,heart,kidney,liver and other tissues thrown directly into liquid nitrogen.This step is to take fresh tissue to do prepalre for subsequent RT-PCR and WB do to prepare3)Collection of the fixed tissuesUse 200ml saline(include heparn 20U/ml)which was Pre-cooling in 4 ?quickly perfusion,and use 4%paraformaldehyde(PH 7.2)slow perfusion 1.5h,ten carefully cut the skull and take brain,cut brain along coronal into 2mm thick slices in the brain mold of rats,put into 4%paraformaldehyde solution and fixed 6h in 4 C state.After the water rinse 2h,gradient alcohol dehydration,xylene transparency,wax dipped embedded.Kidney,heart and liver are equally cut 2mm thick slices,fixed 24h in 4 C state,pure water rinse 2h,gradient alcohol dehydration,xylene transparency,wax dipped embedded,after processing cut 4um of all fixed tissue to make film and dye.4)Plasma angiotensin ?:radioactive immunoassay5)Determination of plasma norepinephrine(NE)levels:ELISA assay The blood urea nitrogen and serum creatinine testing:uses Clinical automatic biochemical analyzer6)Urinary 8-iso-prostaglandin-2 Determination:ELISA assay Urine creatinine and urea nitrogen testing:uses Clinical automatic biochemical analyzer7)The detection of blood brain barrier permeability:inject Evans blue through tail vein,measure per gram of brain tissue Evans blue content 0.5h later,namely,the concentration of brain tissue the permeability higher of rat blood brain barrier.4h urinary protein:use Coomassie brilliant blue-G250 to assay8)The detection of SON,PVN,SFO and other key brain nucleus,RAS and oxidative stress indicators:fresh brain tissue and fixed brain nuclei were collected by 2,3portion respectively use the way WB,RT-PCR and immunohistochemistry to detect the expression of RAS indicators AGT,AT1 and oxidative stress indicators NOX2,NOX49)Detect kidney RAS,oxidative stress,inflammatory index:kidneys were collected by 2,3portion respectively use the way WB,RT-PCR and immunohistochemistry to detect the expression oft RAS indictors indicators AGT,AT1 and oxidative stress indicators NOX2?NOX4 and inflammatory indicators MCP-1.Statistical analysisUse the Image-Pro Plus 7.0 software to analyse immunohistochemical Image,and use Image J software to analyse of protein expression.Data analyzed by SPSS 13.0 statistical package,and data is expressed with a mean ± sd.ANOVA are used between groups when difference was statistically significant with P<0.05 said theResultPart one Study of oxidative stress,inflammation indicators in circulation,brain RAS of non-diabetic rats(Non-DM)and diabetic rats(DM).1)Comparison of blood glucose,weight and blood pressure between Non-DM rats and DM ratsBlood glucose level of DM rats(27.4 ± 3.5mmol/1)was significantly higher than Non-DM rats(5.7± 0.43 mmol/1)(P<0.001),in terms of weight,DM rats(271.3 ±17.5g)was significantly lower than non-DM group(368.7 ± 13.4g)(P<0.001);in terms of blood pressure value,DM rats(124.2±6.6 mmHg)and non-DM rats(124.5± 4.8 mmHg)showed no significant difference(P>0.05).2)The comparison of 24h urine protein,urine albumin,blood Ang?,plasma norepinephrine(NE)levels and urinary 8-hydroxy-2-iso-prostaglandin levelsbetween Non-DM rats and DM rats3.24h urine protein value of DM rats was significantly higher than Non-DM rats(P<0.001);in terms of urine albumin value,DM rats and non-DM rats showed no significant difference(P>0.05);plasma Ang? content of DM rats(4.13± 0.25ng/ml)was significantly higher than Non-DM rats(2.92± 0.25ng/ml)(P<0.001);blood NE levels of DM group was significantly higher than Non-DM rats(P<0.00.1);DM rats urinary 8-iso-prostaglandin-2 levels significantly higher than Non-DM rats(P<0.05)3)Comparison of RAS indicators,neuron activation markers c-fos and oxidative stress indicators of rat brain key nuclei,such as,SON,SFO,PVN,etc.The expression level of RAS indicators AGT,AT1 of DM group rats brain key nervous nucleus,such as SON,SFO,PVN,etc,were significantly higher than Non-DM rats(P<0.05).the expression of neuron activation marker c-fos of DM group rats brain neuron nucleus SON,SFO,PVN and other key nucleus significantly higher than the expression of oxidative stress indicators of Non-DM rats(P<0.05)4)The comparison of Non-DM and DM rats rat back ventrolateral medulla nucleus(RVLM)trosine hydroxylase(TH)expression levels.The expression level of DM rats of tyrosine hydroxylase(TH)was significantly higher than Non-DM rats(P<0.05)5)Comparison of Non-DM rats and DM rats kidney tissue RAS,oxidative stress,inflammatory indicator.DM group RAS kidney tissues RAS indicators AGT,AT1 expression levels were significantly higher than Non-DM rats(P<0.05).the expression levels of DM rats brain and kidney tissue oxidative stress parameters NOX2,NOX4 significantly higher than Non-DM rats(P<0.05);inflammatory markers MCP-1 expression levels in renal tissue of DM group rats was significantly higher in Non-DM rats(P<0.05).6)Comparison in blood brain barrier permeability between DM rats and non-DM ratsDM rat brain barrier permeability was significantly higher than Non-DM rats(P<0.05).Part two Study of oxidative stress,inflammation indicators in circulation,brain RAS)in different groups1)Comparison of blood glucose,weight,blood pressure among different intervention rats groupBlood glucose,weight,blood pressure through statistical comparisons,there was no significant difference(P>0.05)between each intervention group.2)Comparison of urinary albumin,24h urine protein,blood Ang ? and plasma NE levels among different intervention rats groupthe statistical comparison of urinary albumin,24h urinary protein between different interventions group there was no significant difference(P>0.05).IG:50mg/kg/d Losartan group,IG:500mg/kg/d Losartan group,IG:30mg/kg/d Tempol group blood Ang ? levels and blood NE level decreased significantly compared with the other treatment group(P<0.05).Only IG:30mg/kg/d Tempol group urinary 8-iso-prostaglandin-2 level were significantly reduced than other intervention group,although other groups such as IG:50mg/kg/d Losartan group,IG:500mg/kg/d Losartan group also decreased,but it reduces level did not show statistically significant difference3)Comparison of RAS indicators,neuron activation markers c-fos and oxidative stress indicators between different intervention group rat brain SON,SFO,PVN and other key neuron nucleus between each intervention group,only IG:500mg/kg/d Losartan group,ICV:1mg/kg/d Losartan group,ICV:4.5?g/kg/d Tempol Group,IG:30mg/kg/d Tempol group of large rats brain SON,SFO,PVN and other key nucleus RAS indicators AGT,ATI expression levels were significantly reduced(P<0.05).Other intervention group were no significant differences.only in IG:500mg/kg/d Losartan group,ICV:1mg/kg/d Losartan group,ICV:4.5?g/kg/d Tempol group,IG:30mg/kg/d Tempol group Oxidative stress indicators NOX2,NOX4 decreased significantly(P<0.05)in large rats brain SON,SFO,PVN and other key nucleus,the other intervention group were no significant differences.The expression level trends of neuron activation markers c-fos is the same with RAS indicators and oxidative stress.4)Comparison of back ventrolateral medulla nucleus(RVLM)tyrosine hydroxylase(TH)expression level of different interventions groups of ratsbetween each intervention group,IG:500mg/kg/d Losartan group,ICY:lmg/kg/d Losartan group,ICY:4.5?g/kg/d Tempol Group,IG:30mg/kg/d Tempol group TH expression level of rats brain RVLM nuclei decreased significantly(P<0.05).5)Comparison of RAS indicators,oxidative stress and inflammatory indicators among different intervention groups of ratsAmong different intervention groups,,the expression level of kidney RAS indicators AGT,AT1 in IG:50mg/kg/d Losartan group,IG:500mg/kg/d Losartan group,ICV:1mg/kg/d Losartan group,ICV 5.76?g/kg/d Clonidine group,RDX group,ICV:4.5?g/kg/d Tempol group,IG:30mg/kg/d Tempol group has significantly decreased(P<0.05),but the most obvious reducing groups are IG:500mg/kg/d Losartan group and IG:30mg/kg/d Tempol group.Rats kidney tissue Oxidative stress indicators NOX2,NOX4 expression trends same with RAS indicators(P<0.05).nly in IG:50mg/kg/d Losartan group,IG:500mg/kg/d Losartan group,IG:30mg/kg/d Tempol group Inflammatory markers decreased significantly(P<0.05)among each intervention group,other group did not significantly change.6)Comparison of blood brain barrier permeability changes between different intervention groups.The statistical comparison show that the permeability of blood-brain barrier was no significant difference among each intervention group.ConclusionIn summary,this study found that peripheral circulation RAS,sympathetic excitability,the blood-brain barrier permeability of DM rats are higher than non-DM rats.The expression of RAS,oxidative stress markers in part of the central critical nucleus SON,SFO,PVN were significantly increased,The central sympathetic excitability have also been increased,After adopt various means of intervention on DM rats found that although there was no significant difference in body weight,blood glucose,blood pressure,24h proteinuria,urinary albumin concentration,blood-brain barrier permeability and other indicators,but Ang ? levels and blood NE levels has significantly decreased in IG:50mg/kg/d Losartan group,IG:500mg/kg/d Losartan group and IG:30mg/kg/d Tempol groups,only in IG:30mg/kg/d Tempol group urinary 8-iso-prostaglandin-2 had significantly decreased;Intracerebroventricular injection of Losartan,Tempol,fed large doses of Losartan and other methods can significantly reduce the expression level of RAS,oxidative stress indicators and central sympathetic excitability of central SON,SFO,PVN,etc.At the same time,study also found that the expression levels of renal tissue RAS and oxidative stress indicators significantly reduce,besides IG Omg/kg/d Losartan group,IG lmg/kg/d Losartan group and IG 15mg/kg/d Hydralazine group,but only IG:500mg/kg/d Losartan group and IG:30mg/kg/d Tempol group most obvious,only in IG:50mg/kg/d Losartan group,IG:500mg/kg/d Losartan group,IG:30mg/kg/d Tempol group inflammatory markers MCP-1 expression levels were significantly reduced.