The Effect Of TNF-α And The Prevention And Cure Mechanism Of Traditional Chinese Medicine On Kidney Injury Of Diabetic Rat

Diabetic Nephropathy(DN) is one of the diabetic chroniccomplications. DN is very harmful and it is the main cause ofdeath and disfunction of diabetes patient. The basicpathological alteration are proliferation of mesangial cell andincrease of extracellular matrix(ECM). Up to now, detailedonsetting mechanism of DN isn't not clear. In recent years, therelationship between cytokines and DN is paid more attentionto, especially the TNF-α.Cytokine is a low-molecular weight polypeptide, whichhas high activity and many functions. It is produced byactivated immunocytes or relatied cells, which can regulate cellgrowth and function. It has been verified by clinical and animalexperiment that the relationship between cytokine and DN isvery close. TNF is one of an important inflammation factors,which can be divided into two types, TNF-αand TNF-β.TNF-βis mainly produced by T lymph cell, while TNF-αcan be produced by many cells in body, such asmonocyte/macrophage, Mesangial cell etc.Among them,activated monocyte/macrophage are the main source of TNF-α. TNF-αhas many biological effect and take part in manydisease's occurrence and development. Many cytokines, suchas IL-1β,TNF-α,IFN-γcan be released due to advancedglycation endoproducts (AGEs), abnormal oxidative productsor the high-infiltrating state of DM. TNF-αincreasesobviously among them. Release of these cytokines makeinducible nitric oxide synthase(iNOS) have more activity andaccout for more nitric oxide(NO). Excessive NO plays a veryimportant role in the pathological and physiological process ofDN. Whether the NO is the immediate cause of DN or not?Whether there has more important agent to play key role or not?In recent years, the study of peroxynitrite(ONOO-) give us newenlightenment.NO (nitric oxide) and O₂^。(superoxide) can react quicklyto produce ONOO-. L-Arg can be catalyzed by NOS to produceNO. And NOS can be classified into three kinds, includingnNOS (neuronal nitric oxide synthase), eNOS(endothelialnitric oxide synthase) and iNOS(inducible nitric oxidesynthase). nNOS and eNOS called as cNOS can act undernormal condition, but iNOS seldom act. iNOS can be inducedby many cytokines, such as IL-1β, TNF-αand endotoxin. Ifit is induced, its activity can be kept for 20h. And theconcentration of the iNOS will be the 1000 times of theconcentration synthesized by cNOS. iNOS is mainlyresponsible for excessive NO. In body it also produces O₂^。. Theoxidizing activity of ONOO-is higher than NO and O₂^。. Theprotonation product of ONOO-under physical condition(T 37℃, PH 7.4) can be changed to conjugate acid ONOOH.Especially energy-rich type of ONOOH (ONOOH。) and otherderivations of ONOO-can oxidize the sulfydryl of protein,nitrify the residue of tyrosine, stir the peroxidation of lipid,destroy the structure of mitochondrion and destruct DNA.The harmful effect of ONOO-on lung, cardiovascularsystemand nervous system is known at present. Recent foreigninvestigations showed that ONOO-may play an important rolein the occurrence and development of DN. Whether TNF-αreacts through iNOS, ONOO-—oxidative stress pathwayduring kidney damage progress of diabetic rat or not?Nowadays, medicines which can resist TNF-αactivity toprevent and cure DN mainly include pentoxifylline,aminoguanidin(AG), troglitazone etc. With the development oftraditional Chinese medicine, treating DN with traditionalChinese medicine will have wide prospect. Traditional medicalscience considered that yin deficiency is the essence of thirstydiseases, yin deficiency causing thirst has become the mainpathologic basis of most doctors arguing and treating thirstydiseases.And there is a saying that 'thirst must lead tocongestion, congestion can lead to thirst'. This disease belongsto substance deficiency but appearance plenty, deficiencymixes plenty, this disease is also based on deficiency of yin andqi, while the blocking of arteries and veins is its appearance.With above treating principle, the Promoting Blood FlowNourishing yin Recipe(PBFNR) is adopted to prevent and cureDN in order to treat both appearance and substance.With streptozotocin(STZ), six months Sprague Dawley(SD) male rats are used to duplicate DM model, the change ofkidney morphology is observed by HE stain,PASM stain, HE+PASM stain and transmission electron microscopy ; the contentchange of TNF-αin rat plasm is detected withradio-immunity; Other biochemical indexs are detected byroutine method. To investigat the relationship betweenpathogenesis of DN, preventing and curing mechanism ofPBFNR and the change of TNF-α.The morphologic change of kidney is observed by tissuemorphology; the expression of kidney iNOS mRNA is assay byRT-PCR; the content of kidney iNOS protein is detected byimmunohistochemical stain. The relationship of DNpathogenesis , PBFNR preventing and curing mechanism withthe change of iNOS mRNA expression and iNOS protein isinvestigated.The expression of kidney ONOO-is assay byimmunohistochemical stain. The role of ONOO-in DNpathogenesis and preventing and curing effect of PBFNR areinvestigated. Concrete investigation as follows:Objective:To study the relationship between the changeof TNF-αcontent and DN from content change level of TNF-α. Methods: 37 six-month SD male rats are divided into fourgroups randomly, normal control group(the number is eight),aminoguanidine(AG) group(eight), the traditional Chinesemedicine group(twelve) and pathology group (nine). The laterthree groups are injected at one time into abdominal with STZsolution (40mg/kg weight, dissolved with 0.1mol/L sodiumcitrate/citric acid buffer solution. PH=4.4 fresh disposal, withconcentration of 10mg/ml), and make into diabetic modal(blood sugar higher than 13mmol/L, urine sugar +++ andabove). Every group is fed by normal feed, normal controlgroup and pathology group drink freely. The traditionalChinese medicine group is fed medicine with the equivalentdosage converted according to the human and the animal'ssurface areas. Every rat is perfused with 3ml traditionalmedicine (including origin drug 1g/ml) each day. AG group isperfused with 3‰diluted AG solution. For thirteen weeksunder 20～25℃after injection, urine of 24 hours is collectedwith metabolic cage, blood sugar, urine sugar and body weightare detected respectively. After thirteen weeks, the kidney'smorphologic change is observed by histochemisty, the changeof TNF-αcontent is detected meanwhile.Results: Compared with pathology model group, in AGgroup, kidney weight/body weight is decreased (p<0.05),creatinine clearance rate(CCr) is increased (p<0.05), blood ureanitrogen is decreased (p<0.01), urine protein of 24 hours isdecreased (p<0.05), superoxide dismutase (SOD) of renalcortex has no significant difference(p>0.05); malondialdehyde(MDA) of renal cortex is decreased (p<0.05); In PBFNR group,kidney weight/body weight is decreased (p<0.05), CCr isincreased (p<0.01), blood urea nitrogen is decreased (p<0.01),urine protein of 24 hours is decreased (p<0.001), superoxidedismutase (SOD) of renal cortex has no significantdifference(p>0.05), malondialdehyde (MDA) of renal cortex isdecreased (p<0.01); The results of HE, PASM and HE+PASMstaining show that in pathology model group, the volum ofrenal glomerulus is enlarged, renal glomerulus appearsleaf-divided, mesangial cell is proliferative, mesangial area iswider, the renal cell is swelling; Electron microscope show thatin pathology model group, basement membrane of renalglomerulus is thickened homogeneously, mesangial base isincreased, foot process is confluent, meanwhile, plasm TNF-αis increased. In PBFNR group and AG group, kidneymorphology is improved significantly, plasm TNF-αisdecreased.Summary:1.The increase of TNF-αcoincide with kidney morphologic pathologic and kidney function decline, TNF-αmay be an important factor for causing DN2.PBFNR can ameliorate kidney morphologic change and biochemistry index of kidneyfunction3.The preventing and curing effect of PBFNR have relationship with the decrease of TNF- αObjective:To investigate the relationship between iNOSand DN from transcription leveland protein level of iNOS.Methods: Duplication of animal pathology model is samewith above. The rats brood for thirteen weeks are put to deathby femoral artery blood letting after fast twelve hours. Bothkidneys of animal are immediately taken out of abdominalcavity, Three animal's left kidneys in each group are taken. Putpart of them into 4% citromint fixative solution prepared forlight microscope observing, together with the detection ofiNOS'immunohistochemistry. Cut rest of left kidney cortexinto 1×1×1mm, put them into 4% glutaral fixative solutionprepared for electron microscope observing. Immediately putrest of left kidney into liquid nitrogen to preserve and extracttotal RNA using AGPC one-step method. 2μg of total RNA isused for RT-PCR. Each system has two pairs of primer. Inanalysis of result of RT-PCR product electrophoresis, theobjective gene's relative expression is calculated by usingdensity to represent expression. Objective gene's relativeexpression: Objective gene's density/β-actin gene's density.Results: The expression of iNOS mRNA: in normalcontrol group, the expression of iNOS mRNA is none or can'tbe detected. In pathology group, the expression of iNOSmRNA is high; but in PBFNR group and AG group, theexpressions of iNOS mRNA are both significantly lower thanthat in pathology group (P<0.001). There is no significantdifference in PBFNR group and AG group.The immunohistochemistry detection of DM rat's kidneydisplay that the staining of iNOS in pathology group are allstrong orange positive; in normal group, expression of iNOS isnot obvious, cells are stained bice; The staining changes ofiNOS in PBFNR group and AG group are between above twogroups. With the heightening of iNOS, the function of kidney isdeclining and the morphology of kidney is regressive.Summary:1.The abnormal expression of iNOS mRNA and iNOS protein in DM rat kidney caused by TNF-αmay be one of an important pathogenesis of DN2.PBFNR can ameliorate kidney morphologic change and biochemistry index of kidney function,at the same time,it can also decrease the abnormal expression of iNOS mRNA and iNOS protein caused by TNF-α3.Inhibiting the abnormal expression of iNOS mRNA and iNOS protein in kidney may be animportant pathway of PBFNR to prevent and cure DNObjective: To investigate the relationship betweenONOO-and DN from oxidative stress level caused by ONOO-.Methods: Duplication of animal pathology model,materials getting and making section are all same with above.The immunohistochemistry detection of NT is performed.Results: The staining of NT in pathology group are allstrong orange positive, which is coincided with the highpositive staining of iNOS; In normal group, expression of NTis not obvious, cells are stained bice; The staining of ONOO-inPBFNR group and AG group are between above two groups.With the increasing of ONOO-, the function of kidney isdeclining and the morphology of kidney is regressive. PBFNRand AG both play important role in improving function ofkidney and increasing body weight. The results indicate thathigh expression of ONOO-could involve in the occurrence ofDN. PBFNR and AG both can inhibit the activity andexpression of iNOS, decrease the production of ONOO-, whichmay be one of the PBFNR and AG's prevention and curemechanisms. Decreasing the expression of iNOS and theproduction of ONOO-can provide a new way to prevent and...