| Objective: Diabetic nephropathy (DN) is one of thechronic diseases of diabetes, is the main death reason todiabetes. In case diabetic patiences have durative albuminuria,their states of illness can not reverse, then develop to uremia bitby bit, they keep life by dialyse or kedney replant, which givesheavey economy burden to family and society. So preventionsdiabetic nephropathy have become earnest attention problem innephropathy. The mechanisms in diabetic nephropathydeveloping have not completely clarified, now are consideredthat it is the result of integrate effect between the blood flowstudy and no blood flow study, are related to the interact ofRenin-Angiotensin sysetm (RAS), Calcitonin-gene-relatedpeptide (CGRP), Endothelin (ET), et. CGRP is one of thestrongest endogenesis stretching vein peptide now, while ET isone of the strongest contracting vein peptide. CGRP and EThave far-ranging biology effects. Study has showed that CGRPcan prevent the biology effects of ET, it is effectiveendogenesis prevention to ET, the balance of CGRP and ETmaitains the stabilization of blood flowing in kedney and bloodpressure. Investigation has showed that ET expresses highlyduring DN, but the risist relation of CGRP and ET operating inDN has little studied. In this experiment the expression ofCGRP and ET in diabetic nephropathy rats kedneys at 4 weeksand 8 weeks had been studied by immunohistochemistrystaining and RT-PCR, the resisting effect of CGRP and ET hadbeen investigated during DN developing. The pretectingmechanisms of Yimile, Losartan, Lotensin were paralleled,analyzed to diabetic nephropathy.Methods1 Materials: 70 health male Wister rats whose weigh were120-150g were Chose and randomly divided into Normalgroup(group A, n=14), diabetes nsphropathy group(group B,n=14), Yimile group(group C, n=14), Losartan group(group D,n=14), Lotensin group(group E, n=14). Diabetic model making:STZ which was buied from Sigma company, dissolved into0.1mmol/L citron acid cushion liquid(PH=4.5) was injectedinto the rats abdomen of B,C,D,E group's by 65mg/kg once.And equal quantity citron acid cushion liquid was injected intoA group's rats abdomen. After 72 hours blood got from trailvein was measured to 16.65 mmol/L.Then diabetic model wasmade successfully. The second day after model made. C groupwere injected by Yimile (produced by the First PharmacyCompany in Cheng Du) 50mg/kg.d, D group were givenLosartan(produced by Merck Company)by 30mg/kg.d. Egroup were given Lotensin(produced by Novartis Company)by 10mg/kg.d. A group and B group were given equal quantityinjection water and injected with injection water everyday. Allrats were given sufficient food and water, no insulin and theother dropping sugar drugs during experiment. Rats were killedat the end of 4 weeks and 8 weeks, rats urine in 24 hours werecollected by metabolize cage before killed, then rats werenarcosised by 3% hydration chlorin aldehyde injected intoabdomen at that day, spiled into right neck chief artery,measured average artery pression by XY-1 blood pressionmeasuring instrument, collected blood Then the left kidneywhich had sufficiently been watered by NS were weighed,fixed by 10% neutral formalin, embedded by olefin, the rightkidneys were saved by nitrogen.2 biochemistry index: urine in 24h (compare color way),blood sugar, Scr, Bun.3 Immunohistochemistry staining: used to detect theexpression and distribution of ET and CGRP in every groups'kidneys. The procedures were according to the Test Kitintroduction. The paraffin sections were incubated withployclonal antibody of anti-ET and anti-CGRP(1:200 dilution).4 RT-PCR: Used TRIzol reagent to retort total RNA ofevery group's kidney tissue, taken into RT system at 42℃for3 hours; then brought primer of CGRP and ET into PCRsystem, got 30-40 PCR cycles.Results1 biochemistry results: At the end of 4 weeks and 8 weeksThe weighs of A group's rats were higher than C,D,E group'sand B group's (P<0.01), C,D,E group's were higher than Bgroup's (P<0.05); blood pression, kidney/weigh and TP/24hwere lower than C,D,E group's and B group's(P<0.01), C,D,E group's were lower than B group's (P<0.05). There wasnot statistics meaning between 4 weeks and 8 weeks (P>0.05).SCR and BUN weren't different between each group at 4weeks, at 8 weeks SCR and BUN of A group's rats were lowerthan C,D,E group's and B group's(P<0.01), C,D,E group'swere lower than B group's (P<0.05).2 The results of immunohistochemistry staining: At the endof 4 weeks and 8 weeks, the positive particles of CGRP inkidney of A group's were much more than those of C,D,Egroup's and B group's(P<0.01), C,D,E group's were morethan B group's (P<0.05); the positive particles of ET in kidneyof A group's were less more than those of C,D,E group's andB group's(P<0.01), C,D,E group's were less than B group's(P<0.05). There was not statistics meaning between 4 weeksand 8 weeks (P>0.05). CGRP and ET were mainly distributedat the endothelium cells and mesangial cells of neghritiumglobule, epithelium cells of nephridium tubule.3 RT-PCR:At the end of 4 weeks and 8 weeks, CGRPmRNA in the kidneys of A group's expressed higher than C,D,E group's and B group's(P<0.01), C,D,E group's expressedhigher than B group's (P<0.05); ET mRNA of A group'sexpressed lower than C,D,E group's and B group's(P<0.01),C,D,E group's expressed lower than B group's (P<0.05).There was statistics meaning between 4 weeks and 8 weeks... |
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