The Effect Of Shen Kang Pills On The Kidney And CD2AP ZO-1 Expressions In Diabetic Nephropathy Rat

BACKROUND:As we all known,the Diabetic mellitus(DM) has been an epidemic disease of the world in 21st.lt has been regarded as the third chronic and noninfectious disease which seriously threaten the humanrace after the Tumor and Vasculopathy.The features of high-death,high-mutilation and high-cost have been to the common problems about the health which many world countries face.The serious situation of DM in China has even made each one of us into contemplation.On the newly famous MED periodical of the internation<NEW EGLAND MED JOUR>,an investigative acticle named "the case rate of DM in chinese"has announced the result of the recent investigation:the number of DM in China has been up to 92 millions which ranked the 1st in the world.The speed of increasement shocks every one of us.The Diabetic nephropathy(DN) is the most frequent and severious general-mirovascular complications. DN is the capital factor of the dialyse in ESRD of the West,while is not so as in our country,it increases at highly speed every year.DN is a serious threaten to humanrace in subsequent-disease of renal glomerutus because of the most refractoriness,fast-developmence,bad prognosis,and high costs. So,to make clearly of the mechanism and the way to cure are the capital problems at present.The previous study views the main pathological treats of DN are accumulation of the ectocytic base material,the widen mesenterium,the thickening basal lamina,glomerular sclerosis and the fibrosis of renal interstitium.But these can only explain 30%-50% alteration of urine protein's excretory rate and glomerular filteration rate(GFR).The pathogentic mechanism is still indefinite completely. Resent years, the study has been the hot spot, that is the glomerular barrier constitution and functional change,especialy the effect of podocyte which is the component of the barrier in DN. At present,the abnormal change of podocytes is the main pathologic change in DN,with its related protein.Slitdiaphragm(SD) is the main component of the filteration barrier,it works as "static molecular sieve",and it is a functional complex with highly"dynamic".SD participates in the imperative signals'production and transmission of sustaining the podocytes' constitution and curpusular frame.CD2AP(CD2-associated protein)is the main molecule of its signal'transmission,which remains as a protein-complex with podocin and nephrin.Rats with CD2AP gene knocked out could be with the disease of congenital nephritis syndrome.Studies have discovered that it may combine with actin,make nephrine anchorage to the frame of actin cells. ZO-1 (zonula occludens-1) is one of connected proteins,which can make the SD related protein connect to the molecular frame,so it plays a significant role in maintaining the integrity of podocyte.A recent study discovers that it can combine with NEPH1 (podocites'component)which is one of SD,and induces the signal'transmission.While whether CD2AP as the component of protein complex has relationship with ZO-1 by nephrine(NEPHl'isogeneic molecule),or the alteration of ZO-1 and the relationship with the decreasement of podocytes are still unknown.At present,the western medicine treats DN mainly with controlling the high-blood glucose,decreaseing urine protein,controlling HBP,low protein food and retrieve the disorder of fat metabolism.Recently,a study reports that ACEI and Ang II can decrease the proteinuria,protect the kidney,but they still can't stop the development of DN. The TCM has better effect on that while it lacks an unifided typing of clinic and quantitive denom,myri-herb medicine,great changement of the hebal,unifided effective denom,all of which confine its development.DN lacks effective medicine and there's no medicine can stop completely the DN.The TCM has no reports on CD2AP or ZO-1.So,to seek a way of treating DN and the relationship with CD2AP and ZO-1 is of great importance.Chart I the Protective effect of Shenkangwan on the kidney of the DN rat and that is on CD2AP and ZO-1OBJECTIVE:To observe the protection of Shen Kang Pills on rats'kidney of DN,and the influences on CD2AP and ZO-1 Expressions.METHODS:1. The sample's formation of DN:Firstly,injecking STZ with 55mg/kg into rat'left abdominal. After feeding two weeks,measuring the BG,UPD,if the BG>16.6mmol/L,UPD>150%PUPD,proteinurie>30mg/kg.d,the sample of DN sets up.2. Grouping and Administration:Weighing DN rats and making 4 groups:control group(CG),Micardis group(MG),Shenkang group(SG)and normal group(NG)in addition.Drenching the corresponding suspension respectively 8 weeks at once every day.All rats drink unrestrictly without the drug of degrading BG.3. Observation and Detection:①Effects on the general conditions:activity,pelage, ingestion,drinking,urination and defecation,the body weight(BW).②Effects on the kidney weight and relative weight.③Effects on renal function,blood fat,insulin,glucagon,Ucr,BG,UG,GH,proteinurine in 24h.④Effects on the rats'pathology.4. Oberveing the distributions of CD2AP and ZO-1 on normal and DN rats with immunohistochemical method.RESULTS:1.46 rats were used to established the DM models and the rate of successful models was 78.9%(30/38).And the:death rate of DM model rats was 4.67%(2/30).2.During the treating eight weeks,The model rats blood glucose were all over 16.7mmol·L-1,the uric glucose positive experiment were exceeded+++.And they appeared the symptom of the DM such as the quantum of drinking, eating and urinating increased.3.Compare with the normal rats,their following indexes including GHb,24hUpro, Scr, BUN, TC, TG, LDL-c were increased distinctly (P<0.01);4.The HDL-c is decreased distinctly (P<0.01).5.Their kidney weight and relative kidney weight increased distinctly and came forth about the 3 stages renal pathological lesions according to the stages of Mogensen.6. In NG,CD2AP mainly expressed in the podocytes of renal glomerulus,fewly in the tubules and the collecting duct.Comparing with the NG,CD2AP expressed fewer in the CG,while it recovered in AG.Between them,the SG had the highest CD2AP.The ZO-1, with the punctiform, distributes mainly along the capillary loop of renal glomerulus in normal kidneys.It expressed lower apparently in CG,AG's higher,while the SG was the highest.Conclusion:Shenkangwan could treat DN rats and had a certain protective effect on the kidney of DN rats.and it also can increase CD2AP and ZO-1,stop them from changing places.Chart II The effect of Shenkangwan on CD2AP in the rat podocites controlled by high glucoseOBJECTIVE:To explore the effect of the Shenkangwan on CD2AP in the rat podocites controlled by high glucose.METHODS:1.Making the medicated blood serum:Drenching suspension of Shen Kang Pills(large dose,mean dose,low dose)and Micardis respectively with rated concentration,at the same time,drenching the equal volume NS.Then the respective medicated blood serum formed.2.Nourishing and teaming the PC,testing their activities.3.We detected the protein expression of CD2AP with the method of immune histochemistry, and observed the expression of CD2AP mRNA in the podocyte of the rats in every group with the method of the quantitative real-time PCR.4.Testing the cotent of the CD2AP with Western Blot.RESULTS:1.In 48h,the mean dose of Shenkang pills'has the highest activity.2.Comparing with others,the CD2AP's expression of Shenkang pills is the higest,nearly to the normal3.We detected that CD2AP's protein has higher expression in Shenkang and Micards than others.Conclusion:Shenkang pills could ecourage the expression of CD2APmRNA and its protein.ChartⅢThe effect of Shenkangwan on ZO-1 in the rat podocites controlled by high glucoseOBJECTIVE:To explore the effect of the Shenkangwan on ZO-1 in the rat podocites controlled by high glucose.METHODS:the same with aboveRESULTS:1.Comparing with others,the ZO-l's expression of Shenkang pills is the higest,nearly to the normal2.We detected that ZO-l's protein has higher expression in Shenkang and Micards than others.Conclusion:Shenkang pills could ecourage the expression of ZO-1mRNA and its protein.Conclusions of the whole paper:We carried out the experiment on animal and in vitro proved again that Shenkangwan could treat DN and had a certain protective effect on the kidney of DN. Its therapeutic mechanism was related to increase the content of CD2AP and ZO-1, and stop them from changing places, all of which provided the molecular and genie therapeutic mechanism and laboratorial evidence for its clinic treatment on DN.