The Clinical And Experiment Research Of Qi Supplementing, Yin Nourishing, Blood Stasis Dispersing, Collateral Dredging Herbs On Preventing And Treating The Early Diabetic Nephropathy

Objectives: Diabetic nephropathy (DN) is a part of micovasclorcomplication and also the first reason of end stage renal disease. DN ischaracterized by early hypertrophic glomerulus, accumulation of extracellularmatrix (ECM) mesangial expansion and thickening of glomerular basementmembrane, which may cause glomerulosclerosis. DN occurs as a result ofvarious factors including glycometabolic disturbance, haemodynamic change,cytokine,mediators of inflammation and so on. There is no an effectivemethod to prevent and cure DN presently. However, the Traditional ChineseMedicine is potential for its prevention and cure. We summarized thepathogenesis of DN is by the deficiency of qi and yin, and the occurrence ofstagnant blood which result in abdominal mass finally according to thepathogenesis of DN and clinical experience for treating DN. Therefore,qisupplementing,yin nourishing,blood stasis dispersing,collateral dredgingherbs(ZY)are formulated on the basis of the pathogenesis. Furthermore, theformula has been demonstrated by clinical and experimental study that it hasgood effect on the early stage of DN. The study aims to explore the effect ofthe formula compared with irbesartan on DN and the possible mechanism inclinic and experiment, in terms of oxidative stress, c-Jun N-terminal kinase(JNK) signaling pathway and caspase-3.Motheds:1A clinical study of ZY in the treatment of the early diabetic nephropathy.Seventy eight patients with DN of the â…¢stage were randomly dividedinto treatment group (39cases) and control group (39cases). The treatmentgroup received ZY with a dose,2times a day, while the control group received irbesartan150mg/d, for1year. The changes of the therapeutic efficacy,Chinese medicine syndrome scores,urine albumin excretion rate (UAER),serum creatinine (SCr), blood urine nitrogen (BUN), total cholesterol (TC),triglyceride (TG), and the occurrence of end-point events were observed afterone-year treatment.2Renoprotective effect of ZY on diabetic ratsForty-five male Sprague-Dauley (SD) rats weighing120¹50g were fedfor one week after making sure the rats' urine protein negative. The animalswere randomly divided into normal group and DM model group. The normalgroup and the DM model group were fed common diet and high fat dietrespectively. The DM model group was injected intraperitonally with30mg·kg^-1streptozotocin after twelve weeks feeding. Blood glucose levelswere measured for three times continuously (≥16.7mmol·L^-1) on the third dayafter streptozotocin injection to confirm the development of diabetes. Dividingthe model rats randomly into three groups: model group, irbesartan group andZY group. Rats in irbesartan group were orally administered with11.51mg·kg^-1·d^-1irbesartan, the ZY group with1.0g·kg^-1·d^-1, and the left groupswith the same dose of Sodium Chloride everyday for thirty two weeks. At theend of32th week, collecting24h urine, killing rats, obtaining renal cortex,detecting24h urine protein (U Pro), observing the pathomorphology change ofeach group and detecting the expression of Col-I mRNA in the renal cortex byRT-PCR.3Effect of ZY on reactive oxygen species (ROS) mediated JNK signalingpathway in the kidney of diabetic ratsThe method of animal model establishing and the treatment is same asabove. At the end of the32th week, obtaining renal cortex samples, detectingthe content of ROS in the renal cortex by flow cytometry (FCM), anddetecting the content of JNK and p-JNK in the renal cortex by Western-blotand FCM.4Effect of ZY on programmed cell death (PCD) in the kidney of diabetic ratsThe method of animal model establishing and the treatment is same as above. At the end of32th week, obtaining renal cortex samples, detecting theapoptosis rate of renal cortex cells by FCM, detecting the content of caspase-3in the renal cortex by the reverse transcription-polymerase (RT-PCR) andimmunohistochemistry (IHC), and detecting the content of caspase-3andactived caspase-3in the renal cortex by Western-blot and FCM.Results:1A clinical study of ZY in the treatment of the early diabetic nephropathy.1.1Comparison of clinical efficacy between two groupsThe total effective rate in treatment group was83.8%(31/37cases),which was obviously higher than that in control group (60.5%,23/38cases)(χ²=6.131, P<0.05).1.2Comparison of clinical grade of symptoms and signsAfter treatment the Chinese medicine syndrome scores were reducedsignificantly in the treatment group (P<0.05, P<0.01), and showed significantdifference when compared with those in the control group (P<0.05, P<0.01).1.3Comparison of UAER, SCr, BUN, TC and TG in patients of the twogroups before and after treatmentLevels of UAER, SCr, BUN, TC, and TG after treatment in treatmentgroup decreased more significantly than before treatment (P<0.05, P<0.01).The aforesaid indices were also improved in the control group after treatment.The reduction of TC and TG after treatment in the treatment group was moresignificant (P<0.05).1.4Comparison of the incidence rate of end-point events between two groupsThe incidence rate of end-point events (5.4%) of the treatment group wasslightly lower than that of the control group (10.5%), but with no statisticaldifference (P>0.05).2Renoprotective effect of ZY on diabetic rats2.1Comparison of the24h urine protein quantitation (U Pro) of the rats in thedifferent groupsU Pro levels of model group and treated group increased (P<0.05),compared with that of normal group. U Pro levels of irbesartan group and ZY group decreased significantly (P<0.05), compared with that of the modelgroup. But there was no significance statistically between irbesartan group andZY group (P>0.05).2.2Pathomorphology observations on kidney of each groupObservations under light microscope: In the normal group, there were noobvious hypertrophy of renal glomerulus, thickening of GBM and the changeof the MCs quantity. In model group, there were obvious hypertrophy of renalglomerulus, thickening of GBM, expansion of mesangial matrix andvacuolar/granular degeneration of renal tubular cell. In irbesartan group andZY groups, the pathomorphology changes above were improved comparedwith that of the model group. There was no obvious difference between twotreated groups.Observations under transmission electron microscopy: In the normalgroup, the structure of glomerular capillary basement membrane was clear andcomplete, microcirculatary endothelial cell, foot processes was normal. Inmodel group, there were obvious thickening of GBM, the fusion of the footprocesses was extensive, microcirculatary endothelial cell confluence andwindow structure disappeared. In irbesartan group and ZY groups, thepathomorphology changes above were improved compared with that of themodel group. There was no obvious difference between two treated groups.2.3Comparation of Col-I mRNA expression in renal cortexThe Col-I mRNA expression of the model group and the treated groupsincreased (P<0.01), compared with that of the normal group. The Col-I mRNAexpression of the irbesartan group and ZY group decreased (P<0.05, P<0.01),compared with that of the model group. There was no significance statisticallybetween two treated groups (P>0.05).3Effect of ZY on ROS mediated JNK signaling pathway in the kidney ofdiabetic rats3.1Expression of ROS levels in the rats' kidney assayed by FCMThe ROS expression of the model group and the treated groups increased(P<0.01), compared with that of the normal group. The ROS expression of the irbesartan group and ZY group decreased (P<0.01), compared with that of themodel group. There was no significance statistically between two treatedgroups (P>0.05).3.2Expression of JNK and p-JNK protein in the rats' kidney assayed byWestern-blot and FCMThe JNK and p-JNK protein expression of the model group and thetreated groups increased (P<0.01), compared with that of the normal group.The JNK and p-JNK protein expression of the irbesartan group and ZY groupdecreased (P<0.05, P<0.01), compared with that of the model group. Therewas no significance statistically between two treated groups (P>0.05).4Effect of ZY on programmed cell death (PCD) in the kidney of diabetic rats4.1Apoptosis rate of renal cortex cells assayed by FCMThe apoptosis rate of the model group and the treated groups increased(P<0.01), compared with that of the normal group. The apoptosis rate of theirbesartan group and ZY group decreased (P<0.01), compared with that of themodel group. There was no significance statistically between two treatedgroups (P>0.05).4.2Expression of caspase-3mRNA and protein in the rats' kidney assayed byRT-PCR and IHCThe caspase-3expression of the model group and the treated groupsincreased (P<0.01), compared with that of the normal group. The caspase-3expression of the irbesartan group and ZY group decreased (P<0.01),compared with that of the model group. There was no significance statisticallybetween two treated groups (P>0.05).4.3Expression of caspase-3and actived caspase-3protein in the rats' kidneyassayed by Western-blot and FCMThe caspase-3and actived caspase-3protein expression of the modelgroup and the treated groups increased (P<0.01), compared with that of thenormal group. The caspase-3and actived caspase-3protein expression of theirbesartan group and ZY group decreased (P<0.01), compared with that of themodel group. There was no significance statistically between two treated groups (P>0.05).Conclusion:1. Qi supplementing,yin nourishing,blood stasis dispersing,collateraldredging herbs could improve the clinical symptom and physical sign ofDN patients.2. Qi supplementing,yin nourishing,blood stasis dispersing, collateraldredging herbs could regulate lipid metabolism, decrease the UAER, SCrand BUN of DN patients which could retard the progress of DN. However,due to the limitations of follow-up time and sample size, its efficacy hasyet to be confirmed by long-term prognosis.3. Qi supplementing, yin nourishing, blood stasis dispersing, collateraldredging herbs could decrease the proteinuria, extenuate the pathologicallesion and lower the expression of Col-I in order to retard the progress ofDN.4. Qi supplementing, yin nourishing, blood stasis dispersing, collateraldredging herbs could decrease the quantity of apoptosis in the rats' kidney,resist the oxidative stress in the kidney of diabetic rats, inhibit theactivation of the JNK signaling pathway, thus decrease caspase-3andactived caspase-3expression, and then interfere in apoptosis of the rats'kidney, which may be one of the important mechanisms that the herbsprotect the renal.