| Objectives: Diabetic nephropathy(DN),that is, diabetic glomerulosclerosis, is one of the most common and serious chronic micovasclor complication of diabetes,and also a crucial cause of lesion and death for diabetic patients.DN is characterized by early hypertrophic glomerulus, thickening of glomerular basement membrane (GBM) and mesangial expansion, and latter accumulation of extracellular matrix (ECM) which may cause glomerulofibrosis and glomerulosclerosis. DN occurs as a result of an interaction between haemodynamic and metabolic factors,which is influenced by various factors including signaling pathways and cytokines.There is no a specific plan to prevent and cure DN presently.However, TCM is potential for its systemic treatment and personal prescription.We summarized the pathogenesis of DN is by the deficiency of qi and yin, and the occurrence of stagnant blood which result in abdominal mass finally according to the pathogenesis of DN and clinical experience for treating DN. Supplementing qi-nourishing yin -dispersing blood Stasis - dredging collateral herbs are formulated on the basis of the pathogenesis above and effective in clinical practice. Furthermore,the formula has been demonstrated by clinical and experimental study that it is renoprotective. The study aims to explore the effect of the formula on DN and the possible mechanism in vivo and vitro, in terms of oxidative stress and p38MAPK signaling pathway.Motheds:1 1 Renoprotective effect of supplementing qi-nourishing yin-dispersing blood stasis-dredging collateral herbs on diabetic rats110 male Sprague-Dauley(SD) rats weighing 250±20 g (SPF level) were carried out left-nephrectomy after making sure the rats'urine protein negative when fed for one week. Two weeks later, dividing the rats randomly into the simple nephrectomy group and the model group which were injected intraperitonally with 40mg·kg-1 streptozotocin. Blood glucose levels were measured for three times continuously(≥16.7 mmol·L-1) on the third day after streptozotocin injection to confirm the development of diabetes. Dividing the model rats randomly into 5 groups: model group, irbesartan group, TCM low dose group, TCM middle dose group and TCM high dose group. Rats in irbesartan group were orally administered with 15 mg·kg-1·d-1 irbesartan, three TCM groups with 5.29 g·kg-1·d-1,10.58 g·kg-1·d-1,21.16 g·kg-1·d-1 TCM respectively,and the left groups with the same dose of Sodium Chloride everyday for six weeks. At the end of 6th week, collecting 24h urine, killing rats, obtaining renal cortex, detecting 24h urine protein (U Pro),observing the pathomorphology change of each group and detecting the expression of Col-IV(α1) mRNA in the renal cortex by RT-PCR2 Effect of supplementing qi-nourishing yin-dispersing blood stasis-dredging collateral herbs on oxidative stress in the kidney of diabetic ratsThe method of animal model establishing and the treatment is same as above. At the end of 6th week, obtaining renal cortex samples, and detecting the content of MDA and the activity of SOD in the renal cortex by the kits.3 Effect of supplementing qi-nourishing yin-dispersing blood stasis-dredging collateral herbs on p38MAPK signaling pathway in the kidney of diabetic ratsThe method of animal model establishing and the treatment is same as above. At the end of 6th week, obtaining renal cortex samples, and detecting the expression of p38MAPK mRNA in the renal cortex by RT-PCR, and the expression of p38MAPK, p-p38MAPK, STAT3 and p-STAT3 protein by western blot.4 Effect of supplementing qi-nourishing yin-dispersing blood stasis-dredging collateral herbs blood-serum on p38MAPK signaling pathway of in rat mesangial cells cultured under high glucose and AngII4.1 Cultivation and identification of rat mesangial cells(MCs) MCs were cultured and identified by morphology and immunohistochemistry methods. MCs of 5th~7th generation were selected for the experiment.4.2 Medicamentum blood-serum preparationDividing SD rats randomly into 3 groups: TCM blood-serum group, irbesartan blood-serum group and normal blood-serum group. Both of TCM and irbesartan blood-serum groups were orally administered with 15 folds of adult dose /kg two times everyday, 3 mL for each time. After three days, obtaining the blood by the femoral artery sterilely after 1h of the last administration. The blood-serum was separated, deactivated at 56℃for 30min, filtered through 0.22um filter membrane, and reserved at -70℃.At the same time, the normal blood-serum group was orally administered with Sodium Chloride and the method of obtaining the blood-serum is same as above.4.3 Experiment group and index detectionGrouping the MCs and interfering with relative drugs after screening the TCM blood-serum concentration by MTT: control group(D-glucose 5.5mmol·L-1+5% normal blood-serum), high glucose group(D-glucose 25mmol·L-1+5% normal blood-serum), High glucose+AngII group (D-glucose 25mmol·L-1 +AngII 10-6mol·L-1+5% normal blood-serum), Irbesartan group(D-glucose 25mmol·L-1+ AngII 10-6mol·L-1+5% irbesartan blood-serum), TCM group (D-glucose 25mmol·L-1 + AngII 10-6mol·L-1+5% TCM blood-serum). Each group is added with RPMI-1640 containing FCS to make the final blood-serum concentration to be 10%. Detecting the content of Col-IV in MCs supernatant by ELISA kit, and the expression of p38MAPK, p-p38MAPK, STAT3, p-STAT3 protein within the total by western blot after culturing MCs for 48h.Results:1 Renoprotective effect of supplementing qi-nourishing yin-dispersing blood stasis-dredging collateral herbs on diabetic rats1.1 Comparation of 24h urine protein (U Pro) level in the different groupsU Pro levels of model group and treated group increased(P<0.05), compared with that of simple nephrectomy group. U Pro levels of irbesartan group, TCM low group and TCM middle dose group decreased significantly (P<0.05), compared with that of the model group. But there was no significance statistically between TCM high dose group and model group (P>0.05). There was no significance statistically among irbesartan group, TCM low group and TCM middle dose group(P>0.05).1.2 Pathomorphology observations on kidney of each groupLight microscope: In simple nephrectomy group, there were no obvious hypertrophy of renal glomerulus, thickening of GBM and the change of the MCs quantity, mesangial area and renal tubulointerstitium. In model group, there were obvious hypertrophy of renal glomerulus, thickening of GBM, expansion of mesangial matrix and vacuolar/granular degeneration of renal tubular cell. In irbesartan group and TCM treated groups, the pathomorphology changes above were improved compared with that of the model group. There was no obvious difference among the treated groups.1.3 Comparation of Col-IV(α1) mRNA expression in renal cortexThe Col-IV(α1) mRNA expression of the model group and the treated groups increased(P<0.01,P<0.05), compared with that of simple nephrectomy group.The Col-IV(α1) mRNA expression of the irbesartan group and TCM low dose group decreased (P<0.01,P<0.05), compared with that of the model group. There was no significance statistically between the irbesartan group and TCM low dose group(P>0.05). There was no significance statistically between the other treated groups and the model group(P>0.05).2 Effect of supplementing qi-nourishing yin-dispersing blood stasis-dredging collateral herbs on oxidative stress in the kidney of diabetic ratsMDA content of the model and treated groups increased and T-SOD activity levels of them decreased(P<0.05), compared with that of simple nephrectomy group. MDA content of the irbesartan group, TCM low group and TCM middle dose group decreased and T-SOD activity levels of them increased(P<0.05), compared with that of the model group. There was no significance statistically between the TCM high dose group and the model group about their MDA content and T-SOD activity level(P>0.05). There was no significance statistically among irbesartan group, TCM low group and TCM middle dose group(P>0.05).3 Effect of supplementing qi-nourishing yin-dispersing blood stasis-dredging collateral herbs on p38MAPK signaling pathway in the kidney of diabetic rats3.1 Comparation of p38MAPK mRNA expression in renal cortexThe p38MAPK mRNA expression of the model group and the treated groups increased(P<0.01,P<0.05), compared with that of simple nephrectomy group. The p38MAPK mRNA expression of the treated groups decreased(P<0.05), compared with that of the model group.Furthermore, the effect on p38MAPK mRNA expression of TCM low dose group was best among the treated groups(P<0.05). There was no significance statistically among the other treated groups(P>0.05).3.2 Comparation of p-p38 MAPK,p38MAPK,p-STAT3 and STAT3 protein expression in renal cortexThe p-p38 MAPK and p-STAT3 protein expression of the model group and the treated groups increased(P<0.01), compared with that of simple nephrectomy group. The p-p38 MAPK and p-STAT3 protein expression of the irbesartan group, TCM low dose group and TCM middle dose group decreased(P<0.01,P<0.05), compared with that of the model group. There was no significance statistically between the TCM high dose group and the model group about the the p-p38 MAPK and p-STAT3 protein expression(P>0.05). There was no significance statistically among all groups about the p38 MAPK and STAT3 protein expression(P>0.05).4 Effect of supplementing qi-nourishing yin-dispersing blood stasis-dredging collateral herbs blood-serum on p38MAPK signaling pathway of the rat mesangial cells cultured under high glucose and AngII4.1Effect of supplementing qi-nourishing yin-dispersing blood stasis-dredging collateral herbs blood-serum on the content of Col-IV in the MCs supernatant The Col-IV protein content of the other groups increased significantly(P<0.01), compared with that of the control group. The Col-IV protein content of the high glucose+AngII group increased significantly(P<0.01), compared with that of the high glucose group. The Col-IV protein content of the irbesartan blood-serum group and TCM blood-serum group decreased significantly(P<0.01), compared with that of the high glucose+AngII group. There was no significance statistically between the two treated blood-serum groups(P>0.05).4.2Effect of supplementing qi-nourishing yin-dispersing blood stasis-dredging collateral herbs blood-serum on the expression of MCs p-p38MAPK,p38MAPK,p-STAT3 and STAT3 proteinThe p-p38MAPK and p-STAT3 protein expression of the other groups increased significantly(P<0.01), compared with that of the control group. The p-p38MAPK and p-STAT3 protein expression of the high glucose+AngII group increased significantly(P<0.01), compared with that of the high glucose group. The p-p38MAPK and p-STAT3 protein expression of the irbesartan blood-serum group and TCM blood-serum group decreased significantly(P<0.01), compared with that of the high glucose+AngII group. There was no significance statistically between the two treated blood-serum groups(P>0.05). There was no significance statistically among all the groups about the p38MAPK and STAT3 protein expression(P>0.05).Conclusion:1. Supplementing qi-nourishing yin-dispersing blood stasis-dredging collateral herbs could decrease the proteinuria, extenuate the pathological lesion and lower the expression of Col-IV(α1) in order to retard the progress of DN.2. The therapeutic effect of the TCM low and middle dose groups was better than that of the TCM high dose group, which demonstrated the formula was independent on the dose with the short-term intervention.3. Supplementing qi-nourishing yin-dispersing blood stasis-dredging collateral herbs could resist the oxidative stress in the kidney of diabetic rats, inhibit the activation of the p38MAPK/STAT3 signaling pathway, which could be one mechanism of the renoprotective effect of the formula.4. Supplementing qi-nourishing yin-dispersing blood stasis-dredging collateral herbs blood-serum could inhibit the activation of the MCs p38MAPK/STAT3 signaling pathway, lower the expression of Col-IV protein and thereby extenuate the accumulation of extracellular matrix, which conformed to the results of the study in vivo, so the possible mechanism of therapeutic effect of the formula on DN was demonstrated furthermore.
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