| Purpose:Through observing the general effect on pharmacodynamic indexs, pathological changes of rat kidney and mechanism of drug action indexs changes of Yiqi Jiedu Huoluo Chinese Decoction on the early diabetic nephropathy rats induced by high-fat and high-sucrose diet combine injection of STZ, to investigate the prevention effects of Yiqi Jiedu Chinese Decoction on the early diabetic nephropathy and its possible molecular mechanism on the ERK1/2 signal pathway.Material and method:Total healthy 90 Wistar rats (SPF), half males and half females, Weight (280±20) g. After 1 week adaptive fed, divided randomly into normal group 10, and control group 80, according to the rats weight(both half males and half females). The normal group was fed with common diet, the control group were fed with high-fat and high-sucrose diet.1 week later, duplicated diabetes model by injecting STZ (42 mg/kg. bw) in the abdomen in the condition of fasting 12 hours, while those of normal control group were injected citric acid of the same amount. After 72 hours, measure the rats’ fasting blood glucose, and ≥16.7mmol/L was identified as DM mode1.8 rat was deleted.72 DM model rats were divided randomly into model group 16, western medicine group14, traditional Chinese medicine prevention group14, high dose group of traditional Chinese medicine14 and low dose group of traditional Chinese medicine14, according to the blood glucose level. All the above 5 groups were fed with high-fat and high-sucrose diet, drunk water freely. After 2 weeks, Early DN model were identificated according the related document by measuring the microalbuminuria levels. Yiqi Jiedu Huoluo Chinese Decoction contains:sealwort(40g), Astragalus membranaceus(40g), Coptis chinensis(6g), Polygonum cuspidatum(30g),Eupatorium(20g), Leech powder (6g). The western medicine group was givenBenazepril (10mg/pill). The traditional Chinese medicine prevention group was fed traditional Chinese medicine immediately when DM model was established, daily volume was computed by 6.3 times of the equivalent dose of rat-human body (g/kg). The western medicine group, high dose group of traditional Chinese medicine and low dose group of traditional Chinese medicine were intervened when DN model was succeed. Give the same daily volume amount of 6.3 times of the equivalent dose of rat-human body (g/kg) to western medicine group and low dose group of traditional Chinese medicine. The daily volume of high dose group of traditional Chinese medicine was 3 times of the low dose group of traditional chinese medicine. The normal group and the model group were given saline of the same amount (lOmL/kg).1 times a day,10 weeks of prevent group,8 weeks of the other groups, and the same volume of 10 mL/kg. During the experiment, all groups drunk and diet freely, no else medicines or insulin were used for lowering blood glucose. In the end, weighed and collected rats’urine to observe urinary albumin, after anesthetization, drawn blood from abdominal aortic puncture to measure blood glucose, glycosylated hemoglobin, serum β2 microglobulin, BUN, Scr, blood viscosity and FIB. Observed the pathological changes of rats’kidney tissue by light microscopy using HE and PAS staining. Ang Ⅱ and PGE2 were measured by ELISA. Observed the CTGF mRNA by RT-PCR, and protein expressions of CTGF, ERK1/2, p-ERK1/2 by Western Blotting.Results:1. The general condition of ratsThe normal group rats were in a good condition during the experiment, no death, shiny coat, normal dieting and normal defecation. There is a obvious symptom of diabetes, and some complication such as lose hair, tail and ophthalmopathy in model group. Symptoms of all the therapy groups were relieved. Total 8 rats were died during the experiment.2. The comparision of pharmacodynamic indexs2.1 The comparison of rats’weight in each groupThere was no significant statistics difference between the groups weight when DN model was succeed (p>0.05).At the end of the experiment, compared with normal group, the weight of rats in the model group decreased obviously, and there was significant statistics difference between the two groups (p<0.01); Compared with model group, the weight of rats in the treatment groups increased obviously, and the weight of rats’in the western medicine group was the highest, with the statistics difference between the model groups (p<0.05), but there was no statistics difference between in the other treatment groups and the model group (p> 0.05). Compared with traditional Chinese medicine prevention group, there was no statistic difference between with the other treatment groups (p >0.05). The rusults suggest that the experimental drugs can increase the weight of the DN rats.2.2The comparison of rats’fasting glucose in each groupWhen DN model was succeed, compared with normal group, the FBG of rats in the model group increased obviously, and there was significant statistics difference between the two groups (p<0.01).but there was no statistic difference between the treatment groups and model group (p>0.05).After the experiment, compared with normal group, the FBG of rats in the model group increased obviously, and there was significant statistics difference between the two groups (p<0.01). Compared with model group, the FBG of rats in the treatment groups decreased obviously, with significant statistics difference (p<0.01), and the traditional chinese medicine prevention group was the lowest. Compared with traditional chinese medicine prevention group, the FBG of the western medicine group and the low dose group of traditional chinese medicine were higher, with significant statistics difference (p<0.01), and at the same time, the high dose group of traditional chinese medicine was little higher, with statistics difference (p<0.05). The results suggest that the experimental drugs can reduce the DN rats’ FBG, and the traditional Chinese medicine prevention group was the best.2.3The comparison of rats’ HbA1c and β2-MG in each groupCompared with normal group, the HbAlc of rats’ in the model group increased obviously, and there was significant statistics difference between the two groups (p<0.01); Compared with model group, the HbAlc of rats’ in the treatment groups decreased obviously, with significant statistics difference (p<0.01);The HbAlc of rats’ in the traditional chinese medicine prevention group was the lowest,but with no statistics difference compared with the other treatment groups (p>0.05). The rusults suggest that the experimental drugs can reduce the DN rats’HbAlc, and the prevention group was the best.Compared with normal group, the β2-MG of rats’ in the model group increased obviously, and there was significant statistics difference between the two groups (p<0.01); Compared with model group, the β2-MG of rats’ in the treatment groups decreased obviously, with significant statistics difference (p<0.01); There was no statistics difference between treatment groups (p> 0.05).The results suggest that the experimental drugs can reduce the DN rats’ β2-MG.2.4The comparison of rats’ BUN, Scr in each groupCompared with normal group, the BUN of rats’ in the model group increased obviously, and there was significant statistics difference between the two groups (p<0.01); Compared with model group, the BUN of rats’in the treatment groups decreased obviously, with significant statistics difference (p<0.01); There was no statistics difference between treatment groups in BUN (p>0.05). There was statistic difference between the low dose group and the normal group (p<0.05), but no statistic difference between the other treatment group and the normal group (p >0.05).There was no statistics difference between all groups of Scr (p>0.05)2.5The comparison of rats’mALB in each groupWhen DN model was succeed, compared with the normal group, the mALB of the other groups were obviously higher, with significant statistic difference (p<0.01), But there was no statistic difference between the other groups (p>0.05).10 weeks later, compared with normal group, the mALB of rats’in the model group increased obviously, and there was significant statistics difference between the two groups (p<0.01);Compared with model group, the mALB of rats’in the treatment groups decreased obviously, with significant statistics difference (p<0.01); The mALB of rats’in the traditional Chinese medicine prevention group was the lowest, with significant statistics difference compared with the other treatment groups (p<0.01).The results suggest that the experimental drugs can reduce the DN rats’mALB, and the prevention group was best.3. The pathological changes of kidney of rats in each groupHE staining:There was no pathological changes in the normal group, and the structure of glomerular, renal tubule, GBM were intact;In the model group,it was shown that glomerular sclerosis, mesangial cell and mesangial matrix proliferation, glomus atrophy, wall uneven, with varying degrees of swelling, lumen size,portions of the tubular epithelial vacuolation degenerated, and renal tubular withered. After therapy, the pathological lesions have different degrees improved compared with model group.PAS staining:The modality and structure of glomerular and renal tubule were intact, at the same time, no PAS staining was saw in the normal group. The model group take on glomerular diffuse lesions, individual glomerular waste, glomerulus internal occasional nodules, renal arteriole hyaline degeneration, mesangial matrix increased obviously, glomerular basement membrane thickening, a widespread PAS staining in the ministry of centrilobular. After therapy, the pathological lesions have different degrees improved compared with model group, and the changes of traditional Chinese medicine prevention group were relatively better, which showed no changed in renal tubule and GBM, and mild increase in mesangial matrix.In the western medicine group, renal tubular atrophy, the mesangial matrix increased obviously, and glomerular diffuse sclerosis. In the high dose group of traditional Chinese medicine, no changed were happened in the GBM or renal tubule, but mesangial matrix and mesangial cells increased. In the low dose group of traditional Chinese medicine, GBM thicken, mesangial matrix increased and segmental glomerulosclerosis were shown.4. The comparision of of rats’pathological mechanism indexs in each group4.1 The comparision of rats’blood viscosity in each groupCompared with normal group, the blood viscosity of rats’in the model group increased obviously, and there was significant statistics difference between the two groups (p<0.01); Compared with model group, the blood viscosity of rats’in the treatment groups decreased obviously, with significant statistics difference (p<0.01); The blood viscosity of rats’in the traditional Chinese medicine prevention group was the lowest. Compared with the traditional Chinese medicine prevention group, the whole blood viscosity in high shear situation of western medicine group and the traditional Chinese medicine low dose group increased, with significant statistic difference (p<0.01), the high dose group of traditional Chinese medicine with no statistic difference (p>0.05). The whole blood viscosity in medium shear situation of western medicine group and the high dose group of traditional chinese medicine increased than the prevention group with no statistic difference (p>0.05), the low dose group of traditional chinese medicine increased than the traditional chinese medicine prevention group with statistic difference (p< 0.05). Compared with the traditional chinese medicine prevention group, the whole blood viscosity in low shear situation of western medicine group and the low dose group of traditional chinese medicine increased with significant statistic difference (p<0.01), and the same increased in high dose group of traditional chinese medicine with statistic difference (p<0.05). The results suggest that the experimental drugs can reduce the DN rats’blood viscosity.4.2The comparision of rats’FIB in each groupCompared with normal group, the FIB of rats’in the model group increased obviously, and there was significant statistics difference between the two groups (p<0.01);Compared with model group, the FIB of rats’in the treatment groups decreased obviously, with significant statistics difference (p<0.01);The FIB of rats’in the traditional chinese medicine prevention group was the lowest. Compared with the traditional chinese medicine prevention group, FIB of the western medicine group increased, with statistic difference (p<0.05), the high dose group of traditional chinese medicine increased,but with no statistic difference (p>0.05), and the low dose group of traditional chinese medicine increased obviously, with significant statistic difference (p <0.01).The results suggest that the experimental drugs can reduce the DN rats’ FIB, and the prevention group was the best.4.3 The comparision of rats’AngⅡ in each groupCompared with normal group, the Ang Ⅱ of rats in the model group increased obviously, and there was significant statistics difference between the two groups (p<0.01); Compared with model group, the Ang Ⅱ of rats in the treatment groups decreased obviously, with significant statistics difference (p<0.01);Compared with traditional chinese medicine prevention group, Ang Ⅱ of the western medicine group and the high dose group of traditional chinese medicine in rat plasma increased, decreased in the low dose group of traditional Chinese medicine, but the same no statistic difference (p>0.05). Compared with traditional Chinese medicine prevention group, Ang Ⅱ of the western medicine group and high dose group of traditional chinese medicine in rat kidney increased, with significant statistic difference (p<0.01), in the low dose group of traditional chinese medicine increase,but with no statistic difference (p>0.05). The results suggest that the experimental drugs can reduce the DN rats’ AngⅡ.4.4 The comparision of rats’PGE2 in each groupCompared with normal group, the PGE2 of rats in the model group increased obviously, and there was significant statistics difference between the two groups (p<0.01); Compared with model group, the PGE2 of rats in the treatment groups decreased obviously, with significant statistics difference (p<0.01);Compared with traditional chinese medicine prevention group, PGE2 of the western medicine group and high dose group of traditional chinese medicine in rat plasma increased, and decreased in the low dose group of traditional chinese medicine, but all with no statistic difference (p>0.05). Compared with traditional chinese medicine prevention group, PGE2 in the western medicine group and high dose group of traditional chinese medicine of rat kidney increased, with significant statistic difference (p<0.01), but decreased in the low dose group of traditional chinese medicine with no statistic difference (p >0.05). The results suggest that the experimental drugs can reduce the DN rats’PGE2.4.5The comparision of rats’CTGF mRNA and protein expression in each groupCTGF mRNA is a little expression in the normal group. Compared with normal group, the CTGF mRNA of rats in the model group increased obviously, and there was significant statistics difference between the two groups (p<0.01); Compared with model group, the CTGF mRNA of rats in the treatment groups decreased obviously, with significant statistics difference (p <0.01);and the traditional Chinese medicine prevention group was the lowest. Compared with traditional chinese medicine prevention group, CTGF mRNA of the other treatment groups increased with significant statistic difference (p<0.01)CTGF protein is a little expression in the normal group. Compared with normal group, the CTGF protein of rats in the model group increased obviously, and there was significant statistics difference between the two groups (p<0.01); Compared with model group, the CTGF protein of rats in the treatment groups decreased obviously, with significant statistics difference (p<0.01);and the traditional chinese medicine prevention group was the lowest. Compared with the traditional chinese medicine prevention group, CTGF protein of the other treatment groups increased with significant statistic difference (p<0.01)The results suggest that the experimental drugs can reduce the DN rats’CTGF mRNA and protein expression, and the traditional chinese medicine prevention group was the best. 4.6The comparision of rats’phosphorylation of ERK1/2 protein expression in each groupCompared with normal group, the p-ERK1/2 of rats in the model group increased obviously, and there was significant statistics difference between the two groups (p<0.01); Compared with model group, the p-ERK1/2 protein of rats in the treatment groups decreased obviously, with significant statistics difference (p<0.01);and the traditional chinese medicine prevention group was the lowest. Compared with the traditional chinese medicine prevention group, p-ERK1/2 protein of the other treatment groups increased with significant statistic difference (p<0.01).There was no statistics difference of ERK1/2 protein expression among groups. The results suggest that the experimental drugs can reduce the DN rats’ p-ERK1/2 protein expression, and the prevention group was the best.Conclusion:1. There is an obviously preventive effect of Yiqi Jiedu Huoluo Chinese Decoction on early DN, and it can improve the general condition, decrease the mALB, reduce the fasting glucose, β2-MG.2. The possible preventive effect mechanism of Yiqi Jiedu Huoluo Chinese Decoction on early DN is closely related to ERK1/2 signal transduction pathway by reducing the expression of ANGⅡ.
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