| Diabetic nephropathy is a common complication of diabetes, which is the most serious microvascular complications, and the second cause of end-stage renal disease. Modern medicine for the treatment of diabetic nephropathy is mainly symptomatic treatment, but symptomatic treatment faces varying problems, such as poor compliance of patients, drug toxicity, high price, lack of living kiding, and so on. In recent years, the application of Traditional Chinese Medicine in treating diabetic nephropathy shows a definite advantage in clinical application of broad and significant effect. Wan Zheng who is famous veteran doctors of Traditional Chinese Medicine wrote a prescription named Shenluoan decotion. After years of clinical practice, the clinical efficacy of Shenluoan decotion showed significantly, economically, securely, and widespread praise in patients. To investigate the therapeutic action of Shenluoan decotion and to explore the possible mechanisms, the study was conducted.The experiment was adopted diabetic nephropathy rats model with high glucose feeding and low-dose STZ injection, then the rats were given Shenluoan decotion and Irbesartan by gavage. The test was to observe the expression of Integrinβ1 on diabetic nephropathy rats through PI3K/Akt/ m TOR signal transduction system, and the renoprotective effect of Shenluoan decotion on podocytes’ proliferation in high glucose environment, and clinical impact of treating obese patients with early diabetic nephropathy by Shenluoan decoction, and providing trial data for diabetic nephropathy therapy.Part I Renal pathological morphology effect of Shenluoan decotion on diabetic nephropathy ratsObjective: To establish diabetic nephropathy rats model, verified the interference effect of Shenluoan decotion, and observe these indicators: rats general condition, renal pathological morphology, renal function, blood glucose, and blood lipids.Motheds: 90 male Sprague-Dauley(SD) rats were fed for one week after making sure the rats’ urine protein negative. 10 rats were randomly divided into normal group and 80 in model group. The normal group and the model group were fed common diet and high fat diet respectively. The model group rats were injected intraperitonally with 35 mg·kg-1 streptozotocin after 12 weeks feeding. The normal group rats were injected with 0.1mol·L-1 citrate buffer correspondingly. Blood glucose levels were measured 72 h later for three times continuously(≥16.7 mmol·L-1) after streptozotocin injection to confirm the development of diabetes. Dividing the model rats randomly into three groups: 19 rats in model group, 19 rats in Irbesartan group, 19 rats in traditional Chinese medicine(TCM) group, and 20 rats in combination group. After one week of diabetic mellitus models were made successfully, the rats of Irbesartan group were administrated with Irbesartan by gavage, 15.75mg·kg-1.The TCM group were given Shenluoan decotion by gavage, 10.44mg·kg-1. The combination group were given Irbesartan and Shenluoan decotion meanwhile. The normal group and the model group were given physiological saline by gavage. Each group was taken the medicine for 16 weeks. All animals were killed and the blood and the renal tissue were obtained. The general condition of rats, related indicators of kidney, pathomorphology changes, and glomerular basement membrane morphology were observed.Results: 1 General conditions of the experimental rats The experiment results of 70 rats contained 10 rats in the normal group, 14 rats in the model group, 15 rats in the Irbesartan group, 15 rats in the TCM group, and 16 rats in the combination group. 2 Comparation of kidney hypertrophy indexin(KI) in each group Compared with control group, KI of the model group and the treatment groups increased significantly at different weeks(P<0.05). Compared with model group, KI of the treatment groups decresed sharply(P<0.05). However, KI of the treatment groups showed no significant difference(P>0.05). 3 Comparison of fasting blood glucose in each group Compared with the control group, the fasting blood glucose in the model group and the treatment groups was significantly higher(P<0.05). Compared with the model group, the fasting blood glucose in TCM group and combination group was lower at 12 w and 16w(P<0.05). Compared with the Irbesartan group, the fasting blood glucose in the combination group was lower at 16w(P<0.05). 4 Comparison of Hb A1 c in each group Compared with control group, the Hb A1 c in model group and treatment groups were significantly higher at 16w(P<0.05). Compared with model group, the Hb A1 c in TCM group and combination group was lower(P<0.05). Compared with Irbesartan group, the Hb A1 c in combination group was lower sharply(P<0.05). Compared with TCM group, the Hb A1 c in combination group was decreased(P<0.05). 5Comparison of Insulin resistance index of homeostasis model assessment(Homa-IR) in each group Compared with control group, Homa-IR of model group and treatment groups was significantly higher at 16w(P<0.05), while compared with model group, Homa-IR of treatment groups was lower(P<0.05). Homa-IR of combination group was significantly lower than Irbesartan group(P<0.05). Homa-IR of combination group was significantly lower than TCM group(P<0.05). 6 Comparison of 24 h urinary protein quantitative in each group 24 h urinary protein quantitative of control group was the lowest and remained stable(P<0.05). 24 h urinary protein quantitative of model group and treatment groups was higher than control group at 4w, 8w, 12 w, and 16w(P<0.05), while that of model group was increased at different times(P<0.05). 24 h urinary protein quantitative of treatment groups was reduced sharply at 8w, 12 w, and 16 w compared with model groups(P<0.05). Compared with Irbesartan group, 24 h urinary protein quantitative of combination group showed significantly lower at 16w(P<0.05). 7 Comparison of renal function in each group Compared with the control group, the blood uric acid(UA) of model group and treatment groups was higher to varying degrees(P<0.05). Compared with model group, UA in treatment groups was significantly lower(P<0.05). Compared with Irbesartan group, UA in combination group was significantly lower(P<0.05). The serum creatinine of model group and treatment groups was higher than control group(P<0.05). Compared with model group, serum creatinine was significantly lower in treatment groups(P<0.05). The comparison among Irbesartan group, TCM group, and combination group was of no statistically significant difference(P>0.05). Compared with control group, the BUN of the model group and treatment groups was significantly increased(P<0.05). The BUN of combination group was lower than Irbesartan group and TCM group(P<0.05, P<0.05). 8 Comparison of total protein(TP) and albumin(ALB) in each group Compared with the control group, TP of model group and treatment groups was lower to varying degrees(P<0.05). Compared with model group, TP in treatment groups was significantly higher(P<0.05). TP in combination group was increased more significantly compared with Irbesartan group and TCM group(P<0.05). Compared with the control group, ALB of model group and treatment groups was reduced(P<0.05). Compared with model group, ALB in treatment groups was increased more significantly(P<0.05). ALB in combination group was higher than in TCM group(P<0.05). 9 Comparison of serum lipid levels in each group Compared with the control group, TC of model group and treatment groups was increased(P<0.05). Compared with model group, TC in treatment groups was reduced(P<0.05). There was no significant difference among treatment groups on TC level(P>0.05). Compared with the control group, TG of model group and treatment groups was increased(P<0.05). Compared with model group, TG in treatment groups was reduced(P<0.05). Combination group was lower than Irbesartan group on TG level(P<0.05). There was no significant difference among treatment groups on HDL level(P>0.05). Compared with the control group, VLDL of model group and treatment groups was increased(P<0.05). Compared with model group, VLDL in treatment groups was reduced(P<0.05). There was no significant difference among treatment groups on VLDL level(P>0.05). 10 Comparison of fibrosis index in each group Compared with the control group, LN of model group and treatment groups was increased(P<0.05). Compared with model group, LN in treatment groups was reduced(P<0.05). Combination group was lower than Irbesartan group and TCM group on LN level(P<0.05). Compared with the control gorup, the level of serum IV-C was increased in model group and treatment groups(P<0.05). The treatment groups were lower than model group on IV-C level(P<0.05).There was no significant difference among treatment groups on IV-C level(P>0.05). 11 Light microscopy results In the normal group: The structure of renal glomerulus was normal. In the model group: glomerular hypertrophy, thickening of glomerular capillary basement membrane, mesangial matrix increased, mesangial cell proliferation, mesangial area broadening and tubulointerstitial fibrosis were seen. Irbesartan group, TCM group, and combination group showed different degree of pathological changes, but the pathological degree was obviously lighter than the changes of the model group. The combination group improved more significantly in renal pathology. 12 Transmission electron microscopy results In normal group: the structure of glomerular capillary basement membrane was clear and complete, while microcirculatary endothelial cell and foot processes was normal. In the model group: most of the glomerular capillary basement membrane showed significant homogeneous thicking; structure was not clear; mesangial matrix and the fusion of the foot processes was extensive. Irbesartan group, TCM group, and combination group also showed the similar pathological changes, but the pathological degree was improved more than that in model group. The combination group improved the most obvious pathology than that in the other groups.Part ⅡShenluoan decotion up-regulates the expression of Integrinβ1 on diabetic nephropathy rats through PI3K/Akt/m TOR signal transduction systemObjective: This study aims to establish diabetic nephropathy model and observe the intervention of Shenluoan decotion on diabetic nephropathy rats of injured podocytes Integrinβ1 mediated PI3K/Akt/m TOR signal transduction system.Methods: Diabetic nephropathy model was duplicated the same as the above. Integrinβ1, PTEN, p-Akt and p-m TOR were determined by immunohistochemistry. The protein of Integrinβ1, PTEN, Akt, p-Akt, m TOR and p-m TOR were measured by Western blot method. Real-time RT PCR was used to compare the m RNA expression of Integrinβ1, PTEN and IV-C.Results: 1 Comparison of Integrinβ1 protein and m RNA expression in renal tissues The results detected by immunohistochemistry showed: Compared with the control group, the expression of Integrinβ1in the glomerular of model group was significantly decreased, and renal tubular staining was reduced. Compared with model group, the expression of Integrinβ1in treatment groups was significantly deeper, while the combination group was the deepest especially. The results detected by Western blot and real time RT-PCR showed: In control group, Integrinβ1 has the basal expression. Compared with control group, the expression of Integrinβ1in model group and treatment groups was weeker(P<0.05). Compared with model group, Integrinβ1 protein and m RNA expression in treatment groups was significantly higher(P<0.05), while the combination group was the highest expression especially in the treatment groups(P<0.05). 2 Comparison of PTEN protein and m RNA expression in renal tissues The results detected by immunohistochemistry showed: The expression of PTEN in model group was lower than that in control group. The expression of PTEN in treatment groups was stronger than model group. Combination group could increase PTEN expression. The results detected by Western blot and real time RT-PCR showed: PTEN protein and m RNA expression in model group was lighter than control group(P<0.05). PTEN protein and m RNA expression in treatment groups was increased more than model group(P<0.05). Combination group increased obviously(P<0.05). 3 Comparison of Akt protein expression in renal tissues The results detected by Western blot showed: There was no significant difference in the expression of Akt protein in defferent groups(P>0.05). 4 Comparison of p-Akt protein expression in renal tissues The results detected by immunohistochemistry showed: p-Akt positive expression was located in cytoplasm and nucleus which showed brown granules for positive signal. The expression of p-Akt in control group was lower than that in model group and treatment groups. The expression of p-Akt in treatment groups was lighter than model group. Combination group was reduced p-Akt expression especially. The results detected by Western blot showed: In contrast to control group, the expression of p-Akt/Akt in model group and treatment groups was enhanced(P<0.05). Compared with model group, the expression of p-Akt/Akt in treatment groups was decreased(P<0.05), especially combination group was more obviously lower(P<0.05). 5 Comparison of m TOR protein expression in renal tissues The results detected by Western blot showed: There was no significant difference in the expression of m TOR protein in defferent groups(P>0.05). 6 Comparison of p-m TOR protein expression in renal tissues The results detected by immunohistochemistry showed: p-m TOR positive expression was located in glomerular mesangial matrix and tubulointerstitial. The expression of p-m TOR in control group was lower than that in model group and treatment groups. The expression of p-m TOR in treatment groups was lighter than model group. Combination group was reduced p-Akt expression especially. The results detected by Western blot showed: In contrast to control group, the expression of p-m TOR/m TOR in model group and treatment groups was enhanced(P<0.05). Compared with model group, the expression of p-m TOR/m TOR in treatment groups was decreased(P<0.05), especially combination group was more obviously lower(P<0.05). 7 Comparison of IV-C m RNA expression in renal tissues Compared with control group, IV-C m RNA expression in model group and treatment groups was significantly increased(P<0.05). Compared with model group, IV-C m RNA expression in treatment groups was reduced sharply, especially combination group was more lower(P<0.05).PartⅢ Promoting effect of Shenluoan decotion on podocytes’ proliferation in high glucose environmentObjective: This study aims to observe the intervention of Shenluoan decotion on glomerular podocytes’ proliferation in high glucose environment.Methods: 15 SD rats were randomly divided into 3 groups:control group, Shenluoan group, and Irbesartan group. Control group were given distilled water. Shenluoan group and Irbesartan group were administrated with Shenluoan decotion and Irbesartan respectively, while the dosage is 20 times to adults. The treatment lasted for 4 days, and the rats’ blood were obtained.Different groups of 5% drug containing serum were treated podocytes. MTT assay was used to measure the proliferation of podocytes at 12 h, 24 h, 36 h, and 48 h intervals.Results: The podocyte proliferation was significantly lower in high glucose group than in control group at 36h(P<0.05). That showed inhibiting effects of podocytes’ proliferation in high glucose environment. The podocytes’ proliferation was significantly promoted in Irbesartan group compared to high glucose group at 36 h and 48 h, while the podocytes’ proliferation in Shenluoan group was promoted more obviously at 48h(P<0.05). That showed that Shenluoan group and Irbesartan group had promoting effects on the podocytes’ proliferation in high glucose environment. There was no significant differences between Shenluoan group and Irbesartan group at 48 h OD value. The promoting effects on podocytes’ proliferation between Shenluoan group and Irbesartan group had no significant difference with time increased(P>0.05).Part Clinical observation of treating obese patients with early diabetic Ⅳnephropathy by Shenluoan decoctionObjective: To investigate obese patients with early diabetic nephropathy by Shenluoan decoction, and observe efficacy and adverse reactions, all the above that have been done was to provide the foundation in order to generalization and exploitation.Methods: 68 obese patients with early diabetic nephropathy were randomly assigned into the control group(n=34) treated by Irbesartan and the treated group(n=34) treated by Shenluoan decoction combined with Irbesartan, and the conventional western therapies were given to all patients in both groups. The changes of the therapeutic efficacy, Chinese medicine syndrome, waistline, serum creatinine(SCr), blood urea nitrogen(BUN), total cholesterol(TC), triglyeride(TG), fasting blood glucose(FBG), Hemoglobin A1C(Hb A1c), Cystatin C(Cys C), β2-microglobulin(β2-MG), homocysteine(Hcy), urinary monocyte chemoattractant protein-1(UMCP-1), urinary regulated upon activation normal T-cell expressed and secreted(URANTES), urinary podocalyxin(UPCX), urinary nephrin(UNephrin), urinary albumin, and urine creatinine were detected before and after 16 weeks treatment.Results: 1 There is no significant difference between the treatment group and the control group in general condition. 2 Comparison of clinical therapeutic effects 63 cases completed the experiment. The total effective rate in the treatment group was 90.62% while 67.74% in the control group. The total effective rate in the treatment group was significantly higher than that in control group(P<0.05). 3 Comparison of TCM symptoms scores between the two groups There was no significant difference in TCM symptoms between the two groups before treatment(P>0.05). The improvement of the symptoms in the treatment group was significantly better than that in the control group(P<0.05). 4 Comparison of waistline between the two groups There was no significant difference between the two groups(P>0.05). 5 Comparison of SCr, BUN, TC, TG, FBG and Hb A1 c before and after treatment between the two groups There was no significant difference between the two groups before treatment(P>0.05). All the indexes were improved in the treatment group after treatment(P<0.05), of which the SCr, TC, TG, FBG and Hb A1 c was significantly lower than control group after treatment(P<0.05). 6 Comparison of Cys C, β2-MG and Hcy before and after treatment in the two groups There was no significant difference between the two groups before treatment(P>0.05). All the indexes were decreased in the two groups after treatment(P<0.05), but the treatment group was significantly lower than the control group(P<0.05). 7 Comparison of UMCP-1/UCR, URANTES/UCR and UACR before and after treatment in the two groups UMCP-1/UCR, URANTES/UCR and UACR was decreased in the treatment group after treatment(P<0.05), that of which was significantly lower than the control group after treatment(P<0.05). 8 Correlation analysis of UMCP-1/UCR, URANTES/UCR and UACR UMCP-1/UCR was positively related to URANTES/UCR(r=0.561 P=0.000). UACR is positively related to UMCP-1/UCR(r=0.560 P=0.000). UACR is positively related to URANTES/UCR(r=0.489 P=0.000). 9 Comparison of UPCX/UCR and UNephrin/UCR before and after treatment in the two groups UPCX/UCR and UNephrin/UCR was decreased in control group and treatment group after treatment(P<0.05), which was improved more evidently in treatment group after treatment(P<0.05). 10 Correlation analysis of UPCX/UCR, UNephrin/UCR and UACR UPCX/UCR was positively related to UNephrin/UCR(r=0.474 P<0.05). UPCX/UCR is positively related to UACR(r=0.447 P<0.05). UNephrin/UCR is positively related to UACR(r=0.473 P<0.05). 11 Index of security detection in the two groups There was no abnormal changes and adverse events of safety indexes.Conclusion: 1 Shenluoan decotion slowed chronic progressive kidney lesions through improving diabetic nephropathy rats’ general conditions, decreasing urinary protein excretion, improving the renal function, and lightening the renal pathological damage. 2 PI3K/Akt/m TOR pathway was activated in diabetic nephropathy kidney. IV-C collagen synthesis was increased. Extracellular matrix was accumulated. Integrinβ1 expression was weakened. Podocytes’ proliferation was inhibited. Glomerular sclerosis was promoted. Shenluoan decotion slowed down the occurrence and development of glomerular sclerosis through blocking the signaling action of PI3K/Akt/m TOR pathway, inhibiting IV-C collagen synthesis, relieving the damage of podocyte Integrinβ1, and promoting effects on the podocytes’ proliferation. 3 That Shenluoan decotion could significantly improve clinical symptoms and signs of obese patients with early diabetic nephropathy, reduce the UMCP-1, URANTES, UACR, UPCX, and UNephrin excretion, protect the renal function. Those showed us Shenluoan decotion provided a new medicine for the prevention and treatment of diabetic nephropathy. 4 Shenluoan decotion and Irbesartan had good clinical effects in diabetic nephropathy treatment. But as a result of sampling of a few of the limitations of the study need to be in the future to continue to increase sample size, and thereby more eloquent testimony to their security. |
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