| Objective: By observing the effects of extract from Angelica and Hedysari on TGF-β/smad signaling pathway related to cytokines and gene in rats,renal tissue with diabetic nephropathy(diabetic nephropathy,DN),we investigated the influence mechanism of extract from Angelica and Hedysari on DN and confirmed extract from Angelica and Hedysari whether can delay the process of glomerular sclerosis of DN rat, provide the experimental basis for the clinical traditional Chinese Medicine in the treatment of DN, provide theoretical support for the development of prevention and treatment of DN by Chinese herbal medicine.Methods: 72 male Wistar rats of SPF were selected, 6 weeks old and weight 180±20g, after keeping the environment to adapt to 1-2 weeks, 12 rats were randomly selected as normal control group(A group), the remaining 60 rats were all injected intraperitoneally with streptozotocin(STZ), at a dose of 60mg·kg-1(we used when it was dissolved in citrate buffer 0.1mol·L-1), normal group was injected with the same amount of citrate buffer. After 72 h we tested fasting blood glucose(FBG)≥16.7mmol·L-1and after 3 weeks 24 h urine protein>30mg, it suggested success. 60 DN model rats were randomly divided into 5 groups(each group had 12 rats): B group was model group, C group was irbesartan group(it was irrigated into stomach with 17.5mg·kg-1·d-1), D group was low dose group of extract from Angelica and Hedysari(it was irrigated into stomach with 1.8g·kg-1·d-1), E group was middle dose group of extract from Angelica and Hedysari(it was irrigated into stomach with 3.6g·kg-1·d-1), F group was high dose group of extract from Angelica and Hedysari(it was irrigated into stomach with 7.2g·kg-1·d-1), normal group and model group only received the same amount of ultrapure water, intervened for 8 weeks. FBG was measured weekly; at the end of 4 and 8 weeks of administration, we measured quantitative of 24 hour urinary albumin;at the end of 8 weeks the rats were sacrificed, measured blood lipid and renal function through blood taking and get serum; taked kidneys, weighed, calculated the kidey hypertrophy index;and detected kidney histopathology, immunohistochemical staining and reverse transcription polymerase chain reaction(RT-PCR) assay.Results: 1. General condition: After modeling,except normal group, the rats of other groups were all polyuria, polydipsia and apathetic, unresponsive, clustered together and less activity, weight loss, gaunt, fur lost smooth, even appeared abdominal distention, skin ulcerated, cataract and model group was the most obvious. 2. Weight and kidey hypertrophy index: After 8 weeks of treatment, compared with normal group, the weight of model group decreased significantly(P<0.01); compared with model group the weight of rats in each treatment group had a rising trend, but there was no statistical significance(P>0.05).The kidey hypertrophy index of model group rats was significantly higher than that of normal group(P<0.01); the kidey hypertrophy index of irbesartan group, middle and high dose group of extract from Angelica and Hedysari were all lower than these of model group(P<0.05 or P<0.01). 3. 24 h urine protein: 24 h urine protein of model group rats was significantly higher than that of normal group(P<0.01); after administration, on the 4th week,compared with model group, 24 h urine protein of irbesartan group, middle and high dose group of extract from Angelica and Hedysari decreased significantly(P<0.05 or P<0.01), on the 8th week, all the treatment groups were all significantly decreased(P<0.05 or P<0.01); compared with irbesartan group, there was no statistical significance in middle and high dose group of extract from Angelica and Hedysari(P>0.05). 4. FBG: After modeling, FBG of model group rats was higher than that of normal group(P<0.01); after administration 4 weeks, compared with model group, FBG of middle and high dose group of extract from Angelica and Hedysari were significantly decreased(P<0.05), after administration 8 weeks, compared with model group, FBG of all dose groups of extract from Angelica and Hedysari were all significantly decreased(P<0.05 or P<0.01). 5. Blood lipid and renal function: Compared with normal group, triglyceride(TG) and cholesterol(TC) of model group were significantly increased(P<0.01); compared with model group, TC of middle and high dose group of extract from Angelica and Hedysari were significantly decreased(P<0.05 or P<0.01), TG of all the treatment groups were significantly decreased(P<0.05 or P<0.01); compared with irbesartan group, TG of middle dose group of extract from Angelica and Hedysari was more significantly decreased. After 8 weeks, serum creatinine(Scr), blood urea nitrogen(BUN) of model group were significantly higher than these of normal group(P<0.01); Scr, BUN of middle dose group of extract from Angelica and Hedysari were significantly lower than these of model group(P<0.05); Scr of high dose group of extract from Angelica and Hedysari was lower than that of model group(P<0.05). 6. Histopathological detection:(1)HE staining: morphological and size of glomerular and the renal tubules of normal group were all normal;glomerular volume of model group increased,renal capsule narrowed, there was thickening of glomerular basement membrane(GBM), intraglomerular mesangial cells were increased, the epithelial cells of renal tubular were edematous, vacuolar degeneration was found in partially renal tubules, lumen stenosis, irregular structure, there were inflammatory infiltration in renal interstitial;compared with model group, pathological changes of all the treatment groups were all improved at different degree.(2)Masson staining: Glomeruli, renal tubules and renal interstitium of normal group were no abnormalities; glomeruli and renal interstitium of model group shows collagen fibers, part of renal tubules were shrinking; compared with model group, lesions of irbesartan group, middle and high dose group of extract from Angelica and Hedysari were obviously relieved. 7. Immunohistochemistry: Compared with normal group, TGF-β1, Smad2, Smad3 expression of model group renal tissue were significantly increased(P<0.01); compared with model group, TGF-β1, Smad2, Smad3 expression of irbesartan group, middle and high dose group of extract from Angelica and Hedysari were significantly decreased(P<0.05 or P<0.01); compared with irbesartan group, there was no statistical significance in middle and high dose group of extract from Angelica and Hedysari(P>0.05). 8. RT-PCR Detection: TGF-β1, Smad2, Smad3 m RNA expression of model group were significantly higher than these of normal group(P<0.01); TGF-β1, Smad2, Smad3 m RNA expression of irbesartan group, middle and high dose group of extract from Angelica and Hedysari were all lower than these of model group(P<0.05 or P<0.01).Conclusion: 1. Extract from Angelica and Hedysari can improve general condition of DN rats, reduce 24 h urinary protein, decrease blood glucose, decrease the level of serum creatinine and urea nitrogen, improve lipid metabolism, improve renal blood flow, reduce the pathological damage of the kidneys. 2. Extract from Angelica and Hedysari can reduce expression levels of TGF-β1, Smad2 and Smad3 in renal tissue of diabetic nephropathy rats, can delay renal fibrosis. |
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