Effect Of Gui Qi Mixture On The Expression Of Transforming Growth Factorβ₁ And Hepatocyte Growth Factor In The Kidney Of Diabetic Rats

Objective In the present study, the clinical effects of Chinese Medical therapy of YiQiHuoXue method (invigorating Qi-activating the blood) and Gui Qi Mixture (GQM) was investigated and compared with Benazepril and Danggui Buxue Decoction (DBD)in streptozotocin-induced diabetic rats mainly with respect to the effects on the expression of renal transforming growth factor- β₁(TGF- β₁) mRNA and levels of renal and serum hepatocyte growth factor(HGF). It will provide us new insights and therapies for the clinical practices to diabetic nephropathy.Methods Diabetes was induced by the intraperitoneal administration of streptozotocin (STZ, 60mg/kg body weight). Tail vein blood glucose levels were measured after 72h. Animals with blood glucose levels of more than 16. 7mmol/l were selected as diabetic rats and then randomly divided into four groups: (1) Benazepril-treated diabetic rats: received 10mg/kg Benazepril orally once a day by gastric tube; (2) Danggui Buxue Decoction-treated rats: received 3. 6g/kg DBD;(3) Gui Qi Mixture-treated rats: received 4. 5g/kg GQM; (4)diabetic control rats: received an equal volume of drinking water. At 4,6,8 weeks after STZ injection, the animals were housed in individual metabolic cages for 24h to obtain urine for the measurement of albumin and β₂-microglobin(β₂^MG) by radioimmunoassay. The urine creatine (Ucr) was also determined. Plasma was collected under 5% pentobarbital anesthesia (30mg/kg body weight ) and blood glucose, TG, CHO, HDL, Scr and BUN were measured. The two kidney of rats were rapidly removed, carefully decapsulated. The right kidney was weighed and immersed in methyl Carnoy' s fixative, embedded in paraffin, amd sectioned for histology. The sections (6μm) were stained with hematoxylin and eosin (HE) and periodic acid Schiff (PAS), thehistological morphology and average glomerular area were analyzed. A part of left kidney tissue (lmmXlmmXlmm) was immersion fixed in HistoChoice MB for the study of microstructure and mean thickness of glomerular basement membrane. In situ hybridization was adopted to assay the expression of TGF- P , mRNA in renal constitution of rats. Glomerular and tubular positive findings were assessed by medical pathological image analysis system and semi-quantitative method. Concentration of HGF in serum and kidney were determined by enzyme-linked immunosorbent assay (ELISA).Statistical analysis: Results were expressed as mean±S (x±s). Comparisions among 5 groups were analyzed by one-way analysis of variance, followed by either LSD or SNK multiple comparision test to evaluate statistical difference. Value of urinary protein excretion were log transformed before statistical analysis. P < 0.05 is regarded as significant.Results: Diabetic rats with plasma glucose levels higher than 16. 7mmol/l were polyuric and failed to gain weight in the experience. Their level of TG, CHO were significantly higher than those of control rats, and HDL level was decreased(P< 0.01). Both DBD and GQM could decrease the the high plasma glucose level and improve lipid metabolism disorders (P<0.01, P<0.05). Neither hyperglycemia or hyperlipidemia were affected by Benazepril treatment (P>0.05) .Ccr and urinary albumin and P2^MG excretion in diabetic rats were gradually and significantly increased compared with that in control rats at 4 weeks(P < 0. 01, respectively), and increased further with time(P<0.01). There were no significant difference between diabetic group and normal group in level of Scr and BUN at 4 and 6 week (P>0. 05). This two index were increased at 8 week (P<0.01). Daily administration of Benazepril, DBD or GQM improved the renal function and reduced urinary albumin and 0 2^MG toa similar extent(P > 0.05). Kidney weight/body weight ratio, mean glomerular area and mean thickness of glomerular basement membrane were significantly increased in untreated diabetic rats than in control rats(P<0.05, P<0.01) and were reduced by the treatment of diabetic rats with Benazepril and DBD compared with the index in untreated diabetic rats(P<0. 01). The index of hypertrophy of group GQM was lower than that of group Benazepril or group DBD. Compared with findings in control animals, the glomerular expression of TGF- P , genes in diabetic rats was significantly increased (P<0. 01). And In situ hybridization showed that the positive position were major in glomerulus and tubular epithelial cells. Treatment with Benazepril or DBD significantly decreased the expression of TGF-3, mRNA in diabetic rats compared with findings in untreated diabetic rats (/'â– â– CO. 01) and there was no significant difference between this two groups (P>0. 05). The expression of TGF-3 i mRNA in group GQM was lower than those in group Benazepril and DBD(/K0. 01). Diabetic rats have increased serum HGF levels compared with controls (P<0. 01) and the HGF levels were not significantly different between the three stages (P>0. 05). HGF level in the diabetic kidney transiently increased but then declined (P<0. 01). The excretion of renal and serum HGF in GQM , DBD or Bnazepril treated group were significantly higher than those in the diabetic control group (P<0. 01) and there were no significant difference between these three groups(P>0. 05). Conclusion:1. Diabetic mellitus and renal lesions occurred in four diabetic groups after STZ were used.2. GQM could improve lipid metabolism disorders , decrease the high plasma glucose level and the excretion rate of the 24h urine albumin andP2-MG ,and relieve the renal lesion.3. TGF-P! that produced in kidney may serve as major mediators ofthe renal changes observed in experimental diabetes. HGF level in the diabetic kidney transiently increased but then declined. Renal HGF but not serum HGF could reflect the progression of DN.4. GQM could up-regulate the expression of HGF in renal tissue and was superior to Benazepril and DBD in reduction the expression of renal TGF- 3 i mRNA and that could be the reasons for its renal-protecting effects in preventing nephropathy process.