Renal protective effect of xiao-chai-hu-tang on diabetic nephropathy of type 1-diabetic mice

Abstract

Xiao-Chai-Hu-Tang (XCHT), a traditional Chinese medicine formula consisting of seven medicinal plants, is used in the treatment of various diseases. We show here that XCHT could protect type-1 diabetic mice against diabetic nephropathy, using streptozotocin (STZ)-induced diabetic mice and high-glucose (HG)-exposed rat mesangial cell (RMC) as models. Following 4 weeks of oral administration with XCHT, renal functions and renal hypertrophy significantly improved in the STZ-diabetic mice, while serum glucose was only moderately reduced compared to vehicle treatment. Treatment with XCHT in the STZ-diabetic mice and HG-exposed RMC resulted in a decrease in expression levels of TGF-β1, fibronectin, and collagen IV, with concomitant increase in BMP-7 expression. Data from DPPH assay, DHE stain, and CM-H(2)DCFDA analysis indicated that XCHT could scavenge free radicals and inhibit high-glucose-induced ROS in RMCs. Taken together, these results suggest that treatment with XCHT can improve renal functions in STZ-diabetic mice, an effect that is potentially mediated through decreasing oxidative stress and production of TGF-β1, fibronectin, and collagen IV in the kidney during development of diabetic nephropathy. XCHT, therefore merits further investigation for application to improve renal functions in diabetic disorders.

Figure 1

Effects of XCHT on serum levels of glucose (a), BUN (b), and creatinine (c) in normal or STZ-diabetic mice after a 4-week XCHT (200 mg/kg) or vehicle (Veh) treatment. Mice were first induced with STZ for 9 weeks to induce DN and then orally administered with treatments. Values (mean ± SD) were obtained from 6 mice for each test group. ^# P < 0.05 compared to the value of vehicle-treated normal mice. *P < 0.05 values from after 4 weeks of treatment compared to values prior treatment.

Figure 2

Effects of XCHT on kidney hypertrophy of normal or STZ-diabetic mice after a 4-week XCHT (200 mg/kg) or vehicle (Veh) treatment. (a) The kidney/body weight ratio. Values (mean ± SD) were collected from 6 mice for each test group. (b) Representative photomicrographs (original magnification, 200x) of PAS-stained kidney sections from STZ-diabetic mice receiving 4 weeks of XCHT or vehicle treatment; (A) normal mice treated with vehicle, (B) STZ-diabetic mice treated with vehicle, and (C) STZ-diabetic mice treated with XCHT. (c) Expansion of the glomerular matrix was scored as described in the text, and an average value was obtained from analyses of 30 glomeruli in each mouse kidney. ^# P < 0.05  values of STZ-diabetic mice compared to that of the normal mice. *P < 0.05 values of XCHT-treated STZ-diabetic mice compared to values from vehicle-treated STZ-diabetic mice.

Figure 3

Effect of XCHT on TGF-β1, fibronectin, collagen IV, and BMP-7 expression in the kidney of normal or STZ-diabetic mice after 4 weeks of treatment (n = 6 in each group). (a) The mRNA expression of TGF-β1, fibronectin, collagen IV, and BMP-7 in the kidney of mice was detected using RT-PCR. β-actin mRNA expression was included as internal control. (b) Western blot analysis of TGF-β1, fibronectin, collagen IV, and BMP-7 expression in the kidney of the test mice. Representative data are shown.

Figure 4

Renal immunostaining for TGF-β1 (a)–(c), fibronectin (d)–(f), collagen IV (g)–(i), and BMP-7 (j)–(l) expression in normal or STZ-diabetic mice receiving 4 weeks of XCHT treatment (n = 6 per group). Original magnification, 200x. Representative micrographs are shown.

Figure 5

Effect of XCHT on free radicals in glucose-exposed RMCs. (a) Direct free radical scavenging activity of XCHT determined by the DPPH radical scavenging assay. Values represent the data of three independent experiments (SE < 0.05). Vitamin C was used as a positive control compound. (b) Intracellular visualization of ROS in RMCs under different treatments using DHE stains; (A) normal-glucose medium, (B) high-glucose medium, (C) high-glucose medium containing XCHT (20 μg/mL), (D) high-glucose medium containing tiron (antioxidant control; 10 mmol/L). (c) Quantitation of ROS generation in RMCs using CM-H₂DCFDA assay. RMCs were cultured in normal glucose (NG; 5 mmol/L) or high-glucose (HG; 30 mmol/L) medium in the presence of XCHT (1, 10, 20, and 50 μg/mL) or tiron (10 mmol/L) for 24 h. ^# P < 0.05 compared to NG., *P < 0.05 compared to HG.

Figure 6

Effect of XCHT on TGF-β1, fibronectin, collagen IV, and BMP-7 expression in RMCs. RMCs were cultured in normal-glucose (NG; 5 mmol/L) or high-glucose (HG; 30 mmol/L) medium in the presence of 50 μg/mL of XCHT for 24 h. (a) The mRNA expression of TGF-β1, fibronectin, collagen IV, and BMP-7 in RMCs detected by RT-PCR. β-Actin mRNA expression is used as an internal control. (b) Western blot analysis of TGF-β1, fibronectin, collagen IV, and BMP-7 expression in RMCs.