Effect of Huayu Tongluo Herbs on Reduction of Proteinuria via Inhibition of Wnt/ β-Catenin Signaling Pathway in Diabetic Rats

Abstract

The study investigated the expression of Wnt/β-catenin pathway in diabetic rats and the intervention effect of Huayu Tongluo herbs (HTH). Ten rats were randomly selected as control group and the remaining rats were established as diabetic models. The diabetic rats were randomly divided into model group and HTH treatment group. The intervention was intragastric administration in all rats for 20 weeks. At the end of every 4 weeks, fasting blood glucose and 24 h urinary total protein quantitatively were measured. At the end of the 20th week, biochemical parameters and body weight were tested. The kidney tissues were observed under light microscope and transmission electron microscopy. We examined Wnt/beta-catenin signaling pathway key proteins and renal interstitial fibrosis related molecular markers expression. The results showed that HTH could reduce urinary protein excretion and relieve renal pathological damage. Wnt4, p-GSK3β (S9), and β-catenin expression were decreased in the signaling pathway, but GSK3β level was not changed by HTH in diabetic rats. Furthermore, the expressions of TGF-β₁ and ILK were decreased, but the level of E-cadherin was increased in diabetic rats after treatment with HTH. This study demonstrated that HTH could inhibit the high expression of Wnt/β-catenin pathway in kidney of diabetic rats. The effect might be one of the main ways to reduce urinary protein excretion.

Figure 1

FBG and 24 h UTP changes of tendency in different groups. The changes of tendency in rats' FBG every 4 weeks. Compared with C group, ^∗∗P < 0.01 (a). The changes of tendency in rats' 24 h UTP (24 h urine total protein) every 4 weeks. Compared with C group, ^∗∗P < 0.01. Compared with M group, ^△P < 0.05, ^△△P < 0.01 (b).

Figure 2

Effects of HTH on the Wnt/β-catenin signaling pathway of rats. Immunohistochemical staining for Wnt4, phospho-GSK3β (S9), and β-catenin in renal cortex of normal control rat group (C group), diabetic model rat group (M group), and Huayu Tongluo herbs treatment rat group (HTH group), exposed to DM and then administration of Chinese medicine of stasis removing and collaterals dredging herbal granule suspension intragastrically (a). Protein levels of Wnt4, phospho-GSK3β (S9), GSK3β, and β-catenin were determined by Western blot and quantified by densitometry in renal cortex of three groups ((b), (c)). Real-time PCR of Wnt4, GSK3β, and β-catenin mRNA with an internal control of β-actin in three groups (d). Values are expressed as means ± SD. Compared with C group, ^∗P < 0.05, ^∗∗P < 0.01; compared with M group, ^△P < 0.05, ^△△P < 0.01.

Figure 3

Effects of HTH on the Changes in Histopathology of kidney. Histology of kidney tissue under the light microscope from rat of normal control group (C group), diabetic model group (M group), and Huayu Tongluo herbs treatment group (HTH group) (HE staining ×400, PAS staining ×400). Ultrastructural changes in glomeruli under the electron microscope from rat of the three groups (×15000).

Figure 4

Effects of HTH on E-cadherin, ILK, and TGF-β₁ of rats. Immunohistochemical staining for ILK and TGF-β₁ in the kidneys of rat of normal control rat group (C group), diabetic model rat group (M group), and Huayu Tongluo herbs treatment rat group (HTH group), exposed to DM and then administration of Chinese medicine of stasis removing and collaterals dredging herbal granule suspension intragastrically (a). Western blot detection and statistical analyses for renal E-cadherin, ILK, and TGF-β₁ in kidneys of rat of three groups ((b), (c)). Quantification of E-cadherin, ILK, and TGF-β₁ mRNA expression levels in different groups by Real-time PCR (d). Masson trichrome staining revealed extracellular matrix deposition in the kidneys of rat of three groups (stained in blue) (e). Values are expressed as means ± SD. Compared with C group, ^∗P < 0.05, ^∗∗P < 0.01; compared with M group, ^△P < 0.05, ^△△P < 0.01.