Huayu Tongluo Recipe Attenuates Renal Oxidative Stress and Inflammation through the Activation of AMPK/Nrf2 Signaling Pathway in Streptozotocin- (STZ-) Induced Diabetic Rats

Abstract

Diabetic nephropathy (DN), a severe microvascular complication of diabetes, is one of the leading causes of end-stage renal disease. Huayu Tongluo Recipe (HTR) has been widely used in the clinical treatment of DN in China, and its efficacy is reliable. This study aimed to explore the renoprotective effect of HTR and the underlying mechanism. Male Sprague-Dawley rats were fed with high sugar and fat diet combined with an intraperitoneal injection of STZ to establish the diabetic model. Rats in each group were respectively given drinking water, HTR, and irbesartan by gavage for 16 weeks. 24-hour urine samples were collected every 4 weeks to detect the content of total protein and 8-OHdG. Blood samples were taken to detect biochemical indicators and inflammatory markers at the end of 16th week. Renal tissue was collected to investigate pathological changes and to detect oxidative stress and inflammatory markers. AMPK/Nrf2 signaling pathway and fibrosis-related proteins were detected by immunohistochemistry, immunofluorescence, real-time PCR, and western blot. 24h urine total protein (24h UTP), serum creatinine (Scr), blood urea nitrogen (BUN), total cholesterol (TC), and triglyceride (TG) were decreased in the rats treated with HTR, while there was no noticeable change of blood glucose. HTR administration decreased malondialdehyde (MDA) content and increased superoxide dismutase (SOD) activity in kidneys, complying with reduced 8-OHdG in the urine. The levels of TNF-α, IL-1β, and MCP1 and the expression of nuclear NFκB were also lower after HTR treatment. Furthermore, HTR alleviated pathological renal injury and reduced the accumulation of extracellular matrix (ECM). Besides, HTR enhanced the AMPK/Nrf2 signaling and increased the expression of HO-1 while it inhibited the Nox4/TGF-β1 signaling in the kidneys of STZ-induced diabetic rats. HTR can inhibit renal oxidative stress and inflammation to reduce ECM accumulation and protect the kidney through activating the AMPK/Nrf2 signaling pathway in DN.

Figure 1

Effects of HTR on (a) 24h UTP, (b) Scr, and (c) BUN. Data are presented as mean ± SD (n = 7). ^∗P < 0.05, ^∗∗P < 0.01 vs. control; ^#P < 0.05, ^##P < 0.01 vs. DN.

Figure 2

Effects of HTR on (a) RBG and (b) blood lipids. Data are presented as mean ± SD (n = 7). ^∗P < 0.05, ^∗∗P < 0.01 vs. control; ^#P < 0.05, ^##P < 0.01 vs. DN.

Figure 3

Effects of HTR on the renal pathology of STZ-induced diabetic rats. HE, PAS, and Masson's trichrome-stained, magnification×400.

Figure 4

Effects of HTR on the parameters of oxidative stress in urine and kidneys. (a) SOD activity in kidney cortex detected by colorimetric method. (b) MDA content in kidney cortex detected by colorimetric method. (c) 8-OHdG level in urine detected by ELISA. (d) The immunohistochemical stain of 8-OHdG in kidney cortex (×400). Data are presented as mean ± SD (n = 7). ^∗P < 0.05, ^∗∗P < 0.01 vs. control; ^#P < 0.05, ^##P < 0.01 vs. DN.

Figure 5

Effects of HTR on the parameters of inflammation in serum and kidneys. (a) The levels of TNFα, IL-1β, and MCP1 in serum detected by ELISA (n = 7). (b) The mRNA expression of TNFα, IL-1β, and MCP1 in serum detected by qRT-PCR. (c) Total and nuclear NFκB expression of rats in kidney cortex measured by western Blot. (d) Quantitative analyses of total NFκB/β-actin and nuclear NFκB/H3. Data were presented as mean ± SD (n = 3). ^∗P < 0.05, ^∗∗P < 0.01 vs. control; ^#P < 0.05, ^##P < 0.01 vs. DN.

Figure 6

Effects of HTR on Nox4/TGF-β1 signaling pathway and the ECM proteins. (a) Representative western blot is shown for Nox4, TGF-β1, and β-actin. Quantitative analyses are shown for (b) Nox4/β-actin and (c) TGF-β1/β-actin. (d) The immunohistochemical stain of FN and Col IV in kidney cortex (×400). Data were presented as mean ± SD (n = 3). ^∗P < 0.05, ^∗∗P < 0.01 vs. control; ^#P < 0.05, ^##P < 0.01 vs. DN.

Figure 7

Effects of HTR on AMPK/Nrf2 signaling pathway. (a) Representative western blot is shown for total AMPK, pAMPK, total Nrf2, nuclear Nrf2, HO-1, NQO1, β-actin, and histone H3. Quantitative analyses are shown for (b) pAMPK/total AMPK, (c) total Nrf2/β-actin, (d) nuclear Nrf2/H3, (e) HO-1/β-actin, and (f) NQO1/β-actin. (g) The mRNA relative expression of HO-1 in serum detected by qRT-PCR. (h) The mRNA relative expression of NQO1 in serum detected by qRT-PCR. (i) The protein expression detected by immunofluorescence. Data were presented as mean ± SD (n = 3). ^∗P < 0.05, ^∗∗P < 0.01 vs. control; ^#P < 0.05, ^##P < 0.01 vs. DN.

Figure 8

Schematic overview of HTR's effect on renal fibrosis in STZ-induced diabetic rats. ECM accumulation, the characteristic of fibrosis, is a main pathological change in DN. As we all know, oxidative stress and inflammation, which promote each other, are the critical causes of TGF-β1-induced excessive synthesis and deposition of ECM in DN. Interestingly, AMPK acts as the upstream of Nrf2. The activation of AMPK/Nrf2 pathway can inhibit the Nox4-induced oxidative stress and attenuate NFκB-induced inflammation. HTR enhances the activity of AMPK/Nrf2 pathway and suppresses oxidative stress and inflammation, thereby decreasing the ECM deposition and delaying the progression of fibrosis in DN (⟶: activated; ⊣: inhibited).