All animal experiments were performed in accordance with the the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health and approved by Institutional Committee of Health Guide and Care, China Pharmaceutical University. Male C57BL/6 J mice (8 weeks old) were purchased from Yangzhou University Medical Center (Yangzhou, China). Nrf2 knockout mice (B6.129X1-Nfe2l2tm1Ywk/J) were purchased from the Jackson Laboratory (stock No. 017009). Mice were housed under the standard laboratory environmental conditions (temperature 20 ± 2 °C, humidity 50%, and regular light/dark cycle (12 h/12 h)), with free food and water access. The animals were adapted to tested conditions for at least 1 week.

Type 2 diabetes mellitus was induced by feeding with high fat die (HFD, 60% energy from fat). After 12 weeks, HFD animals were received i.p. injection of streptozotocin (STZ, 60 mg/kg in citrate buffer, 10 mmol/l, pH 4.5 on 5 consecutive days) after 4–5 h fasting. Fasting serum glucose (FSG) was checked after 7 days using a glucometer (Contour next EZ, Bayer, ON, Canada) from the tail. FSG exceeded 12 mmol/l was considered diabetic. Body weight was recorded after the model completing. Animals were sacrificed by CO2 gas inhalation and bled rapidly by cutting the carotid arteries to collect the blood sample and bladder tissue.

Organs were fixed with formalin and embedded in paraffin. 5 μm paraffin sections were cut and stained with Masson's Trichrome Stain Kit (Solarbio, Beijing, China).

The Sects. (10 μm) were deparaffinized in xylene and rehydrated in a graded series of ethanols (100%, 95%, 80%, and 75%) for 5 min each, followed by antigen recovery. The sections were then incubated in 3% H2O2 at 37 °C for 15 min, washed with PBS, incubated with goat serum for 30 min at 37 °C, and then incubated with primary anti-Nrf2 antibody (1:100, Abcam, Cat# ab137550) at 4 °C overnight. The next day, the sections were incubated with secondary goat anti-rabbit antibody (ZsBio, Beijing, China) for 45 min at 37 °C followed by an ABC working solution (ZsBio) for 25 min at 37 °C and incubated with 3,3-diaminobenzidine (ZsBio). Ten visual fields were randomly chosen for each sample under a LEICA DM600B automatic microscope (Leica Microsystems, Heidelberg GmbH, Germany). Nrf2 expressions were quantified using Image-Pro Plus 6.0 (Media Cybernetics), and the mean density value (integrated optical density divided by the relevant area) was calculated for each visual field.

Plasma oxidative and anti-oxidative factors in bladder, including advanced glycation end products (AGEs), reactive oxygen species (ROS), superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione (GSH), were assayed using kits obtained from Jiancheng Bioengineering Co., Nanjing, Jiangsu, China, following the instructions.

Bladder tissues were placed in RIPA lysis buffer, containing a protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA). Cytoplasmic and nuclear proteins were extracted from fresh tissues, following the kit instructions (Invent Biotechnologies. Inc., sc-003). Protein samples (30 μg) were separated with SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes (Amersham Biosciences, Piscataway, NJ, USA). The membrane was blocked with blocking buffer (TBS, 0.1% Tween-20, 5% non-fat milk or 2% BSA), for 2 h at room temperature, and incubated with primary antibodies against Nrf2 (1:1000, Abcam, Cat# ab137550), NO-1(1:1000, Abcam, Cat# ab13248), NQO1 (1:1000, Abcam, Cat# ab34174), Caspase3 (1:1000, Cell Signaling Technology, Cat# 9662), Bax (1:1000, Cell Signaling Technology, Cat# 41,162), Bcl-2 (1:1000, Cell Signaling Technology, Cat# 15,071), pro-NGF (1:1000, Abcam, Cat# ab52918), NGF (1:1000, Cell Signaling Technology, Cat# 2046), lamin B (1:1000, Cell Signaling Technology, Cat# 13,435) and β-actin (1:5000, Santa Cruz, Cat# sc-8432), at 4 °C overnight. Goat anti-rabbit IgG horseradish peroxidase (1:10,000, Abcam, Cat# ab6721) or goat anti-mouse IgG horseradish peroxidase (1:5000, Abcam, Cat# ab6789) were incubated for 60 min at room temperature the next day, respectively. The blots were developed with ECL kit (Applygen Technologies Inc, Beijing, China) and finally exposed to X-ray films. Relative protein expression levels were quantified by optical density analysis (Quantity-One software, Bio Rad Gel Doc 1000, Milan, Italy) and normalized to β-actin.

All the data were obtained from at least three independent experiments, and the representative results are presented. All results are expressed as mean ± SD. Statistical analysis was conducted using Prism 7 software (GraphPad Software). Statistical significance was evaluated using the unpaired two-tailed Student’s t-test or one-way ANOVA with post-hoc test among more than two groups. The significant difference was considered if p < 0.05 (n ≥ 3).