Animal models and treatment protocols
The animal study was approved by the Experimental Animal Ethical Committee of Huaihe Hospital of Henan University and performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Male Sprague–Dawley rats (weighing 250 ± 20 g) were purchased from Shanghai Science Academy Animal Center (Shanghai, China) and housed in the standard laboratory conditions. All rats were allowed free access to food and water ad libitum at a controlled temperature room with a constant 12-h light/dark cycle. After a week of adaptive feeding, these rats were randomly divided into 5 groups (n = 10/group): NC group, DN group, DN + BBR (50 mg/kg), DN + BBR (100 mg/kg), and DN + BBR (200 mg/kg). Rats in DN group, DN + BBR (50 mg/kg), DN + BBR (100 mg/kg), and DN + BBR (200 mg/kg) were intraperitoneally injected with 65 mg/kg streptozotocin (STZ, Sigma-Aldrich, St. Louis, MO, USA) dissolved in a 0.1 mM chilled citrate–phosphate buffer (pH 4.5) to induce diabetes . The rats in NC group were injected with chilled citrate–phosphate buffer (0.1 mM, pH 4.4). Control rats in DN group received an equal amount of citrate–phosphate buffer alone by intraperitoneal injection. When the fasting blood glucose levels from tail vein of STZ-induce diabetic rats were over 16.7 mM at 5 days after STZ injection, these rats were considered as diabetes. One week later, the diabetic rats in DN + BBR (50 mg/kg), DN + BBR (100 mg/kg), and DN + BBR (200 mg/kg) were orally treated with BBR dissolved in 0.5% carboxymethyl cellulose at a dose of 50, 100 or 200 mg/kg every day, respectively. The volume of 0.5% carboxymethyl cellulose to dissolve the different doses of BBR (50, 100, and 200 mg/kg) was 4, 2, and 1 ml, respectively. Meanwhile, the rats in NC group and DN group were gavaged with the same volume of 0.5% carboxymethyl cellulose. The fasting blood glucose and body weight were measured every 2 weeks for 8 weeks. The rats were sacrificed at 8 weeks after BBR treatment. Blood samples were collected, and the serum was separated by centrifugation and stored at − 80 °C until analysis. Meanwhile, right kidney samples were rapidly excised, weighted, and stored at − 80 °C until analysis. The ratio of kidney weight to body weight was considered as kidney weight index.
At the end of the experiment, the animals were maintained in metabolic cages for 24 h to harvest urine for assessing 24-h urinary protein with an enzyme-linked immunosorbent assay (ELISA) kit (Runyu Biotechnology Co., Shanghai, China). The fasting blood glucose level was determined based on the glucose oxidase-catalyzed reaction (chemistry analyzer; Auto Analyzer Quik-Lab, Ames, Spain). To assess renal function, blood urea nitrogen and serum creatinine in the serum of blood samples were measured using an automatic biochemistry analyzer (Hitachi, Tokyo, Japan).
Determination of IL-1β, IL-6, and MCP-1 levels
Renal corticals were homogenized and centrifuged at 9000×g for 30 min at 4 °C. The levels of proinflammatory cytokines in kidney homogenate and serum, including IL-1β, IL-6, and MCP-1, were determined using commercially acquired ELISA kits (Abcam Inc., Cambridge, MA, USA).
Cell culture and treatment
Conditionally immortalized mouse podocytes were purchased from Yubo Bio-Technique Co. Ltd (Shanghai, China) and cultured in RPMI 1640 medium (Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone), 100 U/ml penicillin/streptomycin, 5.6 mM glucose (Dingguo Changsheng Biotechnology Co., Ltd., Beijing, China) and 10 U/ml recombinant mouse interferon-γ (IFNγ; Pepro Technology, Rocky Hill, NJ, USA) at 33 °C in a 5% CO2 humidified incubator. To investigate the effect of BBR on DN, podocytes were pre-treated with 30 mM high glucose (HG) for 24 h prior to treatment with BBR at a dose of 10, 30 or 90 μM for 24 h. In some experiment, podocytes were pre-treated with 30 mM HG in the presence of TLR4 antagonist resatorvid (TAK-242, 1 μΜ; ApexBio, Houston, TX, USA), NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC; 50 μM; Sigma), or combined with NF-κB activator phorbol myristate acetate (PMA, 100 ng/ml; Sigma), followed by treated with 30 μM BBR for 24 h.
Quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from treated podocytes with TRIzol reagent (Invitrogen Invitrogen, Carlsbad, CA, USA) and quantified by NanoDrop 2000/2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Complementary DNA (cDNA) was synthesized from 1 µg total RNA by reverse transcription using a high capacity cDNA reverse transcription kit (TaKaRa, Tokyo, Japan). qPCR analysis of interleukin (IL)-1β, IL-6, and MCP-1 mRNA was performed with SYBR Premix ExTaq II kit (TaKaRa) and specific primers on an Applied Biosystems 7900 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). The relative quantification of mRNA levels was calculated based on the 2−ΔΔCt method and normalized to GAPDH. The primers were as follows: GAPDH, forward: 5′-CAG TGC CAG CCT CGT CTA T-3′, reverse: 3′-AGG GGC CAT CCA CAG TCT TC-5′; IL-1β, forward: GTG ATG TTC CCA TTA GAC AGC, reverse: CTT TCA TCA CAC AGG ACA GG; IL-6, forward: 5′-ATG AAC TCC TTC TCC ACA AGC GC-3′, reverse: 5′-GAA GAG CCC TCA GGC TGG ACT G-3′; MCP-1, forward: 5′-TCA GCC AGA TGC AGT TAA CGC-3′, reverse: 5′-TGA TCC TCT TGT AGC TCT CCA GC-3′.
Western blot analysis
Kidney homogenate and podocytes were collected and lysed in cell lysis buffer (Beyotime, Haimen, China) with protease inhibitor cocktail and phosphatase inhibitor (both from Sigma-Aldrich) for protein extraction. Equal amount of protein lysates (30 μg) were separated by 10% serum dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and electrotransferred onto nitrocellulose (NC) membranes (Millipore, Billerica, MA, USA). After being blocked with 5% non-fat dry milk in PBS for 1 h, the membranes were probed with the primary antibodies against TLR4, phosphorylated-p65 (p-p65), p65, p-IκBα, IκBα, Cleaved Caspase-3, Bcl-2 and β-actin (all from Santa Cruz Biotechnology, Santa Cruz, CA) at 4 °C overnight, followed by incubated with a horseradish peroxidase-conjugated secondary antibody (Invitrogen) for 2 h at room temperature. Peroxidase-labeled protein bands were detected by enhanced chemiluminescence reagents (Millipore) and the protein intensity was quantified with Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, MD, USA).
Podocytes were double stained with FITC-Annexin V and propidium iodide (PI) from a FITC Annexin V Apoptosis Detection Kit I (BD Biosciences, San Jose, CA, USA). The apoptotic rats were analyzed using a FACScan flow cytometer (BD Biosciences).
Data are displayed as mean ± standard deviation (SD). Statistical analysis was performed with GraphPad Prism 5 software (GraphPad Software Inc., San Diego, CA, USA). Comparison among experimental groups was performed using unpaired two-tailed Student’s t test and analysis of variance (ANOVA), with a value of P < 0.05 being considered as statistically significant.