Clinically used BHD concentrated Chinese Medicine Granules (CCMG) was obtained from PuraPharm International Ltd. (Hong Kong, China). Antibodies specific for fibronectin, collagen I, TNF-α and IL-1β were purchased from Abcam (Cambridge, UK). Antibodies for NF-κB p65, phosphor-NF-κB p-p65 and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-Smad3 and anti-phospho-Smad3 (Ser423/425) antibodies were purchased from Cell Signaling Technology (CST, Danvers, MA, USA). Antibody anti-Smad7 was purchased from R&D Systems (R&D, Oxford, UK). Antibody for F4/80 was purchased from AbD Serotec (Oxford, UK). Anti-Arkadia antibody was purchased from Thermo Scientific (Waltham, MA, USA). Compounds for quality control analysis, including calycosin-7-glucoside, hydroxysafflor yellow A and amygdalin, were purchased from Nantong FeiYu biological technology Co., Ltd., China. Ferulic acid and paeoniflorin were purchased from Jiangsu Yongjian Pharmaceutical Co., Ltd., China. All standards were of purity greater than 98%.
BHD and compounds solution
BHD concentrated Chinese Medicine Granules (CCMG) (PuraPharm Nongs) were used in our study. For qualitative and quantitative analyses, BHD was dissolved in water (100 mg/ml) under ultrasonic for 10 min at 35 ℃. According to Chinese Pharmacopoeia (2020 edition), the representative active chemical ingredients of Astragali Radix, Angelicae Sinensis Radix Tail, Paeoniae Radix Rubra, Chuanxiong Rhizoma, Persicae Semen and Carthami Flos are calycosin-7-glucoside, ferulic acid, paeoniflorin, ferulic acid, amygdalin and hydroxysafflor yellow A, respectively (Additional file 1: Table S1). The standard chemical ingredients calycosin-7-glucoside (0.05 mg/ml), hydroxysafflor yellow A (0.1 mg/ml), amygdalin (0.35 mg/ml), ferulic acid (0.1 mg/ml), paeoniflorin (0.35 mg/ml) were dissolved together in 50% methanol to prepare a mixed standard working solution.
Quality control analysis
We performed the HPLC fingerprint study on BHD. Chromatographic separations were operated on a Thermo UPLC system with ACE Excel 2 C18 column (100 × 2.1 mm, 2 μm). The following chromatographic parameters were optimized in the study: mobile phase A (acetonitrile) and mobile phase B (0.1% formic acid–water), the flow rate at 0.4 ml/min, column temperature at 25 ℃, and detection wavelength at 227 nm. The whole retention time was 50 min. The percentage of mobile phase A increased from 1 to 10% during the first 10 min, then the ratio of two mobile phases was held for the next 38 min before the percentage of phase A raised to 15% in the last 2 min.
Quantitative quality control analysis
Five standard chemical ingredients were used to perform quantitative analysis. After obtaining the standard curve of each chemical ingredient, mixed standard working solutions were analyzed by HPLC under the same condition as the Quality Control Analysis of BHD. The measurement was conducted 3 times parallelly. According to the standard curve, the content of each standard chemical ingredient was calculated.
Animals and STZ-induced diabetic nephropathy mouse model
Male ICR mice (10–12 weeks old) were supplied from Laboratory Animal Unit at the University of Hong Kong. All experimental protocols of animals were approved by the Committee on the Use of Live Animals in Teaching and Research (CULATR). The mice were maintained on 12-h light/dark cycles in a pathogen-free environment with a constant temperature of 22 ℃. The type I diabetes model was induced in mice according to the low-dose STZ induction protocol recommended by the Animal Models of Diabetic Complications Consortium (https://www.diacomp.org/). Streptozotocin (STZ, S0130, Sigma-Aldrich Corp, St. Louis, MO, USA) was freshly prepared in 0.1 M sodium citrate buffer (pH 4.5). Mice were fasted for 4 h before STZ administration. After 4 h fasting, mice were received a daily intraperitoneal injection of 50 mg/kg STZ or sodium citrate buffer for 5 consecutive days. Fasting blood glucose and urine were collected every 2 or 4 weeks. The mice were sacrificed at week 16 after STZ injection. Kidney tissues were collected and stored at – 80 ºC or paraffin-embedded for subsequent experiments.
Microalbumin and renal function
Renal function was evaluated by urinary albumin excretion (UAE), the ratio of total urinary albumin/creatinine. For urine analysis, 24 h urine samples were collected from metabolic cages every 2–4 weeks. Microalbuminuria was quantified using the competitive ELISA method (Exocell, Philadelphia, PA, USA) and creatinine was detected by the Creatinine Companion kit (Exocell, Philadelphia, PA, USA) according to the manufacturers' instructions.
Renal histology and immunohistochemistry
The kidney tissues were fixed with 4% paraformaldehyde (PFA) and embedded in paraffin, then cut into 5 μm sections as previously described . Mesangial matrix expansion was measured by Periodic acid Schiff (PAS) staining, which was scored by determining the percentage of tubules that displayed tubular necrosis, cast formation, and tubular dilation as follows: 0 = normal; 1 = 1–10%; 2 = 10–25%; 3 = 26–50%; 4 = 51–75%; 5 = 75–95%; 6 = > 96% . Masson Trichrome staining was performed to determine renal fibrosis resulting from extracellular matrix deposition (ECM) by the NovaUltraTM Masson Trichrome Staining kit (IHC World, Woodstock, MD). ECM was examined by ten random view fields (at 400 ×) for each kidney. The immunohistochemical staining for F4/80, fibronectin, IL-1β, phospho-Smad3, phospho-p65 were conducted as described previously .
Cell culture and drug treatments
Mouse mesangial cells (MCs) were used in our study. Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM)/Ham's F12 medium (Invitrogen Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen life Technologies) and 1% penicillin/streptomycin (Gibco) at 37 °C in a humidified atmosphere with 5% CO2. The MCs were cultured in an FBS-free medium for 24 h and then stimulated with BHD or compounds under normal D-glucose (5.5 mM) or high D-glucose (35 mM) conditions for up to 24 h. D-Mannitol (29.5 mM) was used as an osmotic control. MCs were pre-treated with 2 μM SIS3 for 1 h before stimulating with high glucose for positive control groups. Cells were harvested for western blot and real-time PCR analysis. All experiments were conducted three or four times.
BHD treatments in animals
Mice orally received BHD at 0.5 g/kg, 1 g/kg and 2 g/kg daily for 12 weeks from week 4 after STZ injection. Irbesartan (IRB) (50 mg/kg per day) was taken as a positive control.
RNA extraction and RT-qPCR
Total RNA was isolated from harvested cells using an RNA extraction kit (RNeasy; Qiagen, Valencia, CA, USA). Real-time PCR was performed using the Bio-Rad iQ SYBR Green supermix with Opticon 2 (Bio-Rad, Hercules, CA, USA) as previously described [3, 10]. Fibronectin (FN) and IL-1β were analyzed by RT-qPCR analysis. β-actin was used as an internal control. The primers used in this study are listed as follows:
Mouse Fibronectin, forward: 5′-TACCAAGGTCAATCCACACCCC-3′, reverse: 5′-CAGATGGCAAAAGAAAGCAGAGG-3′
Mouse IL-1β, forward: 5′-CTTCAGGCAGGCAGTATCACTCAT-3′, reverse: 5′-TCTAATGGGAACGTCACACACCAG-3′
Western blot analysis
Protein was extracted from renal tissues or cells by RIPA buffer, and western blot analysis was carried out as described previously . Briefly, membranes were incubated with primary antibodies against fibronectin, collagen I, TNF-α, IL-1β, NF-κB p65, phospho-NF-κB p-p65, Smad3, phospho-Smad3 (Ser423/425), Smad7, Arkadia and β-actin at 4 °C overnight, and then the membranes were further incubated in HRP conjugated second antibodies. The signals of proteins were detected by chemiluminescent ECL™ Detection Kit (GE Healthcare), followed by scanning using Gel Doc system (Bio-Rad) and analyzing by Image Lab software (Bio-Rad). Protein levels were quantified using the ImageJ software (NIH, Bethesda, MA, USA).
Data are shown as the mean ± SEM and were analyzed using one-way analysis of variance (ANOVA), followed by Tukey’s post-hoc tests using GraphPad Prism Version 7.0 software (GraphPad Software Inc., CA, USA). A value of p < 0.05 was considered statistically significant.