Sanziguben (SZGB) is composed of four Chinese herbs: G. pentaphyllum (Thunb.) Makino, Chinese R. laevigata Michx, S. chinensis Fructus, and P.emblica and Fructus. The crushed herbs of Schizandrae were purchased from Daxiang Pharmaceuticals Inc. (Guangzhou, China).
The reference standard of glucose and uronic acid were provided by Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). Moreover, metformin hydrochloride tablets were purchased from Sino-American Shanghai Squibb Pharmaceuticals Ltd. (Shanghai, China). STZ was obtained from Sigma-Aldrich (Sacramento, CA, USA). All test assay kits and enzyme-linked immunosorbent assay (ELISA) kits were supplied by NanJing JianCheng Bioengineering Institute (Nanjing, China). Tumor necrosis factor-alpha (TNF-α) was obtained from PeproTech Inc. (Cranbury, NJ, USA). Lipopolysaccharides (LPS) were provided by Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). NF-κB p65 (1:2,000), phospho-NF-κB p65(Ser536; 1:2,000), and phospho-IκBα (Ser32; 1:2,000) were purchased from Cell Signaling Technology (Beverly, MA, USA). The radioimmunoprecipitation assay (RIPA) lysis buffer with protease/phosphatase inhibitor cocktail, bicinchoninic acid (BCA) protein assay, anti-β-actin antibodies (1:2,000), and horseradish peroxidase (HRP)-conjugated secondary antibodies was purchased from Beijing ComWin Biotech Co., Ltd. (Beijing, China). All other chemical reagents were of analytical grade.
Dried SZBG herbs were pulverized and screened through a 50-mesh sieve. The powder was defatted with petroleum ether for 24 h at room temperature under reflux to remove some colored materials and oligosaccharides. The residue was extracted with distilled water at 80°C twice and 2.5 h for each time after filtration. The whole extract was filtered and concentrated in a rotary evaporator under reduced pressure at 60°C for fivefold and then centrifuged at 3,000 rpm for 15 min. Moreover, the extract was then precipitated by the addition of four volumes of 95% ethanol at 4°C overnight. Moreover, the polysaccharide was obtained by centrifugation (4,000 rpm for 10 min). The solution was reprecipitated by the addition of 95% ethanol as described above, and the resultant precipitate was successively washed with anhydrous ethanol and then dried under reduced pressure to obtain SZP.
Male C57BL/6 mice (4 weeks old) were purchased from the Experimental Animal Center of Guangzhou University of Chinese Medicine (Guangdong, China). Mice were housed in a room with a temperature of 23°C ± 1°C and 55% ± 5% relative humidity with a 12-h light/12-h dark cycle. All animal experiments were approved by the Institutional Animal Care and Use Committee of Guangzhou University of Chinese Medicine (license no: SCXK 2013-0020). All animal welfare in this study followed the guidelines for ethical review of animal welfare in the People’s Republic of China.
Induction of mice and drug administration
The HFD plus STZ injections were used to replicate the diabetic models in mice[11, 12]. MET is the first-line drug for the treatment of diabetes and DN. Therefore, MET is selected as a positive control drug. Moreover, the mice were randomly divided into the diabetic and sham groups 1 week after adjusting to the new environment. Mice were fed with either HFD (45% of calories from fat) or standard chow diet (10% of calories from fat) for 10 weeks. Mice in the diabetic group were fed with HFD to accelerate the development of diabetic kidney disease. The diabetic group was intraperitoneally injected with 100 mg/kg STZ dissolved in citrate buffer (pH 4.0). Consequently, mice appeared hyperglycemic with fasting blood glucose (FBG) levels of 11.1–33.3 mmol/L 3 days later. The normal control group was the group that was injected with the citrate buffer without STZ. The following groups were generated for this current study: (1) Sham:normal control mice treated with distilled water (n = 20), (2) DN:diabetic mice treated with distilled water (n = 20 ), (3) SZP-L:diabetic mice treated with SZP (1.01 g/kg body weight/day; n a day for 8 weeks.
= 20), (4) SZP-H:diabetic mice treated with SZP (2.02 g/kg body weight/day; n = 20 ), (5) SZGB:diabetic mice treated with SZGB (4.7 g/kg body weight/day; n = 20 ), and (6)MET: diabetic treated with metformin (300 mg/kg body weight/day; n = 20 ). All mice were treated once
All mice were euthanized at 8 weeks after diabetes induction, and blood samples were collected and centrifuged (3,500 rpm for 10 min) to obtain serum samples. Moreover, the kidneys were removed. The renal cortex of the kidney was isolated immediately and stored in liquid nitrogen for pathological and molecular studies.
Cultivated HG stimulates HK-2 cells
The HK-2 cells were purchased from China Center for Type Culture Collection (Wuhan, China) and grown in Dulbecco’s modified eagle’s medium/nutrient mixture F-12 (DMEM-F12, 1:1; GIBCO, Life Technologies, Carlsbad, CA, USA), which contained 10% fetal bovine serum (GIBCO) and 1% antibiotic–antimycotic solution (GIBCO). Cells were cultured at 37℃ in a humidified atmosphere with 5% CO2. According to the different experiments, different stimuli were used in this study for (1) the cells to be stimulated with normal glucose as normal control, (2) for HG treatment (a final concentration of 90 mmol/L in culture medium), and (3) SZP (a final concentration of 100 or 200 μg/mL in culture medium). Moreover, all cell experiments were repeated thrice. Cell proliferation and morphology were measured using the IncuCyte ZOOM System (Essen, BioScience Inc., Ann Arbor, MI, USA).
Cultivated LPS + TNF-α stimulates HK-2 cells
HK-2 Cells were cultured at 37℃ in a humidified atmosphere with 5% CO2 and were subcultured at 80% confluence using 0.25% trypsin-EDTA solution. The cells were exposed to DMEM-F12 containing 30 ng/mL TNF-α and 30 μg/mL LPS for 16 h to induce an inflammatory milieu. The experiments were divided into the following groups: (1) cells grown in DMEM-F12 complete medium as normal control, (2) cells stimulated by TNF-α (30 ng/mL) and LPS (30 μg/mL) for 16 h to produce inflammatory damage as a model, and (3) 100 or 200 μg/mL SZP simultaneous treatment on the model group. All cell experiments were repeated thrice. Cell proliferation and morphology were measured using the IncuCyte ZOOM System.
Chemical components and monosaccharide composition of SZP
Carbohydrate contents were measured by the phenol-sulfuric acid colorimetric method with glucose as equivalents. Protein contents were measured by Coomassie brilliant blue reaction using bovine serum albumin as the standard. Uronic acid content was determined by the carbazole-sulfuric acid method with glucuronic acid as the standard.
Monosaccharide composition analysis
The SZP monosaccharide compositions were analyzed by gas chromatography (GC) using an Agilent GC-6890A-5975C system equipped with a Hypercarb column. After trifluoroacetate at 121°C for hydrolysis with 2 mol/L trifluoroacetate at 121°C detected with a flame ionization detector (240°C), the column temperature was increased from 170°C for 2 min and 240°C with 6°C/min holding for 60 min. Moreover, the conversion of hydrolysate into distilled water was used as previously described.
Infrared spectroscopic analysis of polysaccharides
SZP was characterized by Fourier transform infrared spectroscopy (FTIR) on a Thermo Nicolet iS 5N infrared spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) at 25°C using KBr pellets. Samples were dried at 60°C in a vacuum drying oven for 48 h before analysis, and spectra were scanned between 4,000 and 400 cm−1 with a resolution of 4 cm−1.
Measurement of body weight, blood glucose, and urinary protein
The bodyweight of mice was measured at 2-week intervals. Blood glucose was detected using the Roche Dynamic Blood Glucose Monitoring System (Roche Inc., Mannheim, Germany) by blood sampling from the tail vein. Mice were kept separately in metabolic cages for 24-h urinary collection. Urinary protein was evaluated using the Bradford method.
At the end of the experiment, blood samples and cell supernatants were centrifuged at 3,000×g for 10 min. Blood samples were separated for the detection of total cholesterol (TC), triglycerides (TG), serum creatinine (Cr) and serum urea (BUN), malondialdehyde (MDA), and catalase (CAT). The levels of the aforementioned biochemical indicators were measured using commercially available kits (Jian Cheng Bioengineering Institute, Nanjing, China). Meanwhile, the serum levels of TNF-α, monocyte chemotactic protein-1 (MCP-1), and interleukin 6 (IL-6) were measured using ELISA kits (Jian Cheng Bioengineering Institute).
The sections of kidney tissues were removed and immediately fixed in 4% paraformaldehyde, dehydrated through a graded alcohol series, and embedded in paraffin. Moreover, 4-µm thick sections were cut using a rotary microtome and stained with hematoxylin & eosin (H&E staining), periodic acid-Schiff (PAS), and Masson’s trichrome to evaluate the pathological changes of the kidney tissue. The stained specimens inspected were placed under a light microscope (Nikon, Tokyo, Japan) and imaged (×400).
Western blot analysis of NF-κB protein expression in renal tissues or HG-treated HK-2 cells
The renal tissues or cells were homogenized in an appropriate amount of protease and phosphatase inhibitor and centrifuged at 12,000 rpm for 10 min at 4℃. The protein concentration in the supernatants was scaled by utilizing the BCA protein assay kit. An aliquot of the supernatant (30 μg protein) was then suspended in a 4× loading buffer. It was then heated at 100°C for 5 min, electrophoresed on 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels at 80 V for 40 min, and then transferred onto polyvinylidene difluoride (PVDF) membranes by utilizing a Trans-Blot semidry transfer cell (Bio-Rad, Hercules, CA, USA) at 300 mA for 60 min. Moreover, 50 μg of the protein samples in each group was separated by 8% SDS-PAGE gel electrophoresis at 80 V for 30 min and then transferred onto the PVDF membrane at 300 mA for 70 min. After transferring, the PVDF membrane was blocked with 5% skim milk under room temperature for 2 h, followed by incubation with specific primary antibodies (NF-κB p65 was diluted at 1:1,000, and β-actin was diluted at 1:1,000) at a temperature of 4°C for 16 h. The samples were then treated with anti-NF-κB p65 and anti-β-actin antibodies (Abcam Inc., Cambridge, MA, USA) under room temperature for 2 h and incubated with secondary antibody (IRDye 800CW Goat Anti-Rabbit or IRDye 800CW Goat Anti-Mouse was purchased from LI-COR, Inc., Lincoln, NE, USA). Ultimately, the bands were analyzed and quantified with Image-Pro Plus 6.0.
Western blot analysis of NF-κB protein expression in LPS + TNF-α stimulates HK-2 cells
Total protein from cells was extracted by RIPA lysis buffer with protease/phosphatase inhibitor cocktail. The BCA protein assay was performed to determine the protein concentration according to the manufacturer’s instructions. The primary antibodies were as follows: NF-κB p65 (1:2,000), phospho-NF-κB p65 (Ser536; 1:2,000), phospho-IκBα (Ser32; 1:2,000), and β-actin (1:2,000). The membrane was stained with the HRP-conjugated secondary antibodies for 1 h at room temperature after being incubated with the primary antibody at 4°C overnight. The bands were presented by reacting with a chemiluminescent HRP substrate (WBKLS0100, Millipore, Germany). The intensities of individual bands were quantified with densitometric analysis using ImageJ (National Institutes of Health, Bethesda, MD, USA), and β-actin expression was considered as the internal reference.
Data are presented as mean ± standard deviation and analyzed using the Statistical Package for the Social Science, version 20.0 (SPSS 20.0, Armonk, NY, USA). Significant differences among groups were compared using analysis of variance, with p < 0.05 being statistically significant.