Plant material and extraction
The tuberous root of L. spicata was purchased from a local market in Pingtung County (Taiwan) on May 2012. Plant identification was done by Professor Hong T.Y. (the Department of Biotechnology, Collage of Pharmacy and Health Care, Tajen University). Random amplified polymorphic DNA analysis of Liriopes Radix supplied was also performed to identify DNA polymorphisms. The voucher specimen (Lot No. LS20120523) has been deposited in our laboratory.
Preparation of PSLS was performed according to the previous study . The powdered material (200 g) was boiled in distilled water three times (1:4, 1:4, 1:2, w/v), 0.5 h each time. Each extract was then filtered and merged together. Phosphate buffer (pH 5.91) and 0.6 g of papain (12 u/mg, Biosharp, USA) were added into the extracts and kept in water-bath (45°C) for 2 h to remove proteins. After boiled for 5 min, the extracts were stored overnight at 4°C and filtered next morning. The filtrate was purified by using the mini pellicon system (ultrafiltration membrane Mw cut-off 1000, Millipore, USA) with 12 l distilled water. The retentate portion was concentrated and then separated in DEAE-cellulose 52 columns to remove pigments. The water eluted solution was concentrated and finally lyophilized (Alpha 2–4, Christ, Germany) and obtained 53.2 g of PSLR from 200 g powdered material. The sugar content of PSLR was 98.1% determined by anthrone-sulfuric acid method at 625 nm using fructose as standard .
Male Wistar rats (8–10 weeks of age, 200–250 g) were obtained from the Animal Center of National Cheng Kung University Medical College. To induce diabetes rats were given a single intravenous injection of 60 mg/kg streptozotocin (STZ; Sigma-Aldrich, Inc., Saint Louis, Missouri, USA). Animals were considered to be diabetic if they had plasma glucose concentrations of 350 mg/dl or greater, in addition to polyuria and other diabetic features. All studies were carried out two weeks after the injection of STZ. All animal procedures were performed according to the Guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health (United States), as well as the guidelines of the Animal Welfare Act. These studies were conducted with the approval of the Institutional Animal Care and Use Committee (IACUC) at Tajen University (approval number: IACUC 100–29; approval date: December 22, 2011).
The selection of dosage regime for the present studies was according to the previous report that demonstrated administration with PSLR at 200 and 300 mg/kg for 4 weeks exerted potential effect in improving hyperglycemia in diabetic mice . In order to obtain the significant effect of PSLR on improvement of DN, STZ-diabetic rats were dosed by oral gavage once per day for eight weeks with PSLR doses of 200 or 300 mg/kg in a volume of 1.5 ml/kg distilled water. A vehicle-treated group of STZ-diabetic rats and normal rats was treated with 1.5 ml/kg distilled water only over the same treatment period. Animals received standard rat diet (Harlan Teklad, Madison, WI, USA; Cat. No. 2018) and water ad libitum throughout the entire treatment period. PSLR treatment was continued even though the plasma glucose of STZ-diabetic rats was lower than 350 mg/dl during the eight-week treatment period. The evening prior to blood sample collection animals were restricted to 3 g of chow (given at 18:00), which was consumed immediately, and thereafter had access to only water. The animals were transferred to metabolic cages (Shineteh Instruments Co., Ltd, Taipei, Taiwan) for urine collection 24 hours before sacrifice. Urine was collected under a layer of toluene (to inhibit bacteria growth) and stored at 4°C until analyzed. Toluene used had no detectable effect on the estimation of albumin and creatinine in the urines collected in the metabolism experiments.
At the end of the eight-week treatment, rats were sacrificed using an intraperitoneal injection of sodium pentobarbital (50 mg/kg). The kidneys were dissected and rinsed with cold isotonic saline and weighed. An index of renal hypertrophy was estimated by comparing the wet weight of the left kidney to total body weight. The cortical tissues from right kidney were stored immediately at -80°C in liquid nitrogen for biochemical determinations and Western blot analyses. Other kidney tissues were fixed in 10% neutralized formalin for histology and immunohistochemistry.
Blood sampling and analysis
Blood sample of rats were centrifuged at 2,000 × g for 10 minutes at 4°C, plasma was removed and aliquot for the respective analytical determinations. The diagnostic kit for determinations for plasma levels of glucose (Cat. No. COD12503) was purchased from BioSystem (Barcelona, Spain). The plasma creatinine (Cr) concentration was determined by the commercial assay kit (Cat. No. 221–30) purchased from Diagnostic Chemicals Limited (Connecticut, USA). Blood urea nitrogen (BUN) was determined by kinetic reagent (Diagnostic Chemicals Limited, Cat. No. 283–30). Commercial enzyme-linked immunosorbent assay (ELISA) kits were used quantify glycosylated hemoglobin (HbA1c) levels (Intergrated Bio Ltd., Taipei, Taiwan; Cat. No. CSB-E08140r). Kits for determination of plasma alanine aminotransferase (ALT; EC 188.8.131.52) (Cat. No. A524-780TM) and aspartate aminotransferase (AST; EC 184.108.40.206) (Cat. No. A559-780TM) concentrations were purchased from Teco Diagnostics (CA). Assays were performed according to the manufacturer’s instructions. All experiments were performed in triplicate to ensure the accuracy of the observations.
Analysis of urine parameters
The 24-h urine collected from each diabetic rat and age-matched control was centrifuged at 2,000 g for 10 min. Urinary albumin concentrations were measured by Nephrat II ELISA kit (Cat. No. NR002) obtained from Exocell, INC. (PA, PUA). The concentration of Cr in pooled urine samples was determined by the commercial assay kit (Diagnostic Chemicals Limited, Cat. No. 221–30). All analyses were performed in triplicate in accordance with the manuals provided by the manufacturers. Creatinine clearance (Ccr) was calculated in individual rats as follows: Ccr = urine creatinine × urine volume/plasma creatinine × time .
Renal cytokines determination
Renal tissue was homogenized in 10 mmol/L Tris–HCl buffered solution (pH 7.4) containing 2 mol/l NaCl, 1 mmol/l EDTA, 0.01% Tween 80, 1 mmol/l PMSF, and centrifuged at 9000 × g for 30 min at 4°C . The resultant supernatant was used for cytokine determination. The levels of proinflammatory and anti-inflammatory cytokines were estimated by performing ELISA using commercial kits. ELISA kits for the determination of tumor necrosis factor (TNF)α (Cat. No. ab46070), interleukin (IL)-6 (Cat. No. ab100772), and IL-10 (Cat. No. ab100764) were obtained from Abcam Inc. (Cambridge, MA, USA). Samples were assayed in triplicate according to manufacturer’s instructions. The protein concentrations of kidney filtrate were determined according to the previous method using BSA as a standard .
Renal histological analysis
Renal tissues were fixed with 10% neutral formalin phosphate buffer, dehydrated through a graded alcohol series and embedded in paraffin, cut into 4 μm sections and stained with hematoxylin and eosin (H&E). The sections were examined with light microscopy by an experienced pathologist and micrographs from six glomerulums were obtained randomly with magnification of 400 ×. Mean glomerular volume was determined from the mean glomerular capillary tuft area (AG) by light microscopy of PAS sections. The areas were determined by light microscopy and analyzed by dedicated software (Analysis 3.0, Soft Imaging System, Münster, Germany) as the average area of 50 glomerular profiles (the capillary tuft omitting the proximal tubular tissue and Bowman’s capsule) for each animal. Glomerular volume (GV) was calculated using the formula GV = β/k × (AG)3/2, where GV is glomerular volume, β = 1.38, which is the shape coefficient for spheres (the idealized shape of glomeruli), k = 1.1, which is a size distribution coefficient, and AG is the glomerular capillary tuft area . The index of mesangial expansion was scored by a quantitative estimate of the mesangial zones width in each glomerulus, expressed as a function of the total glomerular area : 0, normal glomeruli; 1, matrix expansion occurred in up to 50% of a glomerulus; 2, matrix expansion occurred in 50-75% of a glomerulus; 3, matrix expansion occurred in 75-100% of a glomerulus.
Formalin-fixed, paraffin-embedded kidney tissue sections were used for immunohistochemical staining. After deparaffinization and hydration, the slides were washed in Tris-buffered saline (TBS; 10 mmol/L Tris HCl, 0.85% NaCl, pH 7.2). Endogenous peroxidase activity was quenched by incubating with the slides in methanol and 0.3% H2O2 in methanol. After overnight incubation with Mouse monoclonal anti-rat monocyte/macrophage antibody (anti-ED-1) (sc-59103; Santa Cruz Biotechnology Inc. CA, USA) at 4°C, the slides were washed in TBS and horseradish peroxidase (HRP)-conjugated goat anti-mouse secondary antibody was then added and the slides were further incubated at room temperature for 1 h. The slides were washed in TBS and incubated with diaminobenzidine tetrahydrochloride as the substrate, and counterstained with hematoxylin. A negative control without primary antibody was included in the experiment to verify the antibody specificity. Intraglomerular ED-1-positive cells were counted in 200 glomeruli/group under 400-fold magnification.
Protein extraction of isolated kidney was performed as follows . The sample was homogenized in ice-cold in 1 ml of hypotonic buffer A [10 mmol/l HEPES (pH 7.8), 10 mmol/l KCl, 2 mmol/l MgCl2, 1 mmol/l DTT, 0.1 mmol/l EDTA, 0.1 mmol/l phenylmethylsulfonylfluoride]. A solution of 80 μl of 10% Nonidet P-40 was added to the homogenates, and the mixture was centrifuged for 2 min at 14,000 × g. The supernatant was collected as a cytosolic fraction for the assays of CD4, CD8, nephrin, podocin, and inhibitory kappa B kinase (IκB)α. The supernatant containing nuclear proteins was collected for nuclear factor kappa B (NF-κB) p65, p38 mitogen-activated protein kinase (p38 MAPK), and phospho (p)-p38 MAPK (Thr180/Tyr182).
Before immunoblotting, and the protein concentration of each tissue was determined using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Japan) and bovine serum albumin as a standard, to ensure equal loading among lanes. Cytosol (70 μg total protein) and nuclear extracts (50 μg total protein) were separated on a 7.5-15% polyacrilamide gel and electophoretically transferred to nitrocellulose membrane. Membranes were blocked with 5% non-fat dry milk in Tris-buffered saline Tween (20 mmol/l Tris, pH 7.6, 137 mmol/l NaCl, and 0.1% Tween 20) for 3 h at room temperature, followed by an overnight incubation at 4°C with with primary antibodies CD4 (Santa Cruz Biotechnology, Inc., Cat. No. sc-7219), CD8 (Santa Cruz Biotechnology, Inc., Cat. No. sc-7188), nephrin (Santa Cruz Biotechnology, Inc., Cat. No. sc-28192), podocin (Santa Cruz Biotechnology, Inc., Cat. No. sc-21009), NF-κB p65 (Santa Cruz Biotechnology, Inc., Cat. No. sc-109), IκBα (Cell Signaling Technology, Beverly, MA, USA; Cat. No. 9242), p38 MAPK (Cell Signaling Technology; Cat. No. 9212), p-p38 MAPK (Thr180/Tyr182) (Cell Signaling Technology; Cat. No. 9211), or β-actin (Santa Cruz Biotechnology, Inc.; Cat. No. sc-130656). All antibodies were used at a dilution of 1:1000. Three times after washing with Tris-buffered saline Tween 20 (TBST), incubation with appropriate horseradish peroxidase-conjugated secondary antibodies were performed for 1 h at room temperature. After three additional TBST washes, the immunoreactive bands were visualized by enhanced chemiluminescence (Amersham Biosciences, Buckinghamshire, UK) according to the manufacturer’s instructions. Band densities were determined using ATTO Densitograph Software (ATTO Corporation, Tokyo, Japan) and quantified as the ratio to β-actin. The mean value for samples from the vehicle-treated normal rats on each immunoblot, expressed in densitometry units, was adjusted to a value of 1.0. All experimental sample values were then expressed relative to this adjusted mean value. Tissue sections were sampled from 4 independent experiments.
The results are presented as the mean ± standard deviation (SD) for each group of animals at the number (n) indicated. Statistical analysis was performed with one-way analysis of variance (ANOVA). The Dunnett range post-hoc comparisons were used to determine the source of significant differences where appropriate. The renal morphohistology and the morphologic analysis for PAS staining were analyzed statistically using the Kruskal-Wallis Test and Dunn’s Multiple Comparisons Test. Values of P < 0.05 were considered statistically significant.