Animal model of CS
Male Wistar rats weighing 250–300 g were obtained from Japan SLC (Shizuoka, Japan) and housed in a room maintained at a temperature of 23 ± 3 °C and a relative humidity of 55 ± 15% under a 12:12-h light/dark cycle, with free access to food and water. Animal experiments were carried out according to the guidelines for animal use and approved by the Life Science Research Center of Josai University (approval nos. H26027, H26028, H27025, and H27027). Anesthesia was induced by intraperitoneal injection of sodium pentobarbital (50 mg/kg body weight). Body temperature was maintained throughout the experiment using a heating pad. The CS model was established as previously reported [12]. Briefly, a rubber tourniquet was applied to the bilateral hind limb of each rat, wrapped five times around a 2.0 kg metal cylinder, and the end of the band was glued. After a compression of 5 h, the compression was released by cutting the band and removing the tourniquet.
AS preparation
The chemical structure of AS (Carbosynth, Compton, UK) is depicted in Additional file 1: Fig. S1. The probable purity of AS was 99.6 ± 0.8%, as determined using the partial modification HPLC method (HPLC conditions: mobile phase: 50% acetonitrile, flow rate: 1.0 mL/min, column: Inertsil-ODS3 (4.6 mm × 250 mm, φ 5 μm), column temperature: 40 °C, detector wavelength: 203 nm) [13]. A 1,1-diphenyl-2-picrylhydrazyl (DPPH) antioxidant assay of AS was performed as Sharma et al. [14] previously reported. Ascorbic acid (AA) has a high antioxidant capacity and was used as an antioxidant reference for comparison with AS.
Experimental design
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Experimental-1 (AS dosage study) The AS dosages were selected using previous reports by Li et al. [15] to determine the effective dosage in CS rats. To determine the optimal dose of AS, animals were randomly divided into seven groups: (1) sham; (2) CS with no treatment (CS-only group); and (3–7) CS with treatment at 1, 2, 5, 10, and 20 mg/kg AS (named C-1, 2, 5, 10, and 20 AS-one group, respectively) via tail vein bolus injection at decompression just before from a rubber tourniquet.
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Experimental-2 (therapy effect study) To examine the effects of AS in CS, animals were randomly divided into four groups: (8) sham; (9) CS with no treatment (CS-only group); (10) CS with normal saline treatment (C-saline group); and (11) CS with normal saline + 10 mg/kg AS (C-AS group). The C-saline and C-AS groups were subjected to decompression with the rubber tourniquet, immediately followed by 3 h of reperfusion by massive fluid resuscitation at a rate of 30 mL/(kg h−1).
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Experimental-3 (pharmacokinetics (PK) study) We analyzed the blood concentration profile and PK parameters of AS administered by tail vein to sham and CS rats. Animals were randomly assigned to four groups: sham, sham-AS, CS-only, and C-AS groups.
Analysis of mean arterial pressure (MAP), blood gas levels, biochemical parameters, coagulation, and NO and cytokine levels
MAP was recorded using a PowerLab data acquisition system (AD Instruments, Nagoya, Japan). One carotid artery was cannulated with a polyethylene catheter (PE-50 tubing) connected to a pressure transducer. Arterial blood samples from each group were obtained 1, 3, 6, and 24 h after reperfusion using a carotid artery catheter. The pH, partial pressure of oxygen (PaO2), partial pressure of carbon dioxide (PaCO2), bicarbonate (HCO3
−) concentration, and base excess of arterial blood were analyzed using an i-STAT300F blood gas analyzer (FUSO Pharmaceutical Industries, Osaka, Japan).
In each experimental group (0, 1, 3, 6, and 24 h after reperfusion, n = 3–6), venous blood and tissue samples from the gastrocnemius muscles and kidneys were assayed for thiobarbituric acid reactive substances (TBARS), myeloperoxidase (MPO) activity, skeletal muscle edema index, mitochondrial permeability transition (MPT), superoxide dismutase (SOD) activity, Western blotting, and histology at each time point. For histology, tissues were fixed in 10% formalin and embedded in paraffin wax. Sections were cut and stained with hematoxylin and eosin and then carefully examined microscopically. Venous blood samples from each group were obtained using a jugular catheter. Venous blood from the jugular vein was collected and centrifuged to measure plasma levels of potassium (K+), blood urea nitrogen (BUN), creatinine (Cre), and creatine phosphokinase (CPK) (measurements were carried out by SRL Inc., Tokyo, Japan). Red blood cells (RBC), white blood cells (WBC), and platelets were measured using a Celltac hematology analyzer (Nihon Kohden Co., Tokyo, Japan). The nitrite (NO2
−) and nitrate (NO3
−) concentrations in muscle and plasma were measured using a dedicated HPLC system (ENO-20, Eicom, Kyoto, Japan), according to a previously reported method [16]. Interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α) were measured by ELISA according to the manufacturer’s instructions (Rat IL-1β/IL-1F2 Quantikine ELISA Kit and Rat TNF-α Quantikine ELISA Kit, R&D Systems, Inc., MN, USA).
Assessment of kidney function
The bladder was cannulated with PE-50 tubing concomitantly with the jugular vein. Urine samples were obtained starting 1 h prior to decompression until immediately after decompression (0 h). Urine samples were then collected every 1 h for 24 h and centrifuged at 1500×g for 5 min at 20–25 °C. Kidney function was determined based on glomerular filtration rate (GFR), urine volume, urine osmotic pressure (Osmomat 030-D; Gonotec GmbH, Berlin, Germany), N-acetyl-β-d-glucosaminidase (NAG) levels (Shionogi & Co., Osaka, Japan), and urine pH (Pretest 5bII; Wako Pure Chemical Industries, Tokyo, Japan). Kidney injury marker-1 (KIM-1) was measured by ELISA according to the manufacturer’s instructions (Rat TIM-1/KIM-1/HAVCR Immunoassay, R&D Systems, Inc.).
Determination of reactive oxygen species (ROS) production, MPO activity, and mitochondrial function
ROS production in the injured gastrocnemius muscle was determined by measuring the concentration of TBARS. MPO activity in the blood and muscle tissue was measured as previously described [10]. The relative weight (g/100 g body weight) of the injured muscle (gastrocnemius muscle) was determined, which acted as an index of skeletal muscle edema in the affected limbs. Isolation of mitochondria for evaluation of mitochondrial function in crush injury muscle was performed using a mitochondrial isolation kit for tissue (Thermo Fisher Scientific K.K., Kanagawa, Japan). In the MPT, mitochondrial membrane potential (i.e., mitochondrial inner membrane function) was evaluated using a JC-1 mitochondrial membrane potential assay kit (Cayman Chemical Company, Ann Arbor, MI, USA). To evaluate mitochondrial outer membrane function, cytochrome c (cyt c) of crush injured muscle cytoplasm (in the samples that did not include mitochondria) was quantified using a Quantikine® cyt c Immunoassay (R&D Systems, Inc.). SOD activity was determined using a SOD Assay Kit—WST (Dojindo Laboratories, Tokyo, Japan). All operation procedures were performed in accordance with the corresponding instruction manuals.
Western blotting
Western blotting was carried out as previously described [10]. Briefly, rat muscle and kidney tissue were homogenized and centrifuged, and proteins in the lysate were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Proteins were detected on membranes using antibodies against eNOS (Cell Signaling Technology, Tokyo, Japan), phospho-eNOS (Ser1177) (p-eNOS; Cell Signaling Technology), inducible NO synthase (iNOS; Cell Signaling Technology), heme oxygenase (HO)-1 (Thermo Fisher Scientific K.K.), α-tubulin (Cell Signaling Technology), and hypoxia-inducible factor-1 alpha (HIF-1α) (Abcam PLC, Tokyo, Japan). Protein bands were visualized using an enhanced chemiluminescence detection system (SuperSignal West Dura Extended Duration Substrate; Pierce Biotechnology, Tokyo, Japan) with horseradish peroxidase-conjugated secondary antibodies (Pierce Biotechnology). Band intensities were quantified using a ChemiDoc XRS + Molecular Imager with Image Lab software (Bio-Rad Laboratories, Hercules, CA, USA), with α-tubulin used as a loading control.
Sampling and sample preparation for PK parameters
Blood samples from each group were obtained 0 (just before administration), 0.083, 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 7, 8, 9, and 24 h after reperfusion via a jugular catheter. Sample preparations were optimized as follows: 150 µL of plasma and urine samples were extracted with 150 µL methanol. These samples were vortexed and centrifuged at 12,000×g for 20 min at 4 °C. Two hundred microliters of the supernatant was mixed with 790 μL water and 10 μL 0.05 µg/mL digoxin (DIG, internal standard (IS), Wako Pure Chemical Industries, Ltd.), and these samples were subjected to cleanup on an active Strata™ X solid phase extraction column (Shimadzu GLC Ltd., Tokyo, Japan).
Instrumental and conditions for PK parameters of AS
AS concentrations were measured using LC–MS/MS methods with an ACQUITY UPLC TQD (Waters, Milford, MA, USA). Masslynx 4.1 software was used for instrumental control, and acquisition and processing of the data. UPLC separation was performed using an ACQUITY UPLC BEH C18 1.7 µm 2.1 × 100 mm column (Waters) kept at 40 °C. An MS detector with an electrospray ionization (ESI) interface in negative ion mode (ESI−) was used for quantitative analysis, with acquisition in multiple reaction monitoring (MRM) mode. The m/z ratios were as follows: m/z 843.5[M + CH3COO−] > 843.5 for AS, m/z 549.4[M + CH3COO−] > 549.3 for cycloastragenol (CAG), and 839.5[M + CH3COO−] > 839.5 DIG. The following linear gradient program was applied to the analyte, using a mobile phase consisting of deionized water containing 0.1% acetic acid and 10 mM ammonium acetate (A solution) and acetonitrile (B solution): 0–2 min, 60:40–10:90; 2–7 min, 10:90; and 7–12 min, 60:40 (A:B v/v). The sample injection volume was 5 μL. The flow rate was maintained at 0.4 mL/min. The instrumental parameters of the MS detector were optimized as follows: capillary voltage, 3000 V; cone voltage, 30 and 50 V (50 V; AS and 30 V; DIG and CAG); extractor voltage, 3 V; RF lens voltage, 0.2 V; source temperature, 120 °C; desolvation temperature, 350 °C; cone gas flow, 50 L/h; desolvation gas flow, 600 L/h; collision gas flow, 0.15 mL/min; collision energy, 5 eV. The linear calibration curves were plotted between 0.01, 0.05, 0.1, 0.5, 1, 5, and 10 µg/mL (R
2 > 0.999). The minimum limit of quantifiable concentration was less than 0.01 µg/mL. The following PK parameters were calculated: area under the curve (AUC), mean residence time (MRT), total body clearance (CLtot), steady-state volume of distribution (V
ss), elimination rate constant (K
el), biological half-life (T
1/2), maximum drug concentration time (T
max), and maximum blood concentration (C
max) using standard procedures.
Statistical analysis
Results are expressed as mean ± SEM. Differences between groups were assessed by analysis of variance with Tukey’s honest significant difference test or Tukey’s test. Survival curves were generated by the Kaplan–Meier method, and survival was compared by the log-rank test. The PK parameters and blood profiles of AS were compared by the Student’s t test. Differences were considered significant for p values <0.05.