Crush syndrome (CS) is a potentially lethal condition characterized by muscle cell damage resulting from decompression following compression. Patients with CS can develop cardiac failure, kidney dysfunction, shock, systemic inflammation and sepsis. Salvianolic acid B (SalB) has cardiac and kidney protective effects and anti-oxidative, anti-inflammatory, anti-apoptotic and anti-bacterial properties. The present study aimed to demonstrate the survival benefit of SalB in the CS rat model, which comprised anesthetized rats with bilateral hindlimb compression by a rubber tourniquet for 5 h. The rats examined were randomly divided into four groups: i) Sham; ii) sham treated with SalB; iii) CS rat model without treatment; and iv) CS rat model treated with SalB. Under continuous monitoring and recording of arterial blood pressures, blood and tissue samples were collected for biochemical analyses at designated timepoints before and after reperfusion. SalB administration improved the survival rate, kidney function (by treating shock and metabolic acidosis) and inflammation (by reducing mitochondrial dysfunction and endothelial damage). Reduced incidence of cardiac failure due to hyperkalemia was associated with reduced muscle injury via the prevention of mitochondrial dysfunction. Additionally, indirect antibacterial action by the neutrophil extracellular trap system (NETs) was observed. SalB administration to the CS rat model led to a substantial improvement in survival following CS by decreasing kidney and cardiac dysfunctions, inflammation, and endothelial dysfunction by improving the mitochondrial function and through antibacterial effects via NETs.
Crush syndrome (CS) occurs as a result of physical trauma sustained during events such as earthquakes and is associated with high mortality due to circulatory shock, kidney failure, and systemic inflammation (
In general, kidney dysfunction is prevented by hemodialysis and fluid therapy (i.e., kidney replacement therapy) (
Salvianolic acid B (4-[(1E)-3-[(1R)-1-carboxy-2-(3,4-dihydroxyphenyl)ethoxy]-3-oxo-1-propen-1-yl]-2-(3,4-dih-ydroxyphenyl)-2,3-dihydro-7-hydroxy-3-benzofurancarboxylic acid (2S,3S)-3-[(1R)-1-carboxy-2-(3,4-dihydroxyphenyl)ethyl] ester: SalB) is one of the components of
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˚C±3˚C and relative humidity of 55±15% with a 12/12-h light/dark cycle and free access to food and water. All animal experiments were approved by the Institutional Animal Care and Use Committee of Josai University (approval no. JU18030). Anesthesia induction and maintenance was performed using inhaled 2% isoflurane. Body temperature was maintained throughout the experiment using a heating pad. The CS model was established as previously reported (
SalB (Carbosynth) dosages were determined based on previous reports (
The experimental design is shown in
Experiment 2 (observation of vital signs): Among the tested SalB dosages (10, 20, and 50 mg/kg), 20 mg/kg was chosen for this experiment as maximum survival was obtained at this dosage. To examine the effects of SalB in CS, the animals were randomly divided into four groups: vii) sham with vehicle, viii) s-SalB, ix) CS group, and x) CS with 20 mg/kg SalB (CS-SalB). The anesthetized (2% isoflurane inhalation) rats were subdivided above groups, and cannulated a polyethylene catheter (PE-50 tubing) from a carotid artery at 3, 6, 24 and 48 h after reperfusion for sequential sampling (each group; n=6).
Experiment 3 (assessment of therapeutic effects): To examine the effects of SalB in CS, the animals were randomly divided into four groups: xi) sham with vehicle, xii) s-SalB, xiii) CS group, and xiv) CS-SalB. Rats were subdivided at 3, 6, 24, and 48 h after reperfusion for these sampling points (each point; n=6).
The experimental rats (n=210) were monitored for health and behavior every hour until 6 h and every 12 h thereafter. The rats were euthanized (confirmation by pupillary reflex to light) when a no food and/or water intake state, dyspnea (i.e. mouth breathing) state, and autotomy (i.e. bite) occurred in the pressure area. All the rats used in the experiments were euthanized (confirmation by pupillary reflex to light) by administering an overdose of sodium pentobarbital (100 mg/kg body weight, intravenously); Experimental 1: 48 h after reperfusion; Experimental 2: 48 h after reperfusion; Experimental 3: just each sampling time.
Experimental 2: Mean arterial pressure (MAP), heart rate (HR), and rectal temperature (Temp) were recorded using a PowerLab data acquisition system (AD Instruments). A carotid artery was cannulated with PE-50 tubing connected to a pressure transducer (AD Instruments). Arterial blood samples from each mouse were obtained at 3, 6, 24, and 48 h after reperfusion using a carotid artery catheter over time (
Experimental 3: In each experimental group (3, 6, 24, and 48 h after reperfusion, respectively n=6), venous blood and tissue samples from the gastrocnemius muscles were subjected to inflammation, and tissue thiobarbituric acid reactive substance (TBARS), myeloperoxidase (MPO) activity, an index of mitochondrial permeability transition, and superoxide dismutase (SOD) activity (
Experimental 3: The serum levels of high mobility group box 1 (HMGB1), IL-6, IL-8, IL-10, IL-1β, and tumor necrosis factor (TNF)-α were measured using HMGB1 ELISA kit II (Shino-Test Co.). Rat IL-6, IL-8, IL-10, L-1β/IL-1F2, and TNF-α levels were measured using the Quantikine® ELISA kit (RandD Systems, Inc.), and plasminogen activator inhibitor-1 (PAI-1) was measured using the Rat PAI1 ELISA kit (Abcam) according to the manufacturer's instructions.
Experimental 3: In the blood sample, the effect of N-acetyl-β-D-glucosaminidase (NAG) on kidney function was determined using the β-N-acetylglucosaminidase assay kit QuantiChrom (BioAssay Systems Co.), kidney injury marker-1 (KIM-1) using Rat TIM-1/KIM-1/HAVCR Immunoassay (RandD Systems, Inc.), neutrophil gelatinase-associated lipocalin (NGAL) using the Rat Lipocalin-2 ELISA kit (Abcam), and creatinine (Cre) using the Creatinine Assay kit, QuantiChrom (BioAssay Systems Co.). Urine samples were then collected by bladder and centrifuged at 1,500 x g for 5 min at 20-25˚C. Cre using the Creatinine Assay kit, QuantiChrom (BioAssay Systems Co.), urine osmotic pressure (Osmomat 030-D; Gonotec GmbH), urine volume and glomerular filtration rate (GFR). For histological evaluations, tissue samples were fixed in 10% formalin and embedded in paraffin, and sections were cut and stained with hematoxylin and eosin. Microphotographs of the tissue sections were then evaluated by a pathologist (New Histo Science Laboratory). Renal injuries were scored by calculating the percentage of tubules that displayed tubular dilation, cast formation, and tubular necrosis according to a previously described method (
ROS production in the injured gastrocnemius muscle was determined by measuring the concentration of TBARS, MPO activity in the blood and muscle tissue, and mitochondrial function by cytochrome
SOD activity was determined using the SOD Assay kit-WST (Dojindo Laboratories). 1,1-diphenil-2-picryl-hydrazal (DPPH) antioxidant assay of SalB was performed as described by Sharma and Bhat (
NOx [(total nitrite (NO2−) and nitrate (NO3−)] concentrations in the serum were measured using the CII and FX NO2−/NO3− assay kits (Dojindo Laboratories) according to the manufacturer's instructions. Western blotting for inducible nitric oxide synthase (iNOS) and β-actin was carried out as previously described (
The minimum inhibitory concentration (MIC) of SalB was measured using the broth microdilution method. This procedure essentially followed the Clinical and Laboratory Standards Institute guidelines (
Results are expressed as mean ± standard error of the mean. Survival curves were generated using the Kaplan-Meier method, and survival was compared using the log-rank test. Differences between groups were assessed using the one-way analysis of variance with Tukey's honest significant difference test or Tukey's test. Kidney injury score was assessed using the Dunn's nonparametric comparison for post hoc testing after a Kruskal-Wallis test. DPPH antioxidant assay was assessed using the unpaired Student's t test between two groups. Differences were considered significant at P<0.05 (Statcel 2, 2nd ed. OMS Publishing Inc.).
The survival rates of rats in the CS group were 92, 84, 46, 15, and 15% at 1, 3, 6, 24, and 48 h, respectively, and they were significantly lower than those in the sham group at 6, 24, and 48 h after reperfusion (P<0.05). CS rats died of cardiac failure and hypovolemic shock. The cause of death was related to kidney dysfunction and systemic inflammatory response associated with traumatic rhabdomyolysis caused by crush injury. The survival rates of the CS-SalB group at 6, 24, and 48 h after reperfusion (82, 76, and 71%, respectively) were significantly higher than those of the CS group (P<0.05). No mortality was observed in the sham and s-SalB groups (
The parameters of kidney functions are shown in
Endothelial damage with inflammation, and the levels of IL-6, IL-10, IL-1β, TNF-α, HMGB1, and NOx levels are shown in
Endothelial cell damage was assessed using the vWF, PLT, FIB, PAI-1, APTT, and PT levels (
ROS damage was assessed by TBARS production, and muscle SOD activity and mitochondrial damage were assessed using cytoplasm Cyt
We focused in the NETosis process because it is not only a frequent problem associated with infections such as sepsis in patients with CS, but also related to leukocyte activation in the CS rat model. NETosis was evaluated using NEU, IL-8, blood MPO activity, and tissue MPO activity levels (
We demonstrated that a simple therapeutic method of SalB intravenous injection improves severe morbidity and mortality of patients with CS. The 20 mg/kg SalB intravenous injection presented the highest survival rate (
Kidney injury leads to kidney failure, which is a serious complication of CS that results from circulatory shock, renal afferent arteriolar vasoconstriction (urinary concentration), increased urinary myoglobin levels, or metabolic acidosis (urinary acidity) (
Previously, we reported that survival following CS is increased by the anti-inflammatory effects of treatment agents that prevent systemic inflammatory response syndrome, making systemic management difficult, even after the acute phase (
Death due to sepsis associated with CS (i.e., infections) begins to occur 3 days after injury and most often within 2 weeks (
In conclusion, SalB administration to the CS rat model led to a substantial improvement in survival following CS by decreasing kidney and cardiac dysfunction, inflammation, and endothelial dysfunction by improving the mitochondrial function and by antibacterial effects via NETs.
The authors would like to thank Dr Hiroyuki Uchida and Dr Junta Ito for valuable suggestions, and Ms. Shion Terada, Ms. Shiho Morita and Ms. Chikako Murata for technical assistance (all Faculty of Pharmaceutical Science, Josai University, Sakado, Japan).
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
IM led the project and designed and performed most of the experiments. TS and YMu assisted with the survival and biochemical marker analyses. YMi, JK, YI and IK conceived the study, participated in its design and coordination, and helped draft the manuscript. IM, TS and YMu confirm the authenticity of all the raw data. All authors have read and approved the final manuscript.
All animal experiments were approved by the Institutional Animal Care and Use Committee of Josai University (approval no. JU18030; Sakado, Japan).
Not applicable.
The authors declare that they have no competing interests.
Experimental design. CS, crush syndrome; SalB, salvianolic acid B; s-SalB, sham with 20 mg/kg of SalB; SalB10, CS with 10 mg/kg of SalB; SalB20, CS with 20 mg/kg of SalB; SalB50, CS with 50 mg/kg of SalB.
Effect of SalB treatment for varying durations on CS rat viability. Survival curves were obtained using the Kaplan-Meier method (n=15). *P<0.05 vs. sham group, #P<0.05 vs. CS group, †P<0.05 vs. SalB10 group, §P<0.05 vs. SalB20 group (log-rank test). CS, crush syndrome; SalB, salvianolic acid B; s-SalB, sham with 20 mg/kg of SalB; SalB10, CS with 10 mg/kg of SalB; SalB20, CS with 20 mg/kg of SalB; SalB50, CS with 50 mg/kg of SalB.
Effect of SalB on kidney function in the CS rats. (A) Kidney SOD activity, (B) serum NAG level, (C) serum KIM-1 level, (D) serum NGAL level and (E) hematoxylin and eosin-stained kidney sections and kidney injury score following reperfusion for 48 h. (A-D) Bar graph values are presented as the mean ± SEM (n=6). *P<0.05 vs. sham group, †P<0.05 vs. CS group (Tukey-Kramer test). Micrographs are representative of three independent experiments (magnification, x200; scale bar, 100 µm). Black arrowhead, dilated kidney tubule. (E) Box plot for kidney injury score, *P<0.05 vs. sham group, †P<0.05 vs. CS group (Kruskal-Wallis test). CS, crush syndrome; SalB, salvianolic acid B; s-SalB, sham with 20 mg/kg of SalB; CS-SalB, CS with 20 mg/kg of SalB; SOD, superoxide dismutase; NAG, N-acetyl-β-D-glucosaminidase; KIM-1, kidney injury marker-1; NGAL, neutrophil gelatinase-associated lipocalin.
Effect of SalB on inflammatory mediators in the CS rats. (A) Serum IL-6 levels, (B) serum IL-10 levels, (C) serum IL-1β levels, (D) serum TNF-α levels, (E) serum HMGB1 levels, and (F) serum NOx levels and muscle iNOS expression. Values are presented as the mean ± SEM (n=6). *P<0.05 vs. sham group, †P<0.05 vs. CS group (Tukey-Kramer test). CS, crush syndrome; SalB, salvianolic acid B; s-SalB, sham with 20 mg/kg of SalB; CS-SalB, CS with 20 mg/kg of SalB; HMGB1, high mobility group box 1; NOx, nitrogen oxide; iNOS, inducible nitric oxide synthase.
Effect of SalB on the coagulation system in the CS rats. (A) Plasma vWF levels, (B) PLT levels, (C) FIB levels, (D) serum PAI-1 levels, (E) APTT levels and (F) PT levels. Values are presented as the mean ± SEM (n=6). *P<0.05 vs. sham group, †P<0.05 vs. CS group (Tukey-Kramer test). CS, crush syndrome; SalB, salvianolic acid B; s-SalB, sham with 20 mg/kg of SalB; CS-SalB, CS with 20 mg/kg of SalB; PLT, platelet; APTT, activated partial thromboplastin time; PT, prothrombin time; FIB, fibrinogen; vWF, von Willebrand factor; PAI-1, plasminogen activator inhibitor-1.
Effect of SalB on antioxidant action and mitochondrial function in the CS rats. (A) Muscle TBARS levels, (B) muscle SOD activity, (C) cytoplasm Cyt c content and (D) muscle JC-1 fluorescence. Values are presented as the mean ± SEM (n=6). *P<0.05 vs. sham group, †P<0.05 vs. CS group (Tukey-Kramer test). CS, crush syndrome; SalB, salvianolic acid B; s-SalB, sham with 20 mg/kg of SalB; CS-SalB, CS with 20 mg/kg of SalB; TBARS, thiobarbituric acid reactive substance; SOD, superoxide dismutase; Cyt c, cytochrome.
DPPH radical scavenging assay for SalB. Values are presented as the mean ± SEM (n=3). *P<0.05 vs. AA group (Student's t-test). AA, ascorbic acid; SalB, salvianolic acid B; DPPH, 1,1 diphenil-2-picryl-hydrazal.
Effect of neutrophil extracellular trap system of on antibacterial potential in the CS rats. (A) NEU levels, (B) serum IL-8 levels, (C) blood MPO activity and (D) muscle MPO activity. Values are presented as the mean ± SEM (n=6). *P<0.05 vs. sham group, †P<0.05 vs. CS group (Tukey-Kramer test). CS, crush syndrome; SalB, salvianolic acid B; s-SalB, sham with 20 mg/kg of SalB; CS-SalB, CS with 20 mg/kg of SalB; NEU, neutrophil; MPO, myeloperoxidase.
Effect of SalB on kidney function parameters in blood and urine sample in the CS rats.
Reperfusion, h | |||||
---|---|---|---|---|---|
Parameter | Group | 3 | 6 | 24 | 48 |
BUN, mg/dl | sham | 15.3±1.1 | 12.7±1.2 | 14.6±1.1 | 19.5±1.8 |
s-SalB | 14.9±1.3 | 25.7±0.6 |
24.2±10.0 | 17.3±0.2 | |
CS | 29.3±5.6 |
41.3±5.5 |
98.5±3.5 |
88.3±2.9 |
|
C-SalB | 16.2±2.0 |
21.0±1.8 |
33.3±2.3 |
35.1±4.4 |
|
Cre, mg/dl | sham | 0.3±0.3 | 0.2±0.0 | 0.2±0.0 | 0.2±0.0 |
s-SalB | 0.3±0.1 | 0.2±0.0 | 0.2±0.1 | 0.2±0.0 | |
CS | 0.9±0.4 | 1.2±0.3 |
1.5±0.1 |
1.5±0.3 |
|
C-SalB | 0.4±0.2 | 0.5±0.3 |
0.5±0.2 |
0.6±0.3 |
|
Urine osmotic pressure, mOsm/kg • H20 | sham | 1.47±0.02 | 1.37±0.09 | 1.38±0.06 | 1.39±0.07 |
s-SalB | 1.58±0.02 | 1.38±0.35 | 1.31±0.12 | 1.25±0.17 | |
CS | 1.12±0.02 |
0.85±0.22 |
0.66±0.15 |
0.54±0.04 |
|
C-SalB | 1.53±0.01 |
1.51±0.08 |
1.81±0.03 |
1.40±0.11 |
|
Urine volume, ml/l | sham | 0.42±0.02 | 0.40±0.05 | 0.35±0.05 | 0.50±0.06 |
s-SalB | 0.46±0.25 | 0.50±0.06 | 0.60±0.02 | 0.45±0.03 | |
CS | 0.30±0.01 | 0.21±0.02 | 0.16±0.00 | 0.23±0.02 | |
C-SalB | 0.39±0.12 | 0.56±0.02 |
0.54±0.07 |
0.45±0.07 |
|
GFR, ml/min | sham | 1.69±0.22 | 1.50±0.19 | 1.45±0.10 | 1.36±0.23 |
s-SalB | 1.98±0.45 | 1.75±0.24 | 1.35±0.65 | 0.98±0.11 | |
CS | 0.98±0.14 |
0.78±0.24 |
0.55±0.33 |
0.58±0.19 |
|
C-SalB | 1.78±0.45 |
1.53±0.33 |
1.75±0.57 |
1.78±0.22 |
aP<0.05 vs. sham group;
bP<0.05 vs. CS group (Tukey-Kramer test). Values are presented as the mean ± SEM (n=6 each). CS, crush syndrome; SalB, salvianolic acid B; s-SalB, sham with 20 mg/kg of SalB; CS-SalB, CS with 20 mg/kg of SalB; BUN, blood urea nitrogen; Cre, creatinine; GFR, glomerular filtration rate.
Effects of SalB.
Reperfusion, h | |||||
---|---|---|---|---|---|
Parameter | Group | 3 | 6 | 24 | 48 |
CPK, IU/l | sham | 148±20 | 193±46 | 208±40 | 206±43 |
s-SalB | 134±18 | 174±41 | 177±34 | 134±28 | |
CS | 5,305±1,080 |
8,368±1,556 |
12,870±2,281 |
34,562±3,428 |
|
CS-SalB | 3,373±181 | 3,537±307 |
11,251±2,309 | 24,469±2,291 |
|
K+, mEq/l | sham | 4.4±0.3 | 4.3±0.2 | 4.6±0.2 | 3.9±0.2 |
s-SalB | 3.9±0.0 | 3.6±0.1 | 3.8±0.2 | 3.5±0.,1 | |
CS | 5.5±0.2 | 5.9±0.2 | 6.4±0.3 |
7.3±0.7 |
|
CS-SalB | 4.8±0.2 | 5.7±0.2 | 6.6±0.5 | 5.6±0.2 |
|
pH | sham | 7.46±0.04 | 7.48±0.01 | 7.50±0.01 | 7.45±0.01 |
s-SalB | 7.41±0.02 | 7.45±0.05 | 7.44±0.02 | 7.48±0.03 | |
CS | 7.50±0.02 | 7.46±0.02 | 7.24±0.03 |
7.27±0.07 |
|
CS-SalB | 7.46±0.01 | 7.47±0.02 | 7.45±0.03 |
7.47±0.03 |
|
BE, mmol/l | sham | 5.7±0.3 | 6.7±0.7 | 6.7±0.9 | 4.7±0.3 |
s-SalB | 1.3±1.8 | 2.0±2.3 | 2.3±2.2 | 6.3±0.7 | |
CS | 1.3±0.9 | 0.3±1.1 | -5.0±1.6 |
-4.4±2.7 |
|
CS-SalB | 1.0±0.8 | 1.5±1.8 | -4.5±1.5 | 2.0±0.8 | |
MAP, mmHg | sham | 131±5 | 124±5 | 112±11 | 116±9 |
s-SalB | 124±6 | 110±4 | 122±11 | 151±10 | |
CS | 65±6 |
63±7 |
58±6 |
56±14 |
|
CS-SalB | 98±3 |
94±4 |
89±3 |
87±3 |
|
Hct, % | sham | 46.3±0.3 | 44.7±0.3 | 43.3±1.2 | 43.7±0.9 |
s-SalB | 48.3±1.5 | 47.0±1.5 | 48.0±1.2 | 45.0±2.6 | |
CS | 48.6±1.2 | 51.9±1.1 |
51.3±0.9 |
57.2±7.1 |
|
CS-SalB | 45.8±1.7 | 48.5±1.2 | 52.3±1.9 | 46.0±2.1 |
|
HR, bpm | sham | 400±15 | 388±17 | 336±46 | 329±79 |
s-SalB | 337±20 | 309±20 | 329±5 | 385±22 | |
CS | 307±18 |
274±26 |
344±26 | 256±38 |
|
CS-SalB | 333±5 | 374±7 |
443±26 |
416±30 |
|
Temp, ˚C | sham | 36.2±0.4 | 36.4±0.1 | 36.3±0.3 | 36.3±0.6 |
s-SalB | 36.5±0.3 | 37.5±0.7 | 37.8±0.5 | 35.4±0.5 | |
CS | 35.3±0.4 | 34.6±0.6 |
35.0±0.8 |
29.5±1.4 |
|
CS-SalB | 36.2±0.1 | 37.4±0.5 |
38.2±0.8 |
35.0±0.5 |
aP<0.05 vs. sham group;
bP<0.05 vs. CS group (Tukey-Kramer test). Values are presented as the mean ± SEM (n=6 each). CS, crush syndrome; SalB, salvianolic acid B; s-SalB, sham with 20 mg/kg of SalB; CS-SalB, CS with 20 mg/kg of SalB; CPK, creatine phosphokinase; K+, potassium; BE, base excess; MAP, mean arterial pressure; Hct, hematocrit; HR, heart rate.
Antibacterial effects of SalB.
SalB concentration, µg/ml | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Test bacteria | 2000 | 1000 | 500 | 300 | 100 | 50 | 30 | 10 | 1 | 0.5 | 0.3 | 0.1 |
- | - | - | - | - | - | - | - | - | - | - | - | |
- | - | - | - | - | - | - | - | - | - | - | - | |
- | - | - | - | - | - | - | - | - | - | - | - |
-, negative (no antibacterial effect: Bacterial colony growth); SalB, salvianolic acid B.