Aqueous Extract of Whitmania Pigra Whitman Alleviates Thrombus Burden via Sirtuin 1/NF-kB Pathway
Xiao-lan Yao, BS,1 Han Liu, BS,1 Peng Li, BS, Wen-pei Chen, MS, Shi-xia Guan, PhD, Yang Chen, PhD, Yi-Na Wu, MS, and Bao-qin Lin, PhD*
Abstract
Background: Whitmania pigra Whitman (W pigra), a traditional Chinese medicine, has functions of breaking stagnant and eliminating blood stasis. The aim of this study was to investigate the underlying mechanism of W pigra against deep vein thrombosis (DVT). Methods: A rat model of DVT induced by inferior vena cava stenosis was successfully established. Rats were administered vehicle (saline solution, p.o.), three doses of W pigra aqueous extract (34.7, 104.2, or 312.5 mg crude W pigra/kg, p.o.), heparin (200 U/kg, i.v.), or clopidogrel (25 mg/kg, p.o.) once daily for 2 d. Thrombus weight and histopathological changes were examined.Bloodsampleswerecollectedtodeterminebloodcellcounts,bloodviscosity,blood coagulation, blood fibrinolysis, serum levels of interleukin-1b, and tumor necrosis factor-a. Protein expressions of Sirtuin1 (SIRT1), acetylated p65 (Ace-p65), and phosphorylated p65 (pp65) were determined by Western blot. Furthermore, SIRT1-specific inhibitor EX527 was applied to confirm the role of SIRT1 in the antithrombotic effect of W pigra.
Results: W pigra significantly decreased thrombus weight. W pigra had no effects on blood cell counts, whole blood viscosity, blood coagulation, blood fibrinolysis. However, it reduced tissue factor protein expression in the vein wall and thrombus. Moreover, it sharply increased SIRT1 protein expression and decreased leukocytes recruitment in the thrombus and vein wall, serum levels of interleukin-1b and tumor necrosis factor-a, and protein expressions of Ace-p65 and p-p65. Furthermore, the antithrombotic effect of W pigra was significantly abolished by EX527.
Conclusions: Aqueous extract of W pigra effectively reduced DVT burden by inhibiting inflammation via SIRT1/nuclear factor-kappa B signaling pathway. ª 2019 Elsevier Inc. All rights reserved.
Keywords:
Whitmania pigra whitman
Deep vein thrombosis
Inflammation
SIRT1
NF-kB
Introduction
Deep vein thrombosis (DVT) causes significant morbidity and mortality.1 In addition to fatal pulmonary embolism, DVT results in postphlebitic syndrome up to 50% of patients.2 Current treatments of DVT with anticoagulant, antiplatelet, and thrombolytic drugs have a risk of severe hemorrhage.3
From aspects of pathophysiology, three broad categories of factors toward the development of venous thrombosis have been summarized as Virchow’s Triad including endothelial injury, blood stasis, and hypercoagulability.4 A common cause of venous thrombosis is blood stasis rather than endothelial injury in clinic.5,6 Venous stasis results in local hypoxia and distension of vessels, which activates the endothelium and then causes the expression of adhesion molecules on its surface.7,8 Besides, venous stasis fails to clear activated coagulation factors, which precedes platelet activation and aggregation and then induces venous thrombosis.9 Moreover, fibrinolysis system inhibition is a main determination of thrombus resolution. Decrease of tissue plasminogen activator (tPA) or increase of plasminogen activator inhibitor-1 (PAI-1) would alleviate plasminogen converting to plasmin, which inhibit the degradation of fibrin.10
An increasing number of studies have shown that inflammation plays a pivotal role in thrombosis.11-15 Neutrophils infiltration in the vein wall, followed by monocytes and lymphocytes, has been identified as a key event in the process of DVT.16 Neutrophils account for more than 80% of inflammatory cells and release neutrophil extracellular traps.17 Neutrophil extracellular traps activate coagulation via directly binding to vWF and supporting the recruitment and activation of platelets.18 Active monocytes express tissue factor (TF) and release inflammatory cytokines such as interleukin-1b (IL-1b) and tumor necrosis factor-a (TNF-a). IL-1b and TNF-a modulate the anticoagulant system via downregulating thrombomodulin and endothelial protein C receptor.16 Intriguingly, all these molecules are regulated by nuclear factor-kappa B (NF-kB) that is an important transcription factor in inflammation response.19,20
Mammalian sirtuins, also termed silent information regulator 2 (Sir2)-related enzymes, were NADþ-dependent enzymes that deacetylate lysine residues on various proteins. Sirtuinsconsistof SIRT1-7.SIRT1 is theclosesthomologtoSir2 and is essential for cell survival, differentiation, senescence, and metabolism via anti-inflammation or antioxidation.21,22 Acetylated NF-kB subunit p65 (Ace-p65) binds to DNA and promotes transcription of downstream targets.23 SIRT1 displays its antiinflammatory activity through deacetylating Acep65 at Lys310 and then reducing the binding of Ace-p65 to inflammatory genes.24 Inhibition of SIRT1 contributes to arterial thrombosis associated with increased activation of NF-kB, leading to enhanced mRNA and protein expressions of TF.23 Besides, it has been reported that SIRT1 prevents pulmonary thrombus via downregulating platelet-activating factor receptor expression in platelets.25 Our previous study has shown that DVT is associated with inflammation via SIRT1/NF-kB signaling pathway.26
Whitmania pigra Whitman (W pigra) is a traditional Chinese medicine (TCM). It was first recorded in Shen Nong’s Herbal Classic (Shen Nong Ben Cao Jing) about 2400 y ago and is included in Chinese Pharmacopoeia 2015 edition.27 W pigra is the most widely used leeches in China. It has many functions such as removing blood stasis and promoting blood circulation. One of W pigra preparation called Naoxuekang is used in clinic for treatment of cerebral thrombosis with 90.0%-95.6% total efficiency.28,29 Naoxuekang in combination with ahylysantinfarctase prevents DVT and shows a lower incidence of pulmonary embolism than aspirin.30 Although W pigra contains no hirudin, aqueous extract of W pigra (AEW) significantly inhibits inferior vena cava (IVC) ligation-induced thrombosis, and arteriovenous shunt-induced and FeCl3induced arterial thrombosis. Furthermore, antithrombotic effect of AEW was more pronounced in venous compared to arterial thrombosis.31
Therefore, IVC stenosiseinduced DVT rat model was applied to further investigate the anti-VT mechanism of AEW. Platelet aggregation, blood coagulation, blood fibrinolysis and blood viscosity, histopathology and leukocytes recruitment of IVCs, serum levels of inflammatory cytokines, and protein expressions of SIRT1, phosphorylated p65 (p-p65), and Acep65 were observed. Moreover, SIRT1-specific inhibitor EX527 was applied to confirm whether SIRT1 plays a key role in the antithrombotic effect of AEW.
Materials and methods
Animals
Male and female Sprague-Dawley rats (approved no. SCXK 2013-0034), weighing 250-300 g, were provided by the Experimental Animal Center of Guangzhou University of Chinese Medicine. Rats were allowed free access to a standard diet and water and were housed at 24.0 0.5C and with a 12 h/12 h of light/dark cycle. All procedures were performed in accordance withtheAnimalEthicsCommitteeof Guangzhou University of Chinese Medicine.
Materials
The whole body of dried W pigra was provided by Tianxianong (Suqian, China) and identified by Pro. Danyan Zhang from Identification Department of TCM, Guangzhou University of Chinese Medicine. Pentobarbital sodium salt, heparin, and clopidogrelhydrogen sulfate were bought fromMerck(Darmstadt, Germany), Tianjin Biochem Pharmaceutical Co., Ltd. (Tianjin, China),andSalubris(Shenzhen,China),respectively.Adenosine diphosphate (ADP) was purchased from Leagene (Beijing, China). Rat TNF-a and IL-1b enzyme-linked immunosorbent assay(ELISA)kitswereobtainedfromLianke(Hangzhou,China) and Invitrogen (CA), respectively. Rat tPA and PAI-1 ELISA kits, and SIRT1, Ace-p65 (Lys310), and Ly6g (FITC) antibodies were obtained from Abcam (Cambridge, UK). TF antibody was purchased from Santa Cruz (CA). Antibodies against rat p65, p-p65, glyceraldehyde 3-phosphate dehydrogenase, HRP-conjugated secondary antibody, and goat anti-mouse secondary antibody conjugated to Alexa Fluor 555 were bought from Cell Signaling Technology (MA). BCA protein assay kit, Triton X-100, and polyvinylidene difluoride membranes were purchased from CWBIO (Beijing, China), Ameresco (MA), and Millipore (CA), respectively. And 40, 6 diamidino-2-phenylindole dihydrochloride was obtained from Sigma-Aldrich (MO).
Preparation of AEW
Dried W pigra was smashed, and 50 g powder was extracted with 500 mL saline solution. After maintaining at 4C for 2 h, the mixture was heated in a 70C water bath for 1 h and then filtered to obtain liquor A. The residue was extracted with another 250 mL saline solution following the aforementioned procedure to get liquor B. Liquor A and B were mixed and concentrated with rotary evaporator at 70C for 1 h. After lyophilizing (FreeZone; LABCONCO, CA) at 40C for 1 d, 16.24 g freeze-dried powder was obtained. Therefore, 1.00 g freeze-dried powder was equivalent to 3.08 g crude W pigra. And its total protein content measured with a BCA protein assay kit was 11.4%.
IVC stenosiseinduced DVT in rats
IVC stenosis-induced DVT was carried out according to our previous report.25 In brief, rats were anesthetized by intraperitoneal injection of pentobarbital sodium (36 mg/kg, i.p.) and then a 2-cm midline laparotomy was performed. IVC just below the left renal vein was stenosed by tying a 5-0 silk suture tightly around the IVC together with an acupuncture pin (0.35 mm 50 mm). The pin was then removed. Ceftriaxone sodiumwas sprinkledevenlyalong theincisionline to prevent bacterial infection after the abdominal muscle or skin was sutured. The rats in the sham group received the same surgical procedure without IVC stenosis.
Study design
Our previous study has demonstrated that thrombus weight increased to a maximum while SIRT1 protein expression decreased to a minimum at 1 d after IVC stenosis.25 Therefore, 1 d was chosen to investigate the effect and mechanism of W pigra on DVT. This study comprises two parts, and each part has been performed twice independently.
In the first part, rats were randomly divided into seven groups: sham group, model group, low, medium, and high doses of AEW (34.7, 104.2, and 312.5 mg crude W pigra/kg, p.o.) groups, heparin (200 U/kg, i.v.) group, and clopidogrel (25 mg/ kg, p.o.) group. There were four male and four female rats in each group. AEW, heparin, and clopidogrel were administered once daily for 2 d. AEW and heparin was administered at 1 h and 24 h after thrombus induction. Clopidogrel was given 2 h before and 24 h after thrombus induction. The rats in the sham and model groups were orally administered with distilled water. The dose of AEW in humans is 1-3 g/d according to Chinese Pharmacopoeia 2015 edition. According to the conversion of body surface area between humans and rats, the dose of rat is 104.2-312.5 mg crude W pigra/kg/d. Therefore, three doses of AEW (34.7, 104.2, and 312.5 mg crude W pigra/kg) were chosen to explore the effect of AEW on DVT.
In the second part, rats were randomly divided into six groups: normal group, sham group, model group, model þ EX527 group (10 mg/kg/d, i.p.), model þ AEW group (104.2 mg crude W pigra/kg/d, p.o.), and model þ EX527 (10 mg/ kg/d, i.p.) þ AEW (104.2 mg crude W pigra/kg/d, p.o.) group. There were four male and four female rats in each group. AEW was administered at 1 h and 24 h after thrombus induction. EX527 was administered once and was given 20 min before surgery.26,32 The rats in the normal, sham, and model groups were orally administered with distilled water.
Thrombus weight
Sixty minutes after the final administration, rats were anesthetized with pentobarbital sodium. Blood samples were collected from carotid artery into vacuum blood collection tubes with or without anticoagulant. Thrombosed IVCs were then dissected, weighed, and sectioned into two parts. One part was fixed in 4% paraformaldehyde dissolved in phosphate-buffered saline solution (pH 7.4) for 48 h at 4C for histopathologic analysis. The other part was stored at 80C for Western blot and immunofluorescence analyses.
Histopathologic and immunohistochemical analysis
Thrombosed IVCs fixed in 4% paraformaldehyde solution were dehydrated through graded alcohols, embedded in paraffin, and sectioned into slices of 4 mm thickness. After deparaffinization, sections were stained with hematoxylin and eosin (HE). Numbers of neutrophils, monocytes, and lymphocytes within the vein wall and thrombus of each sample were counted and summedfrom five high power fields (HPFs, 400).33
For immunohistochemical staining, slides were retrieved with EDTA solution (pH 9.0), immersed in 3% H2O2, blocked with 10% goat serum, and incubated with antibody to TF (1:200) at 4C overnight. Sections were then incubated with HRP-conjugated secondary antibody (1:125) for 1 h at room temperature and colored with diaminobenzidine for 40 s. Normal goat IgG was used as a negative control. Pathological changes in thrombosed IVCs were observed under an optical microscope (BX53; Olympus, Tokyo, Japan).
Analyses of blood cell counts, whole blood viscosity, blood coagulation, and blood fibrinolysis
Blood anticoagulated by 15% EDTA-K2 was used for blood cell counts with an automatic blood cell analyzer (XT-2000i; Sysmex, Tokyo, Japan). Blood anticoagulated with 3.2% sodium citrate solution (blood/citrate: 9:1, v/v) was applied for blood viscosity test by an automatic hematology analyzer (LBY-N6B; Precil, Beijing, China). Blood anticoagulated with citrate solution was centrifuged at 2000 g for 10 min at 4C to obtain plasma for detections of APTT, PT, TT, and FIB with an automatic coagulation analyzer (CA7000; Sysmex, Tokyo, Japan). Besides, blood samples anticoagulated with citrate solution was centrifuged at 1000 g and 3000 g for 15 min at 4C to obtain plasma for detections of tPA and PAI-1 levels with ELISA kits, respectively.
Platelet aggregation in vivo
Blood samples anticoagulated with citrate solution were centrifuged at 190 g for 10 min at 25C to obtain platelet-rich plasma. Platelet aggregation was induced by 10 mM ADP and measured with an aggregometer (LBY-NJ2; Precil, Beijing, China).
Measurement of inflammatory cytokines
Thirty minutes after blood collection into vacuum blood collection tubes without any anticoagulants or coagulants, the serum was obtained by centrifuging at 1600 g and 4C for 10 min. Serum levels of IL-1b and TNF-a were assayed with ELISA kits.
Western blot analysis
Thrombosed IVCs taken from 80C fridge were lysed with solution containing 98% RIPA, 1% phenyl methyl sulfonyl fluoride, and1% phosphataseinhibitoron ice.Lysates werecentrifugedat 14,000gfor10minat4C,andsupernatantswerethenobtained. TheconcentrationofproteinwasdeterminedwithaBCAprotein assay kit. Proteins were fractionated by 8% polyacrylamide gels andthentransferredontopolyvinylidenedifluoridemembranes. After blocking with 5% bovine serum albumin for 1 h at room temperature, membranes were immunoblotted with primary antibodies against rat SIRT1 (1:8000), Ace-p65 (Lys310, 1:250), p65 (1:1000), p-p65 (1:1000), and glyceraldehyde 3-phosphate dehydrogenase(1:1000)at4Covernight.Afterward,membraneswere incubated with HRP-conjugated secondary antibody (1:2000) for 1hatroomtemperature.Immunoreactivebandswerevisualized withachemiluminescenceapparatus(5200CE;Tanon,Shanghai, China), and quantified using Labwork image analysis software (National Institutes of Health, MD).
Immunofluorescence analysis
Frozen thrombosed IVCs were sectioned into slices of 6 mm thickness. Specimens were fixed with ice-cold acetone at 20C for 15 min and rinsed triple with phosphate-buffered saline solution containing 0.05% Tween 20 (PBST). Sections were permeabilized with 1% Triton X-100 for 20 min at room temperature and blocked with 10% goat serum for 60 min at 37C in a wet box. Thereafter, sections were incubated with anti-SIRT1 antibody (1:2000) overnight at 4C and anti-Ly6g antibody (FITC, 1:1000) for 30 min at 37C, respectively. After rinsing with PBST, sections for SIRT1 were incubated with a goat anti-mouse secondary antibody conjugated to Alexa Fluor 555 for 60 min. All sections were then rinsed with PBST and counterstained with 40, 6 diamidino-2-phenylindole dihydrochloride for 5 min at room temperature. Representative fluorescence images were observed with a laser scanning confocal microscope (LSM800; Carl Zeiss, Jena, Germany). Statistical analysis
Data were expressed as mean SEM (standard error of mean). Statistical significance was calculated by one-way analysis of variance test (SPSS 20.0; SPSS, Inc, Chicago, IL) followed by least-significant difference test. P < 0.05 was considered statistically significant.
Results
AEW reduced thrombus burden
As shown in Figure 1, male and female rats exhibited no difference in thrombus weight after stenosis. AEW at the dose of 104.2 mg crude W pigra/kg inhibited thrombosis by 47.4% (P < 0.01 versus model group).AEW had no effects on blood cell counts, whole blood viscosity, blood coagulation, ADP-induced platelet aggregation, and blood fibrinolysisHeparin sharply increased APTT, PT, and TT (P < 0.01 versus model group). Clopidogrel significantly alleviated ADPinduced platelet aggregation (P < 0.01 versus model group). AEW had no effects on blood cell counts (Table), APTT, PT, TT, FIB, ADP-induced maximum platelet aggregation rate, plasma levels of tPA and PAI-1, and whole blood viscosity (Fig. 2).
AEW reduced TF protein expression
TF was highly expressed in thrombus and vein wall in model rats. AEW at 104.2 and 312.5 mg crude W pigra/kg reduced TF protein expression in the thrombus and vein wall (Fig. 3).
AEW decreased leukocytes recruitment
AEW at 104.2 mg crude W pigra/kg decreased neutrophils by 58.7% and 50.2% in the thrombus and vein wall, respectively(P < 0.01 versus model group, Figs. 4 and 5). AEW at 104.2 mg crude W pigra/kg decreased monocytes by 58.7% (P < 0.01 versus model group) and 39.1% (P < 0.01 versus model group) in the thrombus and vein wall, respectively (Fig. 4).
AEW decreased serum levels of inflammatory cytokines
Serum levels of IL-1b and TNF-a were significantly elevated in the model group (P < 0.01 versus sham group). AEW at 104.2 mg crude W pigra/kg reduced serum levels of IL-1b and TNF-a by 48.8% (P < 0.01 versus model group) and 20.3% (P < 0.01 versus model group), respectively (Fig. 6).
Effect of AEW on SIRT1/NF-kB pathway
As shown in Figure 7A and B, SIRT1 protein was highly expressed in IVCs of sham rats, while it was significantly reduced after IVC stenosis (P < 0.01 versus sham group). However, AEW at 104.2 mg crude W pigra/kg increased SIRT1 protein by 2.4-fold (P < 0.01 versus model group, Fig. 7B). Meanwhile, protein expressions of Ace-p65 (P < 0.01 versus model group, Fig. 7C) and p-p65 (P < 0.01 versus model group, Fig. 7D) were remarkably increased after IVC stenosis. But AEW restored Ace-p65 and p-p65 protein expressions toward normal level.
AEW at 34.7 mg crude W pigra/kg did not upregulate SIRT1 or Ace-p65 protein expressions in the thrombosed vein. AEW at 104.2 and 312.5 mg crude W pigra/kg elevated SIRT1 and decreased Ace-p65 protein expressions while it showed no significant difference between the two groups (Fig. 7A-C). Therefore, AEW at 104.2 mg crude W pigra/kg was chosen to combine with EX527 to explore the effect of AEW on SIRT1/NFkB pathway in DVT rats.
EX527 abolished the antithrombotic effect of AEW
As shown in Figure 8, there were no differences in thrombus weight and protein expressions of SIRT1 and Ace-p65 between normal and sham groups. EX527 alone had no influence on aforementioned parameters in stenosis-treated rats. However, EX527 significantly reversed effects of AEW on thrombus weight, protein expressions of SIRT1, and Ace-p65.
Discussion
According to the TCM theory, thrombotic disorders are described as the blood stasis syndrome.34 W pigra is one of the most representative TCM for the treatment of blood stasis syndrome, for functions of breaking stagnant and eliminating blood stasis. Therefore, parameters that reflect blood coagulation (APTT, PT, TT, and FIB), blood fibrinolysis (tPA and PAI-1), platelet activity (ADP-induced maximum platelet aggregation rate), and blood thickness (RBC and whole blood viscosity) were comprehensively assessed to explore anti-DVT mechanism of W pigra. However, AEW did not influence these parameters (Fig. 2 and Table), which indicates that the antithrombotic mechanism of AEW is not through interrupting coagulation and fibrinolysis system, inhibiting ADP-induced platelet aggregation or alleviating thickness of blood. Moreover, the ineffectiveness of AEW onthese parameters may help it avoid adverse effect of hemorrhage in clinic.
Whitman isolated from the acetone-water extract of W pigra and whitide purified from water extract of W pigra exhibit anticoagulant activity in vitro.36,37 An enzyme purified from enzymatic hydrolysis of W pigra has fibrinolytic activity in vitro.38 A novel peptide named WP-30 from W pigra selectively inhibits thrombin-induced platelet aggregation in vitro and synthetic WP-30 attenuates thrombus formation in rats.39 WP-30 is one kind of peptide in AEW, and it will be hydrolyzed if it is orally administered. Therefore, WP-30 was administered through tail vein to avoid hydrolyzing. Besides, WP-30 is a selective inhibitor to thrombin-induced platelet aggregation rather than other inducers (collagen or U46619) in vitro.39 AEW had no effect on ADP-induced platelet aggregation in vivo in our study (Fig. 2E). Thus, whether WP-30 could inhibit platelet aggregation stimulated with ADP, and whether AEW could inhibit platelet aggregation induced with thrombin, collagen, or U46619, need to be further confirmed. TF is a major initiator of extrinsic coagulation pathway,35 and it reflects local coagulation process. AEW alleviated TF protein expression in the vein wall and thrombus (Fig. 3), which indicates that AEW prevents DVT by inactivating local extrinsic coagulation.
It has been strongly suggested that inflammation contributes to venous thrombosis.14 Participation of leukocytes is a specific feature of thrombosis.17 The NF-kB family is a key player in controlling immune system and inflammatory diseases.40 Releasing from its inhibitor, inhibitory kB (IkB), free NF-kB (p-p65) translocates into the nucleus. AEW decreased neutrophils and monocytes in the thrombus and vein wall (Figs. 4 and 5), serum levels of IL-1b and TNF-a (Fig. 6), and protein expression of p-p65 (Fig. 7), which indicates that the antithrombotic mechanism of AEW may be related to its antiinflammatory activity. However, there was another study that showed that neutrophils depletion leaded to larger thrombi in rats.41 The reason may be that different modeling methods are applied. The prothrombotic effect of neutrophils was observed in the stenosis DVT model,35 whereas neutropeniaresultedin developmentof even largerthrombiin the stasis model.41 Thus, the role of neutrophils in DVT needs to be investigated in future.
Acetylation is one post-translational modification of pp65. Ace-p65 binds to DNA and promotes transcription of downstream targets such as IL-1b, TNF-a, and TF. SIRT1 physically interacts with Ace-p65 and suppresses inflammation-related genes transcription by deacetylating p65 at Lys 310.42 SIRT1 knockdown leads to high expressions of proinflammatory cytokines such as IL-1b and TNF-a.43 Activation of SIRT1 alleviated amyloid-beta-induced inflammation and retinal pigment epithelial barrier disruption via NF-kB pathway in RPE cells.44 In our study, AEW upregulated protein expression of SIRT1 and downregulated protein expression of Ace-p65 (Fig. 7). Furthermore, these effects of AEW were markedly abolished by EX527 (Fig. 8). These results indicate that AEW reduces thrombus burden in a stenosis rat model of DVT via antiinflammation through SIRT1/NF-kB pathway (Fig. 9). However, effective components in AEW were still unclear, so further investigations would be needed.
Conclusions
AEW significantly inhibits DVT. It has no effects on APTT, PT, TT, FIB, ADP-induced maximum platelet aggregation rate, serum levels of tPA and PAI-1, RBC, PLT, and whole blood viscosity. However, AEW remarkably increased SIRT1 protein expression, and decreased serum levels of inflammatory cytokines, leukocytes recruitment and TF protein expression in the thrombus and vein wall, protein expressions of p-p65 and Ace-p65. Furthermore, the antithrombotic effect of AEW was significantly abolished by EX527. Therefore, anti-DVT effect of AEW attributes to its antiinflammatory activity via modulating SIRT1/NF-kB signaling pathway.
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