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Protoscolicidal effects of chenodeoxycholic acid on protoscoleces of Echinococcus granulosus
Accepted Manuscript Protoscolicidal effects of chenodeoxycholic acid on protoscoleces of Echinococcus granulosus Hongjuan Shi, Ying Lei, Bo Wang, Zhuo Wang, Guoqiang Xing, Hailong Lv, Yufeng Jiang PII:
S0014-4894(16)30094-7
DOI:
10.1016/j.exppara.2016.05.004
Reference:
YEXPR 7248
To appear in:
Experimental Parasitology
Received Date: 7 September 2015 Revised Date:
9 May 2016
Accepted Date: 16 May 2016
Please cite this article as: Shi, H., Lei, Y., Wang, B., Wang, Z., Xing, G., Lv, H., Jiang, Y., Protoscolicidal effects of chenodeoxycholic acid on protoscoleces of Echinococcus granulosus, Experimental Parasitology (2016), doi: 10.1016/j.exppara.2016.05.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Protoscolicidal effects of chenodeoxycholic acid on protoscoleces of Echinococcus granulosus
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＊ Hongjuan Shi1, Ying Lei1, Bo Wang2, Zhuo Wang2, Guoqiang Xing2, Hailong Lv2,
and Yufeng Jiang1,*
Department of Histology and Embryology, School of Medicine, Shihezi University,
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1
2
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Shihezi 832008, Xinjiang, China;
Department of Hepatobiliary Surgery, The First Affiliated Hospital, School of
Medicine, Shihezi University, Shihezi 832008, Xinjiang, China First author: Hongjuan Shi [email protected];
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*Corresponding author: Yufeng Jiang [email protected]; Hailong Lv [email protected]
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Email: Hongjuan Shi [email protected];
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Ying Lei [email protected]; Bo Wang [email protected]; Zhuo Wang [email protected]; Guoqiang Xing [email protected]; Hailong Lv [email protected]; Yufeng Jiang [email protected]
ACCEPTED MANUSCRIPT Abstract Dissemination of protoscoleces-rich fluid during surgical operation for cystic echinococcosis is a major cause of its recurrence. Instillation of a scolicidal agent into
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hydatid cysts to reduce the risk of spillage of viable protoscoleces is an integral part of the surgical technique employed by many surgeons. In this study, the protoscolicidal effect of chenodeoxycholic acid (CDCA) was investigated. Freshly
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isolated protoscoleces were subjected to CDCA treatment (500, 1000, 2000, and 3000
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µmol/L), and the effects on protoscoleces were investigated with the help of 0.1% eosin staining, electron microscopy, and colorimetric assay of caspase-3 like activity. Dose-dependent mortality of Echinococcus granulosus protoscoleces was observed within a few days of CDCA treatment. The treated protoscoleces showed loss of
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viability, and morphological changes such as contraction of the soma region, formation of blebs, rostellar disorganization, loss of hooks, destruction of microtriches, and formation of vesicles, lipid droplets, and lamellar bodies. Apoptosis
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was evident in the treated protoscoleces, as compared to the control group, which
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were cultivated for nearly 3 months. Our study indicates a therapeutic potential for CDCA as a protoscolicidal agent against E. granulosus. However, further studies are needed to test the long-term effects of CDCA in animal models.
Keywords Echinococcus granulosus; Protoscoleces; Apoptosis; Cystic echinococcosis; Protoscolicidal; Chenodeoxycholic acid
ACCEPTED MANUSCRIPT Introduction Cystic echinococcosis, caused by the larval stage of the parasitic cestode E. granulosus, affects both humans and animals, and is a major public health and
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economic concern in many countries [1], [2]. The disease is highly prevalent in parts of Eurasia, Africa, Australia, and South America [3]-[6]. Though hydatid cysts may develop in many anatomic sites of the human body, the main organs observed to be
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infected with these cysts in patients are the liver and lungs [3].
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At present, there are three treatment options for patients affected by hydatid disease of liver: surgery, percutaneous aspiration, and medical treatment [7]. The preferred treatment strategies for hydatid disease are surgical resection of the parasitic mass containing hydatid cysts, and percutaneous aspiration involving puncture, aspiration,
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injection, re-aspiration (PAIR) [8], [9]. Which is a minimally invasive techniqueconsisting of percutaneous aspiration of the hydatid cyst under ultrasonographic (US) guidance. Spillage protoscoleces-rich infective fluid of the cyst
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is the major cause of recurrence, which can sometimes also lead to secondary
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disseminated intraperitoneal and brain hydatidosis [10]. Instillation of a scolocidal agent into a hepatic hydatid cyst is the most effective technique to prevent this recurrent infection [11]. Presently, hypertonic saline (HS) solution is the most commonly used
protoscolicidal agent worldwide, but acute hypernatremia that causes convulsions, intracranial bleeding, necrosis, and myelinolysis, has been reported after using HS solution in hydatid cyst surgery [12]-[14]. Another protoscolicidal agent commonly
ACCEPTED MANUSCRIPT used is ethanol. However, the protoscolicidal effect of ethanol depends on its concentration, and the most effective concentration is 95%. Thus, dilution of ethanol during application might reduce its efficacy [15]. Frayha et al. [16] reported that 0.5–1%
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Cetrimide was 100% effective against protoscoleces of the hydatid cyst after 10 min of application. However, it can cause harmful adverse reactions such as convulsions, increased metabolic acidosis, peritonitis, methemoglobinemia, and even coma.
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Besides, benzimidazoles are also recommended against E. granulosus protoscoleces
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as an alternative treatment to surgical operation, especially for inoperable cases [17]. However, adverse effects may occur under long-term chemotherapy, such as diarrhea, nausea, vomiting, aminotransferase elevation, leucopenia [18], [19], and even death [20].
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Clinical studies report that most protoscoleces are found mostly dead in cases of hepatic cystic echinococcosis with biliary fistula, long-term repression of hydatid cysts embedded in the bile ducts can lead to biliary fistula, with bile inducing death of
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protoscoleces in these hydatid cysts., protoscoleces can not develop into hydatid in the
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cavity of biliary tract mucosa, moreover, imaging further confirmed that when cystic echinococcosis evolved into calcified lesion type, the incidence of biliary fistula was gradually increased, and the vitality of capsule protoscoleces was gradually decreased[21], [22]. Kayaalp et al. [23] found that multilocular and degenerated cysts had a higher risk of biliary fistula than single cystic echinococcosis. Therefore bile into the cystic hydatid is one of the causes of cystic echinococcosis death. BAs are a group of water-soluble steroids formed after the catabolism of
ACCEPTED MANUSCRIPT cholesterol, and synthesized in hepatocytes of the liver [24]. There are two primary BAs: cholic acid (CA) and chenodeoxycholic acid (CDCA). CDCA is one of the major constituents of bile acids in cholestasis [25], and is known to be an effective
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agent for the medical dissolution of gallstones [26]. Recently, BAs have also been reported to induce apoptosis in many cancer cell models, such as breast cancer [27], [28], colon cancer [29]-[31], prostate cancer [32], [33], and neuroblastoma [34]. Bile
cells,
in
a
hydrophobicity-dependent
manner
[35].
Physiological
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HepG2
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acids induced endoplasmic reticulum stress, which in turn stimulated apoptosis in
concentrations of bile acids may activate mitogen-activated protein kinase (MAPK) pathway, and affect bile acid metabolism and cell proliferation [36]. In our study, we explore the protoscolicidal effects of CDCA on hydatid cysts of E.
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granulosus.
Materials and methods
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Experimental design
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The study began with the isolation of protoscoleces. Batches of protoscoleces were divided into 2 groups: treated (group 1) and untreated ones (group 2), the influence of CDCA was assessed by light and electron microscopy; apoptosis was determined by the activity of caspase-3 by colorimetric assay. Each experiment was performed in triplicate. In vitro maintenance of E. granulosus protoscoleces Protoscoleces of E. granulosus were collected aseptically from liver and lung
ACCEPTED MANUSCRIPT hydatid cysts of naturally infected sheep slaughtered in an abattoir located in Shihezi of Xinjiang Province, West China. Collected cysts were transported within 30 min and washed several times. Then protoscoleces were harvested as much as possible by
several times in phosphate-buffered saline (PBS, pH 7.2) .
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aspiration with a sterile syringe and collected in sterile centrifuge tubes, and washed
The viability was assessed by 0.1% eosin staining (1 g of eosin powder in 1000 ml
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distilled water) under light microscope. viable, motile and morphological intact
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protoscoleces were cultured under aseptic conditions in culture flasks of medium (RPMI 1640 purchased from Gibco-BRL containing 2 mM glutamine, 200 U/ml of penicillin, 200 mg/ml of streptomycin) supplemented with 10% fetal calf serum (FCS; Gibco-BRL) and phenol red. Cultures were kept in culture flasks (200 mL) placed in
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an upright position in an incubator at 37°C, 5% CO2. We closed the protoscoleces after in vitro cultured for 3 days for drug test, with medium changes every 3–5 days. Treatment of the protoscoleces with CDCA
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CDCA (Sigma) was dissolved in dimethyl sulphoxide (DMSO; Sigma) and added
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to the medium resulting in final concentrations of 500, 1000, 2000, and 3000 µmol/L. Protoscoleces incubated with culture medium containing DMSO was used as control. The protoscoleces after in vitro cultured for 3 days were treated. The viability of protoscoleces was determined by 0.1% eosin staining and followed microscopically everyday. All steps were carried out under sterile conditions. Light microscopical examinations To visualize structural alterations and assay the viability of protoscoleces in
ACCEPTED MANUSCRIPT response to CDCA treatment, 100 µL of pooled protoscoleces was transferred over a slide and mixed with 100 µL 0.1% eosin. After 15 min, we observed that the surviving protoscoleces remained colorless and the dead protoscoleces stained red under an
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inverted microscope (Olympus, Japan). The viability test was carried out from 1 to 10 days.
Examinations by scanning and transmission electron microscopy
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Ultrastructure studies with scanning and transmission electron microscope (SEM
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and TEM, respectively) were performed. Samples were fixed with 2.5% glutaraldehyde in sodium cacodylate buffer for 24 h at 4°C; then several washes in cacodylate buffer were made.
For SEM analysis, the samples were dehydrated by sequential incubations in
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increasing concentrations of ethanol (50–100%) and immersed in hexamethyldisilazane for 5 min, 1 h and then overnight. Finally they were sputtercoated with gold,
kV.
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and inspected on a JEOL JSM-6390 LV scanning electron microscope operating at 15
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For TEM analysis, the samples were post-fixed in 2% OsO4 in cacodylate buffer and followed by several washes in water. the specimens were dehydrated in a graded acetone series, subsequently embedded in Spurr's resin. Polymerization of the resin was carried out at 70°C overnight. Sections 700 Å thick were cut off on an LKB ultramicrotome with diamond knife, stained with uranyl acetate saturated solution (45 min) and lead citrate (20 min) and visualized under a JEOL 100 CXII transmission electron microscope at 80 kV.
ACCEPTED MANUSCRIPT Examinations about the caspase-3 like activity of CDCA-treated protoscoleces Caspase-3 Activity Assay Kit was provided by Applygen Technologies Inc (Beijing, China). Detections were performed according to a previous description using the
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caspase-3 activity colorimetric assay kit instruction. The sediments were resuspended in lysis buffer, ground, kept on ice for 10 min and centrifuged at 12,000g for 10 min at 4°C. The supernatants were harvested and added into the reaction system on an
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assay plate with a control group according to the kit’s instruction. Plates were
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incubated at 37°C for 2 h and detected with a microplate reader for the absorbance at 405 nm (A405). The activated caspase-3 in samples catalyzed colorless substrate AcDEVD-pNA into yellow pNA, which concentrations could be calculated according to pNA standard curve and sample A405, the activity of caspase-3 in samples was
Statistical analysis
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finally deduced based on the pNA concentration.
All experiments were performed in three independent experiments and the data are
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presented as mean±SD. p < 0.05 determined using one-way ANOVA, compared with
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the two groups of the different dose protoscolex by the t test. SPSS 13.0 for windows was used to perform statistical analysis.
Results
Light microscopical examinations The results of the in vitro viability tests are consistent with the tissue damage observed at the structural level (Fig. 1). A 0.1% eosin solution was added to the
ACCEPTED MANUSCRIPT samples in a ratio of 1:1. After 15 min, the viability of protoscolices was determined by observing the change of color under a light microscope. Protoscoleces incubated in the culture medium containing DMSO showed no changes in structure during the
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entire experimental period. Most protoscoleces that did not take up the eosin dye were viable, motile, and morphologically intact, with clear, bright calcareous corpuscles and good light transmittance. There was a marked decrease in the viability of
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protoscoleces following the addition of CDCA. The dead protoscoleces from the
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treated group were red-colored because of the absorbed eosin. Some were shrunken, with weakened light transmittance and blurred calcareous corpuscles. The maximum protoscolicidal effect was found at a concentration of 3000 µmol/L of CDCA. These results demonstrate the dose-dependent protoscolicidal effect of CDCA on E.
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granulosus protoscoleces.
The survival of E. granulosus protoscoleces after exposure to CDCA at different
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concentrations and with different exposure times is shown in Fig. 2. Control
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protoscoleces viability incubated in medium 1640 plus DMSO was near 100% after 10 days of incubation. Treated protoscoleces gradually lost their viability during the experimental period (Fig. 2). After 3 days of incubation, the mortality rates were 4.0%, 12.1%, 16.8%, and 76.3% at 500, 1000, 2000, and 3000 µmol/L of CDCA, respectively. When protoscoleces were exposed to different concentrations of CDCA at 500, 1000, 2000, and 3000 µmol/L for 5 days, the mortality rates were 4.8%, 18.5%, 51.0%, and 98.5%, respectively. After 10 days, the mortality was 100% at
ACCEPTED MANUSCRIPT concentrations of 2000 and 3000 µmol/L, and only 14.3% and 39.5% at 500 and 1000 µmol/L, respectively (Fig. 2). Hence, we can infer that with increase in concentrations
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and exposure time, the protoscolicidal effect of CDCA on E. granulosus increases.
Examinations by scanning and transmission electron microscopy
The results of in vitro vitality and viability tests were consistent with the tissue
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damage observed at the ultrastructural level. No ultrastructural damage was observed
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in the protoscoleces incubated with DMSO (control group) during the entire incubation period (Figs. 3A, B and 4A, B). In contrast, ultrastructural alterations were detected in CDCA treated protoscoleces. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images clearly demonstrated the CDCA-
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induced morphological and structural alterations in the treated protoscoleces (Figs. 3C–F and 4C–F). After 3 days of incubation, the following changes in ultrastructure were observed: a slight contraction of soma region of protoscoleces (Fig. 3C),
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contraction of soma region, loss of microtriches (Fig. 3E), presence of vacuoles in
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distal cytoplasm (Fig. 4C), and an increase in the number of lipid droplets (Fig.4E). After 5 days of incubation, observations by SEM and TEM of protoscoleces incubated with CDCA revealed the presence of tegumental alterations, contraction of the soma region (Fig. 3D), an increase in the number of vacuoles in the distal cytoplasm, and a truncated microthrix into glycocalyx. (Fig. 4D). Formation of tegumental vesicles was observed and loss of morphology was evident in some protoscoleces (Fig. 3F).The internal tissue was affected severely. We detected numerous lamellar bodies, and
ACCEPTED MANUSCRIPT nuclei were absent (Fig. 4F).
Examinations about the caspase-3 like activity of CDCA-treated protoscoleces
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Since the metabolic pathway of programmed cell death is presently not known for E. granulosus. Caspase-3 activity assay was performed to study induced apoptosis in CDCA treated proscoleses. Caspase-3 is an effector molecule, common to all known
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metabolic routes of apoptosis induction. Caspase-3 activity assay was done in the
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treated and control groups, after 24 h and 48 h treatment with different concentrations of CDCA. In our study, it was observed that the specific activity of caspase-3 in CDCA treated groups was higher than that in controls (Fig. 5).
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Discussion
Spillage of the protoscoleces-rich infective fluid from cysts during surgical operation is the major cause of its recurrence[37]-[39]. Instillation of scolicidal agents
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into hydatid cysts to reduce the risk of spillage of protoscoleces is therefore an
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integral part of surgical techniques [11]. But no ideal agent that is both effective and safe has been reported [40]. In this study, we provide report on E. granulosus protoscoleces treated with CDCA.
The morphological changes observed in protoscoleces after treatment were similar to those reported in other studies using other drugs [41]-[45], confirming the inhibitory effect of CDCA on E. granulosus protoscoleces. Our results show not only the protoscolicidal efficacy of CDCA but also its rapid
ACCEPTED MANUSCRIPT action at higher concentrations and on exposure for longer time-periods. Protoscoleces cultured in 3000 µmol/L CDCA showed the maximum mortality rate. The mortality rate was also time-dependent; after 3 days of exposure to CDCA, the
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mortality rate was approximately 76.3% and it increased to 98.5% after 5 days of incubation, and further to 100% after 6 days of exposure to CDCA. In contrast, control groups were not significantly altered.
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The unique ultrastructural deformities observed using SEM and TEM enabled us to
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examine the effects induced by CDCA at different concentrations. Ultrastructural changes induced by CDCA observed in protoscoleces included contraction in soma region, tegumental alterations, rostellar disorganization, loss of hooks, and shedding of microtriches. Increased vacuolization, the presence of lipid droplets and lamellated
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bodies in treated protoscoleces indicated that the internal tissue was severely affected. Alteration of tegumental microtriches probably interferes with protoscoleces nutrition [46] as microtriches are directly associated with nutrient absorption [47]. Pérez-
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Serrano et al. [46] suggested that vacuolation, however, indicates general tissue stress.
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The presence of lipid droplets and lamellated bodies further indicates that protoscoleces might have been approaching a stage prior to necrosis. Programmed cell death (PCD) or apoptosis is the most common form of eukaryotic
cell death [48]. High level of apoptosis has been reported in E. granulosus hydatid cysts [49], [50]. Hanhua et al. [51] reported that hydrogen peroxide (H2O2) and dexamethasone could induce apoptosis in protoscoleces. Our results clearly demonstrate that the activity of caspase-3 in protoscoleces incubated with different
ACCEPTED MANUSCRIPT concentrations of CDCA was significantly higher than that in the controls. This proves that CDCA-induced apoptosis involves the caspase-3-dependent pathway in E.granulosus protoscoleces.
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In conclusion, the data demonstrate that CDCA is very effective as a protoscolicidal agent against E. granulosus. In the next step, we will investigate the in vivo efficacy of CDCA, using mouse as an animal model, whichi will help to provide a rational
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basis for novel drug designing, leading to development of a more effective method for
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treatment of human hydatid disease in the near future.
Abbreviations BAs: Bile acids
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CA: Cholic acid CDCA: Chenodeoxycholic acid DCA: Deoxycholic acid
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DMSO: Dimethyl sulphoxide
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FCS: Fetal calf serum
HS: Hypertonic saline
LCA: Lithocholic acid
PAIR: Puncture, aspiration, injection, re-aspiration PBS: Phosphate-buffered saline SEM: Scanning electron microscopy TEM: Transmission electron microscopy
ACCEPTED MANUSCRIPT WHO: World Health Organization
Conflict of interest
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The authors have declared that no competing interests exist.
Acknowledgments
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This research was supported by grants from the National Natural Science Foundation of China, grant number: U1303121, 81360410, 81560334. We thank Feng Sun and
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Wenjuan Qin for their guidance in the experiments.
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ACCEPTED MANUSCRIPT nitazoxanide on Echinococcus granulosus protoscoleces and metacestodes. The Journal of antimicrobial chemotherapy 54:609-616. 45.
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Hu H, Kang J, Chen R, Mamuti W, Wu G, Yuan W. 2011. Drug-induced apoptosis of Echinococcus granulosus protoscoleces. Parasitology research
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ACCEPTED MANUSCRIPT Fig. 1 Light microscopy of E. granulosus protoscoleces incubated in vitro CDCA at either timepoint 3 days (A,C,E,G) or 5 days (B,D,F,H) (×100). A–B Control groups. C-H Altered protoscoleces incubated with CDCA in concentrations of 1000, 2000,
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and 3000 µmol/L. Fig. 2 Survival of E. granulosus protoscoleces after in vitro exposure to CDCA. Viability was determined by using 0.1% eosin staining. Note the dose-dependent
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Fig. 3 Scanning electron microscopy of E. granulosus protoscoleces incubated in vitro with CDCA after 3 days (A,C,E) or 5 days (B,D,F). A-B Control groups. C-D Protoscolece incubated with CDCA 1000 µmol/L. E-F Protoscolece incubated with CDCA 3000 µmol/L. Note the extensive drug-induced damages.
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Fig. 4 Transmission electron microscopy of E. granulosus protoscoleces incubated in vitro with CDCA after 3 days (A,C,E) or 5 days (B,D,F). A Control group (dc distal cytoplasm, n nucleus, nu nucleolus; ×15000). B Sucker region of an invaginated
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untreated control (g glycocalyx; ×2500). C-D Protoscoleces with CDCA 1000 µmol/L. C Note the vacuolation of the distal cytoplasm (v vacuoles; ×5000); D Note the
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presence of large vacuolation (v) in the distal cytoplasm and the truncated microthrix into glycocalyx (arrowhead; ×10000). E-F Protoscoleces incubated with CDCA 3000 µmol/L. E Note the presence of lipid droplets (l; ×8000 ); F The internal tissue was severely affected. Note the presence of lamellar bodies (b; ×12000). Fig. 5 Caspase-3 activity of E. granulosus protoscoleces at different doses of CDCA. Bar graph indicates the mean±SD from three experiments in duplicate. compared with the control; ▼P<0.05 compared with the control.
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ACCEPTED MANUSCRIPT Highlights: ► We evaluated the protoscolicidal effect of CDCA on E. granulosus protoscoleces. ► Light microscope and electron microscopy showed morphological changes in CDCA-treated.
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► Colorimetric assay of caspase-3 like activity induced apoptosis in protoscoleces.
S0014-4894(16)30094-7
DOI:
10.1016/j.exppara.2016.05.004
Reference:
YEXPR 7248
To appear in:
Experimental Parasitology
Received Date: 7 September 2015 Revised Date:
9 May 2016
Accepted Date: 16 May 2016
Please cite this article as: Shi, H., Lei, Y., Wang, B., Wang, Z., Xing, G., Lv, H., Jiang, Y., Protoscolicidal effects of chenodeoxycholic acid on protoscoleces of Echinococcus granulosus, Experimental Parasitology (2016), doi: 10.1016/j.exppara.2016.05.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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ACCEPTED MANUSCRIPT Protoscolicidal effects of chenodeoxycholic acid on protoscoleces of Echinococcus granulosus
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＊ Hongjuan Shi1, Ying Lei1, Bo Wang2, Zhuo Wang2, Guoqiang Xing2, Hailong Lv2,
and Yufeng Jiang1,*
Department of Histology and Embryology, School of Medicine, Shihezi University,
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1
2
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Shihezi 832008, Xinjiang, China;
Department of Hepatobiliary Surgery, The First Affiliated Hospital, School of
Medicine, Shihezi University, Shihezi 832008, Xinjiang, China First author: Hongjuan Shi [email protected];
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*Corresponding author: Yufeng Jiang [email protected]; Hailong Lv [email protected]
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Email: Hongjuan Shi [email protected];
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Ying Lei [email protected]; Bo Wang [email protected]; Zhuo Wang [email protected]; Guoqiang Xing [email protected]; Hailong Lv [email protected]; Yufeng Jiang [email protected]
ACCEPTED MANUSCRIPT Abstract Dissemination of protoscoleces-rich fluid during surgical operation for cystic echinococcosis is a major cause of its recurrence. Instillation of a scolicidal agent into
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hydatid cysts to reduce the risk of spillage of viable protoscoleces is an integral part of the surgical technique employed by many surgeons. In this study, the protoscolicidal effect of chenodeoxycholic acid (CDCA) was investigated. Freshly
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isolated protoscoleces were subjected to CDCA treatment (500, 1000, 2000, and 3000
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µmol/L), and the effects on protoscoleces were investigated with the help of 0.1% eosin staining, electron microscopy, and colorimetric assay of caspase-3 like activity. Dose-dependent mortality of Echinococcus granulosus protoscoleces was observed within a few days of CDCA treatment. The treated protoscoleces showed loss of
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viability, and morphological changes such as contraction of the soma region, formation of blebs, rostellar disorganization, loss of hooks, destruction of microtriches, and formation of vesicles, lipid droplets, and lamellar bodies. Apoptosis
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was evident in the treated protoscoleces, as compared to the control group, which
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were cultivated for nearly 3 months. Our study indicates a therapeutic potential for CDCA as a protoscolicidal agent against E. granulosus. However, further studies are needed to test the long-term effects of CDCA in animal models.
Keywords Echinococcus granulosus; Protoscoleces; Apoptosis; Cystic echinococcosis; Protoscolicidal; Chenodeoxycholic acid
ACCEPTED MANUSCRIPT Introduction Cystic echinococcosis, caused by the larval stage of the parasitic cestode E. granulosus, affects both humans and animals, and is a major public health and
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economic concern in many countries [1], [2]. The disease is highly prevalent in parts of Eurasia, Africa, Australia, and South America [3]-[6]. Though hydatid cysts may develop in many anatomic sites of the human body, the main organs observed to be
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infected with these cysts in patients are the liver and lungs [3].
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At present, there are three treatment options for patients affected by hydatid disease of liver: surgery, percutaneous aspiration, and medical treatment [7]. The preferred treatment strategies for hydatid disease are surgical resection of the parasitic mass containing hydatid cysts, and percutaneous aspiration involving puncture, aspiration,
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injection, re-aspiration (PAIR) [8], [9]. Which is a minimally invasive techniqueconsisting of percutaneous aspiration of the hydatid cyst under ultrasonographic (US) guidance. Spillage protoscoleces-rich infective fluid of the cyst
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is the major cause of recurrence, which can sometimes also lead to secondary
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disseminated intraperitoneal and brain hydatidosis [10]. Instillation of a scolocidal agent into a hepatic hydatid cyst is the most effective technique to prevent this recurrent infection [11]. Presently, hypertonic saline (HS) solution is the most commonly used
protoscolicidal agent worldwide, but acute hypernatremia that causes convulsions, intracranial bleeding, necrosis, and myelinolysis, has been reported after using HS solution in hydatid cyst surgery [12]-[14]. Another protoscolicidal agent commonly
ACCEPTED MANUSCRIPT used is ethanol. However, the protoscolicidal effect of ethanol depends on its concentration, and the most effective concentration is 95%. Thus, dilution of ethanol during application might reduce its efficacy [15]. Frayha et al. [16] reported that 0.5–1%
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Cetrimide was 100% effective against protoscoleces of the hydatid cyst after 10 min of application. However, it can cause harmful adverse reactions such as convulsions, increased metabolic acidosis, peritonitis, methemoglobinemia, and even coma.
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Besides, benzimidazoles are also recommended against E. granulosus protoscoleces
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as an alternative treatment to surgical operation, especially for inoperable cases [17]. However, adverse effects may occur under long-term chemotherapy, such as diarrhea, nausea, vomiting, aminotransferase elevation, leucopenia [18], [19], and even death [20].
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Clinical studies report that most protoscoleces are found mostly dead in cases of hepatic cystic echinococcosis with biliary fistula, long-term repression of hydatid cysts embedded in the bile ducts can lead to biliary fistula, with bile inducing death of
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protoscoleces in these hydatid cysts., protoscoleces can not develop into hydatid in the
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cavity of biliary tract mucosa, moreover, imaging further confirmed that when cystic echinococcosis evolved into calcified lesion type, the incidence of biliary fistula was gradually increased, and the vitality of capsule protoscoleces was gradually decreased[21], [22]. Kayaalp et al. [23] found that multilocular and degenerated cysts had a higher risk of biliary fistula than single cystic echinococcosis. Therefore bile into the cystic hydatid is one of the causes of cystic echinococcosis death. BAs are a group of water-soluble steroids formed after the catabolism of
ACCEPTED MANUSCRIPT cholesterol, and synthesized in hepatocytes of the liver [24]. There are two primary BAs: cholic acid (CA) and chenodeoxycholic acid (CDCA). CDCA is one of the major constituents of bile acids in cholestasis [25], and is known to be an effective
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agent for the medical dissolution of gallstones [26]. Recently, BAs have also been reported to induce apoptosis in many cancer cell models, such as breast cancer [27], [28], colon cancer [29]-[31], prostate cancer [32], [33], and neuroblastoma [34]. Bile
cells,
in
a
hydrophobicity-dependent
manner
[35].
Physiological
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HepG2
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acids induced endoplasmic reticulum stress, which in turn stimulated apoptosis in
concentrations of bile acids may activate mitogen-activated protein kinase (MAPK) pathway, and affect bile acid metabolism and cell proliferation [36]. In our study, we explore the protoscolicidal effects of CDCA on hydatid cysts of E.
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granulosus.
Materials and methods
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Experimental design
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The study began with the isolation of protoscoleces. Batches of protoscoleces were divided into 2 groups: treated (group 1) and untreated ones (group 2), the influence of CDCA was assessed by light and electron microscopy; apoptosis was determined by the activity of caspase-3 by colorimetric assay. Each experiment was performed in triplicate. In vitro maintenance of E. granulosus protoscoleces Protoscoleces of E. granulosus were collected aseptically from liver and lung
ACCEPTED MANUSCRIPT hydatid cysts of naturally infected sheep slaughtered in an abattoir located in Shihezi of Xinjiang Province, West China. Collected cysts were transported within 30 min and washed several times. Then protoscoleces were harvested as much as possible by
several times in phosphate-buffered saline (PBS, pH 7.2) .
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aspiration with a sterile syringe and collected in sterile centrifuge tubes, and washed
The viability was assessed by 0.1% eosin staining (1 g of eosin powder in 1000 ml
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distilled water) under light microscope. viable, motile and morphological intact
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protoscoleces were cultured under aseptic conditions in culture flasks of medium (RPMI 1640 purchased from Gibco-BRL containing 2 mM glutamine, 200 U/ml of penicillin, 200 mg/ml of streptomycin) supplemented with 10% fetal calf serum (FCS; Gibco-BRL) and phenol red. Cultures were kept in culture flasks (200 mL) placed in
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an upright position in an incubator at 37°C, 5% CO2. We closed the protoscoleces after in vitro cultured for 3 days for drug test, with medium changes every 3–5 days. Treatment of the protoscoleces with CDCA
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CDCA (Sigma) was dissolved in dimethyl sulphoxide (DMSO; Sigma) and added
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to the medium resulting in final concentrations of 500, 1000, 2000, and 3000 µmol/L. Protoscoleces incubated with culture medium containing DMSO was used as control. The protoscoleces after in vitro cultured for 3 days were treated. The viability of protoscoleces was determined by 0.1% eosin staining and followed microscopically everyday. All steps were carried out under sterile conditions. Light microscopical examinations To visualize structural alterations and assay the viability of protoscoleces in
ACCEPTED MANUSCRIPT response to CDCA treatment, 100 µL of pooled protoscoleces was transferred over a slide and mixed with 100 µL 0.1% eosin. After 15 min, we observed that the surviving protoscoleces remained colorless and the dead protoscoleces stained red under an
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inverted microscope (Olympus, Japan). The viability test was carried out from 1 to 10 days.
Examinations by scanning and transmission electron microscopy
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Ultrastructure studies with scanning and transmission electron microscope (SEM
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and TEM, respectively) were performed. Samples were fixed with 2.5% glutaraldehyde in sodium cacodylate buffer for 24 h at 4°C; then several washes in cacodylate buffer were made.
For SEM analysis, the samples were dehydrated by sequential incubations in
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increasing concentrations of ethanol (50–100%) and immersed in hexamethyldisilazane for 5 min, 1 h and then overnight. Finally they were sputtercoated with gold,
kV.
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and inspected on a JEOL JSM-6390 LV scanning electron microscope operating at 15
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For TEM analysis, the samples were post-fixed in 2% OsO4 in cacodylate buffer and followed by several washes in water. the specimens were dehydrated in a graded acetone series, subsequently embedded in Spurr's resin. Polymerization of the resin was carried out at 70°C overnight. Sections 700 Å thick were cut off on an LKB ultramicrotome with diamond knife, stained with uranyl acetate saturated solution (45 min) and lead citrate (20 min) and visualized under a JEOL 100 CXII transmission electron microscope at 80 kV.
ACCEPTED MANUSCRIPT Examinations about the caspase-3 like activity of CDCA-treated protoscoleces Caspase-3 Activity Assay Kit was provided by Applygen Technologies Inc (Beijing, China). Detections were performed according to a previous description using the
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caspase-3 activity colorimetric assay kit instruction. The sediments were resuspended in lysis buffer, ground, kept on ice for 10 min and centrifuged at 12,000g for 10 min at 4°C. The supernatants were harvested and added into the reaction system on an
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assay plate with a control group according to the kit’s instruction. Plates were
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incubated at 37°C for 2 h and detected with a microplate reader for the absorbance at 405 nm (A405). The activated caspase-3 in samples catalyzed colorless substrate AcDEVD-pNA into yellow pNA, which concentrations could be calculated according to pNA standard curve and sample A405, the activity of caspase-3 in samples was
Statistical analysis
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finally deduced based on the pNA concentration.
All experiments were performed in three independent experiments and the data are
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presented as mean±SD. p < 0.05 determined using one-way ANOVA, compared with
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the two groups of the different dose protoscolex by the t test. SPSS 13.0 for windows was used to perform statistical analysis.
Results
Light microscopical examinations The results of the in vitro viability tests are consistent with the tissue damage observed at the structural level (Fig. 1). A 0.1% eosin solution was added to the
ACCEPTED MANUSCRIPT samples in a ratio of 1:1. After 15 min, the viability of protoscolices was determined by observing the change of color under a light microscope. Protoscoleces incubated in the culture medium containing DMSO showed no changes in structure during the
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entire experimental period. Most protoscoleces that did not take up the eosin dye were viable, motile, and morphologically intact, with clear, bright calcareous corpuscles and good light transmittance. There was a marked decrease in the viability of
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protoscoleces following the addition of CDCA. The dead protoscoleces from the
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treated group were red-colored because of the absorbed eosin. Some were shrunken, with weakened light transmittance and blurred calcareous corpuscles. The maximum protoscolicidal effect was found at a concentration of 3000 µmol/L of CDCA. These results demonstrate the dose-dependent protoscolicidal effect of CDCA on E.
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granulosus protoscoleces.
The survival of E. granulosus protoscoleces after exposure to CDCA at different
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concentrations and with different exposure times is shown in Fig. 2. Control
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protoscoleces viability incubated in medium 1640 plus DMSO was near 100% after 10 days of incubation. Treated protoscoleces gradually lost their viability during the experimental period (Fig. 2). After 3 days of incubation, the mortality rates were 4.0%, 12.1%, 16.8%, and 76.3% at 500, 1000, 2000, and 3000 µmol/L of CDCA, respectively. When protoscoleces were exposed to different concentrations of CDCA at 500, 1000, 2000, and 3000 µmol/L for 5 days, the mortality rates were 4.8%, 18.5%, 51.0%, and 98.5%, respectively. After 10 days, the mortality was 100% at
ACCEPTED MANUSCRIPT concentrations of 2000 and 3000 µmol/L, and only 14.3% and 39.5% at 500 and 1000 µmol/L, respectively (Fig. 2). Hence, we can infer that with increase in concentrations
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and exposure time, the protoscolicidal effect of CDCA on E. granulosus increases.
Examinations by scanning and transmission electron microscopy
The results of in vitro vitality and viability tests were consistent with the tissue
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damage observed at the ultrastructural level. No ultrastructural damage was observed
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in the protoscoleces incubated with DMSO (control group) during the entire incubation period (Figs. 3A, B and 4A, B). In contrast, ultrastructural alterations were detected in CDCA treated protoscoleces. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images clearly demonstrated the CDCA-
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induced morphological and structural alterations in the treated protoscoleces (Figs. 3C–F and 4C–F). After 3 days of incubation, the following changes in ultrastructure were observed: a slight contraction of soma region of protoscoleces (Fig. 3C),
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contraction of soma region, loss of microtriches (Fig. 3E), presence of vacuoles in
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distal cytoplasm (Fig. 4C), and an increase in the number of lipid droplets (Fig.4E). After 5 days of incubation, observations by SEM and TEM of protoscoleces incubated with CDCA revealed the presence of tegumental alterations, contraction of the soma region (Fig. 3D), an increase in the number of vacuoles in the distal cytoplasm, and a truncated microthrix into glycocalyx. (Fig. 4D). Formation of tegumental vesicles was observed and loss of morphology was evident in some protoscoleces (Fig. 3F).The internal tissue was affected severely. We detected numerous lamellar bodies, and
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Examinations about the caspase-3 like activity of CDCA-treated protoscoleces
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Since the metabolic pathway of programmed cell death is presently not known for E. granulosus. Caspase-3 activity assay was performed to study induced apoptosis in CDCA treated proscoleses. Caspase-3 is an effector molecule, common to all known
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metabolic routes of apoptosis induction. Caspase-3 activity assay was done in the
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treated and control groups, after 24 h and 48 h treatment with different concentrations of CDCA. In our study, it was observed that the specific activity of caspase-3 in CDCA treated groups was higher than that in controls (Fig. 5).
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Discussion
Spillage of the protoscoleces-rich infective fluid from cysts during surgical operation is the major cause of its recurrence[37]-[39]. Instillation of scolicidal agents
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into hydatid cysts to reduce the risk of spillage of protoscoleces is therefore an
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integral part of surgical techniques [11]. But no ideal agent that is both effective and safe has been reported [40]. In this study, we provide report on E. granulosus protoscoleces treated with CDCA.
The morphological changes observed in protoscoleces after treatment were similar to those reported in other studies using other drugs [41]-[45], confirming the inhibitory effect of CDCA on E. granulosus protoscoleces. Our results show not only the protoscolicidal efficacy of CDCA but also its rapid
ACCEPTED MANUSCRIPT action at higher concentrations and on exposure for longer time-periods. Protoscoleces cultured in 3000 µmol/L CDCA showed the maximum mortality rate. The mortality rate was also time-dependent; after 3 days of exposure to CDCA, the
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mortality rate was approximately 76.3% and it increased to 98.5% after 5 days of incubation, and further to 100% after 6 days of exposure to CDCA. In contrast, control groups were not significantly altered.
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The unique ultrastructural deformities observed using SEM and TEM enabled us to
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examine the effects induced by CDCA at different concentrations. Ultrastructural changes induced by CDCA observed in protoscoleces included contraction in soma region, tegumental alterations, rostellar disorganization, loss of hooks, and shedding of microtriches. Increased vacuolization, the presence of lipid droplets and lamellated
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bodies in treated protoscoleces indicated that the internal tissue was severely affected. Alteration of tegumental microtriches probably interferes with protoscoleces nutrition [46] as microtriches are directly associated with nutrient absorption [47]. Pérez-
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Serrano et al. [46] suggested that vacuolation, however, indicates general tissue stress.
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The presence of lipid droplets and lamellated bodies further indicates that protoscoleces might have been approaching a stage prior to necrosis. Programmed cell death (PCD) or apoptosis is the most common form of eukaryotic
cell death [48]. High level of apoptosis has been reported in E. granulosus hydatid cysts [49], [50]. Hanhua et al. [51] reported that hydrogen peroxide (H2O2) and dexamethasone could induce apoptosis in protoscoleces. Our results clearly demonstrate that the activity of caspase-3 in protoscoleces incubated with different
ACCEPTED MANUSCRIPT concentrations of CDCA was significantly higher than that in the controls. This proves that CDCA-induced apoptosis involves the caspase-3-dependent pathway in E.granulosus protoscoleces.
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In conclusion, the data demonstrate that CDCA is very effective as a protoscolicidal agent against E. granulosus. In the next step, we will investigate the in vivo efficacy of CDCA, using mouse as an animal model, whichi will help to provide a rational
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basis for novel drug designing, leading to development of a more effective method for
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treatment of human hydatid disease in the near future.
Abbreviations BAs: Bile acids
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CA: Cholic acid CDCA: Chenodeoxycholic acid DCA: Deoxycholic acid
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DMSO: Dimethyl sulphoxide
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FCS: Fetal calf serum
HS: Hypertonic saline
LCA: Lithocholic acid
PAIR: Puncture, aspiration, injection, re-aspiration PBS: Phosphate-buffered saline SEM: Scanning electron microscopy TEM: Transmission electron microscopy
ACCEPTED MANUSCRIPT WHO: World Health Organization
Conflict of interest
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The authors have declared that no competing interests exist.
Acknowledgments
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This research was supported by grants from the National Natural Science Foundation of China, grant number: U1303121, 81360410, 81560334. We thank Feng Sun and
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Wenjuan Qin for their guidance in the experiments.
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ACCEPTED MANUSCRIPT Fig. 1 Light microscopy of E. granulosus protoscoleces incubated in vitro CDCA at either timepoint 3 days (A,C,E,G) or 5 days (B,D,F,H) (×100). A–B Control groups. C-H Altered protoscoleces incubated with CDCA in concentrations of 1000, 2000,
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and 3000 µmol/L. Fig. 2 Survival of E. granulosus protoscoleces after in vitro exposure to CDCA. Viability was determined by using 0.1% eosin staining. Note the dose-dependent
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Fig. 3 Scanning electron microscopy of E. granulosus protoscoleces incubated in vitro with CDCA after 3 days (A,C,E) or 5 days (B,D,F). A-B Control groups. C-D Protoscolece incubated with CDCA 1000 µmol/L. E-F Protoscolece incubated with CDCA 3000 µmol/L. Note the extensive drug-induced damages.
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Fig. 4 Transmission electron microscopy of E. granulosus protoscoleces incubated in vitro with CDCA after 3 days (A,C,E) or 5 days (B,D,F). A Control group (dc distal cytoplasm, n nucleus, nu nucleolus; ×15000). B Sucker region of an invaginated
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untreated control (g glycocalyx; ×2500). C-D Protoscoleces with CDCA 1000 µmol/L. C Note the vacuolation of the distal cytoplasm (v vacuoles; ×5000); D Note the
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presence of large vacuolation (v) in the distal cytoplasm and the truncated microthrix into glycocalyx (arrowhead; ×10000). E-F Protoscoleces incubated with CDCA 3000 µmol/L. E Note the presence of lipid droplets (l; ×8000 ); F The internal tissue was severely affected. Note the presence of lamellar bodies (b; ×12000). Fig. 5 Caspase-3 activity of E. granulosus protoscoleces at different doses of CDCA. Bar graph indicates the mean±SD from three experiments in duplicate. compared with the control; ▼P<0.05 compared with the control.
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ACCEPTED MANUSCRIPT Highlights: ► We evaluated the protoscolicidal effect of CDCA on E. granulosus protoscoleces. ► Light microscope and electron microscopy showed morphological changes in CDCA-treated.
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► Colorimetric assay of caspase-3 like activity induced apoptosis in protoscoleces.