Schistosomiasis poses a serious threat to human health and remains a major tropical and parasitic disease in more than 70 countries. Praziquantel (PZQ) has been the primary treatment for schistosomiasis for nearly 4 decades.
KEYWORDS: Schistosoma japonicum, migratory-stage schistosomula, radicicol, schistosomicidal efficacy
ABSTRACT
Schistosomiasis poses a serious threat to human health and remains a major tropical and parasitic disease in more than 70 countries. Praziquantel (PZQ) has been the primary treatment for schistosomiasis for nearly 4 decades. However, its efficacy against migratory-stage schistosomula is limited. Radicicol (RAD), a β-resorcylic acid lactone derived from Paecilomyces sp. strain SC0924, was investigated as an alternative treatment for Schistosoma japonicum. In vitro tests showed that within 72 h, RAD (10 μmol/liter) completely killed schistosomula of both skin and liver stages with an efficacy significantly higher than that of PZQ, although it was less potent against adult worms than PZQ. In vivo, RAD reduced worm burdens and liver eggs by 91.18% and 86.01%, respectively, by killing migratory-stage schistosomula. Optical microscopy and scanning electron microscopy revealed that RAD damaged the epiderm and tegument morphology of S. japonicum worms at various stages and altered their motility to different degrees. RAD exhibited schistosomicidal effects at different stages in vitro and in vivo, especially at the migratory stage, implying that its mechanism could be different from that of PZQ. Collectively, these results showed that RAD is promising as a lead for the development of drugs to control the migratory-stage schistosomula of S. japonicum.
INTRODUCTION
Schistosomiasis, also called snail fever or bilharzia, is one of the most devastating parasitic diseases, causing more than 280,000 deaths yearly (1, 2). Currently, chemotherapy is the most direct and effective intervention that reduces the prevalence of this disease. Praziquantel (PZQ) and artemisinin are the primary drugs used to treat schistosomiasis. PZQ has been used to treat adult worms, whereas artemisinin has been used to treat liver-stage schistosomula (3). However, there is no effective drug against migratory-stage schistosomula (i.e., skin-stage and lung-stage schistosomula), especially for Schistosoma japonicum. Studies have demonstrated incidences of PZQ resistance (4–8). This indicates that there is a need to develop alternative treatments. Notably, miltefosine and mefloquine have been reported to be highly effective against different developmental stages of S. japonicum. Although miltefosine is an effective antitumor and anti-Leishmania drug, it is associated with significant side effects. In schistosomiasis, it is associated with a longer course of treatment (lasting several days) (9). Mefloquine is an effective antimalarial drug, but its side effects limit its application in the prevention and treatment of schistosomiasis (10, 11). Therefore, there is an urgent need to develop novel antischistosomal drugs, especially for migratory-stage schistosomula.
β-Resorcylic acid lactones (RALs) are benzomacrolides with different biological activities, like cytotoxicity, antifungal, and antimalarial activities (12, 13). Previously, we isolated and identified 40 compounds from Paecilomyces sp. strain SC0924 metabolites, which included 21 new RALs (13–16). Among these compounds, radicicol (RAD) exhibited highly lethal schistosomicidal activity against migratory-stage schistosomula of S. japonicum. Therefore, this study was carried out to investigate the inhibitory activity of RAD on schistosomula at different developmental stages both in vivo and in vitro.
RESULTS AND DISCUSSION
In vitro schistosomicidal activity against S. japonicum. (i) In vitro schistosomicidal activity of RALs.
RALs obtained from Paecilomyces sp. SC0924 were evaluated for in vitro schistosomicidal activity against skin-stage schistosomula of S. japonicum. As shown in Table 1, 11 RALs, including RAD, showed potent schistosomicidal activity against skin-stage schistosomula of S. japonicum. Among them, three RALs containing an α/β-unsaturated ketone group, RAD, monorden D, and monocillin II, together with two zearalenone derivatives, dehydrozearalenone and trans-7′,8′-dehydrozearalenol, showed death rates higher than 90% and 95% after 48 h and 72 h of treatment at a dose of 5 μg/ml.
TABLE 1.
In vitro schistosomicidal activities of RAD and its analogs (5 μg/ml) against skin-stage schistosomula of S. japonicum
| Compound | Death rate (%) |
||
|---|---|---|---|
| 24 h | 48 h | 72 h | |
| RAD | 92.9 | 95.2 | 100.0 |
| Monorden D | 95.9 | 96.6 | 98.1 |
| Monocillin I | 83.8 | 79.0 | 80.6 |
| Monocillin II | 87.2 | 96.2 | 100.0 |
| Monocillin III | 79.7 | 82.5 | 66.8 |
| Monocillin IV | 86.2 | 80.0 | 76.2 |
| Zearalenone | 91.9 | 69.7 | 83.9 |
| Dehydrozearalenone | 92.3 | 93.5 | 97.5 |
| trans-7′,8′-Dehydrozearalenol | 92.3 | 93.5 | 97.6 |
| Hypothemycin | 79.3 | 80.5 | 90.8 |
| Paecilomycin P | 75.0 | 83.4 | 90.1 |
| PZQ | 90.5 | 70.1 | 68.9 |
| DMSO | 5.0 | 8.3 | 9.7 |
| RPMI 1640 medium | 4.9 | 5.9 | 8.9 |
(ii) In vitro schistosomicidal activity of RAD against different developmental stages of S. japonicum.
RAD showed strong in vitro schistosomicidal activity against the schistosomula of S. japonicum, with a 50% lethal concentration (LC50) value of 1.92 μg/ml (95% confidence interval [CI], 0.786 to 3.29 μmol/liter) based on two parallel tests. Furthermore, RAD exhibited significant schistosomicidal effects on different stages after 24 h of treatment in vitro. As shown in Table 2, RAD (10 μmol/liter) completely killed schistosomula of both skin and liver stages within 72 h and showed worm reduction rates of 78.2% and 62.3% for liver-stage schistosomula and 30-day-old adult worms, respectively. However, significant differences were observed in the inhibitory effects of RAD and the positive control PZQ on liver-stage schistosomula and adult worms. Here, RAD showed a higher inhibitory effect on liver-stage schistosomula than that of PZQ (P < 0.01) but showed a significantly lower effect than that of PZQ against adult worms (P < 0.05). Interestingly, the dynamic effects of the two drugs were different against migratory-stage schistosomula. PZQ treatment induced movements of the worms that seemed to be convulsive, and the worms curled up 2 h later, as though in a state of death. This contraction was probably triggered by rapid Ca2+ influx in the schistosome (17). Methylene blue-stained specimens showed that some worms appeared to have resuscitated after 24 h, and the number of living worms increased significantly after 72 h (Fig. 1C2). In contrast, the administration of RAD suppressed the worm activity, but no convulsions and contractions were observed (Fig. 1B1). The number of living worms remained unchanged even after culturing for a further 72 h (Fig. 1B2). PZQ treatment resulted in reversible inhibition of the worms, while RAD exhibited irreversible inhibition. These results indicated that the inhibitory activity of RAD was more persistent than that of PZQ. Further studies are required to investigate the mechanisms of these effects.
TABLE 2.
In vitro schistosomicidal activities of RAD and PZQ against S. japonicum at different developmental stagesa
| Parameter | Value for treatment |
||||
|---|---|---|---|---|---|
| RAD (+DMSO+medium) at 40 μmol/liter | RAD (+DMSO+medium) at 10 μmol/liter | PZQ (+DMSO+medium) at 15 μmol/liter | DMSO+medium | Medium | |
| Skin-stage schistosomula | |||||
| No. of specimens | 316 | 288 | 345 | 268 | 305 |
| Death rate (%) at time after treatment | |||||
| 24 h | 99.7† | 92.6† | 92.6† | 4.7 | 2.5 |
| 48 h | 100.0†Δ# | 95.3†Δ# | 66.8‡ | 8.5 | 4.0 |
| 72 h | 100.0†Δ# | 100.0†Δ# | 63.2‡ | 9.7 | 6.3 |
| Lung-stage schistosomula | |||||
| No. of specimens | 182 | 168 | 153 | 176 | 148 |
| Death rate (%) at time after treatment | |||||
| 24 h | 100.0†Δφ | 92.0†Δφ | 45.6 | 35.4 | 20.1 |
| 48 h | 100.0†Δφ | 96.8†Δφ | 38.2 | 34.2 | 26.4 |
| 72 h | 100.0†Δφ | 100.0†Δφ | 33.6 | 30.0 | 28.5 |
| Liver-stage schistosomula | |||||
| No. of specimens | 69 | 72 | 76 | 91 | 85 |
| Rate of decline in viability (%) at time after treatment | |||||
| 24 h | 75.2‡ | 64.1‡ | 72.6 | 32.4 | 31.2 |
| 48 h | 80.3‡ | 70.3‡ | 65.1 | 38.6 | 37.3 |
| 72 h | 87.6‡ | 78.2‡ | 55.8 | 40.2 | 38.8 |
| 30-day-old adult worms | |||||
| No. of specimens | 36 | 31 | 42 | 37 | 35 |
| Rate of decline in viability (%) at time after treatment | |||||
| 24 h | 80.3† | 62.3‡ | 97.6†∇ | 17.6 | 14.5 |
| 48 h | 81.2† | 60.5‡ | 94.8†∇ | 21.2 | 17.6 |
| 72 h | 80.3† | 62.3‡ | 89.2†∇ | 23.8 | 17.6 |
†, P < 0.001; ‡, P < 0.05 (compared to the DMSO group). Δ, P < 0.05 (compared to PZQ in terms of schistosomicidal activity). ∇, P < 0.05 (compared to PZQ in terms of schistosomicidal activity). #, P < 0.05; φ, P < 0.01 (compared to PZQ in terms of the death rate). Medium indicates RPMI 1640 medium.
FIG 1.
Morphology and motility differences of skin-stage schistosomula treated with RAD and PZQ in vitro (magnification, ×100). DS, dead schistosomula without staining; AS, alive schistosomula with purple staining.
(iii) Effects of RAD on the morphological structure of S. japonicum at different stages of development.
After exposure to RAD in vitro, schistosomula (skin stage, lung stage, and liver stage) were observed under a microscope. RAD significantly decreased the viability and growth of worms at all stages. The teguments appeared discontinuous or peeled off, and the morphology of the worms was disrupted at high magnification. In contrast, worms from the solvent control group generally grew larger, with smooth teguments and well-defined morphology throughout the culture cycle. Subsequently, the teguments from liver-stage schistosomula were observed by scanning electron microscopy (SEM). After culturing for 24 h, the teguments from the worms exhibited the following characteristics: disordered or no fold structures, erosion and ulceration, and shedding in some parts of the tegument, thereby exposing the subcutaneous tissue (Fig. 2B1). In the control group, spongelike folds and depressions on the tegument appeared in a more regular cord pattern (Fig. 2A1). Moreover, results from the in vivo experiments showed that RAD damaged the worms’ teguments in mice (Fig. 2B2).
FIG 2.
SEM of liver-stage schistosomula exposed to RAD in vitro and in vivo.
Schistosomicidal effects of RAD against migratory-stage schistosomula and adult S. japonicum in mice.
The migratory and developmental stages in the life cycle of S. japonicum and Schistosoma mansoni differ slightly when their cercariae penetrate the skin of human or mammalian hosts. Here, it was noted that the cercariae of S. japonicum will remain underneath the skin for about 1 day after penetrating the skin of mice and will then transform into a schistosomulum before migrating to the lungs along with blood circulation on the third day (18). By around days 5 to 8, most schistosomula will have migrated to the intrahepatic portal vein, and 2 weeks later, these liver-stage schistosomula will develop into paired adult worms (19). Experiments A and B were performed to evaluate the therapeutic effects of RAD against the migratory stage and adult worms of S. japonicum in mice, respectively. The data shown in Table 3 indicate that the intraperitoneal administration of RAD was effective against migratory-stage schistosomula and adult S. japonicum. Particularly, RAD was effective against worms from the migratory stage at days 1 to 4 after the penetration of cercariae into the host skin and reduced the worm burden by 91.18% at a dose concentration of 400 mg/kg of body weight. Besides, RAD showed no obvious inhibitory activity on liver eggs; that is, the rates of reduction of worm burdens and liver egg counts in different adjuvant groups were the same. Effective drugs currently in use are derivatives of artemisinin and PZQ. The former is particularly effective against 7-day-old schistosomula and 35-day-old adult worms, whereas the latter is effective against adult worms (over 21 days old) (20, 21). To date, no drug inhibits migratory-stage schistosomula (1 to 6 days’ juvenile after infection). Therefore, RAD is a promising lead drug with great potency to compensate for the deficiency of artemisinin or PZQ on migratory-stage schistosomula.
TABLE 3.
Worm burdens and egg burdens after RAD treatment in mice infected with S. japonicuma
| Exptl grouping and drug dose | Route of administration | No. of mice | Mean worm load ± SD (reduction rate [%]) | Mean egg load (LEPG) ± SD (% decrease) |
|---|---|---|---|---|
| Batch A: treatment at days 1–4 postinfection for killing schistosomula | ||||
| RAD at 50 mg/kg · day | Intraperitoneal injection (once a day for 4 days) | 6 | 12.80 ± 5.64 (37.25)† | 1,100 ± 496 (20.28) |
| RAD at 100 mg/kg · day | 6 | 9.60 ± 3.61 (52.29)† | 600 ± 179 (56.52)† | |
| RAD at 200 mg/kg · day | 6 | 11.80 ± 2.93 (42.16)† | 460 ± 273 (66.67)† | |
| RAD at 400 mg/kg · day | 6 | 1.80 ± 1.79 (91.18)‡Δ | 192 ± 103 (86.01)‡Δ | |
| DMSO + RPMI 1640 | 7 | 20.40 ± 3.93 | 1,380 ± 299 | |
| RPMI 1640 | 6 | 26.20 ± 5.18 | 1,812 ± 343 | |
| Batch B: treatment at day 30 postinfection for killing adult worms | ||||
| RAD at 100 mg/kg · day | Intraperitoneal injection (once a day for 4 days) | 5 | 13.80 ± 4.70 (10.97) | 5,580 ± 3,949 (36.71) |
| RAD at 200 mg/kg · day | 5 | 7.83 ± 2.79 (49.48)† | 3,800 ± 1,364 (56.70)† | |
| RAD at 400 mg/kg · day | 6 | 6.91 ± 3.15 (55.42)†§ | 3,272 ± 2,142 (62.87)† | |
| DMSO + RPMI 1640 | Single oral injection | 7 | 15.50 ± 3.35 | 8,816 ± 2,149 |
| PZQ at 300 mg/kg · day | 6 | 6.60 ± 2.73 (57.42)† | 2,583 ± 1,282 (70.70)‡ | |
| Batch C: treatment at day 30 postinfection for killing adult worms | Oral injection | |||
| RAD at 200 mg/kg · day | Once a day for 2 days | 5 | 21.21 ± 4.05 (33.75)†§ | |
| PZQ at 300 mg/kg · day | Single day | 6 | 12.70 ± 4.20 (60.31)† | |
| PZQ at 300 mg/kg · day | Once a day for 2 days | 6 | 0.80 ± 0.75 (97.50)‡Δ | |
| Tween 80 | 7 | 32.00 ± 3.19 | ||
†, P > 0.05; ‡, P > 0.01 (compared to the solvent control group). Δ, P > 0.05 (compared to mice exposed to low-dose drugs). §, P < 0.05 (two routes of administration were compared).
Conclusions.
RAD had been reported to lack in vivo antitumor activity as it is unstable in serum (22). Further investigation found that its oxime derivatives had not progressed to clinical trials for their toxicity in the eye. However, as a naturally occurring inhibitor of HSP90, RAD and its derivatives still solicited much interest from biologists and pharmacologists (23, 24).
This study demonstrates for the first time that RAD shows strong schistosomicidal activity against migratory-stage schistosomula (skin stage and lung stage) of S. japonicum both in vivo and in vitro. Additionally, we present clear evidence that RAD damages the morphology and teguments of S. japonicum worms at different stages. Therefore, RAD is worthy of further study in order to discover lead compounds for the early prevention and treatment of schistosomula.
MATERIALS AND METHODS
Source of RAD.
Radicicol (RAD) (purity, >95%) was isolated from the fermentation broth of a soil fungus (Paecilomyces sp. SC0924) (16).
Laboratory animals and parasites. (i) Experimental animals.
Male Kunming mice aged 7 to 8 weeks were obtained from the Department of Experimental Zoology, Central South University, China. This study was approved by the Animal Ethics Committee of Hunan Province (syxk-Xiang, 2011-0001). Animals were housed at the Experimental Animal Center of Central South University.
(ii) Source of S. japonicum at different developmental stages.
The cercariae of S. japonicum (mainland strain) were obtained from infectious Oncomelania hupensis purchased by the Hunan Institute of Schistosomiasis Control. Skin-stage schistosomula were prepared as described previously (25). Lung-stage and liver-stage schistosomula and adult worms were harvested as previously described (26).
(iii) In vitro schistosomicidal test.
Freeze-dried RAD powder was dissolved in dimethyl sulfoxide (DMSO) (Gibco) to prepare a stock solution of 2 mg/ml. Next, the stock solution was diluted to concentrations of 80 μmol/liter and 20 μmol/liter in RPMI 1640 medium (containing 1,000 IU/ml penicillin and 1,000 μg/ml streptomycin). Living S. japonicum worms were suspended in the control solution (containing 1,000 IU/ml penicillin, 1,000 μg/ml streptomycin, and 10% fetal calf serum in RPMI 1640 medium) at concentrations of 250 to 300 worms/ml for skin-stage schistosomula, 100 to 120 worms/ml for lung-stage schistosomula, 60 to 80 worms/ml for liver-stage schistosomula, and 34 to 50 worms/ml for adult worms. Subsequently, 0.5 ml of the test solution or control solution was added to a 24-well plate to prepare final concentrations of test chemicals of 40 μmol/liter and 10 μmol/liter. The plates were then placed in a 0.5% CO2 humidified incubator at 37°C. For each treatment, four parallel wells were prepared. One of the plates was used to observe the motility and morphological changes of the parasites, whereas the rest were used for assessment of either death or vitality rates of the parasites. The worms without movement that were light blue or colorless were judged to be dead. For the vitality of lung-stage schistosomula, the criteria for judgment are described in the literature (26). Briefly, there are four levels of judgment for vitality: level 3 (active, soft, transparent, and shiny worm body), level 2 (slight activity, slightly stiff body, translucent, and lackluster), level 1 (only the head end or tail end of the worm is slightly active, and the worm body is stiff and white), and level 0 (complete immobility, dark and opaque body color, and morphological changes). The positive-control group was exposed to 15 μmol/liter of PZQ (batch number 20100109; Nanjing Pharmaceutical Co., Ltd.). Culture medium containing a similar concentration of DMSO was used as a solvent control, and RPMI 1640 medium containing both double antibiotics and fetal bovine serum served as a control for the culture medium.
(iv) Assessment of S. japonicum worm viability.
The effect of test compounds on the viability of migratory-stage schistosomula was assessed under an inverted microscope 24 and 48 h after administration. After incubation for 72 h, schistosomula were stained using 0.05% methylene blue, and the total number of dead worms was determined as described previously (26). Next, the viability of liver-stage schistosomula and adult worms was determined as described in the literature (27). Rates of death and decline in viability (percentages) were calculated as follows: death rate = 3 parallel wells (total number of worms − number of living worms)/total number of worms × 100%, and rate of decline in viability = 3 parallel wells (total viability score − decline in viability score)/total viability score × 100. Each treatment was reiterated in triplicate.
(v) Motility and morphology of S. japonicum worms.
(a) Motility and morphology of migratory-stage schistosomula. An inverted microscope (Motic-BA400) was used to observe the motility and morphological changes of migratory-stage schistosomula in all groups (i.e., skin-stage and lung-stage schistosomula) at intervals of 2, 6, 12, 24, and 48 h after administration.
(b) Tegument changes in liver-stage schistosomula observed by electron microscopy. In the in vitro test, liver-stage schistosomula were transferred into Eppendorf tubes after exposure to 10 μg/ml of RAD for 24 h. Next, they were washed 3 times with phosphate-buffered saline (PBS) and then fixed with 4% glutaraldehyde (Wuhan Organic Chemical Co., Ltd., China). For scanning electron microscopy (SEM) (catalog no. S-3400N; Hitachi), the fixed worms were rinsed three times with 0.1 M PBS (15 min each time) and then fixed with 1% osmium acid (Beijing Greenlite Trade Development Co., Ltd., China) for 2 h. Subsequently, these worms were washed, dehydrated, dried, and gilded through a vacuum. Finally, the worms’ teguments were observed by SEM.
For the in vivo test, mice were intraperitoneally injected daily with RAD (100 mg/kg) 4 days after they were infected for 18 days with S. japonicum. Next, these mice were anesthetized with ether, and 24 h after the last injection, they were sacrificed. The S. japonicum worms were harvested from the portal vein and observed using SEM. Finally, the worms from mice that were not treated with RAD were used as controls.
(vi) Assessment of the median lethal concentration of RAD against schistosomula of S. japonicum in vitro.
The skin-stage schistosomula of S. japonicum were distributed equally in 24-well culture plates (about 100 to 150 worms/well). Next, RAD was added to these plates to a final concentration of 1.25, 2.5, 5.0, 10, or 20 μmol/liter. Finally, after 24 h, the live and dead worms were counted, and probit analysis (parametric procedure) was used to calculate median lethal dose (LC50) values.
In vivo antischistosomal tests. (i) Drug preparation and use.
RAD was administered either intraperitoneally or intragastrically. First, for intraperitoneal injection, a stock solution of RAD (50 mg/ml) dissolved in DMSO was prepared. The stock solution was diluted in RPMI 1640 medium to a final concentration of 0.1 ml/10 g of body weight. For intragastric administration, mice fasted for 12 h before administration. Here, the required amount of dry powder from either RAD or PZQ was calculated according to the body weight of the mouse. The resulting value was then added to a 2.5-ml homogenate tube, and 0.5 ml Tween 80 was then added to the mixture for grinding. Next, PBS was added dropwise to the mixture to ensure thorough emulsification, and a concentration of 0.1 ml/10 g of body weight of the emulsified solution was administered orally. Finally, adverse reactions were recorded within 1 h of administration. Of note, the solvent control was a combination of DMSO and RPMI 1640 medium or just normal saline.
(ii) Animal experiments.
The animal experiments were divided into three independent tests. First, mice were infected with the cercariae of S. japonicum at room temperature (25°C) as previously described (28). Next, the cercariae of S. japonicum were counted under a microscope and applied onto the shaved abdominal skin of mice for 15 min. Notably, the number of cercariae required for infection was determined as described above (29).
To begin with, mouse experiment A was designed to observe the schistosomicidal effect of RAD against migratory-stage schistosomula at days 1 to 4 after mice were infected with cercariae of S. japonicum. After infection (40 ± 2 cercariae/mouse), 37 mice were randomly divided into 6 groups: 4 groups were exposed to different doses of RAD (50, 100, 200, or 400 mg/kg · day), 1 group was exposed to DMSO plus RPMI 1640 medium and used as the solvent control, and 1 group was exposed to RPMI 1640 medium and used as a blank control. Subsequently, RAD was administered via intraperitoneal injection, and the respective doses were calculated according to animal body weight. The first administration was carried out 6 h after infection of mice and then injected once every 24 h for a total of 4 times. Finally, parasitic indicators were observed at day 30 after infection with cercaria.
Second, mouse experiment B was designed to observe the schistosomicidal effect of RAD against adult worms at day 30 after infection with S. japonicum cercariae. Thirty mice were randomly divided into 5 groups after they were infected with S. japonicum cercariae (30 ± 1 cercariae/mouse). Here, three groups were injected intraperitoneally once a day with different concentrations of RAD (100, 200, or 400 mg/kg · day) for a total of 4 days, one group was given 0.2 ml of DMSO once a day, and one group was administered a single oral dose of PZQ (300 mg/kg · day). Finally, parasitic indicators were observed at day 46 after the mice were infected with cercaria.
Third, mouse experiment C was designed to observe the differences in the schistosomicidal effects between intragastrically administered RAD and two PZQ doses against the worms of S. japonicum. First, 24 mice were randomly divided into 4 groups after they were infected with cercariae (30 ± 1 cercariae/mouse) of S. japonicum. Next, these four groups were treated with RAD (400 mg/kg · day) (once daily for 4 days), PZQ (300 mg/kg · day) (in a single oral dose), PZQ (600 mg/kg · day) (orally given once daily for 2 days), or saline-Tween 80 (once daily for 4 days). Finally, parasitic indicators were observed at day 46 after infection with cercaria.
(iii) Observation of parasitic indicators.
Mice were sacrificed, and their worms and eggs were collected as described above (30). Briefly, these mice were anesthetized using ether and then dissected to expose the abdominal and thoracic cavities. Next, a solution of sodium citrate (1.0%) was used to perfuse the left ventricle, and worms were collected from the portal vein. Additionally, the worms were also checked in the mesenteries. The numbers of female, male, and paired worms obtained from each mouse were recorded. Subsequently, an observation was made on the differences in the growth status and density of egg granulomas on the liver surface. Liver eggs were counted as outlined previously by Cai et al. (31). Briefly, 0.5 g of mouse liver was cut into pieces and placed in a 10.0-ml graduated tube. Next, the cells were disrupted using a disperser (Ningbo, China) and then digested overnight with 1% trypsin. The obtained solution was then dissolved in 5 ml of 1.2% sodium chloride, and 1.0 ml of the fully shaken solution was pipetted and smeared on multiple slides. Finally, the number of S. japonicum eggs was counted under a microscope, and the results were multiplied by 10 to give the total number of eggs per gram of liver (LEPG).
(iv) Statistical analysis.
SPSS version 17.0 software was used for analysis. P values below 0.05 were considered to be statistically significant. For the in vitro setup, schistosomicidal data from the number of worms under the same treatment conditions were the sum of data for triplicate wells. Differences in the death rate or viability between groups were analyzed using the chi-square test. The number of worms detected in each mouse is reported as the mean ± the standard deviation (SD), and the differences among groups were analyzed using analysis of variance (ANOVA).
ACKNOWLEDGMENTS
We are grateful to Wu Xiaoying of the Ultra Microstructure Laboratory of Xiangya School of Medicine, Central South University, for assisting with scanning electron microscopy (SEM).
This work was funded by NSFC grants (no. 20672114, 31772032, and 30901856) and the Open-End Fund for the Valuable and Precision Instruments of Central South University (CSUZC201538).
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