Effects of individual compounds on the viability of S. japonicum in vitro
In order to select the optimal concentration of PZQ and DW-3-15 for combination therapy, the antischistosomal activity of PZQ and DW-3-15 individually was assayed at concentrations of 25–100 μM. As shown in Additional file 1: Tables S1–S3, with ascending concentrations of PZQ, the viability of juveniles, males, and females was significantly reduced compared with the control group (juveniles: F(10, 319) = 56.97, P < 0.0001; males: F(10, 319) = 98.59, P < 0.0001; females: F(10, 319) = 90.66, P < 0.0001), with PZQ at 100 μM showing the highest viability reduction rate in juveniles, males, and females. However, as the incubation period continued, juvenile, male, and female worms gradually recovered after 48 h exposure to PZQ, in accord with our previous study [14]. For DW-3-15, unlike PZQ, the antischistosomal effect was both concentration- and time-dependent, with DW-3-15 at 100 μM demonstrating the most potent effect. However, neither compound was capable of killing all the worms. In order to investigate whether combination therapy could enhance the antischistosomal performance of the two compounds, juveniles, males, and females were incubated with a constant dose ratio (1:1) of the lower concentration (50 μM, PDa) and the higher concentration (100 μM, PDb) of PZQ combined with DW-3-15.
The combination compounds significantly reduced the viability of S. japonicum in vitro
As shown in Fig. 1, there was a statistically significant difference in terms of viability among the different groups against juveniles (F(6, 203) = 109.3, P < 0.0001), males (F(6, 203) = 181.2, P < 0.0001), and females (F(6, 203) = 217.8, P < 0.0001). In comparison with the PZQ monotherapy group, a combination of PZQ and DW-3-15 at different concentrations induced a more significant reduction in viability of juveniles (PDa: F(6, 203) = 109.3, P < 0.0001; PDb: F(6, 203) = 109.3, P < 0.0001), males (PDa: F(6, 203) = 181.2, P < 0.0001; PDb: F(6, 203) = 181.2, P < 0.0001), and females (PDa: F(6, 203) = 217.8, P < 0.0001; PDb: F(6, 203) = 217.8, P < 0.0001). There was also a more significant reduction in viability of males (PDa: F(6, 203) = 181.2, P = 0.0189 ) and females (PDa: F(6, 203) = 217.8, P = 0.0053) treated with the combination of PZQ and DW-3-15 compared with DW-3-15 monotherapy. Notably, exposure to the higher-concentration combination resulted in 100% reduction in viability of juvenile and male adult worms after 72 h (Additional file 1: Tables S1–S3), indicating that the combination of the two compounds enhanced the anthelmintic performance of the individual compounds. No significant difference in viability was observed between the two concentrations of the combinations (Fig. 1).
The CI of the lower-concentration combination of PZQ and DW-3-15 (PDa) against juveniles, males, and females was 0.57, 0.63, and 0.65, respectively; for the higher-concentration combination group (PDb), the CI was 0.28, 0.27, and 0.53, respectively (Fig. 2a–c). Based on the results, we concluded that the combination of the two compounds induced a synergistic effect against S. japonicum, and the higher-concentration combination group showed stronger synergistic effects in males and juveniles. In addition to the bright-field microscopic assessment, the viability of the worms was assessed by a red-fluorescent dye propidium iodide (PI) that objectively detects dead parasites during in vitro culture. Following 72 h incubation with individual or combined PZQ and DW-3-15 at different concentrations, dead worms were stained with PI showing red fluorescence signals. As expected, worms treated with combination compounds (PDa and PDb) displayed the brightest red fluorescence intensity among juveniles, males, and females (Fig. 3a–c). The fluorescence intensity of the worms in the PZQ group was similar to that of the untreated control groups (Fig. 3a–c). In addition, worms incubated with DW-3-15 alone were also stained by PI, and the fluorescence intensity was stronger than in worms with PZQ monotherapy (Fig. 3a–c).
Morphological analysis by SEM
Males from the control group showed normal ultrastructural features (Fig. 4). The oral and ventral suckers and gynecophoral canals showed no obvious morphological alterations, with apically directed spines in the suckers (Fig. 4a–c). The tegument of the mid-body showed numerous crests with sensory papillae that were distributed along the body (Fig. 4d–f).
Males exposed to 100 μM PZQ showed various changes in the tegument and gynecophoral canal, including extensive blebs, shallow peeling, pit-shaped erosion, and the rupture of the crests (Additional file 2: Figure S1a, d–f). In the oral and ventral suckers, disarrangement of the inner wall and a loss of spines were observed (Additional file 2: Figure S1b, c). Alterations in the tegument caused by 100 μM DW-3-15 were more significant than those from 100 μM PZQ. Along the gynecophoral canal and tegument of worms, sensory papillae exhibited severe damage, with the tegumental structures showing marked lysis, disorganization, and collapse, with pronounced blisters and hole-shaped erosions (Additional file 3: Figure S2a, d–f). Fusion of the inner wall and a loss of spine structure were observed in the oral and ventral suckers of males, with hole-shaped formations evident in the oral suckers (Additional file 3: Figure S2b, c). It was noteworthy that changes in the worms in the PZQ group were confined to the tegument, with no damage observed in the muscle tissue layer (Additional file 2: Figure S1). In contrast, DW-3-15 induced the exposure of the subtegument layer of the muscle tissue in some regions (Additional file 3: Figure S2).
When the two compounds were combined, extensive tegumental damage appeared along the whole tegument and in the gynecophoral canal (Additional file 4: Figure S3; Fig. 5). PDa-treated males showed severe tegumental damage in the form of hole-shaped erosions, blisters, and extensive sloughing, causing exposure of the subtegumental muscle layer and muscle injury (Additional file 4: Figure S3a, d–f). Images showing tegument swelling of the oral and ventral suckers with focal areas of disintegration and fusion are shown in Additional file 4: Figure S3b and c. More significant tegumental changes and sucker deformity were observed in worms exposed to PDb. Extensive sloughing of the tegument, with almost complete subtegumental muscle layer exposure, blisters, muscle injury, and muscle dissolution were observed in the whole worm tegument and gynecophoral canal (Fig. 5a, d–f). All the subtegumental structures of the oral suckers were exposed, with hole-shaped erosions on the muscle layer (Fig. 5b). The ventral sucker showed extensive swelling and collapse at the tegument, spine loss, and complete fusion, and long, irregular disorganized splits appeared (Fig. 5c).
Significant reduction in worm burden by combination chemotherapy in vivo
Data for worm and egg burden assessment in the study subgroups are shown in Fig. 6 and in Additional file 1: Tables S4–S6. Oral administration of the two-dose combination, PDc (100 mg/kg PZQ combined with 200 mg/kg DW-3-15) and PDd (200 mg/kg PZQ combined with 400 mg/kg DW-3-15), to infected mice against 14-day-old juvenile, 28-day-old adult, and multiple parasitic stages resulted in a statistically more significant reduction in the mean total worm burden (Fig. 6; juveniles: F(6, 98) = 192.6, P < 0.0001; adults: F(6, 98) = 229.1, P < 0.0001; multiple parasitic stages: F(6, 98) = 123.1, P < 0.0001) compared with the infected but untreated control group. Mice treated with a higher-dose combination (PDd) against 14-day-old juveniles, 28-day-old adults, and multiple parasitic stages showed the highest reductions in total worm burden, at 83.8, 97.3, and 83.0%, respectively (Additional file 1: Tables S4–S6). Respective reductions in total worm burden induced in the corresponding PDc group against 14-day-old juveniles, 28-day-old adults, and multiple parasitic stages were 67.5, 92.1, and 65.9% (Additional file 1: Tables S4–S6). As expected, PZQ monotherapy was effective against adult worms but was less effective against juveniles. Reductions induced by PZQ monotherapy at the highest dose (400 mg/kg) against juvenile, adult, and multiple-stage worms was 43.3, 96.9 and 64.1%, respectively (Additional file 1: Tables S4–S6). For the DW-3-15 monotherapy group, the efficacy against juvenile, adult, and multiple-stage worms was dose-dependent, with increasing dose resulting in a more significant decrease in total worm burden. Reductions induced by DW-3-15 against the corresponding developmental stages at the highest dose (400 mg/kg) were 70.3, 60.0, and 74.5%, respectively (Additional file 1: Tables S4–S6). Compared with PZQ monotherapy, a statistically significant reduction in total worm burden was observed for treatment with the two-compound combinations against juveniles (Fig. 6; F(6, 98) = 192.6, P < 0.0001) and multiple parasitic stages (Fig. 6; F(6, 98) = 123.1, P < 0.0001). Although there was no significant difference in the reduction of total worm burden between the higher-dose combination compounds and PZQ alone at 200 mg/kg against adults, the combination groups displayed a higher reduction than PZQ alone. Compared with DW-3-15 monotherapy, a statistically significant reduction in total worm burden was found with combination-group treatment against juveniles and adult worms (Fig. 6; juveniles: F(6, 98) = 192.6, P = 0.0001; adults: F(6, 98) = 229.1, P < 0.0001). With regard to the efficacy against multiple parasitic stages harbored in one host, although there was no statistically significant difference between combination therapy and DW-3-15 monotherapy at the highest dose, the reduction in total worm burden was higher using combination therapy than DW-3-15 monotherapy (Fig. 6). The results indicated that the PZQ and DW-3-15 combined application was synergistically active against multiple developmental stages of S. japonicum. Synergism was observed for a higher-dose combination of PZQ and DW-3-15 (PDd) against 14-day-old juveniles, 28-day-old adult worms, and multiple-parasitic-stage worms. The CI was 0.49, 0.43, and 0.59, respectively (Fig. 7).
Significant decrease in hepatic granulomas by combination chemotherapy
The histopathological features of granulomas in Masson trichrome-stained mouse liver sections are shown in Additional file 5: Figure S4. The livers of untreated infected mice showed typical hepatic schistosomal granulomas, surrounded by large numbers of inflammatory cells. Pronounced reductions in S. japonicum-associated hepatic granuloma size were observed in all developmental stages in infected mice treated with PZQ and DW-3-15 combinations (Additional file 5: Figure S4; juveniles: PDc: F(4, 10) = 11.5, P = 0.0018, PDd: F(4, 10) = 11.5, P = 0.0010; multiple parasitic stages: PDc: F(4, 10) = 14.9, P = 0.0011, PDd: F(4, 10) = 14.9, P = 0.0002; adults: PDc: F(4, 10) = 9.731, P = 0.0027, PDd: F(4, 10) = 9.731, P = 0.0007). By calculating the egg burden per gram of liver tissue, all treatments were found to induce statistically significant reductions in liver egg burden when compared with the infected untreated control (juveniles: F(8, 81) = 184.6, P < 0.0001; multiple parasitic stages: F(8, 83) = 114.8, P < 0.0001; adults: F(8, 82) = 134.8, P < 0.0001). The group treated with the higher-dose combination of PZQ and DW-3-15 (PDd) showed the greatest decrease in egg burden (Additional file 1: Tables S4–S6).