Ana-1 cell line is a well-established murine macrophage cell line, and it can be transformed into either M1 or M2 macrophage with some administration (Chen et al., 2012). It was purchased from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China. The cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum in an incubator at 37°C, 5% CO₂ and saturated humidity.

According to the previously published paper (Li et al., 2016), we evaluated cell viability using the CCK8 assay. The Ana-1 cells were plated at a density of 1 × 10⁴/mL cells in 96-well culture plates with different concentrations of corilagin (400, 200, 100, 50, and 25 mg/mL). The Ana-1 cells without corilagin administration was set as a positive control, and a blank well-containing the medium culture was set as a negative control. After 24 h, the cell morphology was observed, and 10 μl of CCK-8 solution was added to each well, which was then incubated for 2 h before the absorbance was measured at 450 nm using a microplate reader. Three wells in each group were measured.

Recombinant IL-13 was diluted to 5 μg/ml with RPMI-1640 medium containing 5% fetal bovine serum and stored at −20°C. Praziquantel was diluted to 5 pg/ml with PBS and stored at 4°C. Corilagin was diluted to 2 mg/ml with PBS and stored at 4°C. To explore the anti-fibrogenic effect of corilagin with the praziquantel intervention via the IL-13α1 signaling pathway, Ana-1 cells were divided into six groups. The high- (100 μg/ml), medium- (50 μg/ml), and low-concentration (25 μg/ml) corilagin groups were stimulated with rIL-13 (50 ng/ml) for 24 h, followed by treatment of corilagin for 24 h, and no praziquantel was administered. The praziquantel group was stimulated with rIL-13 (50 ng/ml) for 24 h, followed by treatment of praziquantel (5 pg/ml) for 24 h. The model group was incubated with rIL-13 (50 ng/ml) only. The untreated group was the control group.

IL-13Rα1 was up-regulated by means of lentivirus transfection containing pre-IL-13Rα1 and green fluorescent protein. Ana-1 cells were cultured in enhanced infection solution with the vehicle for 96 h at a MOI of 40. The transfection efficiency was observed under a fluorescence microscope (Olymbus IX2 series microscope). To further confirm the effect of corilagin on the IL-13α1 pathway, Ana-1 cells were divided into seven groups for an up-regulation experiment. The group stimulated with rIL-13 alone without IL-13Rα1 up-regulation was included in the previous experiments. Therefore, we set up this part of the experiment without the group of rIL-13 stimulation alone. In the high- (100 μg/ml), medium- (50 μg/ml), low-concentration (25 μg/ml) corilagin up-regulation groups, the IL-13Rα1 up-regulated Ana-1 cells were stimulated with rIL-13 (50 ng/ml) for 24 h before corilagin was added. In the model up-regulation group, the IL-13Rα1 up-regulated Ana-1 cells were incubated with rIL-13 (50 ng/ml) only. In the up-regulation group, the IL-13Rα1 up-regulated Ana-1 cells were cultured without any treatment. In the empty-vector group, the Ana-1 cells were cultured in enhanced infection solution with the empty vector for 96 h only. The untreated group was the control group.

IL-13Rα1 was down-regulated by means of siRNA transfection. The siRNA was purchased from RiboBio company (Guangzhou, China), and the specificity of siRNA was checked by Primer BLAST. The target sequence of the siRNA was CCTGCACTGGAAGAAGTAT, and the concentration of the siRNA was 0.02 nmol/μl. The siRNA contained cy3 dye. Ana-1 cells were incubated in RPMI-1640 medium containing 5% fetal bovine serum with 0.25% (5 μl Lipofectamine™ 2000 in 2 ml cell culture medium) Lipofectamine™ 2000 (purchased from Invitrogen) and 100 pmol of siRNA for 6 h, then cultured with RPMI-1640 medium containing 5% fetal bovine serum that did not contain siRNA or Lipofectamine™ 2000 for 48 h. Subsequently, the transfection efficiency was observed under a fluorescence microscope. To further confirm the effect of corilagin on the IL-13α1 pathway, Ana-1 cells were divided into seven groups in the down-regulation experiment. In the high- (100 μg/ml), medium- (50 μg/ml), low-concentration (25 μg/ml) corilagin down-regulation groups, the IL-13Rα1 down-regulated Ana-1 cells were stimulated with rIL-13 (50 ng/ml) for 24 h before corilagin was added. In the model down-regulation group, the IL-13Rα1 down-regulated Ana-1 cells were incubated with rIL-13 (50 ng/ml) only and cultured without any treatment. In the empty-vector group, the Ana-1 cells were transfected by the empty vector. The untreated group was the control group.

The animal experiments were approved by the Tongji Medical College, HUST Institutional Animal Care and Use Committee ([2016] IACUC Number: 598). A total of 42 male C57BL/6 mice, 6 to 8 weeks old and weighing 18–22 g, were purchased from the Hubei Provincial Center for Disease Control and Prevention (Wuhan, China). The mice were randomized into seven groups, with six mice per group. The mice were fed an untreated diet and given access to water under standard laboratory conditions at a temperature of 25 ± 2°C, a relative humidity of 50 ± 15% and a normal circadian rhythm (12-h dark/12-h light). All study protocols were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals of Huazhong University of Science and Technology and approved by the Ethics Committee of Union Hospital, Tongji Medical College, Huazhong University of Science and Technology. The control group served as a blank control without any administration or infection. The animals in all groups except the control group were infected with 25 ± 5 Schistosoma japonicum cercaria via the abdominal-patch method (Huang et al., 2013). After anaesthesia with 2% pentobarbital sodium (intraperitoneal injection, 45 mg/kg), the abdominal skin without hair was exposed to the cercaria carried by Oncomelania hupensis for 30 min to infect the mice. Four weeks after infection, the animals in all groups except the control group were administered praziquantel orally at 500 mg/kg·d for 5 days to kill the adult schistosomes. Subsequently, the high- (80 mg/kg·d), medium- (40 mg/kg·d), and low-concentration (20 mg/kg·d) corilagin groups were administered corilagin orally for 21 days. The praziquantel group as a positive control continued to be administered praziquantel orally at 500 mg/kg·d for 21 days after the initial 5 days of praziquantel treatment. The levofloxacin group as a negative control was administered levofloxacin orally at 100 mg/kg·d for 21 days. The model infection group was administered with no more drugs after 5-day praziquantel treatment. Finally, the mice were euthanized after anesthetization of 4% chloral hydrate by intraperitoneal injection and the specimens were collected. Blood was collected via retro-orbital route, centrifuged at 3,500 rpm for 10 min and sera were stored at −20°C for ELISA. Part of the liver tissue was fixed in 10% formalin for tissue examination. The rest was stored in liquid nitrogen for PCR and Western-blot assays. During the dissection of mice, the adult schistosomes were not observed in the liver of mice or in the portal system, which indicated the 5-day praziquantel treatment was effective on killing the adult schistosomes.

The expression of PPARγ, KLF4, SOCS1 and STAT6 was quantified by real-time qPCR. The operation and analysis was performed according to our previously published protocol (Guo et al., 2015b). Total RNA in Ana-1 cells was isolated using RNAiso Plus (TaKaRa Company). The A260/280 ratio of the mRNA was detected by spectrophotometer (UV751GD UV/VIS spectrophotometer, Implen company, German), and only the samples whose A260/280 ratios were between 1.8 and 2.2 were used. The cDNAs were produced with the PrimeScript™ RT reagent kit (TaKaRa company) and incubated at 37°C for 15 min and 85°C for 5 s. Real-time PCR reactions were performed using a StepOne Plus device (Applied Biosystems) at 95°C for 10 s, followed by 40 cycles of 95°C for 5 s and 60°C for 20 s according to the instructions for the SYBR Premix Ex Taq kit (TaKaRa company). The standard curves indicated that all the slopes ranged from −3.3515 to −3.222 and the R² values were greater than 0.99. Therefore, the data were analyzed by the 2^−ΔΔCT method. The number of PCR cycles varied according to the expression level of the target gene. An appropriate primer concentration and number of cycles was determined to ensure that the PCR was taking place in the linear range and thereby guaranteed a proportional relationship between input RNA and the cycles readout. All primers were synthesized by TSINGKE (Wuhan, China). GAPDH and β-actin were used as reference genes. In our study, the trends of KLF4 expression were consistent in the case of two reference genes (GAPDH and β-actin). Therefore, we chose GAPDH as the reference gene for the following qPCR assay. Three sets of duplicate data were included for statistical analysis. The primers were constructed and purchased from Tsingke Biological Technology Company (Beijing, China). The specificity of primer was checked by Primer Blast. The standard curves and amplification curves could be seen in Supplementary Data Sheet 1.

The abundance of PPARγ, KLF4, SOCS1, STAT6 and p-STAT6 was detected by Western blot. The operation and analysis were performed as described in a previously published paper (Wang et al., 2017). The protein was separated on SDS-PAGE gels and then transferred to nitrocellulose filter membrane, which was blocked overnight with 5% non-fat milk in TBST. Subsequently, the membrane was probed with the indicated antibody at 4°C before being washed three times in TBST and then incubated with an HRP-labeled secondary antibody. The dilutions of the primary antibodies were as follows: PPARγ (Proteintech Company, Wuhan, China), 1:1,000; KLF4 (Santa Cruz Biotechnology Company, Dallas, TX, US), 1:300; SOCS1 (Abclonal Technology, Woburn, MA, US), 1:500; p-STAT6 (CST, Boston, MA, US), 1:1,000.

The TGF-β levels in the cell supernatant or in the sera of the mice were determined by ELISA. The sera were assayed for TGF-β with Mouse TGF-β ELISA kits (Elabscience, Wuhan, China). The procedures were conducted according to the instruction manuals for the kits. Sterile PBS was used as control.

Hepatic tissue samples were obtained, formalin-fixed and paraffin-embedded. As in our previously published paper, haematoxylin and eosin (HE) staining (Jin et al., 2015) and Masson's trichrome staining (Li et al., 2016) were performed. Masson's trichrome stains hepatic cells in red and collagen in blue, which could help to clarify the degree of fibrosis. CD206 was a cell surface marker which could distinguish M2 macrophages from M1 macrophages. Immunohistochemistry for CD206 was performed to observe the M2 macrophages distribution in the liver tissue sections. The operation and analysis was performed according to our previously published protocol (Du et al., 2016). CD206 antibody was purchased from Proteintech Group, INC (Chicago, USA), catalog number 18704-1-AP, and its dilution was 1:200. The dilution of CD206 antibody was 1:200. The granuloma area and IOD of CD206 was measured by Imag-Pro Plus 6.0.

The data are presented as the means ± SD. Referring to a previously published statistical approach (Ding et al., 2015), Kolmogorov-Smirnov (K-S) test and Shapiro-Wilk (S-W) test were performed to assess the normalcy. The significance of K-S and S-W test indicated that the data were normally distributed (p > 0.05). Levene's test was performed to assess the equality of variances. The significance of Levene's test indicated the variances of the data from different groups were equal (p > 0.05). The measurement data were compared between the two groups with Student's t-test. Multiple comparisons between multiple groups were performed with one-way ANOVA tests, and a post-hoc test was performed by the Student-Newman-Keuls (S-N-K) method. The statistical analyses were conducted with SPSS 12.0 software. Statistical significance was defined at p < 0.05.

The viability of Ana-1 cells with corilagin administration was illustrated in Figure 1. As illustrated, the viabilities of all corilagin groups decreased compared with the 0 μg/ml corilagin group (p < 0.05, Student's t-test, n = 3). In our study, we chose the concentrations which could result in a cell viability more than 70% (100, 50, 25, μg/ml). With the corilagin concentration ranging from 50 to 400 μg/ml, the viability decreased with increasing concentrations of corilagin (p < 0.05, one-way ANOVA, post-hoc: S-N-K method, n = 3), suggesting a dose-dependent effect.

PPARγ, KLF4, SOCS1, and p-STAT6 are important regulators of M2 macrophage polarization in the IL-13α1 signaling pathway (Sica and Mantovani, 2012). Therefore, we investigated the expression of genes besides IL-13Rα1 to explore the anti-fibrogenic effect of corilagin. As illustrated in Figure 2, the mRNA levels of PPARγ, KLF4 and SOCS1 were detected by real-time qPCR (Figures 2A–C,G), and the TGF-β levels in the cell supernatant were detected by ELISA (Figure 2F). Meanwhile, we detected mRNA levels of IL-13Rα1 and STAT6 by real-time qPCR (Figures 2D,E). The abundance of PPARγ, KLF4, SOCS1, and p-STAT6 proteins was detected by Western blot (Figure 3). The levels of PPARγ, KLF4, SOCS1, and p-STAT6 in the model experimental groups were increased compared with levels in the control group (p < 0.01, Student's t-test, n = 3), which revealed the model is successful and the expression of these molecules increased with IL-13 stimulation. Meanwhile, with corilagin treatment after IL-13 stimulation, the levels of these molecules decreased significantly compared with the levels in the model group (p < 0.01 or 0.05, Student's t-test, n = 3). And the levels decreased with increasing concentrations of corilagin (p < 0.05, one-way ANOVA, post-hoc: S-N-K method, n = 3), which confirmed a dose-dependent inhibitory effect of corilagin on the cytokines in IL-13Rα1 signaling pathway in vitro. With praziquantel treatment after IL-13 stimulation, the levels of these molecules showed no significant difference compared with the model group (p > 0.05, Student's t-test, n = 3), which indicated praziquantel had little effect on the cytokines in IL-13Rα1 signaling pathway. There were no significant differences in the levels of IL-13Rα1 and STAT6 among groups (p > 0.05). As illustrated in Figures 2B,G, the trends of KLF4 mRNA expression were showed to be consistent when real-time qPCR was performed with two reference genes in our study.

To further explore the anti-fibrotic effect of corilagin in hepatic schistosomiasis, we up-regulated the expression of IL-13Rα1 by lentivirus transfection. The transfection effect was observed under a fluorescence microscope and examined by real-time qPCR, ELISA and Western blot. The Ana-1 cells were observed under a fluorescence microscope 96 h after lentivirus transfection (Figure 4A). The mRNA levels of IL-13α1 were determined by real-time qPCR, and the expression of IL-13Rα1 in up-regulation group increased by 130% compared to the control group (Figure 4B). The abundance of IL-13α1 protein was determined by Western blot, and the abundance of IL-13Rα1 protein in up-regulation group increased by 115% compared to the control group (Figure 4C). As illustrated, the fluorescence was strong and most cells were infected successfully. The levels of IL-13Rα1 mRNA and protein in the up-regulation group were increased significantly compared with the control groups (p < 0.01, Student's t-test, n = 3). The levels in the empty-vector group showed no significant difference compared to the control group (p > 0.05, Student's t-test, n = 3).

We down-regulated the expression of IL-13Rα1 by siRNA transfection. The transfection effect was observed under a fluorescence microscope and examined by real-time qPCR, ELISA and Western blot. The Ana-1 cells were observed under a fluorescence microscope 48 h after the siRNA transfection (Figure 5A). As illustrated, the fluorescence was strong and most cells were infected successfully. The mRNA levels of IL-13α1 were determined by real-time qPCR, and the expression of IL-13Rα1 in down-regulation group dropped 61% compared to the control group (Figure 5B). The abundance of IL-13α1 was determined by Western blot, and the abundance of IL-13Rα1 in down-regulation group dropped 41% (Figure 5C). As shown in Figure 5, the level of IL-13Rα1 in the down-regulation group was decreased significantly compared with the empty-vector and control groups (p < 0.01, Student's t-test, n = 3). The level in the empty-vector group showed no significant differences compared with that in the control group (p > 0.05, Student's t-test, n = 3).

Real-time qPCR was performed to examine the mRNA expression levels of PPARγ, KLF4 and SOCS1 in IL-13Rα1 up-regulated Ana-1 cells (Figures 6A–C) and down-regulated Ana-1 cells (Figures 8A–C). The TGF-β levels in the cell supernatant were determined by ELISA (Figures 6D, 8D). The abundance of PPARγ, KLF4, SOCS1 and p-STAT6 was determined by Western blot (Figures 7, 9). As illustrated, the levels of these molecules in the up- or down-regulation group correspondingly increased or decreased significantly compared with the control group (p < 0.01), indicating that IL-13Rα1 up- or down-regulation had a significant effect on the IL-13α1 signaling pathway. With IL-13 stimulation, the levels in the model up- or down-regulation group were increased significantly compared with those in the up- or down-regulation group (p < 0.01, Student's t-test, n = 3). The levels of these molecules with corilagin treatment of different concentrations were decreased compared with the model up- or down-regulation group (p < 0.01, Student's t-test, n = 3), and the inhibitory effects increased with the increasing concentration of corilagin (p < 0.05, one-way ANOVA, post-hoc: S-N-K method, n = 3), which revealed the dose-dependent inhibitory effect of corilagin on IL-13Rα1 signaling pathway still persisted when IL-13Rα1 was up- or down-regulated. The levels of these molecules in the empty-vector group showed no significant differences compared with the control group (p > 0.05, Student's t-test, n = 3).

In the animal model, we quantified the mRNA expression of PPARγ, KLF4 and SOCS1 in liver tissue using real-time qPCR (Figures 10A–C). The level of TGF-β in mice serum was measured by ELISA (Figure 10D). The abundance of PPARγ, KLF4, SOCS1 and p-STAT6 in liver tissue was measured by Western blot (Figure 11). As shown, the levels of these molecules in the model infection group were increased compared with the control group (p < 0.01, Student's t-test, n = 6). The levels of these molecules with corilagin treatment of different concentrations were decreased compared with the model infection group (p < 0.01 or p < 0.05, Student's t-test, n = 6), and the inhibitory effects increased with increasing concentrations of corilagin (p < 0.05, one-way ANOVA, post-hoc: S-N-K method, n = 6), which confirmed the dose-dependent inhibitory effect of corilagin on IL-13Rα1 signaling pathway in vivo. The levels of these molecules in the praziquantel group (positive control) and levofloxacin group (negative control) were increased compared with the control group (p < 0.01, Student's t-test, n = 6) and showed no significant differences compared with the model infection group (p > 0.05, Student's t-test, n = 6), which indicated praziquantel had little effect on IL-13Rα1 signaling pathway.

The effect of corilagin on pathological changes of schistosomiasis liver fibrosis in mice liver was observed with HE, Masson staining and immunohistochemistry. According to the results, all the liver tissue of the infected mice showed varying degrees of fibrosiss, which indicated the model establishment was successful. As illustrated in Figure 12, significant fibrosis and granuloma were observed in the model infection, praziquantel and levofloxacin groups, and the area of granuloma was remarkably larger than that in the control group (p < 0.01, Student's t-test, n = 6). The area of granuloma with corilagin treatment of different concentrations compared was significantly smaller with the model infection groups (p < 0.05, Student's t-test, n = 6), and the area decreased with increasing concentrations of corilagin (p < 0.05, one-way ANOVA, post-hoc: S-N-K method, n = 6), which showed a dose-dependent anti-fibrogenesis effect. For pathological scores, apart from normal group, the inflammation score of high-concentration corilagin group and medium-concentration corilagin group was G2, while low-concentration corilagin group was G2-G3, and praziquantel group was G3. The inflammation score of levofloxacin group and model infection group was G3-G4. The fibrosis score of corilagin groups was S2 and praziquantel, levofloxacin group and model infection group was S3. As illustrated in Figure 13, and the area of fibrosis in the model infection, praziquantel and levofloxacin groups was remarkably larger than that in the control group (p < 0.01, Student's t-test, n = 6). The area of fibrosis with corilagin treatment of different concentrations compared was significantly smaller compared with the model infection groups (p < 0.05, Student's t-test, n = 6), and the area decreased with increasing concentrations of corilagin (p < 0.05, one-way ANOVA, post-hoc: S-N-K method, n = 6), which showed a dose-dependent anti-fibrogenesis effect. As illustrated in Figure 14, the levels of CD206 staining in the model infection, praziquantel and levofloxacin groups were significantly higher than that in the control group (p < 0.01, Student's t-test, n = 6). The levels of CD206 staining were significantly lower in the corilagin groups compared with the model infection groups (p < 0.05, Student's t-test, n = 6), and the area decreased with increasing concentrations of corilagin (p < 0.05, one-way ANOVA, post-hoc: S-N-K method, n = 6). The less area of fibrosis and distribution of M2 macrophages in corilagin treatment group indicated the anti-fibrogenic effect of corlagin in vivo.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.