Introduction

Schistosomiasis, caused by parasitic flatworms of the genus Schistosoma, is a tropical disease that occurs in subtropical regions. There are almost 260 million people affected by schistosomiasis (WHO 2016). Humans are infected by one of the following six species of schistosomes: S. guineensis, S. haematobium, S. japonicum, S. mansoni, S. intercalatum, and S. mekongi, of which S. haematobium, S. japonicum, and S. mansoni are the predominant causes of disease. China is mainly stricken by S. japonicum. By the end of 2013, 184,943 schistosomiasis japonica cases were calculated in China, and a total of 29,796 advanced schistosomiasis cases were reported (Lei et al. 2014).

Schistosomiasis is distributed mainly in four provinces, Jiangxi, Hubei, Hunan, and Anhui, and accounts for 96.34% of the total number of cases (178,180/184,943) (Lei et al. 2014). The adult worms, with the exception of S. haematobium, inhabit the mesenteric veins, where they produce eggs. The pathology of schistosomiasis is caused by the eggs that are arrested in the host tissue, particularly in the liver and intestine. The lodged ova generate sustained antigenic stimulation, which is followed by the recruitment of inflammatory and immune cells to the sites of infection, resulting in the formation of granuloma and eventually chronic fibrosis in some infected individuals (Chuah et al. 2014).

The transcription factors of the nuclear factor kappa B (NF-κB) family are key regulators of a variety of inflammatory genes in immune and inflammatory processes (Napetschnig and Wu 2013). NF-κB is retained as a dormant molecule in the cytoplasm by binding tightly to the IκB inhibitor protein. Upon stimulation, phosphorylation of IκB by the IκB kinase (IKK) separates IκB from NF-κB, leading to NF-κB translocation and activation (Hayden and Ghosh 2008). One vital component of the canonical NF-κB signaling pathway is p65, also known as RelA, which binds as a homodimer or heterodimer to target specific sequences in the promoter or enhancer regions of target genes (Kumar et al. 2004). It has been reported that activation of the NF-κB signaling pathway is closely related to hepatic granuloma and fibrosis formation (He et al. 2014; Liu et al. 2014). The size of hepatic granuloma and fibrosis is determined by the Th1 and Th2 cytokines (Caldas et al. 2008). The maintenance of TNFα at high levels in the serum of patients and mice promotes the formation of liver granuloma and fibrosis (Joseph and Boros 1993; Shahat et al. 2007). Transforming growth factor-β (TGF-β), as one of the major profibrotic cytokines, plays a pivotal role not only in the inflammatory process but also in the pathogenesis of hepatic fibrosis caused by the infection of S. japonicum (Li et al. 2015).

Genistein, also called 4′,5,7-trihydroxyisoflavone, is the principal isoflavone found in soy products, and it has potential effects in a multitude of disease states due to its biological activities. Growing evidence has shown that genistein is a promising agent for the inhibition of carcinogenesis, angiogenesis, and metastasis (Banerjee et al. 2008; Guo et al. 2007; Huang et al. 2014; Xiao et al. 2015). Genistein has many health benefits through the modulation of some important cell signaling pathways, such as the NF-κB signaling pathway, AKT signaling pathway, and ROS/Nrf2 signaling pathway (Davis et al. 2001; El Touny and Banerjee 2007; Liu et al. 2016; Wang et al. 2013). It has been shown that genistein inhibits the NF-κB signaling pathway, as evidenced by the inhibition of phosphorylation of IκB, which prevents the translocation of NF-κB to the nucleus and represses NF-κB downstream genes (Davis et al. 2001). Furthermore, it is demonstrated that genistein protects against methionine-choline-deficient (MCD) diet-mediated hepatic inflammation and fibrosis in db/db mice (Yoo et al. 2015). However, the effect of genistein on schistosomiasis has not been elucidated. This encouraged us to investigate the function and mechanism of genistein in S. japonicum egg-induced liver granuloma and later progression of hepatic fibrosis in detail.

Materials and methods

Chemicals, parasites, and animals

Genistein (98% purity, Sigma-Aldrich, Saint Louis, MO, USA) was dissolved in 0.5% sodium carboxymethylcellulose (CMC-Na; Sangon Biotech, Shanghai, China). Oncomelania hupensis snails, infected with the Chinese strains of S. japonicum, were provided by Hunan Provincial Institute of Parasitic Disease. Six- to eight-week-old male BALB/c mice weighing 18–25 g were purchased from the China Three Gorges University Laboratory Animal Center. All animals were maintained under controlled temperature, humidity, and a 12-h light/dark cycle and had free access to food and water in the China Three Gorges University Laboratory Animal Center. All experimental procedures involving animals were carried out in accordance with NIH Guidelines for the Care and Use of Laboratory Animals. Animal care and use was approved by the Experimental Animal Management Committee of China Three Gorges University (permit number 2015100A; 11/10/2015).

Infection of mice with S. japonicum

The cercariae, shed when placing Oncomelania in chlorine-water under light for 30 min, were examined and counted by light microscopy. The animals were infected percutaneously with 18 ± 3 S. japonicum cercariae per mouse.

Animal treatment

For early/late phase genistein treatment, the mice were randomly divided into four groups: (1) control, (2) infection, (3) infection + genistein (25 mg/kg), and (4) infection + genistein (50 mg/kg), with six mice each group. All the groups were infected with S. japonicum cercariae except group (1). The group (2) was infected with S. japonicum cercariae without administration of genistein. For the early phase, mice were treated intragastrically with genistein (25 or 50 mg/kg) daily from week 4 to week 6 after infection in groups (3) and (4). At the same time, mice were treated intragastrically with 0.5% CMC-Na in groups (1) and (2). Mice were sacrificed 24 h after the last administration. For the later phase, the infected mice were orally administered praziquantel (0.5 g/kg) on week 6 for 2 days to remove the adult worms. Then, mice were treated intragastrically with genistein (25 or 50 mg/kg) daily from week 6 to week 10 after infection in groups (3) and (4). Mice were treated intragastrically with 0.5% CMC-Na in groups (1) and (2) from week 6 to week 10 after infection. The diagram of the experiment design is shown in Fig. 1.

Fig. 1
figure 1

Diagrammatic overview of S. japonicum cercarial infection and genistein administration in a murine model of hepatic schistosomiasis japonica. BALB/c mice were infected with 18 ± 3 cercariae received treatment with genistein (25 or 50 mg/kg) after infection. G25 mice treated with 25 mg/kg genistein, G50 mice treated with 50 mg/kg genistein

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Histology and immunohistochemistry analysis

After 24 h of the last genistein administration, the mice were sacrificed and the livers were removed. Small pieces of liver tissue were fixed with 4% paraformaldehyde overnight at 4 °C. The fixed tissues were further processed histologically through an increasing alcohol series, embedded in paraffin, and then sectioned at 4-μm thickness. The tissue sections were stained with hematoxylin and eosin (H&E) or Masson’s trichrome, respectively. The extent of granulomas and hepatic fibrosis was observed by light microscopy (Olympus, Japan). Some paraffin sections of livers were used to stain for p65, α-smooth muscle actin (α-SMA), and TGF-β, using primary antibodies including anti-p65 (Abcam, Cambridge, UK) and anti-α-SMA (Bioss Inc., Beijing, China). IHC was performed with the Ultrasensitive™ SP (Mouse/Rabbit) IHC Kit (Mai-xin Biotechnology Co., Fuzhou, China). The results were observed by light microscopy (Olympus, Japan) and analyzed using morphometric software (Image-Pro Plus software), and results were expressed as percentage of the total cells.

RNA extraction, RT-PCR, and quantitative real-time PCR

Total RNA was extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) and reverse transcribed with ReverTra Ace (Toyobo, Dalian, China) to produce cDNA. The sequences of the primers listed in Table 1 were used for quantitative real-time PCR. Real-time PCR was performed using SYBR Green-based detection in StepOnePlus™ (ABI, CA, USA) according to the manufacturer’s instructions. The relative messenger RNA (mRNA) levels for specific genes were normalized to β-actin levels.

Table 1 Primers used for quantitative real-time PCR
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Western blot

Mouse liver lysates were prepared with RIPA lysis buffer followed by centrifugation. Western blot analysis of p65 (Abcam, Cambridge, UK; 1:3000), phospho-p65 (S536) (CusAb Inc., Wuhan, China; 1:1000), IκB (CusAb Inc., Wuhan, China; 1:2000), phospho-IκB (S32/S36) (CusAb Inc., Wuhan, China; 1:1000), α-SMA (Bioss Inc., Beijing, China; 1:2000), TGF-β (Bioss Inc., Beijing, China; 1:2000), and β-actin (Sigma-Aldrich, Saint Louis, MO, USA; 1:10,000) in mouse liver was performed according to the standard protocols. The blots were detected by Immobilon Western Chemiluminescent HRP Substrate Kit (Millipore), followed by exposure to Kodak-X-Omat film (Shanghai, China).

Statistical analysis

All statistical analyses were performed with the GraphPad Prism software. Values are expressed as the mean ± SEM. Pairwise comparisons were performed with Student’s t test (two-tailed), and multiple-group comparisons were performed with one-way ANOVA with Bonferroni’s post hoc test. A P value <0.05 was considered to be significant.

Results

Genistein inhibited S. japonicum egg-induced liver granuloma formation

To evaluate the effect of genistein on S. japonicum egg-induced liver granuloma, mice were infected with 18 ± 3 cercariae of S. japonicum and treated intragastrically with genistein (25 or 50 mg/kg) every day from week 4 to week 6 after infection (Fig. 1). Liver samples were harvested 24 h after the last treatment. The mice infected with S. japonicum cercariae exhibited enlarged livers and had dark brown, sharp, and hard edges and a large number of miliary gray nodules scattered on the surface of the liver (Fig. 2a). After the genistein treatment (25 mg/kg), a dark red liver was observed in Fig. 2a. Pink, smooth, and soft liver tissue appeared in mice treated with 50 mg/kg genistein (Fig. 2a). The livers were fixed and stained with H&E. The results demonstrated that eggs are encompassed by a mass of immune cells, resulting in the formation of a mature granuloma, and granulomatous inflammation is regressed in the groups with increased doses of genistein (Fig. 2b). Obviously, the area of liver granuloma was significantly attenuated in the group treated with 25 or 50 mg/kg genistein (Fig. 2c). To detect whether the above difference in the area of liver granuloma is due to dissimilarity of parasite pairs or parasite eggs, parasite pairs and parasite eggs in livers were examined. Changes in parasite pairs and parasite eggs were negligible upon genistein treatment or vehicle treatment after the infection (Fig. 2d, e).

Fig. 2
figure 2

Effect of genistein on S. japonicum egg-induced liver granuloma formation. Mice were infected with 18 ± 3 cercariae, treatment with genistein (25 or 50 mg/kg) from week 4 to week 6 after infection. G25 mice treated with 25 mg/kg genistein, G50 mice treated with 50 mg/kg genistein after infection. Asterisk indicates G25 or G50 vs control. Values represent the means ± SE. n = 6; ***P < 0.001. a Gross morphology of liver tissue. b Representative images of liver tissue in different groups stained with H&E. Magnification ×100; scale bar indicates 200 μm. c Analysis of hepatic granuloma size using Image-Pro Plus. d Worm pairs. e Egg burden in host livers

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The administration of genistein repressed the NF-κB signaling pathway

To analyze the changes of the NF-κB signaling pathway upon genistein treatment in the formation of liver granuloma, first, we analyzed the expression of p65 in liver tissue by IHC and western blot, which is a pivotal member of NF-κB signaling pathway. The results demonstrated that p65 expression increased significantly by 6 weeks post-infection (Fig. 3a, b). The observation of nuclear staining of p65 indicated the translocation of NF-κB. The p65 expression in the liver tissues of genistein-treated animals was markedly reduced compared with the infected non-treated group (Fig. 3a, c). To further address the NF-κB signals, we investigated the expression levels of phospho-IκB, IκB, and phospho-p65, which are parameters of NF-κB activity. Certainly, the expression levels of phospho-IκB and phospho-p65 increased significantly, and the expression levels of IκB decreased markedly after infection with cercariae of S. japonicum (Fig. 3c–f). Phospho-IκB and phospho-p65 expressions in genistein-treated animals were significantly reduced compared with the infection group. At the same time, IκB expression increased significantly (Fig. 3c–f).

Fig. 3
figure 3

Effects of genistein administration on NF-κB pathway activation in the liver tissue of mice infected with S. japonicum. Mice were infected with 18 ± 3 cercariae, treatment with genistein (25 or 50 mg/kg) from week 4 to week 6 after infection. G25 mice treated with 25 mg/kg genistein, G50 mice treated with 50 mg/kg genistein after infection. Pound sign indicates the infected group vs control; asterisk indicates the G25 or G50 vs the infected group. Values represent the means ± SE. n = 6; *** or ### P < 0.001, ** P < 0.01, * P < 0.05. a Representative images of p65 expression in liver, as determined by IHC. Magnification ×400; scale bar indicates 100 μm. b The analysis of the expression of p65 detected by IHC using Image-Pro Plus. c Representative western blots for IκB, phosphor-IκB, p65, and phospho-p65 expressions in mouse liver tissues. d Densitometric analysis of the ratio of pIκB/IκB by ImageJ. Densitometric analysis of p65 (e) and p-p65 (f) by ImageJ; β-actin was used for calibration

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Early phase administration of genistein reduced MCP1, TNFα, IL1β, IL4, and IL10 mRNA levels

To explore the molecular mechanism of the anti-granuloma effects mediated by early genistein treatment, we tested for MCP1, TNFα, IL1β, IL4, CXCL1, and IL10 mRNA expressions in the liver tissue of genistein-treated or vehicle-treated mice by real-time PCR. The results demonstrated that infection with cercariae of S. japonicum stimulates the upregulation of MCP1, TNFα, IL1β, CXCL1, IL4, and IL10 mRNA levels (Fig. 4a–d, f). MCP1, TNFα, IL1β, IL4, and IL10 mRNA expressions in the liver tissues of genistein-treated animals were markedly decreased compared with the infection group (Fig. 4a–d, f). There was no significant difference in the CXCL1 mRNA expression in the liver tissues of genistein-treated animals compared with the infection group (Fig. 4e).

Fig. 4
figure 4

Early phase genistein treatment markedly decreases MCP1, TNFα, IL1β, IL4, and IL10 mRNA levels. Mice were infected with 18 ± 3 cercariae, treatment with genistein (25 or 50 mg/kg) from week 4 to week 6 after infection. G25 mice treated with 25 mg/kg genistein, G50 mice treated with 50 mg/kg genistein after infection. Pound sign indicates the infected group vs control; asterisk indicates the G25 or G50 vs the infected group. Values represent the means ± SE. n = 6; ** or ### P < 0.05, *** or ### P < 0.001. The mRNA levels were expressed relative to the levels in control group; β-actin was calibrated

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Late phase administration of genistein reverses S. japonicum egg-induced liver fibrosis

To examine the effect of genistein on S. japonicum egg-induced liver fibrosis, all infected mice were cleared of worms with praziquantel for 2 days on week 6, followed by treatment with genistein (25 or 50 mg/kg) or 0.5% CMC-Na for the following 33 days (Fig. 1). From gross morphology, we observed that the liver fibrosis had been ameliorated significantly upon the administration of genistein in comparison to the infection group (Fig. 5a). Masson’s trichrome staining exhibited severe hepatic fibrosis, and genistein treatment resulted in a dramatic reduction in the area of fibrosis in the liver sections in comparison to the infected mice in a dose-dependent manner (Fig. 5b, c). Compared with the infection group, fewer fibrous septa were formed, and less collagen was formed in hepatic tissue of genistein-treated groups. Furthermore, we investigated the expression of α-SMA using western blot, which is an indicator of stellate cell activation and prediction of liver fibrosis (Akpolat et al. 2005). As expected, cercariae of S. japonicum infection resulted in a markedly increased α-SMA expression (Fig. 6a, b). Moreover, genistein treatment significantly decreased the α-SMA expression compared with the infection group. Simultaneously, IHC assay was used to detect the expression of TGF-β. The results revealed that the cercariae of S. japonicum significantly upregulated TGF-β in the late phase, and in the genistein-treated animals, it was markedly decreased compared with the infection group (Fig. 6c, d).

Fig. 5
figure 5

Effect of genistein on S. japonicum egg-induced liver fibrosis formation. Mice were infected with 18 ± 3 cercariae; mice were orally administered praziquantel on week 6 for 2 days, following treatment with genistein from week 6 to week 10. Asterisk indicates the G25 or G50 vs the infection group. G25 mice treated with 25 mg/kg genistein, G50 mice treated with 50 mg/kg genistein after infection. Values represent the means ± SE. n = 6; *** P < 0.001. a Gross morphology of liver tissue. b Representative images of liver tissue in different groups stained with Masson’s trichrome. Magnification ×100; scale bar indicates 200 μm. c Analysis of hepatic fibrosis size using Image-Pro Plus

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Fig. 6
figure 6

Effect of genistein on α-SMA expression and TGF-β expression in S. japonicum egg-induced liver fibrosis formation. Mice were infected with 18 ± 3 cercariae; mice were orally administered praziquantel on week 6 for 2 days, following treatment with genistein from week 6 to week 10. Pound sign indicates the infected group vs control; asterisk indicates the G25 or G50 vs the infected group. Values represent the means ± SE. n = 6; ### or *** P < 0.001. a Representative western blots for α-SMA expression in mouse liver tissues. b Densitometric analysis of the α-SMA expression by ImageJ. β-actin was calibrated. c Representative images of TGF-β expression in liver, as determined by IHC. Magnification ×400; scale bar indicates 100 μm. d The analysis of the expression of TGF-β detected by IHC using Image-Pro Plus

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Late phase genistein treatment markedly decreases MCP1, TNFα, IL1β, IL4, and IL10 mRNA levels

To examine the molecular mechanism of the anti-fibrosis effect mediated by late phase genistein treatment, we tested for MCP1, TNFα, IL1β, IL4, CXCL1, and IL10 mRNA expressions in the liver tissue of genistein-treated or 0.5% CMC-Na-treated mice by real-time PCR. The results demonstrated that the cercariae of S. japonicum stimulate the upregulation of MCP1, TNFα, IL1β, CXCL1, IL4, and IL10 in the late phase of S. japonicum infection (Fig. 7a–d, f). MCP1, TNFα, IL1β, IL4, and IL10 mRNA expressions in the liver tissues of genistein-treated animals were markedly decreased compared with the infection group (Fig. 7a–d, f). There was no significant difference in the CXCL1 mRNA expression in the liver tissues of genistein-treated animals compared with the infection group (Fig. 7e).

Fig. 7
figure 7

Late phase genistein treatment markedly decreased MCP1, TNFα, IL1β, IL4, and IL10 mRNA levels. Mice were infected with 18 ± 3 cercariae; mice were orally administered praziquantel on week 6 for 2 days, following treatment with genistein from week 6 to week 10. Pound sign indicates the infected group vs control; asterisk indicates the G25 or G50 vs the infected group. Values represent the means ± SE. n = 6; ** or ### P < 0.05, *** or ### P < 0.001. The mRNA levels were expressed relative to the levels in control group; β-actin was calibrated

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Discussion

Genistein, the predominant isoflavone in soy products, has attracted more and more interest. Numerous favorable biological effects of genistein consumption have been revealed with respect to beneficial health. Dietary genistein has been shown to repress endotoxin-induced inflammatory reaction in the liver and intestine (Paradkar et al. 2004). Gupta et al. (2015) demonstrated that genistein lowers streptozotocin-induced cardiac inflammation and oxidative stress. Multiple studies from the last few decades have provided definitive evidence of the inhibitory effects of genistein on various cancer cells (Huang et al. 2014). One year of treatment with genistein improves surrogate endpoints associated with risk for diabetes and cardiovascular disease in post-menopausal women with metabolic syndrome (Squadrito et al. 2013). Administration of genistein to fructose-fed rats reduced inflammation, fibrogenesis, and NF-κB activation in the kidney (Palanisamy et al. 2011). Similarly, Li et al. (2013) have demonstrated that genistein decreases the expression of fibrogenic genes to attenuate the formation of fibrosis. It is well established that the etiopathology of schistosomiasis is primarily due to an excessive or unregulated inflammatory response to the parasite, in particular to the eggs that become trapped in the liver and induce hepatic granuloma and fibrosis. Here, we addressed the effect of genistein on S. japonicum egg-induced liver granuloma and fibrosis. The results showed that genistein treatment markedly attenuated S. japonicum egg-induced liver granuloma and fibrosis and markedly improved hepatic gross morphology. H&E and Masson’s trichrome staining have shown that genistein significantly ameliorates liver granuloma and fibrosis. Then, we wanted to investigate in detail the potential mechanism of the positive effects of genistein on S. japonicum egg-induced liver granuloma and fibrosis.

In resting cells, NF-κB is bound to IκBs in the cytoplasm, preventing the transcriptional factor’s nuclear translocation. Upon stimulation by pathogens, IκB is phosphorylated by IκB kinase α/β, resulting in proteasome-dependent degradation of the IκBs. Released NF-κB is then phosphorylated and translocated to the nucleus to activate target gene expression. Consistent with previous reports (Liu et al. 2014; Napetschnig and Wu 2013), p65, phospho-p65, and phospho-IκBα expressions were significantly stimulated, and IκBα expression was significantly decreased after infection with cercariae of S. japonicum. p65, phospho-p65, and phospho-IκBα expressions were markedly decreased, and IκBα expression was significantly increased in the liver of genistein-treated mice in comparison to the infection group. The p65 staining intensities in liver granuloma from genistein-treated groups were reduced compared with the infection group. Together, these data provide evidence that genistein suppresses NF-κB signaling in the liver tissues after S. japonicum infection.

Multiple studies have indicated that activation of NF-κB signaling plays a crucial role in the pathology of hepatic fibrosis by modulating hepatocyte, hepatic stellate cells (HSCs), and Kupffer cell function. Recently, He et al. (2014) reported that NF-κB signaling-activated HSCs are present in the periphery of S. japonicum egg granulomas in murine and human infections and are likely effector cells contributing to the granuloma-associated fibrosis. Suppression of NF-κB signaling is closely associated with the inhibition of hepatic fibrosis (He et al. 2014; Liu et al. 2014; Xu et al. 2012). Consistent with those findings, genistein markedly decreased the area of the liver granuloma and fibrosis in the present study, which occurred, at least in part, due to the repression of NF-κB signaling pathway.

HSCs are the primary sources of collagen and play a key role in hepatic fibrogenesis. Under pathological conditions, quiescent HSCs are activated and transdifferentiate into myofibroblast-like cells, characterized by the expression of the myofibroblast marker and α-SMA, and exhibit upregulated collagen synthesis (Xu et al. 2012). Thus, the expression of α-SMA has been considered to be one of the primary characteristics of HSC activation and has become an important evaluation index for hepatic fibrosis. TGF-β1 participates not only in the inflammatory process but also in the fibrotic process (Li et al. 2015). TGF-β1, which has been identified as the key profibrotic cytokine, can regulate the activation and transdifferentiation of HSCs (Cassiman et al. 2002). Furthermore, blockade of TGF-β1 signal transduction successfully inhibits the production of ECM and ameliorates hepatic fibrosis (Sun et al. 2015). Similar to the results of previous studies, we found that TGF-β1 and α-SMA expressions are significantly stimulated after the infection, and the expression is significantly repressed upon genistein treatment compared with the infection group at the late phase. However, a few of the published results are contradictory. Kaviratne et al. (2004) have shown that hepatic fibrosis is independent of TGF-β1 in the S. mansoni-infected mouse model. Therefore, we could only conclude that genistein significantly suppresses HSC activation, associated with the decrease of TGF-β1 expression, which is anticipated to contribute to the inhibition of hepatic fibrogenesis. Notably, consistent with our findings, Li et al. (2015) strongly suggested that TGF-β1 is involved in the pathogenesis of human schistosomal hepatic fibrosis. The specific role of TGF-β1 in schistosomal hepatic fibrosis requires further study.

The involvement of the CD4+ T helper (Th) cell response in the progress of hepatic schistosomiasis is clearly demonstrated. A Th1 response is initiated at the early stages of the infection, which is characterized by increased expression of Th1 proinflammatory cytokines, such as IL1, IL12, and TNFα (Wilson et al. 2007). The immune response switches to a pronounced Th2 response at 6 to 8 weeks post-infection following the onset of egg deposition, which is characterized by stimulating the levels of Th2 cytokines, such as IL4, IL5, IL10, and IL13 (Pearce and MacDonald 2002). Cheever et al. (1999) provides evidence that TNFα alone can reconstitute granuloma formation or hepatic fibrosis in schistosome-infected mice lacking functional B and T cells. Anti-IL4 treatment can inhibit the development of T cells and decrease the S. mansoni egg-induced hepatic fibrosis (Cheever et al. 1994). Moreover, lower expression of IL1β is associated with the decreased granulomatous inflammation (Zhang et al. 2012). MCP1 and CXCL1 are significantly upregulated at transcriptional levels during S. japonicum infection (Bartley et al. 2006; Burke et al. 2010). The increased CXCL1 is involved in HSC recruitment to the granulomas, and the increased MCP1 correlates with peak fibrosis in the same model. It has also been demonstrated, both by in vivo and in vitro observations, that genistein possesses immunomodulatory activity by tuning cytokine expression (Cui et al. 2016; Kim et al. 2014; Valsecchi et al. 2008).

Our data have shown that in the early and late phases, the mRNA levels of MCP1, TNFα, IL1β, IL4, CXCL1, and IL10 are significantly decreased upon genistein treatment after the infection; the genes of these mRNAs are reported to be under the control of NF-κB (Kumar et al. 2004). We postulated that repression of egg-induced immunopathology in schistosomiasis by genistein includes at least in part the blockade of the induction of cytokines and chemokines, such as MCP1, TNFα, IL1β, IL4, CXCL1, and IL10, possibly by reducing the inhibition of Th1 and Th2 response.

In summary, this study demonstrated that genistein protects against S. japonicum egg-induced hepatic granuloma and fibrosis. The effects may involve the NF-κB signaling pathway and at least in part involve the blockade of the induction of cytokines and chemokines. These results provide evidence for the promising potential of therapeutic use of genistein in S. japonicum egg-induced hepatic granuloma and fibrosis.