New insight into the antifibrotic effects of praziquantel on mice in infection with Schistosoma japonicum

Abstract

Background: Schistosomiasis is a parasitic disease infecting more than 200 million people in the world. Although chemotherapy targeting on killing schistosomes is one of the main strategies in the disease control, there are few effective ways of dealing with liver fibrosis caused by the parasite infection in the chronic and advanced stages of schistosomiasis. For this reason, new strategies and prospective drugs, which exert antifibrotic effects, are urgently required.

Methods and findings: The antifibrotic effects of praziquantel were assessed in the murine models of schistosomiasis japonica. Murine fibrosis models were established by cutaneous infection with 14 ± 2 Schistosoma japonicum cercariae. Then, the mice of both chronic (8 weeks post-infection) and advanced (15 weeks post-infection) schistosomiasis were treated by gavage of praziquantel (250 mg/kg, once daily for 3 days) to eliminate worms, and followed by praziquantel anti-fibrosis treatment (300 mg/kg, twice daily for 30 days). The fibrosis-related parameters assessed were areas of collagen deposition, content of hydroxyproline and mRNA expressions of Col1α1, Col3α1, α-SMA, TGF-β, MMP9, TIMP1, IL-4, IL-10, IL-13 and IFN-γ of liver. Spleen weight index, alanine aminotransferase activity and liver portal venous pressure were also measured. The results showed that anti-fibrosis treatment improved liver fibrosis, splenomegaly, hepatic function, as well as liver portal hypertension. In order to confirm the anti-fibrotic properties of praziquantel, we established a CCL(4)-induced model and revealed that CCL(4)-induced liver fibrosis was inhibited by PZQ treatment for 30 days. Furthermore, we analyzed the effects of praziquantel on mouse primary hepatic stellate cells (HSCs). It is indicated that mRNA expressions of Col1α1, Col3α1, α-SMA, TGF-β, MMP9 and TIMP1 of HSCs were all inhibited after praziquantel anti-parasite treatments.

Conclusions: The significant amelioration of hepatic fibrosis by praziquantel treatment validates it as a promising drug of anti-fibrosis and offers potential of a new chemotherapy for hepatic fibrosis resulting from schistosomiasis.

Figure 1. Anti-fibrosis treatment with prizaquantel reduced areas of liver Sirius Red staining and liver hydroxyproline contents of mice 8 weeks post infection with Schistosoma japonicum.

(A) Representative images of Sirius Red staining by light microscope (×100). Ai, uninfected; Aii, infected; Aiii, anti-parasite; Aiv, anti-fibrosis. (B) Statistical analysis of scanned images in Sirius Red staining by software IPP6.0. (C) Measurement of liver hydroxyproline content. (* p<0.05, ** p<0.01, *** p<0.001).

Figure 2. Anti-fibrosis treatment with prizaquantel reduced spleen weights in mice 8 weeks post infection with Schistosoma japonicum.

(A) images of representative spleens in different groups. The bar is 1 cm. (B) Statistical analysis of spleen weights. (C) Spleen weight index (spleen weight/body weight). (* p<0.05, ** p<0.01, *** p<0.001).

Figure 3. Anti-fibrosis treatment with prizaquantel reduced serum alanine aminotransferase (ALT) levels in mice 8 weeks post infection with Schistosoma japonicum.

Serum ALT was assessed by Olympus AU5400 automatic biochemistry analyzer. (* p<0.05, ** p<0.01, *** p<0.001).

Figure 4. Anti-fibrosis treatment with prizaquantel inhibited expressions of liver profibrotic genes and α-SMA protein expression in mice 8 weeks post infection with Schistosoma japonicum.

The expressions of mRNA were detected by Real Time PCR. (A) Expression of α-SMA. (B) Expression of Col1α1. (C) Expression of Col3α1. (D) Expression of TGF-β. (E) Expression of MMP9. (F) Expression of TIMP1. α-SMA protein expression was assessed by immunohistochemistry (G) Representative images of immunohistochemistry by light microscope (×100). Gi, uninfected; Gii, infected; Giii, anti-parasite; Giv, anti-fibrosis. Arrows showed the positive staining. (H) α-SMA positive areas were measured by software IPP6.0. (* p<0.05, ** p<0.01, *** p<0.001).

Figure 5. Anti-fibrosis treatment with prizaquantel reduced areas of liver Sirius Red staining and liver hydroxyproline content in mice 15 weeks post infection with Schistosoma japonicum.

(A) Representative images of Sirius Red staining by light microscope (×100). Ai, uninfected; Aii, infected; Aiii, anti-parasite; Aiv, low dose; Av, high dose. (B) Statistical analysis of scanned images in Sirius Red staining by software IPP6.0. (C) Measurement of Liver hydroxyproline content. (* p<0.05).

Figure 6. Spleen weights were not changed after Anti-fibrosis treatment with low dose or high dose prizaquantel compared with anti-parasite treatment in mice 15 weeks post infection with Schistosoma japonicum.

Spleen weights were reduced in anti-parasite, anti-fibrosis low dose and high dose groups compared with infected group, but no significant changes were found between treated groups. (** p<0.01).

Figure 7. Anti-fibrosis treatment with prizaquantel reduced hepatic portal venous pressure.

Hepatic portal venous pressure was measured by PowerLab8SP and analyzed by chart5 software. (A) Representative recording images were showed: Ai, uninfected; Aii, infected; Aiii anti-parasite; Aiv, low dose; Av, high dose. (B) Statistical analysis of each group hepatic portal venous pressure. Although anti-parasite treatment reduced portal venous pressure compared with infected group, anti-fibrosis treatment (both low dose and high dose group) further reduced the pressure to nearly uninfected level. (** p<0.01, *** p<0.001).

Figure 8. Anti-fibrosis treatment with prizaquantel reduced serum alanine aminotransferase (ALT) level in mice 15 weeks post infection with Schistosoma japonicum.

Serum ALT was assessed by Olympus AU5400 automatic biochemistry analyzer. Levels of serum ALT were significantly decreased nearly to the normal level after anti-fibrosis treatment with high dose prizaquantel. (* p<0.05).

Figure 9. Anti-fibrosis treatment with prizaquantel inhibited expressions of profibrotic genes and α-SMA protein expression in liver, but increased MMP9 mRNA expressions of mice 15 weeks post infection with Schistosoma japonicum.

(A)α-SMA; (B)Col1α1; (C)Col3α1; (D)TGF-β were reduced by anti-fibrosis treatment with high dose prizaquantel. (E)MMP9 levels in high dose group were increased about 10 fold compared with uninfected group, whereas (F) TIMP1 levels were decreased nearly to uninfected level. α-SMA protein expressions was assessed by immunohistochemistry. (G) Representative images of immunohistochemistry by light microscope (×100). Gi, uninfected; Gii, infected; Giii, anti-parasite; Giv, low dose; Gv, high dose. Arrows showed the positive staining. (H) α-SMA positive areas were measured by software IPP6.0. Expression of IL-4 (I), IL-10 (J), IL-13(K) and IFN-γ (L) were significantly reduced in high dose group compared respectively with infected, anti-parasite or low dose group. (* p<0.05, ** p<0.01, *** p<0.001).

Figure 10. Treatment of prizaquantel for 3 days inhibited expressions of HSC profibrotic genes of mice 12 weeks post infection with Schistosoma japonicum.

Prizaquantel (250 mg/kg/day) was administrated to mice for 3 days. Primary HSCs were isolated and transcriptional levels were assessed by Real Time PCR. Transcriptional level of (A) Col1α1, (B) α-SMA,(C) Col3α1, (D) TGF-β, (E) MMP9 and (F) TIMP1 in HSC of treated mice were significantly reduced compared with untreated mice. (* p<0.05).