Anti-angiogenic and anti-proliferative activity of ziziphus leaf extract as a novel potential therapeutic agent for reducing hepatic injury in experimental hamster schistosomiasis

Abstract

Background: Schistosomiasis is one of the most prevalent helminthic infections worldwide. Praziquantel (PZQ) resistance poses a possible danger to the disease's ability to be controlled. Little is known about the role of Ziziphus spina-christi leaf extract (ZLE) in the treatment of hepatic schistosomiasis. However, no study has explored ZLE's anti-angiogenic and anti-proliferative activity as a possible mechanism for reducing hepatic injury in this context. Therefore, this study aimed to evaluate the therapeutic potential of ZLE as an anti-angiogenic, and anti-proliferative agent in hamsters infected with S. mansoni.

Methods: Fifty hamsters were used and divided into 5 groups (10 hamsters each); noninfected untreated (controls), noninfected treated with ZLE, infected untreated, infected treated with PZQ- and infected treated with ZLE. Anti-angiogenic and anti-fibrotic effects of the drugs were assessed pathologically through the immunohistochemical expression of VEGF, Ki-67, and TGF β1 in liver sections. Some oxidative stress parameters were measured in hepatic homogenates (NO, GSH, GST, and SOD), and serum liver enzymes were also assessed.

Results: A significant decrease in worm burden, granuloma size, granuloma area, and numbers in the ZLE- and PZQ-treated groups compared to the infected untreated group, and the decrease in granulomas number and tissue egg load was significantly lower in PZQ treated group compared to ZLE treated group (p<0.05). ZLE exhibited significant anti-angiogenic and anti-fibrotic effects on granulomas, illustrated by significantly lower expression of VEGF and TGF-β1 than infected untreated and PZQ-treated groups. ZLE exhibits antiproliferative activity evidenced by a significant reduction of positive Ki-67 hepatocytes percentage compared to the infected untreated group. Moreover, ZLE exhibits potent antioxidant effects evidenced by a significantly lowered NO and conservation of hepatic GSH, GST, and SOD in hepatic homogenates compared to infected untreated and PZQ-treated groups (p<0.05).

Conclusion: Our results point to ZLE as a promising hepatoprotective therapeutic tool in the treatment of schistosome hepatic fibrosis as it has anti-angiogenic, anti-proliferative, anti-fibrotic, and antioxidant effects in hamsters infected with S. mansoni, providing scientific support for its use in conventional medicine.

Fig 1. Dispersion graph illustrating a significant reduction of the number of hepatic tissue egg load with PZQ and ZLE therapy compared to infected untreated hamsters with S. mansoni (p< 0.001 for both).

However, PZQ therapy significantly reduced the hepatic tissue egg load compared to ZLE therapy (p = 0.03). The dispersion graph data of 10 hamsters/group. PZQ, praziquantel; ZLE, Ziziphus leaf extract.

Fig 2

(A) The Liver histopathology section stained with H&E shows egg granulomas in infected untreated hamsters. (B) The Liver histopathology section stained with H&E shows egg granulomas in infected hamsters treated with PZQ. (C) The Liver histopathology section stained with H&E shows egg granulomas in infected hamsters treated with ZLE. (D) PZQ and ZLE therapy significantly reduces the mean number of granulomas in hamsters infected with S. mansoni compared to the infected untreated group (p < 0.001, 0.007, respectively). This reduction in granuloma number was significantly lower among the PZQ regimen compared to the ZLE regimen (p = 0.002). The dispersion graph data of 10 hamsters/group. PZQ, praziquantel; ZLE, Ziziphus leaf extract.

Fig 3

A) The Liver histopathology section stained with H&E shows egg granuloma diameter in infected untreated hamsters. B) The Liver histopathology section stained with H&E shows egg granuloma diameter in infected hamsters treated with PZQ. C) The Liver histopathology section stained with H&E shows egg granuloma diameter in infected hamsters treated with ZLE. (D) PZQ and ZLE therapy significantly reduces the mean diameter of hepatic granulomas in hamsters infected with S. mansoni compared to the infected untreated group (p < 0.001 in both cases) with more reduction with ZLE therapy (p. < 0.001). The dispersion graph data of 10 hamsters/group. PZQ, praziquantel; ZLE, Ziziphus leaf extract.

Fig 4

A) The Liver histopathology section stained with H&E section shows a congested central vein and egg granulomas in infected untreated hamsters. B) The histologic activity index (HAI) score was significantly reduced with PZQ and ZLE therapy in hamsters infected with S. mansoni (p< 0.001 for both). HAI score was categorized from 12–18 for the infected livers. The dispersion graph data of 10 hamsters/group.

Fig 5

A) Immunohistochemistry for VEGF in Infected untreated hamsters shows positive VEGF highlights the endothelial cells of liver sinusoids with positive reactions within granulomas (x200) (++++). B) Immunohistochemistry for VEGF in infected hamsters treated with PZQ shows positive VEGF highlighting the endothelial cells of liver sinusoids with focal positive reaction within granulomas (x200) (++). C) Immunohistochemistry for VEGF in infected hamsters treated with ZLE shows positive VEGF lightening the endothelial cells of liver sinusoids with negative within granulomas (x200) (—). D) There was a significant reduction in VEGF immunohistochemical expression in the hepatic tissues of hamsters infected with S. mansoni treated with PZQ and ZEL, however, ZLE therapy caused a potent significant reduction in VEGF hepatic tissue expression (negative expression) compared to the PZQ-treated group. VEGF, vascular endothelial growth factor; PZQ, praziquantel; ZLE, Ziziphus leaf extract. -The intensity was expressed as + (weak immunoreactivity), ++ (moderate immunoreactivity), +++ (strong immunoreactivity), or ++++ (very strong immunoreactivity). -The intensity was expressed as + (weak immunoreactivity), ++ (moderate immunoreactivity), +++ (strong immunoreactivity), or ++++ (very strong immunoreactivity).

Fig 6

A) Immunohistochemistry for TGF-β1 in Infected untreated hamsters shows strong positive reaction within the granulomas and the fibrous capsule (x200) (++++). B) Immunohistochemistry for TGF-β1 in infected hamsters treated with PZQ shows moderate reaction within the granulomas and the fibrous capsule (x200) (++). C) Immunohistochemistry for TGF-β1 in infected hamsters treated with ZLE shows weak reaction within the granulomas and within the fibrous capsule (x200) (+). D) There was a significant reduction in TGF-β1 immunohistochemical hepatic tissues expression of hamsters infected with S. mansoni treated with PZQ and ZEL, however, ZLE therapy caused a more significant reduction in TGF-β1 hepatic expression compared to the PZQ-treated group. TGF- β1, Transforming growth factor- β1; PZQ, praziquantel; ZLE, Ziziphus leaf extract. -The intensity was expressed as + (weak immunoreactivity), ++ (moderate immunoreactivity), +++ (strong immunoreactivity), or ++++ (very strong immunoreactivity).

Fig 7

A) Immunohistochemistry for Ki-67 in infected untreated hamsters shows positive reaction (x400). B) Immunohistochemistry for Ki-67 in infected hamsters treated with PZQ the negative reaction (x400). C) Immunohistochemistry for Ki-67 in infected hamsters treated with ZLE shows the negative reaction (x400) (<1%). D) The percentage of immunohistochemical expression of Ki-67-positive hepatocytes was low. (2±0.47) in the hepatic tissue of the infected untreated group and significantly reduced 0.48±0.09) in both praziquantel treated groups(0.47±0.11) and ZLE treated group (p < 0.001). -The Ki-67 staining pattern was in the nuclei of hepatocytes in a directional pattern between granuloma and central veins. -The error bar data are mean ± SD of 10 hamsters/group. PZQ, praziquantel; ZLE, Ziziphus leaf extract.