Introduction
The zoonotic disease schistosomiasis is caused by parasites belonging to the Schistosoma species. With a global prevalence of over 120 million symptomatic cases, 20 million of which exhibit severe morbidity, this parasitic disease ranks as the second most prevalent worldwide. It results in more than 200,000 mortalities and the loss of 70 million disability-adjusted life years (DALYs) annually (Kamdem et al. Reference Kamdem, Moyou-Somo, Brombacher and Nono2018; Liu et al. Reference Liu, Zhang, Liang and Lu2022). Furthermore, as per the projections provided by the World Health Organization, schistosomiasis is prevalent in 78 countries and presents a potential risk of infection to approximately 236.6 million individuals (WHO 2022).
Schistosoma mansoni is a digeneic intravascular worm that inhabits the venous portal-mesenteric system. The liver is particularly susceptible to pathogenic insult due to its location (Andrade Reference Andrade2009). After adult female schistosomiasis worms deposit eggs, the pathology commences as the eggs attempt to develop outside the host. Antigens released by eggs that become entrapped in tissues stimulate a vigorous Th2-directed immune response, resulting in the formation of granulomatous lesions that surround the eggs to segregate the spread of their antigens (Pearce and MacDonald Reference Pearce and MacDonald2002; Burke et al. Reference Burke, Jones, Gobert, Li, Ellis and McManus2009; Fairfax et al. Reference Fairfax, Nascimento, Huang, Everts and Pearce2012). The resulting hepatic injury stimulates the transformation of quiescent hepatic stellate cells (HSCs) into myofibroblasts, which are responsible for collagen production and, subsequently, fibrosis of the granulomatous parenchyma. This fibrosis results in compression destruction of the portal vasculature that extends from the small to the larger portal spaces, ending in portal hypertension and its life-threatening sequelae (Andrade Reference Andrade2009; El Ridi and Tallima Reference El Ridi and Tallima2013; Carson et al. Reference Carson, Ramm, Robinson, McManus and Gobert2018).
Reversibility of liver fibrosis is maintained under stable, quiescent conditions. However, persistent harm and inflammatory stimulation have the potential to progress liver fibrosis to cirrhosis and potentially malignancy (Elbaz and Esmat Reference Elbaz and Esmat2013). Unfortunately, the primary antischistosomal drug PZQ exhibits only limited efficacy against laid eggs, as they persist in releasing their damaging antigens (Vale et al. Reference Vale, Gouveia, Rinaldi, Brindley and Gärtner2017). Moreover, the therapeutic dose of PZQ used cannot markedly affect the occurring fibrosis (El Ridi and Tallima Reference El Ridi and Tallima2013). Therefore, it is critical to develop antifibrotic therapies for schistosomiasis in order to improve the prognosis of patients with the disease. GA is one of the substances that has demonstrated encouraging anti-inflammatory and antifibrotic characteristics.
GA, a polyphenol with a low molecular weight found in various plants including pineapple, lemon, bananas, and grapes, possesses potent antioxidant properties (Ola-Davies and Olukole Reference Ola-Davies and Olukole2018). Furthermore, it exhibited promising activities in cardiac, pulmonary, and hepatotoxic fibrosis (Jin et al. Reference Jin, Sun, Ryu, Piao, Liu, Choi, Kim, Kim, Kee and Jeong2018; Rong et al. Reference Rong, Cao, Liu, Li, Chen, Chen, Liu and Liu2018; Hussein et al. Reference Hussein, Anwar, Farghaly and Kandeil2020). There are currently no published studies regarding the antiparasitic properties of GA.
Consequently, the current work aimed to investigate GA as an antiparasitic and antifibrotic therapy for experimental S. mansoni, as well as the outcomes of its combination with PZQ, the standard anti-schistosomiasis therapy.
Methodology
Ethics statement
All animal experiments and sample size were approved by the Institutional Committee (ethics number 3/2023PARA18) and conformed to the international ethical guidelines of care of experimental animals. Mice were bred in standard housing environment of food and temperature in the animal house of Theodor Bilharz Research Institute, TBRI (Giza, Egypt). All efforts were made to minimize animal suffering throughout the experiment.
Study design
Two experiments were conducted as part of this study. The initial step was to determine the optimal dosage of GA. The second objective was to evaluate the efficacy of GA in conjunction with PZQ, the standard antischistosomal treatment. In experiment I, four S. mansoni-infected groups were present. The infected group that did not receive treatment was designated Group I (GI; control1). Group II (GII; GA10) received 10 mg/kg of GA treatment. Group III (GIII; GA20) received 20 mg/kg of GA treatment. Group IV (GIV; GA40) received 40 mg/kg of GA treatment. Experiment II included four S. mansoni-infected groups. Group I (GI; control2) served as the infected untreated control. Group II (GII; PZQ) served as the PZQ-treated group. Group III (GIII; GA40) received 40 mg/kg of GA treatment. Group IV (GIV; GA40+PZQ) received GA and PZQ. Each of the studied groups included five mice.
Animals and S. mansoni infection
Forty male pathogen-free BALB/c mice (6–8 weeks old, 18–20 g) were obtained from the Schistosome Biological Supply Program, TBRI and subcutaneously infected by 100 ± 10 S. mansoni cercariae/mouse (Peters and Warren Reference Peters and Warren1969).
Drug therapy
Praziquantel (Egyptian International Pharmaceutical Industries Company, A.R.E., E.I.P.C.O.) was suspended in 2% cremophore (Sigma Aldrich, USA) and orally administered 45 days post-infection (dpi) at a dose of 500 mg/kg for 2 consecutive days (El-Lakkany et al. Reference El-Lakkany, Hammam, El-Maadawy, Badawy, Ain-Shoka and Ebeid2012).
The mice in the treatment groups received oral GA dissolved in distilled water by Sigma-Aldrich at concentrations of 10, 20, or 40 mg/kg (Ola-Davies and Olukole Reference Ola-Davies and Olukole2018). Treatment started at 45 dpi and continued for 30 days.
Euthanizing mice and sampling
Mice in both experiments were decapitated 75 dpi. The serum was separated from the collected blood through centrifugation for 3 min at 3,000 rounds per minute (rpm). Livers were dissected to retrieve adult worms and divided into three parts to perform egg count, cytokine assay, and histopathology studies. Duplicate tests were conducted for immunological studies and liver enzyme assessment. The average values of each test were then calculated and presented for further statistical analysis.
Evaluation of the antiparasitic activity of drugs
Evaluation of worm load changes
Adult worms were recovered by saline perfusion of hepatic and porto-mesenteric veins through cannulation of the inferior vena cava of euthanized mice according to Duvall and DeWitt (Reference Duvall and DeWitt1967). Retrieved worms were categorized into male, female, and coupled worms.
Evaluation of hepatic egg load changes
One gram of each liver was digested by 5% potassium hydroxide incubation at 37 °C for 16 h. Eggs were collected and counted using Olympus light microscope at ×40 magnification. Data were expressed as egg/g of liver tissue (Herbert et al. Reference Herbert, Orekov, Roloson, Ilies, Perkins, O’Brien, Cederbaum, Christianson, Zimmermann, Rothenberg and Finkelman2010).
Evaluation of changes in hepatic granulomas and fibrosis
Samples of livers were fixed in formalin 10%, paraffinized, and stained with hematoxylin and eosin and Masson’s trichrome stains. Granulomas were counted and digitally measured using a multi-head microscope (Olympus SC100) and analySIS getIT software considering only the diameter of single-ovum granulomas. The percentage of fibrosis was measured in the photos of Masson’s trichrome-stained slides using the image J software program version 1.47v (Amin and Mahmoud-Ghoneim Reference Amin and Mahmoud-Ghoneim2011).
Immune staining of HSCs
Activated HSCs were identified using mouse anti-smooth muscle action-alpha (SMA-α) antibody (Abcam, USA) (Mustafa et al. Reference Mustafa, El Awdan, Hegazy and Abdel Jaleel2015). Positive staining was determined when the cell membrane alone or with the cytoplasm stained brown. The histo score (H-score) was used to calculate the degree of HSCs activation where the percentage of positive cells was multiplied by the intensity of SMA-α expression, which was given a number from (0, 1+, 2+, & 3+) for example [1 × (% cells 1+) + 2 × (% cells 2+) + 3 × (% cells 3+)] (Fraser et al. Reference Fraser, Reeves, Stanton, Black, Going, Cooke and Bartlett2003).
Evaluation of liver functions
Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) serum levels were colorimetrically measured using Alanine Aminotransferase Activity Assay Kit (catalog number MAK052) and Aspartate Aminotransferase (AST) Activity Assay Kit (catalog number MAK055), Sigma-Aldrich, USA. All procedures were performed according to the manufacturer’s instructions.
Estimation of fibrosis-regulating cytokine levels
Each mouse’s liver tissue sections were homogenized in 0.9% saline (Bakery et al. Reference Bakery, Allam, Abuelsaad, Abdel-Latif, Elkenawy and Khalil2022). Homogenates were centrifuged at 3,000 rpm for 15 min. Then, the supernatants were used to estimate TGF-β1, IL-4, IL-13, and IL-10 levels using mouse TGF-β1, IL-4, IL-13, and IL-10 ELISA kits (Abcam, USA). The manufacturer’s protocols were followed for assessment.
Statistical analysis
The data were analyzed using SPSS statistical package version 26 (SPSS Inc. Released 2019. IBM SPSS statistics for windows, version 23.0, Armnok, NY: IBM Corp.). The variables were expressed in mean (x̅), standard deviation (SD), median, and range. ANOVA (with Homogeneity testing) test was used for comparison of quantitative variables between more than two groups of normally distributed data with Tuckey test as post hoc test while; Kruskal Wallis test was used for comparison of quantitative variables between more than two groups of not normal distributed data with Tamhane’s test as post hoc test. The student’s t test was used to compare means of normally distributed variables between two groups, while Mann Whitney test was used for not normally distributed ones. Two-sided P- value of < 0.05 was considered statistically significant.
Results
Experiment I
Effect of GA treatment on worm load
In comparison to the control group (21.60 ± 1.67), all GA-treated groups showed statistically significant decreases in the total worm loads (p<0.01). As the GA dose increased, the percentage of reduction (PR) increased. The GA40 group had the lowest total worm load (12.40 ± 1.67; PR = 42.38 ± 8.16) followed by GA20 and GA10 (18.20 ± 2.48 and 18.20 ± 1.09, respectively). When compared to the control group (9.80 ± 0.83) and other GA doses (GA10: 9.0 ± 1.22; GA20: 9.0 ± 2.0), only GA40 demonstrated a statistically significant decrease in the number of coupled worms (6.80 ± 1.09; PR= 29.79 ± 14.34; p<0.05). (Figure 1a).
Column chart presentations of the results of the parasitological tests of experiment I. N.B. columns with similar letters or symbols refer to unsignificant difference, while different letters or symbols demonstrate significance. a. Effect of GA on worm load. The lowest numbers of total worm load and coupled worms were detected in GA40, with statistically significant differences compared with other groups. b. Effect of GA on hepatic egg load. The GA40 group had the lowest number of hepatic egg loads, with statistically significant differences compared with other groups. c. Effect of GA on number of hepatic granulomas. GA40 treatment showed the lowest number of hepatic granulomas, with statistically significant differences compared with other groups. d. Effect of GA on diameter of hepatic granulomas. The lowest diameter of hepatic granulomas was detected in GA40, with statistically significant differences compared with other groups.
Effect of GA treatment on hepatic egg load
In all GA-treated groups, statistically significant decreases in hepatic egg loads were found when compared with the control group (3970.0 ± 416.73), just like with worm load. When compared to the other groups (GA10: 3490.0 ± 238.22, PR=10.93 ± 14.15; GA20: 2020.0 ± 201.86, PR=48.88 ± 5.17), the GA40 group experienced the most significant reductions (2020.0 ± 201.86; PR=63.15 ± 4.44) with statistically significant differences (p<0.01). (Figure 1b).
Effect of GA treatment on hepatic granulomas
The three GA-treated groups displayed statistically significant reductions in hepatic granulomas’ number and diameter compared to the control-infected group (16.0 ± 2.34 and 264.60 ± 12.85, respectively). Compared to other groups (granuloma numbers of GA10 and GA20: 11.80 ± 2.28 and 10.0 ±1.22, respectively, and diameters of GA10 and GA20: 230.40 ± 13.50 and 204.0 ± 19.28, respectively), the GA40 group had the lowest granuloma number and diameter (6.0 ±1.0; PR=62.16 and 119.20 ± 25.37; PR= 55.14 ± 8.15, respectively) with statistically significant differences (p<0.05) (Figure 1c,d).
Effect of GA treatment on hepatic fibrosis
These differences were statistically significant when comparing the percentage of hepatic fibrosis in the GA40 group to that of the other groups (13.60 ± 2.30; PR = 62.46 ± 8.66). GA20 placed second (23.20 ± 2.38; PR = 35.82 ± 13.71). In contrast to the higher doses, no statistically significant difference was observed between the control (37 ± 5.56) and GA10 (28.60 ± 3.04) groups. (Figure 2a).
Column chart presentations of the results of the pathological tests of experiment I. N.B. columns with similar letters refer to unsignificant difference, while different letters demonstrate significance. a. Effect of GA on hepatic fibrosis percentage. GA40-treatment was associated with the lowest percentage of fibrosis, with statistically significant differences compared with other groups. b. Effect of GA on expression of SMA-α. The lowest H-score of SMA-α expression was detected in GA40, with statistically significant differences compared with other groups.
Effect of GA treatment on HSC activation
In comparison to the control group (113.80 ± 15.97), the three GA-treated groups showed statistically significant decreases in HSC activation. As the dose was increased, the level of activation decreased. The GA40 group (17.80 ± 2.77) had the lowest H-score of SMA-α, which was followed by GA20 (48.0 ± 5.14) and GA10 (73.20 ± 10.08). (Figure 2b).
Effect of GA treatment on hepatic enzymes
The ALT and AST scores of the GA40 group were the lowest (70.60 ± 8.41 and 71.20 ± 6.22, respectively), followed by those of the GA20 group (AST: 87.80 ± 8.43; ALT: 99.00 ± 7.0; p<0.05). These results were significantly different from those of the GA10 and control groups. In contrast to the results obtained from GA20 and GA40, no statistically significant disparities were observed between GA10 (ALT: 136.20 ± 12.04; AST: 128.80 ± 7.82) and the control group (ALT: 151.60 ± 11.97 AST: 137.60 ± 15.96) (p>0.05) (Figure 3).
Column chart presentation of the results of the ALT and AST in the studied groups. N.B. columns with similar letters or symbols refer to unsignificant difference, while different letters or symbols demonstrate significance. The GA-treated group presented the lowest levels of the ALT and AST enzymes, with statistically significant differences compared with other groups.
Experiment II
Effect of treatment on worm load
The total worm load in all treated groups was found to be significantly lower compared to the control group (19.60 ± 1.51). The PZQ (1.40 ± 0.89; PR=92.65 ± 5.21) and PZQ+GA40 (1.0 ± 0.70; PR=94.71 ± 3.94) groups exhibited the lowest total worm load. These two groups were found to be statistically comparable (p>0.05). GA40 was the only group to place second (13.20 ± 2.58; PR=32.61 ± 12.33). Additionally, the number of coupled worms was significantly lower in the PZQ and PZQ+GA40 groups (0.40 ± 0.54 and 0.20 ± 0.44, respectively; p>0.05) than in the GA40 group (6.40 ± 1.34), which ranked second, and the control group (9.60 ± 1.14) (Figure 4a).
Column chart presentations of the results of the parasitological tests of experiment II. N.B. columns with similar letters or symbols refer to unsignificant difference, while different letters or symbols demonstrate significance. a. Effect of drug therapy on worm load. The lowest numbers of total worm load and coupled worms were detected in GA40+PZQ and PZQ groups, with statistically significant differences compared with other groups. b. Effect of drug therapy on hepatic egg load. GA40+PZQ group showed the lowest number of hepatic egg loads, with statistically significant differences compared with other groups. c. Effect of drug therapy on number of hepatic granulomas. The lowest number of hepatic granulomas was detected in GA40+PZQ and PZQ groups, with statistically significant differences compared with other groups. d. Effect of drug therapy on diameter of hepatic granulomas. The granulomas of the GA40+PZQ group had lowest diameter, with statistically significant differences compared with other groups.
Effect of treatment on hepatic egg load
The hepatic egg load was lowest in the combined therapy GIV group (501.60 ± 92.37; PR=87.05 ±1.81), with statistically significant differences compared to the other study groups (p<0.05). The PZQ-treated group came in second (696.0± 79.24; PR=81.95 ± 1.83), followed by the sole GA-treated group (1535.0 ± 92.33; PR=60.09 ± 3.65) that was significantly lower than the infected control group (3862.0 ± 296.17). There were significant statistical differences among all the studied groups (p <0.05) (Figure 4b).
Effect of treatment on hepatic granulomas
The two PZQ-treated groups showed the fewest hepatic granulomas, either alone (3.60 ± 1.14; PR=77.35 ± 8.92) or in combination with GA40 (1.80 ± 1.30; PR=89.29 ± 7.44), with no statistically significant difference between the two groups (p>0.05). With a statistically significant difference from the control group (16.40 ± 1.81), the sole GA40-treated group (6.60 ± 1.14; PR= 59.45 ± 7.60) came in second (Figure 4c).
The changes in diameter of the granulomas did not correspond to the changes in their number. The GA40 treatment achieved the second highest rank with an average value of 116.60 ± 17.54 and a PR value of 54.73 ± 6.29. Similarly, the PZQ treatment, with an average value of 189.40 ± 20.51 and a PR value of 26.23 ± 9.52, also dropped to the third rank (p<0.01). However, both treatments were statistically lower than the infected control group, with an average value of 257.40 ± 9.96. Nevertheless, the combined PZQ+GA40-treated group (33.20 ± 12.67; PR= 87.12 ± 4.72) continued to exhibit the lowest values (Figure 4d and Figure 5).
H & E-stained liver sections of experiment II (Scale bar = 100μm). a. large sized granuloma of infected control group (referred by the yellow arrow) with single central ovum (referred by the green arrow) surrounded by epithelioid cells, fibroblast, and lymphocytes. b. moderate-sized granuloma of PZQ group (referred by the yellow arrow) with single central ovum (referred by the green arrow) surrounded by epithelioid cells, fibroblast, and lymphocytes. c. small- to moderate-sized granuloma of GA40 group (referred by the yellow arrow) with single central ovum (referred by the green arrow) surrounded by epithelioid cells, fibroblast, and lymphocytes. d. small-sized granuloma of GA40+PZQ group (referred by the yellow arrow) with single central ovum (referred by the green arrow) surrounded by epithelioid cells, fibroblast, and lymphocytes.
Effect of treatment on hepatic fibrosis
Among the groups under study, the lowest incidence of hepatic fibrosis (3.40 ± 1.67; PR= 90.95 ± 4.17) was observed in the group treated with a combination of GA40 and PZQ. This was followed by the group that received GA40 only (13.60 ± 2.07; PR=63.50 ± 7.97), which ranked second. The PZQ-treated group (25.40 ± 1.67; 32.43 ± 5.31) came in third place with statistically significant differences compared with other studied groups, including the infected control group (37.80 ± 4.02) (p<0.01) (Figure 6 and Figure 7a).
Masson’s trichrome-stained liver sections of experiment II (Scale bar = 100μm). a. massive hepatic fibrosis and large-sized granuloma of infected control group. b. moderate hepatic fibrosis of PZQ group. c. moderate hepatic fibrosis of GA40 group. d. mild hepatic fibrosis and small-sized granuloma of GA40+PZQ group.
Column chart presentations of the results of the pathological, immunological, and functional tests of experiment II. N.B. columns with similar letters or symbols refer to unsignificant difference, while different letters or symbols demonstrate significance. a. Effect of drug therapy on hepatic fibrosis percentage. The lowest percentage of fibrosis was detected in GA40+PZQ group, with statistically significant differences compared with other groups. b. Effect of drug therapy on expression of SMA-α. GA40+PZQ group showed the lowest SMA-α H-score, with statistically significant differences compared with other groups. c. Effect of drug therapy on fibrosis-regulating cytokines. The lowest levels of the fibrosis-enhancing cytokines (IL-4, IL-13, and TGF-β1) were detected in GA40+PZQ group, with statistically significant differences compared with other groups. Also, the highest levels of the anti-inflammatory cytokine IL-10 was detected in GA40+PZQ group, with statistically significant differences compared with other groups. d. Effect of drug therapy on ALT and AST levels. GA40+PZQ treatment was associated with the lowest levels of the ALT and AST enzymes, with statistically significant differences compared with other groups.
Effect of GA treatment on HSC activation
Differences in HSC activation were in line with the percentage of hepatic fibrosis where the lowest H-score of SMA-α was detected in the combined PZQ+GA40 group (4.20 ± 1.09) followed by sole GA40 (18.0 ± 2.91) and sole PZQ (65.40 ± 10.57) with statistically significant differences among them and when compared with the infected control (116.20 ± 12.11) (p<0.01). (Figure 7b and Figure 8).
SMA-α IHC stained liver tissue of the studied groups of experiment II. a. strong SMA-α expression of infected control mice. b. moderate SMA-α expression of PZQ group. c. mild to moderate SMA-α expression of GA group d. mild SMA-α expression of GA40+PZQ group.
Effect of treatment on fibrosis-regulating cytokines
The combination therapy group GIV exhibited statistically significant reductions in fibrosis-promoting cytokines when compared to the other research groups (p<0.001). Specifically, the levels of TGF-β1, IL-4, and IL-13 were significantly decreased in the combination therapy group (TGF-β1: 57.40 ± 3.94; IL-4: 31.80 ± 8.10; IL-13: 13.80 ± 4.61). The group that received GA40 treatment achieved second place (TGF-β1: 77.80 ± 5.49; IL-4: 82.0 ± 7.17; IL-13: 30.60 ± 4.61). The group that received only PZQ treatment exhibited the highest levels of TGF-β1, IL-4, and IL-13 compared to the other treated groups. Specifically, the levels of TGF-β1, IL-4, and IL-13 in the PZQ-treated group were measured to be 118.20 ± 7.15, 214.0 ± 14.47, and 60.40 ± 5.31, respectively. These values were found to be statistically significantly different from the levels observed in the infected control group (TGF-β1: 143.20 ± 6.61; IL-4: 283.40 ± 29.49; IL-13: 81.60 ± 6.26)
However, it is noteworthy that the group receiving combination therapy, denoted as GIV, exhibited the most elevated levels of the anti-inflammatory cytokine IL-10 (625.60 ± 64.70). These findings were found to be statistically significant when compared to the other groups involved in the study (p<0.001). The GA and PZQ groups came in the second and third ranks, respectively, with mean values of 443.80 ± 33.82 and 240.80 ± 7.69. These values showed statistically significant differences compared to the infected control group (175.40 ± 16.97) (Figure 7c).
Effect of treatment on hepatic enzymes
The combined PZQ+GA40 group had the lowest levels of the ALT and AST enzymes (ALT: 41.80 ± 6.83; AST: 42.20 ± 8.07), whereas the solitary GA40 group came in second (ALT: 71.00 ± 6.85; AST: 74.80 ± 6.68). PZQ had the highest ALT and AST values among the treatment groups (ALT: 98.40 ± 5.54; AST: 91.80 ± 7.94), but statistically lower than the infected control (ALT: 147.20 ± 14.09; AST: 142.00 ± 14.47). All group differences were statistically significant (p<0.01). (Figure 7d).
Discussion
The current research was an experiment to discover a treatment for liver fibrosis caused by Schistosoma, which is the main contributor to all potentially deadly consequences from this parasite. The primary anti-schistosomiasis treatment, PZQ, has a low antifibrotic efficacy and only partially kills laid eggs (El Ridi and Tallima Reference El Ridi and Tallima2013; Vale et al. Reference Vale, Gouveia, Rinaldi, Brindley and Gärtner2017), which results in chronic release of antigens and exacerbated disease (Elbaz and Esmat Reference Elbaz and Esmat2013). Finding efficient antifibrotic treatments for schistosomiasis is therefore urgently needed to enhance the prognosis of more than 120 million schistosomiasis patients globally. Additionally, the exclusive dependence of schistosomiasis treatment on PZQ implies a potential increase in the emergence of PZQ resistance (Liu et al. Reference Liu, Zhang, Liang and Lu2022). Therefore, the identification of an additional efficacious anti-schistosomiasis treatment is necessary.
Our decision to adopt GA as a potential anti-schistosomal and antifibrotic therapy was based on the substance’s promising antibacterial and antifibrotic characteristics, which have been documented in several investigations (Ola-Davies and Olukole Reference Ola-Davies and Olukole2018; Rong et al. Reference Rong, Cao, Liu, Li, Chen, Chen, Liu and Liu2018; Jin et al. Reference Jin, Sun, Ryu, Piao, Liu, Choi, Kim, Kim, Kee and Jeong2018; Hussein et al. Reference Hussein, Anwar, Farghaly and Kandeil2020) Based on the substance’s reported antibacterial and antifibrotic properties, we decided to utilize GA as a prospective anti-schistosomal and antifibrotic therapy (Ola-Davies and Olukole Reference Ola-Davies and Olukole2018; Rong et al. Reference Rong, Cao, Liu, Li, Chen, Chen, Liu and Liu2018; Jin et al. Reference Jin, Sun, Ryu, Piao, Liu, Choi, Kim, Kim, Kee and Jeong2018; Hussein et al. Reference Hussein, Anwar, Farghaly and Kandeil2020).
The GA dose of 40 mg/kg in our first experiment provided the most potent antiparasitic effects. Simoes et al. (Reference Simoes, Bennett and Rosa2009) linked the antimicrobial efficacy of GA to its capacity to prevent microbes from controlling their internal environment and eliminating hazardous chemicals and metabolites by inhibiting the efflux pumps. The documented antiparasitic efficacy of GA40 could be accounted for by this hypothesis, given that Schistosoma homeostasis also relies on pumps of a similar nature to eliminate waste products (Kasinathan et al. Reference Kasinathan, Morgan and Greenberg2010).
Although GA40 was found to have antiparasitic properties, its effectiveness was still inferior to that of PZQ. Furthermore, its combination with PZQ in GIV failed to enhance the antiparasitic properties of PZQ. On the contrary, GA demonstrated the capacity to enhance the efficacy of drugs distinct from PZQ. According to Rajamanickam et al. (Reference Rajamanickam, Yang and Sakharkar2019), GA increased the antibacterial activity of tulathromycin against Mannheimia haemolytica and Pasteurella multocida, two critical pathogens responsible for bovine respiratory illness.
Sarjit et al. (Reference Sarjit, Wang and Dykes2015) also reported comparable findings concerning the restricted antibacterial efficacy of GA. The authors asserted that the effectiveness of GA against Campylobacter (C.) is strain-specific. The substance exhibited bactericidal effects against only two strains of C. coli, while it demonstrated growth inhibition against five strains of C. coli and three strains of C. coli. In addition, Lima et al. (Reference Lima, Oliveira-Tintino, Santos, Morais, Tintino, Freitas, Geraldo, Pereira, Cruz, Menezes and Coutinho2016) reported that GA did not demonstrate any observable antibacterial activity against Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa, as well as the fungi Candida albicans and Candida tropicalis.
The present investigation revealed that GA exhibited greater efficacy in reducing the burdens of eggs compared to adult worms. The decrease in coupled worms, as observed by Lu et al. (Reference Lu, Sessler, Holroyd, Hahnel, Quack, Berriman and Grevelding2016), plays a crucial role in the maturation of female worms and the subsequent deposition of eggs. This observation can be utilized to account for the higher percentage of reduction in egg burden observed in the GA40 group. The co-administration of the schistosomicidal PZQ exhibited an additive effect. Notably, a statistically significant reduction in egg burden was observed compared to the group that solely received PZQ treatment. This finding illustrates the effectiveness of GA in enhancing PZQ’s efficacy in egg load but not in worm load.
The variations in egg loads across the examined groups, where PZQ outperformed GA, were reflected in the variations in granuloma number. The size of granulomas was shown to be affected in the opposite way, with GA significantly reducing their size more than PZQ. This could be a result of GA’s anti-inflammatory activity, which was noted in the cytokines we studied, which modulated the strong inflammatory response that typically takes place in response to tissue trapped eggs, as found in the control infected group (Bai et al. Reference Bai, Zhang, Tang, Hou, Ai, Chen, Zhang, Wang and Meng2021; Llanwarne and Helmby et al. Reference Llanwarne and Helmby2021). This conclusion was supported by the combination of therapy group’s results, which showed the lowest granuloma diameter as a result of a PZQ-enhanced decrease in the number of trapped eggs and a corresponding decrease in immune system induction (Llanwarne and Helmby et al. Reference Llanwarne and Helmby2021). The small size of granulomas in GA does not represent a deficiency. In the study conducted by Damian et al. (Reference Damian, Roberts, Powell, Clark, Lewis and Stirewalt1984), it was observed that smaller granulomas in S. mansoni-infected baboons exhibited a higher degree of efficacy in capturing egg antigens compared to larger granulomas.
HSC activation, which was considerably lower in both GA40-treated groups than in the only PZQ-treated group, may have contributed to the observed considerable reduction in the percentage of hepatic fibrosis in GA-treated groups. Therefore, the GA-treated group had a more significant percentage of hepatic egg load. The resulting fibrosis was comparatively less severe than the group treated with PZQ, suggesting that GA exhibits significant antifibrotic properties.
Under typical circumstances, HSCs, which make up 5–8% of all liver cells, remain dormant in the Disse space of the liver sinusoids, where they serve as a source of vitamin A and erythropoietin as well as maintenance for the extracellular matrix. When the liver is damaged, these cells become activated, expressing more of the profibrotic gene SMA-α, before trans-differentiating into the collagen-producing myofibroblasts, the principal cell type responsible for hepatic fibrosis. To treat liver fibrosis, it is therefore important to inhibit HSC activity (Puche et al. Reference Puche, Lee, Jiao, Aloman, Fiel, Muñoz, Kraus, Lee and Friedman2013; Kamdem et al. Reference Kamdem, Moyou-Somo, Brombacher and Nono2018; Liu et al. Reference Liu, Zhang, Liang and Lu2022). The observed decrease in SMA-α expression, which was statistically significant, in the groups treated with GA40 and demonstrated superior outcomes compared to the group treated solely with PZQ provides evidence to support the significant role of GA40 in inhibiting HSC activation. The observed synergistic effect in the combined therapy group, which resulted in a reduction of fibrosis to approximately 3%, can be attributed to the diminished hepatic damage caused by PZQ in conjunction with GA40. The results of our study support the findings reported by Hussein et al. (Reference Hussein, Anwar, Farghaly and Kandeil2020), who used a rat model of thioacetamide-induced liver fibrosis. They illustrated that GA prevented HSC activation signals from compromising the integrity of liver tissue. Similarly, several organs other than the liver have been observed to benefit from GA’s antifibrotic activity. According to Jin et al. (Reference Jin, Sun, Ryu, Piao, Liu, Choi, Kim, Kim, Kee and Jeong2018), transverse aortic constriction-induced cardiac hypertrophy, dysfunction, and fibrosis were reduced by GA therapy. Moreover, it reduced the expression of fibrosis-related genes and deposition of collagen type I in TGF-β1-treated cardiac fibroblasts. Likewise, GA ameliorated the pathological changes in the cardio-renal system of Wistar rats induced by the endocrine-disrupting chemical Bisphenol A (Ola-Davies and Olukole Reference Ola-Davies and Olukole2018).
The study conducted by Rong et al. (Reference Rong, Cao, Liu, Li, Chen, Chen, Liu and Liu2018) demonstrated that the administration of GA treatment resulted in a significant reduction in the percentage of fibrosis, as well as a decrease in the pathology and infiltration of inflammatory cells in individuals with idiopathic pulmonary fibrosis. The researchers ascribed their findings to the antioxidative properties of GA, the inhibition of the TGF-1/Smad2 signaling pathway, and the resulting decrease in the expression of SMA-α, a significant indicator of idiopathic pulmonary fibrosis and a crucial factor for fibroblast transition into myofibroblasts.
Numerous cytokines are responsible for regulating HSC activation and, consequently, hepatic fibrosis. The immune system is stimulated to activate M2 macrophages and HSCs by the Th2 cytokines IL-4 and IL-13 (Kamdem et al. Reference Kamdem, Moyou-Somo, Brombacher and Nono2018). Additionally, the fibrogenic peptide TGF-β1 is primarily linked to the activation of HSCs, which leads to the buildup of extracellular matrix proteins (Wahl et al. Reference Wahl, Frazier-Jessen, Jin, Kopp, Sher and Cheever1997; Nallagangula et al. Reference Nallagangula, Nagaraj, Venkataswamy and Chandrappa2018). However, the production of IL-10 plays a significant role in the downregulation of various inflammatory responses, which typically result in periportal fibrosis. In numerous experimental and human research, its inhibition was linked to exacerbated pathology and fibrosis (Booth et al. Reference Booth, Mwatha, Joseph, Jones, Kadzo, Ireri, Kazibwe, Kemijumbi, Kariuki, Kimani and Ouma2004; Mentink-Kane et al. Reference Mentink-Kane, Cheever, Wilson, Madala, Beers, Ramalingam and Wynn2011; Franco et al. Reference Franco, de Amorim, Santos, Rollemberg, de Oliveira, França, Santos, Magalhães, Cazzaniga, de Lima, Benevides, Carregaro, Silva, Brito, Fernande, da Silva, de Almeida and de Jesus2021). The rationale for selecting cytokine estimation as the primary focus of our study is outlined, which elucidates the observed significant decrease in profibrotic cytokines (IL-4, 13, TGF-β1) and the concurrent increase in the anti-inflammatory cytokine IL-10 in the GA40-treated groups. This cytokine modulation effectively inhibited HSCs, leading to a reduction in fibrosis and the preservation of liver integrity.
Jin et al. (Reference Jin, Sun, Ryu, Piao, Liu, Choi, Kim, Kim, Kee and Jeong2018), Rong et al. (Reference Rong, Cao, Liu, Li, Chen, Chen, Liu and Liu2018), and Hussein et al. (Reference Hussein, Anwar, Farghaly and Kandeil2020) have reported comparable impacts of GA on TGF-β1, as well as the inhibition of the TGF-1/Smad2 signaling pathway. These studies have established a correlation between these effects and the decrease in cardiac, pulmonary, and hepatic fibrosis.
Similar results were reported by Zhu et al. (Reference Zhu, Gu and Shen2019), who demonstrated the efficacy of GA in mitigating the pathology associated with ulcerative colitis. The anti-inflammatory effect of GA was ascribed to an elevation in the anti-inflammatory cytokine IL-10 and a reduction in several proinflammatory cytokines, such as IL-6, IL-12, IL-17, IL-23, TGF-β, and TNF-α.
Our study showed improved levels of the liver enzymes ALT and AST, observed in both GA40-treated groups and superior to the PZQ-treated group, clinically demonstrated intact hepatic integrity (Pratt and Kaplan Reference Pratt and Kaplan2000).
Hussein et al. (Reference Hussein, Anwar, Farghaly and Kandeil2020) also reported on this protective effect of GA. Based on the results of their study, the administration of GA treatment led to a significant reduction in serum ALT and AST levels, approaching typical values, in a liver fibrosis model induced by thioacetamide. Additionally, GA treatment enhanced the activities of hepatic antioxidant enzymes.
Conclusion
GA slightly enhanced the antischistosomal activity of PZQ in comparison to PZQ. However, it was linked to decreased fibrosis and preserved integrity of liver tissue, particularly when administrated with PZQ. This finding can be attributed to the upregulation of the fibrosis-promoting cytokines IL-4, IL-13, and TGF-β1. Our pilot study suggests GA as a natural antifibrotic medication that can be used in conjunction with PZQ to lessen fibrosis, the main contributor to the complications associated with schistosomiasis.
Data availability
All data generated or analyzed during this study are included in this published article.
Funding
No funding was received to assist with the preparation of this manuscript.
Competing interest
The author(s) declare none.
Ethics approval
All procedures involving animals were in compliance with international ethical guidelines, and ethical approval was granted by the Faculty of Medicine, Menoufia University Ethics Committee (No. (ethics number 3/2023PARA18), Menoufia, Egypt).
Financial interest
The authors declare they have no financial interests.
