Enhanced surgical tolerance of schistosomiasis-induced fibrotic liver following treatment with Ziziphus plant extract

Abstract

BACKGROUND

Ziziphus spina-christi leaf extract (ZSCLE) can be used to treat hepatic schistosomiasis. However, its role as an anti-inflammatory and anti-proliferative agent remains unexplored.

AIM

To assess the therapeutic potential of ZSCLEs in hamsters infected with Schistosom mansoni (S. mansoni) undergoing 50% liver resection (LR).

METHODS

Fifty hamsters were divided into five groups (10 hamsters each), with group I serving as the control; group II received ZSCLE treatment only; group III was infected with S. mansoni but was untreated; group IV was infected with S. mansoni and received ZSCLE treatment; group V was infected with S. mansoni and underwent ZSCLE treatment and 50% LR. Each group was analyzed using DNA flow cytometry, and oxidative stress (nitric oxide levels), inflammatory markers (tumor necrosis factor-α and interferon-γ), and liver biomarkers were assessed. Histopathological examination was conducted for all groups.

RESULTS

Compared with the infected untreated group, the ZSCLE-treated group showed a significant 70.2% reduction in hepatic tissue egg load (3459.5 ± 191.3 vs 1032 ± 25.1, P < 0.001), and the ZSCLE-treated group that underwent surgical resection showed a 71.8% decrease (2021.7 ± 190.2 vs 7193.3 ± 103.4, P < 0.001). Alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and bilirubin levels were higher in group III than in group I, group IV, and group V (P < 0.0001). The liver regeneration rate (%) was significantly higher in group IV and group V than in group III (median values: 45.18%, 47.93% vs 28.31%). Pathological examination revealed fewer granulomas in group V.

CONCLUSION

ZSCLE-LR is a potent agent against schistosomiasis-induced hepatic damage, exhibiting a significant role in promoting hepatic regeneration. Further molecular-level studies are warranted to investigate the phytochemical properties of ZSCLE and its potential applications in managing schistosomiasis-induced hepatic fibrosis.

Key Words: Schistosomiasis; Oxidative stress; Ziziphus spina-christi leaf extract; Liver resection; Tumor necrosis factor-α; Interferon-γ

Core Tip: The present research strongly suggests that Ziziphus spina-christi leaf extract is a powerful agent in protecting the liver from damage induced by Schistosoma mansoni. Furthermore, this study’s data revealed that liver damage is severely influenced by Schistosoma mansoni infection. The Ziziphus spina-christi leaf extract treatment improved biochemical parameters, pro-inflammatory cytokine levels, DNA integrity, and pathological liver outcomes.

INTRODUCTION

Schistosomiasis is a major global cause of disease and mortality. In tropical endemic regions, rivers, lakes, reservoirs, and irrigation canals serve as the infection sources. Schistosoma mansoni (S. mansoni) causes intestinal schistosomiasis, a debilitating disease prevalent in 52 countries. These comprise most African nations, the Caribbean, the Eastern Mediterranean, and South America[1,2]. In ≥ 74 countries, approximately 207 million people have an active schistosomal infection. Approximately 60% are clinically impacted, with malnutrition and chronic anemia being the major conditions. Over 20 million people suffer from serious illnesses caused by schistosomal infection. Factors contributing to the spread of infection include poverty with inadequate sanitation, contaminated freshwater irrigation, and widespread schistosomal infestation by animal, snail, and human populations.

Analyzing human samples (blood, tissue, and stool) is crucial to extrapolate findings from animal trials to the human system[3], providing valuable insights into the pathophysiology of schistosome infection. However, chimpanzees and baboons provide the most realistic representations of human schistosomiasis, including intestinal lesions and peri-portal fibrosis[4,5]. Numerous cytokine genes, such as those for interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ, are also expressed in the affected tissue; however, details regarding the function of these proinflammatory cytokines remain controversial[6,7].

In biological studies, herbal plants have been recognized for their potential as a valuable source of bioactive metabolites, offering preventative and therapeutic benefits for managing various illnesses. The genus Ziziphus (family: Rhamnaceae) includes almost 53 species that can withstand heat and drought in Africa, Australia, and Asia. An evergreen tree indigenous to Sudan, Ziziphus spina-christi L. (Christ’s thorn Jujube) is found in Khartoum along the Nile banks[8,9].

The wild plants primarily grow in Egypt, particularly Sinai, and Saudi Arabia, and are commonly identified as Sidr and Nabka[10]. Phytochemical investigations have revealed that Ziziphus spina-christi leaf extract (ZSCLE) comprises essential oils, tannins, cyclopeptide alkaloids, triterpenoid sapogenins, flavonoids, phytosterols, and saponins[11]. ZSCLE has been extensively used in herbal and traditional medicine owing to its hepatoprotective, hypoglycemic, antimicrobial, immunostimulant, and tonic effects[12,13]. However, to our knowledge, there has not been any study focused on schistosomacidal activity of ZSCLE. In addition, recent data from previous studies have revealed that this plant extract has antioxidant, antinociceptive, anticancer, anti-inflammatory, antidiarrheic, antispasmodic, antimicrobial, and antileishmanial effects[14].

Biological experiments were conducted to explore ZSCLE’s potential physiological effects and therapeutic action. The plant roots’ ability to lower ulcer size, colitis parameters, inflammatory mediators, and mucosal damage in vivo supported its traditional anti-inflammatory use[15]. Additionally, the anti-inflammatory properties of its leaves were evidenced by their ability to prevent spleen damage in vivo and inhibit sepsis-induced liver damage[16].

This study aims to measure biochemical parameters and proinflammatory cytokines and evaluate the physiological and anti-inflammatory effects, including the promising hepatoprotective potential of ZSCLE against severe S. mansoni infection. ZSCLE treatment was followed by partial liver resection (LR) to evaluate the fibrotic liver’s tolerance for LR after ZSCLE treatment. Additionally, histopathological examination was performed to evaluate the hepatoprotective effects of ZSCLE and its potential to enhance liver regeneration. The study compared the efficacy of ZSCLE alone vs ZSCLE with partial LR in ameliorating liver fibrosis and promoting cellular repair.

MATERIALS AND METHODS

Ethics statement

National and institutional guidelines for the use and care of laboratory animals were followed when handling the animals. The Ethics Committee Board of Faculty of Medicine, Al-Baha University, Kingdom of Saudi Arabia approved the study protocol (Approval No. REC/SUR/BU-FM/2022/59).

Animals

Golden hamsters were acquired from the animal residence of King Abdulaziz University for scientific research (Jeddah, Kingdom of Saudi Arabia). Animals were housed in a pathogen-free environment in the animal house of Faculty of Medicine, Al-Baha University, Kingdom of Saudi Arabia. Throughout the 12-hour light-dark cycle, they had free access to food and drink. Experiments were performed following the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals, United States. Additionally, experiments were conducted following ethical standards[17]. Fifty golden hamsters weighing 130-150 g were used in this study. The resource equation method developed by Arifin and Zahiruddin[18] was used to calculate the study sample size. The hamsters’ abdomens were shaved under anesthesia, and they were percutaneously exposed to a suspension containing approximately 250 live cercariae in a metal ring for 30 minutes using a micropipette, as described by Smithers and Terry[19]. The hamsters were housed in an animal house with a 12-hour light/dark cycle and a regulated temperature of 20 °C-22 °C. The animals were given water and a typical lab diet.

ZSCLE preparation

ZSCLE was prepared as described previously[20]. Ziziphus spina-christi leaves were cleaned, allowed to air dry in the shade, and selected and confirmed by a skilled taxonomist from the Department of Botany, Faculty of Science of Al-Baha University. Subsequently, they were dried, crushed into a fine powder, and stored at 4 °C for 48 hours in 70% (volume/volume) methanol. The extract was evaporated to a semi-dry state using a rotary evaporator set at 45 °C and liquefied in distilled water. Subsequently, ZSCLE treatment began 49 days after infection, and it involved daily gavage with 100 μL of 500 mg/kg ZSCLE for 10 consecutive days[21]. Hamsters were used to assess the acute oral toxicity of ZSCLE. After a 12-hour fast, the animals received a single dose of extracts dissolved in distilled water, and their mortality was evaluated (short-term toxicity) over 48 hours. The next dose was determined following Organization for Economic Co-operation and Development recommendations for short-term toxicity[22].

Phytochemical analysis

Primary phytochemical analysis of ZSCLE was performed to identify and quantify phytochemicals, including saponins, tannins, alkaloids, glycosides, and flavonoids, based on the method described in a previous study[23]. The analysis used Mg and HCl for flavonoid detection; a 1% gelatin solution containing 10% NaCl for tannin detection; Mayer’s and Dragendorff’s reagents (Loba Chemie Private Limited, India) for alkaloid detection; an H₂SO₄-FeCl₂ mixture of for glycoside evaluation; and a suds-based assay for saponin detection.

Overall phenolic content was determined following the colorimetric Folin-Ciocalteau’s chemical method, with gallic acid (Twinkle Chemi Lab PVT. Ltd., India) as the standard[24]. The test involved mixing 0.2 mL ZSCLE with 2 mL Folin-Ciocalteu solution, followed by adding 2 mL sodium carbonate (7%) to the reagent. After 30 minutes of incubation in the dark, absorbance was measured at 750 nm using a spectrophotometer (Mettler Toledo, OH, United States). Total phenolic content was expressed as milligrams of gallic acid equivalents per dry weight.

The overall flavonoid content was determined using the colorimetric aluminum chloride method, with quercetin (Sigma-Aldrich, MO, United States) as the standard[24]. The assay involved mixing 0.2 mL of the standard solution with 0.2 mL of aluminum chloride, 0.1 mL of 33% aqueous acetic acid, and 90% ethanol. After 30 minutes incubation at room temperature (20 °C-22 °C), the absorbance was measured at 430 nm.

Tannin contents were estimated using the vanillin-HCl assay, with catechin as the standard. The extract was mixed with 5 mL vanillin-HCl and incubated for 200 minutes. Absorbance was measured at 500 nm, and tannin content was expressed as milligrams of catechin equivalents per gram of dry weight[24].

Experimental design

Fifty hamsters were divided into five groups (10 per group): Group I (control), group II (ZSCLE-treated), group III (S. mansoni-infected and untreated), group IV (S. mansoni-infected and ZSCLE-treated), and group V (S. mansoni-infected, ZSCLE-treated, and 50% LR. Hamsters received all treatments orally 49 days after exposure to cercariae. Nine weeks post-infection, the animals were sacrificed, and their organs were dissected. Appropriate anesthetic and euthanasia techniques were used to ensure that animals did not suffer during the experiments, following our Ethics Committee’s legal ethical guidelines, which are based on the American Veterinary Medical Association’s Guidelines for the Euthanasia of Animals. After blood sampling, hamsters were sacrificed under anesthesia using an overdose of thiopental sodium administered intraperitoneally[25].

Assessment of parasitological criteria

Worm recovery: Adult S. mansoni were extracted from hamsters using a hepatic-portal-mesenteric perfusion approach. Worm loads were tallied, and the percentage change in overall number of worms was calculated[26].

Ova counts in feces and hepatic tissue egg load: Feces were collected 1 week before treatment began to facilitate the examination of parasite eggs under a microscope. Treatment effectiveness was assessed after 1 week of treatment. Specific liver slices from each hamster were used to analyze hepatic tissue egg load. Cheever’s method was used to calculate the quantity of ova per gram of tissue. The hamster liver was digested in 4% potassium hydroxide overnight at 37 °C. After digestion, supernatants were extracted from tissue suspensions through centrifugation for 5 minutes at 1500 × g. Eggs were counted using a light microscope in two 100 mL aliquots following three cycles of centrifugation and washing. The results were expressed as the mean number of eggs per gram liver tissue[27].

Surgical procedures

The liver was removed via a midline laparotomy after general anesthesia was administered. Ketamine 40 mg/kg, buprenorphine 0.05 mg/kg, and intramuscular acepromazine 0.8 mg/kg were used to induce general anesthesia. The surgical resection procedure was performed following Higgins and Anderson’s recommendations[28]. Midline laparotomy exposed and mobilized the liver. Subsequently, the lobes were resected by ligating the lobe origin with 3-0 silk sutures, which were loosely tied at the root over the superior lobe border. Dissecting scissors were used to carefully remove the lobes distal to the ligated sutures. The abdominal wall was closed with a 3-0 polyglactin suture, and the skin was subsequently closed with a polyamide suture.

Biochemical analysis

On the day of euthanasia, routine analysis was performed at the Department of Clinical Biochemistry, Faculty of Medicine, Al-Baha University. After euthanasia, blood was collected from the heart, processed, and stored for analysis at -80 °C. Bilirubin, alkaline phosphatase, aspartate aminotransferase, and alanine aminotransferase levels were measured on a Cobas 6000 (Roche Diagnostics, Basel, Switzerland) following a standard procedure[29].

Nitric oxide levels: Two stable nitric oxide (NO) oxidative metabolites, nitrite and nitrate, were biochemically evaluated to estimate NO production. The amounts of nitrite and nitrate were measured using the Griess method[30]. The study’s data reveal the sum of the NO metabolites (nitrite and nitrate).

Proinflammatory cytokine levels: The proinflammatory cytokines (TNF-α and IFN-γ) in hamster serum were analyzed using commercial enzyme-linked immunosorbent assay (ELISA) kits (Platinum ELISA, eBioscience, Waltham, MA, United States) following the manufacturer’s instructions. Quantitative cytokine analysis was performed using ELISA, which involves using monoclonal antibodies against the corresponding cytokine. Test standards, internal controls, and diluted rat serum samples (1:2) were added to a solid phase for TNF-α and IFN-γ detection. Absorption, which is calorimetrically measured at 450 nm and 620 nm on an ELISA reader, is directly related to the cytokine concentration. Cytokine concentration (pg/mL) is determined by constructing a standard curve[31].

Assessment of hepatic regeneration

The hepatic regeneration ratio has been defined in a previous study[32]. Hepatic regeneration ratio was calculated for each animal. Net regeneration was defined as the total liver weight at sacrifice, liver weight predicted before surgery, and the weight of the removed liver at hepatectomy. Hepatic regeneration ratio was calculated as the liver weight per 100 g body weight at sacrifice divided by preoperative projected liver weight per 100 g body weight (preoperative projected liver weight: Weight of resected liver at hepatectomy/0.7).

DNA fragmentation analysis by flow cytometry

The liver samples from each group were defrosted, cut with a scalpel in a cold phosphate-buffered saline solution, and incubated at 4 °C for 15 minutes. Subsequently, samples were filtered through a nylon mesh filter. The cells were collected and incubated with a solution containing ribonucleases (1 mg/mL; Sigma, MA, United States) and propidium iodide (10 μg/mL; Sigma, MA, United States) after centrifugation and washing in volume solution. The tubes were stored at 4 °C in the dark for ≥ 30 minutes before undergoing flow cytometry analysis. The propidium iodide fluorescence of individual nuclei was measured with a Coulter Epics XL (Kraemer Blvd, CA, United States). Cells (≥ 5 × 10³) were measured for each sample. Apoptotic cells were detected using six-color flow cytometry on a FACS Canto II (BD Biosciences, CA, United States) with the Flow Jo software (Tree Star, Inc., OR, United States), in which a distinct sub-diploid cell peak in the red fluorescence channel, separate from the cell peak with the diploid DNA content, indicated apoptosis. The percentage of apoptotic cells was calculated based on the proportion of cells with sub-diploid DNA content[33].

Histopathology and granuloma measurement

Liver tissue samples from each group were dehydrated using escalating ethanol concentrations, embedded in paraffin, and fixed for 24 hours with 10% neutral buffered formalin. After deparaffinization and staining with hematoxylin and eosin, 5-μm-thick sections were observed under an Olympus light microscope (Olympus Corporation, Japan). Area and mean granuloma diameter were measured for granuloma morphometric analysis. Additionally, stained sections were used to assess the related histological alterations. A calibrated ocular micrometer was used to measure granuloma diameter with a single egg in the middle. Two lesion diameters were measured perpendicular to each other to determine mean granuloma diameter. An ocular micrometer was used to quantify the minimum diameters of up to 50 egg-induced granulomas per animal. Following a 100 × magnification examination of 100 granulomas from each group, the mean ± SD was reported in micrometers. A Sony analog RGB camera (Sony Group Corporation, Japan) with 640 pixels × 480 pixels was used to capture the granuloma region. Subsequently, the images were analyzed using Scion Image software (Scion Corporation, MD, United States).

Statistical analysis

Statistical analyses were performed using the statistical software application SPSS version 21 (IBM, Chicago, IL, United States). Data are presented as mean ± SD. The Shapiro-Wilk test was used to assess normality in the distribution of the variables. The means of the two groups were compared using the Student’s t-test. After using the Kruskal-Wallis test (for non-normally distributed data) or one-way analysis of variance (for normally distributed data), the Bonferroni post hoc test was used for comparing three or more groups. The threshold for statistical significance was set at P < 0.05.

RESULTS

We performed the following assessments to validate the results of the analysis of variance and independent t-test before they were obtained. Analysis of data revealed that data were normally distributed, as evidenced by skewness and kurtosis data, which revealed values within the normal range (-1.96 ± 1.96). In addition, the Shapiro test for evaluating data normality yielded a value of 0.911, confirming that the results were normally distributed. We analyzed variance homogeneity with a 95% confidence interval (95%CI), and the assumption was met. Furthermore, we performed Levene’s test for variance quality using an F-value of 3.167, which was not significant. Hence, an independence t-test was conducted.

Hepatic tissue egg load and granulomas

The ZSCLE-treated group exhibited a 70.2% decrease in hepatic tissue egg load (3459.5 ± 191.3 vs 1032 ± 25.1, P < 0.001), while those who underwent surgical resection had a 71.8% decrease (2021.7 ± 190.2 vs 7193.3 ± 103.4, P < 0.001) compared with the infected untreated group (Table 1).

Table 1 Comparison between hepatic tissue egg load between infected untreated and Ziziphus spina-christi leaf extract-treated and infected untreated and surgically treated groups, mean ± SD.

Parameter	Infected untreated and treated group			Infected and surgically treated group
	Infected untreated group	ZSCLE-treated group	P value	Infected untreated group	ZSCLE-surgically treated group	P value
Hepatic tissue egg load	3459.5 ± 191.3	1032 ± 25.1 (70% decrease)	< 0.001	7193.3 ± 103.4	2021.7 ± 190.2	< 0.001

ZSCLE: Ziziphus spina-christi leaf extract.

Biochemical analysis

Hamster serum samples from each experimental group were assessed. Blood alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and bilirubin levels were significantly higher in group III than in group I, owing to infected bilharziasis (BLZ) in the former (87.44 ± 2.62 U/L, 99.85 ± 2.17 U/L, 144.41 ± 2.39 U/L, and 0.93 ± 0.05 U/L, respectively). Hamsters in group IV and group V, treated with ZSCLE and undergoing 50% resection, showed significant improvement and reduced infection levels compared with those in group III (P < 0.0001, Table 2). Moreover, the liver regeneration rate (%) was significantly higher in group IV and group V (median values: 45.18% and 47.93%, respectively) than in group III (28.31%), which was only infected with BLZ.

Table 2 Serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and bilirubin in different hamster groups, mean ± SD.

	Control negative group I	Control negative ZSCLE-treated group II	Infected BLV group III	Infected BLV and ZSCLE-treated group IV	Infected BLZ, ZSCLE-treated, and 50% resected group V	P value
Serum ALT (U/L)	27.01 ± 2.86	29.01 ± 3.75	87.44 ± 2.62a,b	39.90 ± 2.62a,b,c	38.08 ± 2.09a,b,c	< 0.0001
Serum AST (U/L)	30.75 ± 1.07	31.58 ± 0.96	99.85 ± 2.17a,b	42.44 ± 1.96a,b,c	41.18 ± 2.10a,b,c	< 0.0001
Serum ALP (U/L)	65.69 ± 1.63	68.35 ± 1.99	144.41 ± 2.39a,b	79.72 ± 4.45a,b,c	77.45 ± 1.39a,b,c	< 0.0001
Serum bilirubin (mg/dL)	0.22 ± 0.03	0.26 ± 0.03	0.93 ± 0.05a,b	0.41 ± 0.03a,b,c	0.39 ± 0.05a,b,c	< 0.0001

^aP < 0.05 vs group I.

^bP < 0.05 vs group II.

^cP < 0.05 vs group III.

Statistical tests: One way ANOVA followed by post hoc Tukey’s multiple comparison test. ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; ALP: Alkaline phosphatase; BLV: Bovine leukemia virus; ZSCLE: Ziziphus spina-christi leaf extract.

Effect of ZSCLE on serum NO

The mean NO level of the infected BLZ group III hamsters (60.32 ± 1.66 μmol/mL) was significantly lower than that of the control group I (85.20 ± 2.27 μmol/mL). However, the ZSCLE treatment (group IV) and ZSCLE treatment with resection (group V) groups had significantly higher mean NO levels (76.92 ± 1.85 μmol/mL and 79.29 ± 2.11 μmol/mL, respectively; P < 0.0001) than group III with infected BLZ (P < 0.0001; Tables 2 and 3).

Table 3 Serum nitric oxide, tumor necrosis factor-α, and interferon-γ levels and apoptosis in different hamster groups, mean ± SD.

	Control negative group I	Control negative ZSCLE-treated group II	Infected BLV group III	Infected BLV and ZSCLE-treated group IV	Infected BLV, ZSCLE-treated, and 50% resected group V	P value
Serum NO (μmol/mL)	85.20 ± 2.27	86.34 ± 2.68	60.32 ± 1.66a,b	76.92 ± 1.85a,b,c	79.29 ± 2.11a,b,c	< 0.0001
Serum TNF-α level (pg/mL)	17.57 ± 1.38	20.39 ± 1.31	189.27 ± 3.38a,b	45.21 ± 2.95a,b,c	39.85 ± 1.73a,b,c,d	< 0.0001
Serum IFN-γ level (pg/mL)	28.72 ± 1.33	31.81 ± 1.09	185.25 ± 4.91a,b	72.30 ± 2.92a,b,c	74.73 ± 3.26a,b,c	< 0.0001
Apoptosis, %	7.6	7.6	62.9	28.7	23.6

^aP < 0.05 vs group I.

^bP < 0.05 vs group II.

^cP < 0.05 vs group III.

^dP < 0.05 vs group IV.

Statistical test: One way ANOVA followed by post hoc Tukey’s multiple comparison test. NO: Nitric oxide; TNF: Tumor necrosis factor; INF: Interferon; BLV: Bovine leukemia virus; ZSCLE: Ziziphus spina-christi leaf extract.

TNF-α and IFN-γ serum levels in different hamster groups

Table 3 reveals that group I and group II that were treated or untreated with ZSCLE had normal TNF-α and IFN-γ levels (17.57 ± 1.38 pg/mL and 28.72 ± 1.33 pg/mL, respectively) compared with group III with infected BLZ, which had elevated TNF-α and IFN-γ levels (189.27 ± 3.38 pg/mL and 185.25 ± 4.91 pg/mL, respectively). The ZSCLE-treated hamsters (group IV) and those treated with ZSCLE after undergoing resection (group V) showed a significant decrease in TNF-α and IFN-γ levels compared with the infected group III (P < 0.0001; Tables 2 and 3).

Flow cytometry for liver cell apoptosis

DNA histograms were used to calculate the proportion of apoptotic cells with hypodiploid DNA content. ZSCLE-treated normal hamster group I and group II displayed a peak pattern of 7.6% and 7.6%, respectively, indicating no liver damage (Figure 1A and B). Conversely, hamsters with infected BLZ (group III) displayed and represented a high percentage of liver damage (62.9%; Figure 1C). However, the ZSCLE-treated liver (group IV) displayed a shift and a relative decrease in peak size by 28.7% (Figure 1D). Furthermore, group V, which received ZSCLE treatment and underwent LR 23.6% of the time, showed a notable parallel shift towards decreased fluorescence intensity. This reduction in intensity and shift may indicate nuclear apoptosis and fragmentation reduction (Figure 1E; Table 3).

Figure 1 Apoptosis analysis. A and B: Liver cell damage, represented by apoptosis, was normal in group I and I group II, respectively; C: A higher percentage of liver damage (62.9%) was observed in group III hamsters with infected Bovine leukemia virus; D and E: A marked decline in apoptosis (28.7% and 23.6%, respectively) in the groups that received Ziziphus spina-christi leaf extract (group IV) and Ziziphus spina-christi leaf extract + 50% liver resection (group V). Apoptosis is identified by the dark, black-stained area upon propidium iodide staining, which labels DNA; the stained area denotes marked DNA fragmentation. FL2-H: Fluorescence channel 2 height.

Histopathology parameters

Hamsters infected with S. mansoni (group III) had multiple bilharzial granulomas comprising bilharzial ova, eosinophils, epithelioid cells, and fibrous capsule with cytoplasm vacuolization and hepatocyte degradation. Granulomas contained trapped eggs surrounded by concentric fibrosis with numerous fibroblasts, and abundant granulomas contribute to disorganized liver architecture. Additionally, the livers had enlarged hepatic sinusoids with more Kupffer cells. ZSCLE-treated group IV exhibited a moderate number of granulomas and an accompanying inflammatory response. However, a notable reduction in granulomas was observed in group V, which received ZSCLE treatment and underwent 50% tumor removal, although congestion and focal inflammatory cell infiltration persisted (Figure 2).

Figure 2 Hematoxylin and eosin micrograph of liver sections. A and B: Normal hepatic structure with hepatocytes arranged in strands alternating with blood sinusoids forming a network around central veins. No neuroinflammatory injury or granulomas were detected. Control negative group I (A); control negative Ziziphus spina-christi leaf extract (ZSCLE)-treated group II (B); C: Group III with infected Bovine leukemia virus (BLV), showing multiple bilharzial granulomas formed of bilharzial ova, eosinophils, epithelioid cells, and fibrous capsule; D: Infected BLV and ZSCLE-treated group IV, showing moderate granuloma formation accompanied by inflammatory reaction; E: Infected BLV and ZSCLE-treated + 50% resected group V, showing few granulomas with remaining focal inflammatory cell infiltration and congestion.

DISCUSSION

Previous studies have revealed that several plants, including ZSCLE, have therapeutic potential against parasitic diseases[34]. In this study, we evaluated ZSCLE as a treatment for S. mansoni infections using a hamster model. ZSCLE significantly reduced S. mansoni counts. The 50% resection data in group V are similar to those of the control hamster group I; however, these results also reveal that ZSCLE treatment restored S. mansoni-induced reduction in group IV and group V, which underwent treatment and surgery. These findings are consistent with those of Almeer et al[21], who showed that S. mansoni infection caused by ZSCLE reduced elevated serum liver function. This is also consistent with the findings of Mostafa et al[35], who showed that S. mansoni increased bilirubin levels and impaired liver function, whereas the naturally occurring polyphenol resveratrol mitigated these effects and improved liver function. Infected hamsters treated with ZSCLE showed significant improvement after surgery, possibly owing to the liver’s regenerative ability following surgery, particularly after partial hepatectomy (50% liver removal). However, even with recent advances, liver surgery requires careful patient selection and meticulous preoperative planning to ensure a sufficient future liver remnant, as emphasized by Del Basso et al[36].

Recent studies have revealed that the bioactive components of medicinal plants are a promising substitute for current clinical therapy. As deworming does not completely halt the progression of liver fibrosis, anti-fibrotic treatment is crucial for patients with chronic schistosomiasis[37]. Some studies have reported the use of various natural products to treat schistosomiasis-associated liver fibrosis. These products may alter fibrotic factors [such as IL-13, growth stimulation expressed gene 2, transforming growth factor (TGF)-β1, and TNF-α levels] and anti-fibrotic factors (such as IL-10, regulatory T cells, and major histocompatibility complex class II) by influencing intracellular signaling pathways such as the nuclear factor kappa-light-chain-enhancer of activated B cells, phosphoinositide 3-kinase/protein kinase B, and TGF-β1/Smad pathways[38].

Serum NO, IL-6, IFN-γ, and TNF-α end products are currently being investigated. ZSCLE treatment (group IV) reversed S. mansoni-induced changes in Goblet cell numbers, muscle thickness, intraepithelial lymphocytes, and villus heights. Our findings indicate that, for the first time, LR can be used to assess fibrotic liver tolerance in hamsters infected with S. mansoni. Similarly, a 50% surgical resection should be considered an effective management approach for the infected hamster liver (group V). These outcomes are attributed to the pharmacological action of ZSCLE, which reduces profibrotic proinflammatory markers and molecules[39]. This is consistent with earlier findings by Alghamdi et al[40], who reported that ZSCLE demonstrated anti-hepatic fibrosis activity and reduced granuloma formation in mice infected with Schistosoma haematobium. This is also consistent with the findings of Almeer et al[21], who confirmed that 400 mg/kg ZSCLE treatment reduces hepatic granuloma area in mice infected with S. mansoni and reduces hepatic fibrosis by blocking TGF-β1, vascular endothelial growth factor, α-smooth muscle actin, tissue inhibitor of metalloproteinase-1, and matrix metalloproteinase-9 expression. ZSCLE inhibits inflammation and oxidative stress by upregulating the transcription factor nuclear factor erythroid-2-related factor 2, which is involved in regulating the oxidative stress response. Furthermore, Vatankhah et al[41] confirmed that ZSCLE decreased TGF-β and α-smooth muscle actin levels. Studies have also corroborated the findings of this investigation and demonstrated that ZSCLE administration activated the antioxidant signaling pathway in response to intoxication. ZSCLE may exhibit antiapoptotic properties by scavenging reactive oxygen species, consistent with previous studies demonstrating its ability to downregulate proinflammatory mediators, including TNF-α, IL-1β, nitric oxide synthase 2, and NO[42,43]. Notably, group V, which received ZSCLE treatment and LR, showed significant improvement in these parameters, approaching near-normal levels. This group outperformed group III (infected) and ZSCLE-only treated groups, supporting our hypothesis and confirming that LR regulates and improves the proinflammatory cytokines that contribute to liver damage. These data are consistent with a study by Fathi et al[44], who revealed that LR significantly affected and reduced TNF-α and IL-6 serum levels (P < 0.0001).

S. mansoni infection exacerbates oxidative stress, primarily owing to the metabolic burden imposed by eggs lodged in the liver and the ensuing granulomatous inflammatory response. Therefore, hepatocytes are continuously exposed to oxidative stress during infection, potentially damaging the DNA[45]. This is consistent with our findings; group III, which was infected with S. mansoni, showed notable changes of 62.9% (Figure 1C) compared with group I. ZSCLE-treated and ZSCLE + LR-treated group IV and group V, respectively, showed improvement following treatment, with 50% LR by 28.7% and 23.6%, respectively (Figure 1C and D). This reduction in intensity and shift may indicate nuclear apoptosis and fragmentation reduction (Figure 1D). In rats exposed to aflatoxin M1, ZSCLE (1 mg) rescued the elevation of liver functions, oxidative markers (malondialdehyde), inflammatory markers (TNF-α), and DNA damage. These findings are consistent with those of Badr et al[46], who showed that Ziziphus or encapsulated probiotics may successfully protect the liver from DNA damage and oxidative stress.

The clinical-pathological profile of schistosomiasis, a common neglected tropical disease, is intricate and multidimensional. We examined molecular processes after S. mansoni infection in the liver, the organ most severely impacted in patients with schistosomiasis, using hamsters as final hosts. In the present study, liver sections from group III revealed multiple bilharzial granulomas containing Schistosoma ova, accompanied by significant hepatocyte degeneration. These findings are consistent with those of Ghezellou et al[47], who revealed the intricacy of liver pathology by describing the hepatic response to S. mansoni infection. Additionally, this is consistent with the findings of Gharib et al[48], who reported that lipid peroxide production increases as a result of reduced hepatic antioxidant capacity caused by oxidative processes at sites of granulomatous inflammation associated with schistosomiasis infection. The pathology may be largely caused by the ensuing imbalance between pro-oxidant and antioxidant processes. Conversely, group IV exhibited less inflammation and granuloma. Group V received 50% LR after ZSCLE treatment and displayed mild granulomas with limited congestion. The pathological improvement underscores the potential value of combined treatment approaches. Although the data collected are not directly comparable to those of other related studies, the results suggest that combining systemic therapy with surgery may be a future direction for improving survival in this model, particularly given the advances in targeted therapies and immune-checkpoint inhibitors with biochemical targeting parameters.

The study was conducted in a hamster model, which necessitates further investigation in humans to determine its relevance and potential translation to clinical practice. In addition, pharmacokinetic data, potential risks of ZSCLE and liver surgery, and the phytochemical characteristics of each ZSCLE constituent against biochemical effects of schistosomiasis require further investigation. Furthermore, the study findings are limited by our incomplete understanding of ZSCLE’s mechanism of action during the schistosomiasis-induced liver fibrosis pathogenesis. Future investigations should focus on the biological and pharmacological properties of ZSCLE, which may support its future use in human clinical trials.

CONCLUSION

The present study revealed that ZSCLE treatment in schistosomiasis significantly improved biochemical parameters, proinflammatory cytokine levels, DNA integrity, and pathological liver outcomes. These results indicate that ZSCLE-LR is a strong protective agent against schistosomiasis-induced liver and plays a significant role in hepatic regeneration after resection. Further molecular-level investigations into the biological and pharmacological aspects of ZSCLE, including dose-response evaluations and phytochemical properties, are warranted to explore its therapeutic potential in schistosomiasis-induced hepatic fibrosis.