Introduction
Inflammatory bowel disease (IBD) is a severe worldwide autoimmune illness affecting the intestine, its prevalence is going on in Egypt over the past decade (Mosli et al. Reference Mosli, Alawadhi, Hasan, Abou Rached, Sanai and Danese2021). Ulcerative colitis (UC) is a chronic recurrent colonic inflammation influenced by genetics, epithelial barrier abnormalities, immunological responses, and environmental factors (Oraby et al. Reference Oraby, Mageed, Raouf, Abdelshafy, Ahmed, Khalil, Mangoura and Fadaly2024).
Complications such as stenosis, abscess, and an increased risk of colorectal cancer have been attributed to the progressive and destructive nature of IBD (Neurath Reference Neurath2019). However, Sulfasalazine (SLZ), corticosteroids, immunomodulators, and anti-tumor necrosis factor (TNF)-α agents are the main drugs that induce and maintain clinical remission of IBD (Park and Cheon Reference Park and Cheon2022), its restricted response and significant side effects making the finding of new therapeutic alternatives a critical priority (Na and Moon Reference S-Y and Moon2019).
Regulatory T cells (Tregs), a specific subset of T cells, induce immunomodulation through suppressive cytokines, inhibitory receptor expression, and cell-contact-dependent processes (Grant et al. Reference Grant, Liberal, Mieli-Vergani, Vergani and Longhi2015). Treg abnormalities can lead to immune-related disorders such as IBD, rheumatoid arthritis (RA), multiple sclerosis (MS), and systemic lupus erythematosus (SLE) (Pereira et al. Reference Pereira, Gomes, Ishak and Vallinoto2017). The number of Tregs in the bloodstream and colonic mucosa of IBD patients has been elevated using anti-tumor necrosis factor (TNF)-α treatment (Li et al. Reference Li, Vermeire, Bullens, Ferrante, Van Steen, Noman, Bossuyt, Rutgeerts, Ceuppens and Van Assche2015), demonstrating that the development of IBD may be attributed to Treg dysregulation. Therefore, Treg modulation is thought to be a potentially effective therapeutic strategy for treating autoimmune illnesses such as IBD.
Forkhead box P3 (FOXP3) is a transcription regulator that belongs to the forkhead-winged helix family that influences the development and function of Tregs and facilitates the transformation of naïve T cells into Treg-like cells with suppressive activity. FOXP3 modulates the expression of immunosuppressive molecules by binding to their corresponding target genes, such as cytotoxic T-lymphocyte antigen 4 (CTLA4) (van Nieuwenhuijze and Liston Reference Van Nieuwenhuijze and Liston2015). It was previously observed that ectopic FOXP3 expression in CD4+ CD25-T cells might induce a Treg phenotype and prevent IBD. It has been demonstrated that absent FOXP3 expression impairs Treg function (Huehn and Beyer Reference Huehn and Beyer2015). However, several studies linked FOXP3 overexpression to various immune-related illnesses like MS, RA, and SLE (Pesenacker et al. Reference Pesenacker, Cook and Levings2016). It seems that oxidative stress has a major pathogenic role in the development and progression of IBD. Increased inflammatory cells, particularly neutrophils, that penetrate intestinal tissue and upset cellular homeostasis are the hallmarks of inflammatory bowel disease (IBD). This accelerates the inflammatory process and causes cell damage (Piechota-Polanczyk and Fichna Reference Piechota-Polanczyk and Fichna2014). According to Dröge (Reference Dröge2002), an imbalance between oxidant and antioxidant compounds was also an important characteristic of experimentally induced colitis using acetic acid. Epidemiological research indicates a strong correlation between the high prevalence of helminthic infections in developing countries and the immune system’s fight against inflammatory disorders like IBD (Yeshi et al. Reference Yeshi, Ruscher, Hunter, Daly, Loukas and Wangchuk2020). As a result, many studies on helminths showed that worms, their ova, or derived excretory or secretory products have great potential for modulating and downregulating the host immune system in autoimmune disease models, including IBD, RA, and MS (Alghanmi et al. Reference Alghanmi, Minshawi, Altorki, Zawawi, Alsaady, Naser, Alwafi, Alsulami, Azhari and Hashem2024). This could involve Schistosoma egg therapy (Ma et al. Reference Ma, Sun, Zheng, Li, Zhang, Huang, Lu, Li, Nouhoum and Yu2022), Trichuris suis larvae (Huang et al. Reference Huang, Zeng, Chen, J-P and Zhu2018), and Necator americanus inoculation (Croese et al. Reference Croese, O’neil, Masson, Cooke, Melrose, Pritchard and Speare2006). Therefore, novel anti-inflammatory therapies may be applied to humans and animals (Ben-Ami Shor et al. Reference Ben-Ami Shor, Lachnish, Bashi, Dahan, Shemer, Segal, Shovman, Halpert, Volkov and Barshack2019). E. granulosus is the cause of cystic hydatid disease. Human E. granulosus cysts are typically found in the liver or lung (Gessese Reference Gessese2020). The cyst comprises an outer acellular laminated layer (LL) and a germinal layer containing a hydatid cyst fluid (HCF) that fills a vesicle. The primary components of E. granulosus’s LL are lipids, proteins, and carbohydrates (Sadr and Borji Reference Sadr and Borji2023) that act as both an immunological and a mechanical barrier to the host immune system. The parasite uses evasion mechanisms to remain in the host, which may be facilitated by the LL-induced suppression of NO production. In addition, LL pretreated mice expressed a systemic increase in IL-10 with concomitant localized attenuation in the expression of iNos and NF kβ in colonic tissues (Soufli et al. Reference Soufli, Toumi, Rafa, Amri, Labsi, Khelifi, Nicoletti and Touil-Boukoffa2015). Helminth-produced antigens influence the immunological response by stimulating the production of Tregs, generating anti-inflammatory cytokines, and eventually favoring a T helper cell type 2 (Th-2) biased immune response (Alghanmi et al. Reference Alghanmi, Minshawi, Altorki, Zawawi, Alsaady, Naser, Alwafi, Alsulami, Azhari and Hashem2024). As a result, the Th1 cell subset—mostly linked to autoimmune illnesses like IBD—was downregulated (Sun et al. Reference Sun, Hao, Wu, Luo, El-Ashram, Alouffi, Gu, Liu, J-J and Zhu2024). Therefore, this study aims to explore the potential therapeutic effects of HCF and hydatid LL-derived crude extract of E. granulosus, in combination with the standard medication sulfasalazine, for treating experimental colitis by targeting Foxp3+ Treg cells.
Materials and methods
Animals
Forty-two laboratory-bred male Swiss albino mice, free of parasites and weighing between 20 and 25 g, were acquired from the Animal House at the Faculty of Medicine, Zagazig University, Egypt.
Ethical concerns
This research was conducted according to the guidelines of the Zagazig University Institutional Animal Care and Use Committee (ZU-IACUC) (Approval No. ZU-IACUC/3/F/113/2024).
Drug preparation
Acetic acid (AA) and sulfasalazine ((5-[4-(2-Pyridylsulfamoyl) phenylazo] salicylic acid)) were acquired from Sigma Aldrich (Sigma Co., MO, USA) and Pfizer Pharmaceutical Industries (New York City, USA), respectively. Suppliers of reagents, kits, and antibodies were emphasized during the biochemical analysis. After dissolving AA in sterile filtered water to a final concentration of 5%, colitis was produced in mice by injecting 150 μl intrarectally (Luo et al. Reference Luo, Zhong, J-C and Cui2011). Sulfasalazine was taken orally (100 mg/kg/day) (Khajah et al. Reference Khajah, Fateel, Ananthalakshmi and Luqmani2016). Mice were injected with 50 μg/mL of HCF (Hajizadeh et al. Reference Hajizadeh, Jabbari, Spotin, Hejazian, Mikaeili Galeh, Hassannia, Sahlolbei, Pagheh and Ahmadpour2024) and 200 μl of a crude extract of a HLL (i.p) intraperitoneally (i.p) (Soufli et al. Reference Soufli, Toumi, Rafa, Amri, Labsi, Khelifi, Nicoletti and Touil-Boukoffa2015).
Preparation of the hydatid cyst-derived HLL crude extract and HCF collection
Hydatid cysts from the human liver have been obtained from Zagazig University Hospital’s Advanced Hepato-Pancreatico-Biliary Center and General Surgery Department. Each patient provided written informed consent for the research. The procedure previously outlined by Steers et al. (Steers et al. Reference Steers, Rogan and Heath2001) was used to prepare the crude extract of HLL. First, the infectivity of cysts was proved through the detection of protoscolices. Next, the fluid was aseptically aspirated using a needle and syringe, and it was eventually collected at −20°C (Rahimi et al. Reference Rahimi, Sadjjadi and Sarkari2011). After that, to aid in removing the LL, the hydatid cyst membrane was repeatedly cleaned in PBS supplemented with 300 UI penicillin and 0.3 mg/ml streptomycin, and it was allowed to freeze for an additional night. After being removed, the LL was sonicated for 10 seconds on ice in PBS containing a cocktail of protease inhibitors (Invitrogen, Life Technologies, Carlsbad, CA, USA) until a particulate solution was formed. 200 μl of HLL crude extract and 50 μg/mL were intraperitoneally administered to mice once a day, three days before colitis induction and continued for one week.
Induction of acute colitis in mice
Mice were given free access to tap water but were deprived of food for 24 hours before the colitis induction. Colitis was produced in mice by injecting 150 μl intrarectally of 5% AA using plastic catheters once under a light anesthetic. Only sterile filtered water was given to the controlled animals. Mice were held head-down for 15–20 seconds to prevent the intracolonic instillation from leaking too soon (Randhawa et al. Reference Randhawa, Singh, Singh and Jaggi2014).
Experimental Design
Forty-two male albino mice were randomly divided into 7 groups, each with 6 mice. G1 (control healthy mice) received 1 ml/kg of sterile filtered water three days before being given the sterile filtered water intrarectally and continued for one week (Ghasemi-Dehnoo et al. Reference Ghasemi-Dehnoo, Safari, Rahimi-Madiseh, Lorigooini, Moradi and Amini-Khoei2022). G2 (colitis model) mice received AA without any treatment. G3: mice with colitis received HCF intraperitoneally (i.p). G4: Group with colitis treated with a crude extract of a LL (i.p). G5: mice with colitis received SLZ orally. G6: mice with colitis treated with a combination of SLZ orally and HCF (i.p). G7: mice with colitis treated with a combination of SLZ orally and crude extract of LL (i.p). Groups 3 through 7 received a treatment regimen three days before the induction of colitis by acetic acid and continued for one week after that (Figure 1).
A design to clarify the experimental process and the time schedule of administration of drugs and antigens.
Evaluation of colitis severity
On the 8th day following the establishment of the colitis model, the mice were sacrificed. Several indicators were used to monitor the severity of colitis, including the following:
Survival rate
Following the induction of colitis: The deaths of the mice in each group were recorded daily, and the survival rates were calculated (Silva et al. Reference Silva, Solas, Pinto and Mateus2022).
Disease Activity Index (DAI)
The mice were observed daily to follow up on weight loss, stool consistency, and bleeding feces. Mice were given DAI ranging from 0 to 4 (Jeengar et al. Reference Jeengar, Thummuri, Magnusson, Naidu and Uppugunduri2017). The mice’s fecal occult blood was assessed by the FOB kit (Biodiagnostic CAT No. OB24 12).
Inflammatory cytokines and oxidative stress markers assessment
Using commercial enzyme-linked immunosorbent assay (ELISA) kits and following manufacturer guidelines (Sun Red Biological Technology Co., China, Shanghai), serum samples were separated and used to measure systemic levels of TNF-α and IL-10. Malondialdehyde (MDA) and total antioxidant capacity (TAC) were measured using the colorimetric method (Biodiagnostic CO., USA CAT).
Macroscopic inflammation score
Following scarification, the colons were aseptically isolated and cleaned with PBS, and the presence of adhesions, wall thickness, degree of mucosal edema, and colonic ulcerations were the four parameters used to assess the colonic damage macroscopically. A score ranging from 0 (normal) to 3 (severe) was assigned to each parameter. A minimum of 0 and a maximum of 12 made up the overall score (Menachem et al. Reference Menachem, Trop, Kolker, Shibolet, Alper, Nagler and Ilan2005). To measure the level of inflammation, the colon’s length was also measured (Moreels et al. Reference Moreels, Nieuwendijk, De Man, Winter, Herman, Van Marck and Pelckmans2004).
Histopathology and immunohistochemistry (Foxp3+ in colon and spleen tissue)
For histological analysis, colon segments were fixed in 4% neutral-buffered formalin, embedded in paraffin, and stained with hematoxylin and eosin (H&E) (Suvarna et al. Reference Suvarna, Layton and Bancroft2018). The colonic inflammation was assessed using the following criteria based on the histological damage score from (Mojtahed et al. Reference Mojtahed, Khanna, Sandborn, D’haens, Feagan, Shackelton, Baker, Dubcenco, Valasek and Geboes2014). Other paraffin-embedded sections of the colon and spleen were prepared for immunostaining using an anti-Foxp3+ rabbit polyclonal antibody kit (Servicebio Technology Co., China, Hu Bei, Wuhan). Various staining patterns were produced by the antibodies that specifically targeted different Foxp3+ epitopes. The percentage of positive lymphocytes was computed using 10 high-power fields. The following score was used: 0. Foxp3 is not immunoreactive. 1. Only around 25% of lymphocytes had hot patches that showed inflammation. 2. Between 25% to 50% of stained lymphocytes. 3. The percentage of stained lymphocytes was more than 50%. The staining in the necrotic and suppurative areas was disregarded while scoring. The mean value (x) ± standard deviation (SD) represented the mean score for each group (Ashour et al. Reference Ashour, Othman, Shareef, Gaballah and Mayah2014).
Statistical analysis
The data were computerized and statistically analyzed using SPSS version 27.0. Qualitative data was expressed in numbers and percentages. Quantitative data were expressed in mean and standard deviation (SD). Paired sample t-test, two-way ANOVA, Fisher’s exact test, and Tukey post hoc test were used to compare data. The threshold for statistical significance was set at P < 0.05.
Results
Survival rate
The colitis model (33.33%) showed the most remarkable significant reduction in survival rates among studied groups, followed by GIII (50%), GIV and GV (66.67%), GVI and GVII (83.33%) compared with the control negative group (100%). GVI and GVII received SLZ + HCF and SLZ + LL crude extract, respectively, and showed the highest survival rates compared with GII.
Weight changes and DAI scores at different time intervals post-colitis
There were highly statistically significant differences on the 3rd, the 5th, and 7th days regarding weight changes (Figure 2a) and DAI scores (Figure 2b) between the studied groups. Weight loss score is considered a part of the clinical disease score (DAI) in addition to stool score and blood stool score. Colitis models showed the typical severe manifestations, including a high DAI score represented by progressive weight loss, soft or diarrheic bloody stool, hair loss, and apathetic behavior. In contrast, mice that received therapeutic doses of SLZ + HCF and SLZ + LL crude extract showed the most significant improvement in these clinical manifestations, including lower DAI scores compared to colitis models. Also, there were substantial drops in colon weight/length (CW/L) (Figure 3a) and colon weight/body weight (CW/BW) (Figure 3b) ratios within the same therapeutic groups compared with other ones.
Weight change (a); DAI score (b) among the studied groups.
Colon weight/length (a) and colon weight/body weight (b) ratios of the studied groups. Results are representative as means ± SD, * P<0.05, ** P<0.01, *** P<0.001.
Inflammatory cytokines and oxidative stress markers levels
The therapeutic groups that received SLZ + HLL exhibited lower levels of TNF-α (Figure 4a) and a significant upregulation of IL-10 levels (Figure 4b). The ratio between TNF-α/IL-10 was evaluated (Figure 4c). There were highly statistically significant variations in levels between studied groups compared to the colitis model. Regarding oxidant and antioxidant levels, mice in the therapeutic groups administered SLZ + HLL showed significant downregulated MDA levels (Figure 5a) and upregulated levels of TAC (Figure 5b).
Inflammatory cytokines among the studied groups: a: TNF-α; b: IL-10; c: Ratio between TNF-α/IL-10. Results are representative as means ± SD, * P<0.05, ** P<0.01, *** P<0.001.
Oxidative stress markers among the studied groups: a: MDA; b: TAC. Results are representative as means ± SD, * P<0.05, ** P<0.01, *** P<0.001.
Macroscopic, histopathological, and immunohistochemistry study
Regarding macroscopic assessment, the colonic wall of GVII (SLZ + HLL) was normal with no discernible abnormalities. At the same time, GVI (SLZ+HCF) exhibited edema without congestion compared to GII (colitis) where the colonic wall displayed edema, congestion, and typical hemorrhagic regions according to macroscopic score shown in (Figure 6a). Microscopic examination of the colonic wall showed significant improvement of the histological appearance (restoration of villous structure, presence of goblet cells, and minimal to moderate inflammatory infiltrate) in the treated groups compared with the colitis alone group (GII) in which the colonic wall showed dense inflammatory infiltrate and degeneration of villous structure (Figure 6b–i). In immunohistochemical assessment, the colonic wall of GII (colitis alone) showed negative expression of FOXP3. In contrast, treated groups showed a positive expression of FOXP3, with a strong positive expression detected in GVII (SLZ+LL crude extract) (Figure 7a–h).
Macroscopic scores (a), microscopic scores (b) among the studied groups. Results are representative as means ± SD, * P<0.05, ** P<0.01, *** P<0.001. Histopathological examination of the colon sections of the studied groups c: The negative control group showed normal intestinal villi (V) with abundant goblet cells (G) with clear cytoplasm and underlying regular smooth muscle fibers (M). No villous degeneration or inflammatory infiltrate. (H&Ex100). d: Colitis models showed degenerated villi (DV) with loss of apical brush border (arrow) surrounded by cryptitis and crypt abscess (dense lymphocytic inflammatory infiltrate among the villi and in the submucosa) (arrowhead). (H&Ex100). e: Treated group with HCF showed degenerated villi (DV) with absent goblet cells surrounded by dense lymphocytic inflammatory infiltrate among the villi (arrowhead) and in the submucosa (star) and bands of fibrosis (F) indicating minimal improvement. (H&Ex100). f: Treated group with LL crude extract showed restoration of villous structure (V) with focal villous degeneration (DV) surrounded by moderate lymphocytic inflammatory infiltrate (arrowhead) indicating mild improvement. (H&Ex100). g: Treated group with SLZ showed restoration of villous structure with focal villous degeneration (DV) surrounded by bands of fibrosis (F) and mild lymphocytic inflammatory infiltrate (arrowhead) indicating moderate improvement. (H&Ex100). h: Treated group with SLZ combined with HCF showed restoration of villous structure and presence of goblet cells surrounded by moderate lymphocytic inflammatory infiltrate (arrowhead) indicating mild to moderate improvement. (H&Ex100). i: Treated group with SLZ combined with LL crude extract showed restoration of villous structure with prominent clear goblet cells (G) surrounded by minimal lymphocytic inflammatory infiltrate (arrowhead) indicating best improvement. (H&Ex100).
Immunohistochemical examination of colon sections a: Foxp3/HPF score of colon sections among the studied groups. Results are representative as means ± SD, * P<0.05, ** P<0.01, *** P<0.001. b: The negative control group showed normal intestinal villi (V) with few scattered regulatory T cells for FOXP3 immunohistochemical staining. (FOXP3 IHCx200). c: Colitis models showed damaged intestinal villi with absent regulatory T cells detected by negative FOXP3 immunohistochemical staining. (FOXP3 IHCx200). d: Treated group with HCF showed damaged intestinal villi with few regulatory T cells detected by focal positive FOXP3 immunohistochemical staining. (FOXP3 IHCx200). e: Treated group with LL crude extract showed restoration of intestinal villi with minimal regulatory T cells detected by focal positive FOXP3 immunohistochemical staining. (FOXP3 IHCx200). f: Treated group with SLZ showed restoration of intestinal villi with increased regulatory T cells detected by focal positive FOXP3 immunohistochemical staining. (FOXP3 IHCx200). g: The treated group with SLZ combined with HCF showed restoration of intestinal villi with a moderate amount of regulatory T cells detected by focal positive FOXP3 immunohistochemical staining. (FOXP3 IHCx200). h: Treated group with SLZ combined with LL crude extract showed normal intestinal villi with strong positive regulatory T cells for FOXP3 immunohistochemical staining. (FOXP3 IHCx200).
Discussion
Acetic acid-induced colitis in experimental mice is a simple form of intestinal inflammation resembling human IBD in pathology, histology, and clinical features. It results in intestinal lining bleeding, ulceration, ischemia, inflammation, and edema (Ahmed et al. Reference Ahmed, Abdel-Halim, Farid and Elamir2022). Despite significant emergence in IBD treatments, conventional medicine is unable to provide a satisfactory treatment for this condition; thus, the search for alternatives has become an urgent need. ‘Helminth therapy’ is considered one of the most promising agents for the treatment of numerous autoimmune diseases, including IBD, due to their immunomodulatory elements (Maruszewska-Cheruiyot et al. Reference Maruszewska-Cheruiyot, Donskow-Łysoniewska and Doligalska2018). Studies have been conducted to evaluate the effects of helminths and their products, including Schistosoma ova (Taghipour et al. Reference Taghipour, Aghdaei, Haghighi, Mossafa, Tabaei and Rostami-Nejad2014), Necator americanus (Croese et al. Reference Croese, O’neil, Masson, Cooke, Melrose, Pritchard and Speare2006), Trichuris suis (Summers et al. Reference Summers, Elliott, Urban, Thompson and Weinstock2005), Trichuris trichiura ova (Broadhurst et al. Reference Broadhurst, Leung, Kashyap, Mccune, Mahadevan, Mckerrow and Loke2010), and Trichinella spiralis, on various experimentally induced colitis (Fawzy et al. Reference Fawzy, Selim, Mostafa, Abdelhameed, Darwish, Yousef, Alabiad, Ibrahim, Fawzy and Hamed2024) and autoimmune encephalomyelitis (EAE) (Sofronic-Milosavljevic et al. Reference Sofronic-Milosavljevic, Radovic, Ilic, Majstorovic, Cvetkovic and Gruden-Movsesijan2013). Echinococcus granulosus is the parasitic cause of lung or liver cystic hydatid illness. The cyst comprises an outer, acellular laminated layer (LL) and a germinal layer within a fluid-filled vesicle (Gessese Reference Gessese2020). Long-term host survival of E. granulosus indicates that the parasite can efficiently modulate the host’s immunological response (Aimulajiang and Guo Reference Aimulajiang, Guo, Aimulajiang and Guo2024). This could be attributed to hydatid cyst fluid (HCF) that reduces macrophage inflammatory responses (Wang et al. Reference Wang, Lin, Fu, Yang, Dong, Wu, Chen, Wang and Hou2023). HCF is a blend of proteins, lipids, and carbohydrates. For example, two of the main compounds the cyst synthesizes are antigen B and antigen 5. AgB is a thermostable polymeric lipoprotein that is highly immunogenic and has a high sensitivity and specificity for the serological diagnosis of cystic hydatid disease. One of the most immunogenic and common components of HCF is antigen 5 (Ag5). Ag5 and AgB, the most studied antigens in the serodiagnosis of CE, make up its 57 and 67 kDa components (Sarkari and Rezaei Reference Sarkari and Rezaei2015). Also, it has been shown that treating mice with hydatid LL crude extract from the E. granulosus cyst reduces inflammation in a mouse model of DSS-induced colitis (Soufli et al. Reference Soufli, Toumi, Rafa, Amri, Labsi, Khelifi, Nicoletti and Touil-Boukoffa2015). Mice that received SLZ/HLL and SLZ/ HCF showed the highest survival rate (83.33%) compared to the colitis models (33.33%). This may be explained by the derived molecules acting as a unique therapeutic regulator of inflammation when combined with sulfasalazine. This is consistent with the findings of Xie et al. (Reference Xie, Wu, Wu, Liu, Chen, Shi, Wei, Li, Lv and Li2022), who reported that treatment with Clonorchis sinensis cysteine protease significantly improved the survival rate of mice with DSS-induced colitis by reducing inflammation in the colon. Moreover, Fawzy et al. (Reference Fawzy, Selim, Mostafa, Abdelhameed, Darwish, Yousef, Alabiad, Ibrahim, Fawzy and Hamed2024) noted that T. spiralis antigens loaded on nanomaterials improved experimental colitis, enhancing the survival rate in groups that received treatment. In the current study, the intraperitoneal administration of E. granulosus HLL and HCF along with SLZ resulted in a significant decrease in the clinical score for colitis including decreased colon W/L and CW/BW ratios, improved body weight, decreased DAI, and less diarrhea. This might be explained by the fact that the colitis group’s colon weight dramatically rose because of tissue edema and inflammatory cellular infiltrate, while their colon length was reduced as a result of longitudinal muscle spasm. This led to a significantly larger colon W/L ratio in the colitis group. Intestinal inflammation may also contribute to the weight loss of animals in the colitis group by causing malabsorption of several nutrients, which raises the CW/BW ratio in the colitis group. Fawzy et al. (Reference Fawzy, Selim, Mostafa, Abdelhameed, Darwish, Yousef, Alabiad, Ibrahim, Fawzy and Hamed2024) demonstrated a reduction in the clinical score (DAI) when T. spiralis larval Ags/Ca-BTC MOFs were used as a preventative and therapeutic measure as compared to the colitis group. This was in agreement with Soufli et al. (Reference Soufli, Toumi, Rafa, Amri, Labsi, Khelifi, Nicoletti and Touil-Boukoffa2015), who reported that the clinical score (DAI) was considerably lower after pretreatment with a hydatid laminated layer (HLL/DSS) than for the DSS group. Moreover, there were no deaths or moribund symptoms in the LL/DSS group. Furthermore, Xu et al. (Reference Xu, Liu, Yu, Wu and Lu2019) found that experimental groups treated with recombinant proteins or Trichinella-derived antigens, whether as a preventative measure or a therapeutic intervention, significantly improved their DAI scores when compared to control-positive groups. According to Abdelmegid et al. (Reference Abdelmegid, Abdo, Ahmed and Kattaia2019), weight loss may also be related to the catabolic cytokine tumor necrosis factor (TNF) and modifications in metabolic hormone levels that affect appetite and cause anorexia and subsequent rectal bleeding. Furthermore, Ige et al. (Reference Ige, Adeniyi, Olayinka and Kehinde2020) noted that when investigating the therapeutic effects of dietary maize formula for experimental colitis, intrarectal instillation of AA produces an increase in colon W/L and CW/BW ratios. They explained their findings by immunomodulating regulatory T cells, which reduce TNBS-induced colitis in mice by producing regulatory and anti-inflammatory cytokines and inhibiting pro-inflammatory cytokines. Oxidative stress plays a major pathogenic role in IBD (Muro et al. Reference Muro, Zhang, Li, Zhao, Jin, Mao and Mao2024). The group that received SLZ/HLL had the highest levels of antioxidant enzymes (TAC) and decreased oxidative stress markers (MDA) in serum samples followed by the group that received SLZ/HCF when compared to colitis models. This is primarily because of the inhibition of the recruitment of inflammatory cells, particularly neutrophils, into colon tissue. This could be explained by the connection between oxidative stress and inflammatory responses, which have been connected to the onset and aggravation of IBD. In colitis models, mucosal tissue is invaded by activated phagocytic immune cells, which produce reactive oxygen species (ROS) and nitrogen species. It causes cellular homeostasis to be disturbed, increases mucosal barrier permeability, damages cells, and speeds up the inflammatory process (Piechota-Polanczyk and Fichna Reference Piechota-Polanczyk and Fichna2014). Our findings are in agreement with the study, which found that colitis induction in mice led to an increase in MDA levels as well as decreases in GSH-Px and superoxide dismutase (SOD) activity (Arda-Pirincci and Aykol-Celik Reference Arda-Pirincci and Aykol-Celik2020; Shaaban et al. Reference Shaaban, El-Menshawy, Salem and Othman2021). The severity of TNBS-induced colitis in mice was reported to be decreased by T. spiralis secretory products-induced macrophage 2, according to Antoniou et al. (Reference Antoniou, Margonis, Angelou, Pikouli, Argiri, Karavokyros, Papalois and Pikoulis2016). Additionally, Joshi et al. (Reference Joshi, Vyas, Shah, Shah, Shah and Gandhi2011) reported that IBD patients have increased levels of reactive oxygen species (ROS), including hydrogen peroxide, superoxide anion, hypochlorous acid, and hydroxyl radical, as well as nitrogen species. Conversely, Kuralay et al. (Reference Kuralay, Yildiz, Ozutemiz, Islekel, Caliskan, Bingol and Ozkal2003) demonstrated a significant increase in SOD levels in injected acetic acid-fed female Swiss rats. Gender differences may be the cause of the disparity between these results and the study’s conclusions. In this regard, studies in both preclinical and clinical settings have demonstrated that the pathogenesis of colitis involves an excess of oxidative stress markers (Balmus et al. Reference Balmus, Ciobica, Trifan and Stanciu2016). Concerning the novelty of our work, SLZ, in conjunction with crude extracts of E. granulosus LL and HCF, at least partially counteracted the opposing effects of colitis through the reduction of inflammatory cytokines and the attenuation of oxidative stress. To pinpoint other underlying mechanisms influencing these antigens’ impact on colitis, further investigation is yet required. The primary mechanism by which helminth-secreted proteins influence host immunomodulation is hypothesized to be Th2/Treg cell upregulation, which results in the production of anti-inflammatory cytokines like IL-4 and IL-10 and downregulates Th1/Th17 cells and other pro-inflammatory cytokines like TNF-α and IFNγ implicated in tissue inflammation. Treg cell function usually inhibits inflammatory illnesses like IBD (Xu et al. Reference Xu, Liu, Yu, Wu and Lu2019). According to our findings, colitis models had higher serum levels of Th1 pro-inflammatory cytokines (TNF-α) and low levels of anti-inflammatory cytokines (IL-10). Mice who received SLZ/HLL and SLZ/HCF showed TNF-α greatly decreased, while Th2 cytokine (IL10) was significantly elevated. This immunomodulation is hypothesized to be Th2/Treg cell upregulation and downregulating Th1/Th17 cells, leading to improvements in clinical and histological characteristics when comparing treated mice to nontreated mice. According to Soufli et al. (Reference Soufli, Toumi, Rafa, Amri, Labsi, Khelifi, Nicoletti and Touil-Boukoffa2015), proinflammatory cytokines like TNF-α, IFN-γ, and IL-6, as well as elevated NO generation, are characteristics of colitis brought on by DSS. These variables cause the intestinal mucosa to erode and infiltrate and the epithelium to be destroyed (Aboumosalem et al. Reference Aboumosalem, Mokhbatly, Goda, Ghazy, Abou Elazab, Abdelatty, Elbialy and Assar2025). Studies show that IL-10 inhibits the synthesis of proinflammatory cytokines such as TNF-α, IL-12, and IFN-γ, which in turn inhibits the expression of iNOS. It has also been suggested that one potential way IL-10 carries out its regulatory duties is through NF-κB inhibition. Similar findings were made by Pang et al. (Reference Pang, Ding, Zhang, Zhang, Yang, Bai, Liu, Jin, Guo and Yang2020), who discovered that recombinant serine protease from the adult stage of T. spiralis reduced the production of cytokines related to Th1 and Th17 while enhancing the expression of cytokines related to Th2 and Foxp3+T-reg cells in TNBS-induced colitis in mice. Conversely, Díaz et al. (Reference Díaz, Casaravilla, Allen, Sim and Ferreira2011) report that mice receiving hydatid LL alone exhibited increased plasma levels of TNF-α and IFN-γ but no effect on the mice’s health. This may be attributed to the complex variety of chemicals in the hydatid LL crude extract, which may provoke immunological reactions in mice. Thus, locating and isolating the bioactive molecule is likely to result in longer-acting and more potent anti-inflammatory actions. Consequently, our next study will be to identify the bioactive chemicals within the LL that were extracted from the E. granulosus cyst wall. The hydatid LL plays a crucial role in the host-parasite relationship. It provides a strong defense against the immune system of the host. The parasite induces Th2 and regulatory T cell (Treg) responses to survive and then integrates these responses into the immune evasion strategy. This action could be mediated by LL antigens. Therefore, the reduction in NO and proinflammatory cytokine levels in the plasma of pretreated animals is likely connected to the immunomodulatory effect of the cystic extract. The impact of cystic extract therapy appears to have been mediated by IL-10’s immunoregulatory role, as seen by the considerable elevation of IL-10 during the treatment. In the current study, mice treated with E. granulosus Ags or in combination with SLZ had a significantly higher proportion of Foxp3+ Treg cells in their colonic tissues. Consequently, there were improvements in the clinical status and the absence of an inflammatory reaction on the immunohistochemically stained slides. Consistent with our findings, Fawzy et al. (Reference Fawzy, Selim, Mostafa, Abdelhameed, Darwish, Yousef, Alabiad, Ibrahim, Fawzy and Hamed2024) suggested that T. spiralis E/S Ags could decrease the severity of inflammatory colitis in mice by immunomodulating Treg cells to produce regulatory and anti-inflammatory cytokines while obstructing pro-inflammatory cytokines. According to Negi et al. (Reference Negi, Saini, Tandel, Sahu, Mishra and Tyagi2021), patients with IBD had lower percentages of Foxp3+ T-reg cells or poorer activity, which prevented them from controlling excessive inflammation. Zheng et al. (Reference Zheng, Xu and Wang2019), however, stated that IFN-γ has a paradoxical involvement in the pathophysiology of IBD since it also protects against T cell-mediated colitis by controlling the Treg/Th17 response. The epithelium in colitis is more susceptible to bacterial endotoxins like lipopolysaccharide (LPS). TLR4 on the gut wall can identify these LPS. The interaction between LPS and TLR4 initiates a signaling pathway that eventually causes NF-κB to become active. Proinflammatory cytokines, including TNF-α, are produced as a result of this process (Miao et al. Reference Miao, Shan, SaH, Akhtar, Geng, Ning and Wang2021). Mice in the colitis group showed a grossly apparent shortening of the colon with numerous ulcerative, hemorrhagic, and edematous changes. Microscopically, the mucosal lining showed degenerative and necrotic changes, goblet cell depletion, edema, and a marked inflammatory infiltration of cells throughout the mucosa and submucosa, with the muscularis propria occasionally involved. Furthermore, extravasated blood between the crypts, crypt abscess, and cryptitis were detected. The mice given a combination of SLZ and HCF showed the greatest improvements in the microscopic score, followed by the groups given a combination of SLZ and crude extract of E. granulosus LL, which demonstrated regeneration of superficial epithelium and reactive goblet cell metaplasia. These results were consistent with Soufli et al. (Reference Soufli, Toumi, Rafa, Amri, Labsi, Khelifi, Nicoletti and Touil-Boukoffa2015), who demonstrated that pretreatment of mice with E. granulosus LL reduced colon shortening and prevented the loss of crypt glands and destruction of epithelium that was shown in the DSS-alone group as a sign of mucosal injury. Furthermore, compared to the DSS-treated animals, the LL/DSS group had a regular mucosal structure with goblet cells secreting mucus, according to a histological investigation employing Alcian blue staining. This finding implies that LL pretreatment could protect mice from colitis brought on by DSS by increasing mucus secretion. Moreover, Sarazin et al. (Reference Sarazin, Dendooven, Delbeke, Gatault, Pagny, Standaert, Rousseaux, Desreumaux, Dubuquoy and Capron2018) discovered notable improvements in the colonic tissues of mice treated with T. spiralis adult E/S products after they had been experimentally induced with colitis in terms of both macroscopic and microscopic inflammatory scores. The same outcomes were obtained by Fawzy et al. (Reference Fawzy, Selim, Mostafa, Abdelhameed, Darwish, Yousef, Alabiad, Ibrahim, Fawzy and Hamed2024) and Xu et al. (Reference Xu, Liu, Yu, Wu and Lu2019) using antigens produced from Trichinella. Analysis of our data showed that the combination of SLZ with E. granulosus-derived LL crude extract and HCF appears to be a promising therapy for the treatment of IBD.
Conclusion
The findings of our study indicate that molecules derived from E. granulosus (HCF and LL crude extract) exhibit immunomodulatory, antioxidant, and anti-inflammatory properties in the context of acetic acid-induced colitis. The therapeutic efficacy of these molecules was enhanced when administered in combination with the standard treatment (sulfasalazine). However, additional investigations are essential to elucidate the precise mechanisms of action and to evaluate their safety profile.
Financial support
There was no financial support was obtained.
Competing interests
None.
