Introduction
Ulcerative colitis (UC) is one of the most common autoimmune diseases affecting the intestine with a chronic relapsing nature (McDowell et al., Reference McDowell, Farooq and Haseeb2023) and increasing risk of colorectal cancer (Stidham and Higgins, Reference Stidham and Higgins2018). It is diagnosed during the most active years of adulthood and may last throughout life, leading to substantial morbidity (Beard et al., Reference Beard, Franco and Click2020). Risk factors may include a combination of hereditary, genetic, environmental, and immunologic abnormalities (Lee and Chang, Reference Lee and Chang2021). The incidence of inflammatory bowel disease (IBD) has been increasing worldwide in the past 10 years, including Egypt (Mostafa et al., Reference Mostafa, Metwally and Hussein2018). The highest rates were reported in developed countries. It may be attributed to a high prevalence of helminthic infections in developing areas and their immunomodulation against autoimmune diseases (Yeshi et al., Reference Yeshi, Ruscher, Hunter, Daly, Loukas and Wangchuk2020).
Therefore, current researchers employ parasites or their derived products to treat various autoimmune diseases, including IBD, such as Necator americanus (Buitrago et al., Reference Buitrago, Pickering, Ruscher, Caceres, Jones, Cooper and Loukas2021), Trichuris suis ova or larvae (Corfixen, Reference Corfixen2017), and schistosome-derived antigens (Cleenewerk et al., Reference Cleenewerk, Garssen and Hogenkamp2020). Many studies have shown that therapy with T. spiralis infection or antigens generated from it has a positive impact on experimentally produced colitis (Li et al., Reference Li, Li, Chen, Zhang and Lu2020). The study of these molecules is critical for developing new therapies (Cleenewerk et al., Reference Cleenewerk, Garssen and Hogenkamp2020).
The anti-inflammatory effect of helminths is primarily attributed to cytokine-mediated immunoregulation because they shift the host immune response from Th1/Th17-promoted inflammation, which is mostly linked with autoimmune diseases, to Th2/T regulatory (Treg) cells by stimulating the secretion of anti-inflammatory cytokines interleukin (IL)-4, IL-5, IL-10, and IL-13 (Kingston Reference Kingston2022). According to Langer et al. (Reference Langer, Vivi, Regensburger, Winkler, Waldner, Rath and Stürzl2019), interferon (IFN)-γ is a primary cytokine that promotes IBD pathogenesis by disrupting the vascular barrier, resulting in increased permeability of intestinal arteries, increased inflammation, and disease development.
Despite substantial advances in IBD therapy, the vast majority of patients do not benefit from medication, leaving surgery as the only therapeutic option. As a result, finding innovative therapeutic alternatives with few side effects has become a critical priority (Na and Moon, Reference Na and Moon2019).
T. spiralis-derived antigens were used to develop novel nanomedical approaches for more effective and promising options in the treatment of autoimmune diseases by enhancing immunomodulatory processes (Ilic et al., Reference Ilic, Kosanović, Gruden-Movsesijan, Glamočlija, Sofronić-Milosavljević, Čolić and Tomić2021). The use of nanoparticles (NPs) to transport immunomodulatory molecules to Dendritic cells (DCs) leads to the production of tolerogenic DCs, the modulation of broad immunological activities via the down-regulation of Th1 and Th17 responses, or the increase in the number of Treg cells, boosting Foxp3+ Treg development (Kwiatkowski et al., Reference Kwiatkowski, Stewart, Cho, Avram and Keselowsky2020). Metal-organic frameworks (MOFs) are among the numerous developed nanocarriers of biological interest (Hidalgo et al., Reference Hidalgo, Simón-Vázquez, González-Fernández and Horcajada2022). They have been widely used to treat a wide range of ailments, including allergies, autoimmune diseases, and various infections (Quijia et al., Reference Quijia, Alves, Hanck-Silva, GalvãoFrem, Arroyos and Chorilli2022). Calcium MOFs are a unique subclass of MOFs with high stability, low density, and low toxicity. When compared to MOFs made of other metal groups, they have much potential for biological applications because of their high biocompatibility (Xian et al., Reference Xian, Lin, Wang and Li2021). Thus, we present an in-depth study of the prophylactic and therapeutic effects of T. spiralis larvae E/S Ags/Ca-BTC MOFs on acetic acid (AA)-induced colitis in mice.
One of the key contributing elements implicated in the etiology and worsening of IBD is oxidative stress, which may be due to inflammation (Hamouda et al., Reference Hamouda, Zakaria, Ismail, Khedr and Mayah2011). Malondialdehyde (MDA) is the end product of lipoperoxidation and is employed as a lipid peroxidation marker. It was computed to reflect inflammation-induced oxidative damage to membranes (Bou-Fersen et al. Reference Bou-Fersen, Anim and Khan2008). MDA proved to be an excellent marker of Crohn’ disease (CD), with 91% overall accuracy in distinguishing CD patients from controls (Boehm et al., Reference Boehm, Krzystek-Korpacka, Neubauer, Matusiewicz, Paradowski and Gamian2012). Foxp3 is a key transcription factor that governs the formation and differentiation of Treg cells (Li et al., Reference Li, Li, Tsun and Li2015). Foxp3-expressing Treg cells can inhibit and modify the excessive immune response.
The AA-induced colitis model used in this study is an easily generated model that closely reflects clinical IBD in terms of pathophysiology, histology, inflammatory mediators, oxidative stress indicators, and proinflammatory cytokines (Chamanara et al., Reference Chamanara, Abdollahi, Rezayat, Ghazi-Khansari, Dehpour, Nassireslami and Rashidian2019).
Materials and Methods
Animals and ethical statement
One hundred laboratory-bred, parasite-free male Swiss albino mice weighing 20 to 25 g were procured from Theodor Bilharz Research Institute’s Animal House. They were cared for in accordance with the study protocols and the recommendations of the Zagazig University Institutional Animal Care and Use Committee.
Induction of colitis
Except for the normal control animals, which received a 0.9% phosphate-buffered saline (PBS) solution, colitis was produced in all mouse groups. The mice were gently sedated with thiopental sodium before being placed in a Trendelenburg position during the induction phase. Through the catheter, AA (5%, pH 2.5, 0.15 mL) (Sigma-Aldrich, St. Louis, MO, USA) was fed into the colon lumen. To prevent early leakage of the intracolonic instillation, mice were held in a head-down position for 15 to 20 seconds (Randhawa et al., Reference Randhawa, Singh, Singh and Jaggi2014).
Preparation of T. spiralis larvae E/S Ags
Living muscle larvae were cultured in serum-free RPMI 1640 (Biowest, DNo: MS009E100p) supplemented with antibiotics (penicillin 100 U/mL and streptomycin 100 g/mL) for 24 hours under controlled conditions of 37°C, 95% air, and 5% CO2. The E/S products were filtered through a 0.2-mμ membrane into a 50-mL conical tube, then centrifuged at 4°C and 10,000 rpm for 30 minutes. The supernatant containing larvae (E/S Ags) was dialysed against deionized water at 4°C for 2 days (Wang et al., Reference Wang, Cui, Hu, Liu and Wang2014), and protein concentration was estimated in accordance with Bradford (Reference Bradford1976).
Synthesis of nanoporous support (Vakiti et al., Reference Vakiti, Garabato, Schieber, Rucks, Cao, Webb and Yan2012)
All reagents and chemicals used in this study for production of Ca-BTC MOFs, including 1,3,5-benzene dicarboxylate (BTC) (C9H6O6; 95%), ethanol, and Ca-acetate, were obtained from Merck (Darmstadt, Germany) and used without additional purification. A mixture of BTC (0.042 g), Ca-acetate (0.0316 g), distilled H2O (3.0 mL), and ethanol (1.0 mL) was stirred for 20 minutes at 25oC to produce a homogeneous solution. The reaction mixture was kept in the oven for 24 hours at 90°C. Next, the Ca-BTC was collected and filtered out using Whatmann filter paper before being rinsed with 99.9% ethanol. The produced Ca-BTC MOFs (42.32 mg) were air-dried for 1 hour at 25°C.
Sample characterisation (El-Shahat and Abdel-Hameed, Reference El-Shahat and Abdelhameed2022)
All of the reagents were purchased commercially and used precisely as instructed. Powder X-ray diffraction patterns for Cu K radiation (= 1.5406) were recorded on a Phillips Panalytical diffractometer with a scan speed of 2° min-1 and a step size of 0.02° in 2. The surface area of a Brunauer-Emmett-Teller (BET) was measured using an Autosorbi Q-C automated gas adsorption device (Quantachrome Instruments, USA). Fourier transformed infrared (FTIR) spectra were acquired in the 600–4000 range using a Mattson FTIR instrument.
Antigens loading
T. spiralis larvae E/S Ags were loaded into Ca-BTC MOFs by dissolving the antigens in 10 mL of PBS at various concentrations (100–1000 ppm). One gram of Ca-BTC was added to the antigen solutions and stirred for 90 minutes at room temperature using a magnetic stirrer at 600 rpm. The solution was then refrigerated overnight, undisturbed. The suspension was then centrifuged at 5,000 rpm for 5 minutes to separate the supernatant and precipitate. The amount of loaded antigens was calculated by comparing the concentration of antigens in the solution before and after antigen loading (Figure 1). The following equation was used to compute the percentage of antigen loading: Antigen loading percentage = [(AB)/A] 100, where A and B represent the beginning and final antigen concentrations, respectively.
Figure 1. a: FTIR of Ca-BTC used that the BTC molecule is linked to Ca ions, and the C=O stretching frequency of free aromatic carboxylic acid is shifted to lower frequencies (Ca. 1550 cm-1).; b: thermogravimetric analysis of Ca-BTC was used to characterise its thermal stability. Three weight-loss phases of Ca-BTC MOFs were obtained as the temperature increased (25–800°C). The weight loss in step 3 (450°C-700°C) can be attributed to calcium oxide production as the temperature rises. Ca-BTC has a BET-specific surface area of 376.805 m2 g-1 and a pore size of 1.68 nm. c: The equilibrium between the concentrations of the various antigens and the loading quantity. d: The maximum antigen loading was attained at 100 minutes. e: E/S Ags/Ca-BTC TEM pictures after antigen loading, the morphology of the nanoparticle changed significantly, with the E/S Ags appearing as white dots surrounding the Ca-BTC particles. f: Kaplan-Meier curves prophylactic. g: Kaplan-Meier curves among therapeutic groups. h: CW/BW ratio (prophylactic and Therapeutic groups). i: CW/L ratio (prophylactic and Therapeutic groups). Results are representative as means ± SD (h,i). * P < 0.05, ** P < 0.01, *** P < 0.001.
Experimental Design
One hundred male albino mice were randomly allocated into two groups: prophylactic (a) and therapeutic (b), each subdivided into five subgroups of 10 mice as follows: GIa (the negative control group) received 50 μL of PBS by intraperitoneal injection (i.p.) three times at 5-day intervals before being given 100 μL of PBS intrarectally. GIIa (colitis model): mice were given acetic acid intrarectally; GIIIa: mice were given 25 μL Ca- BTC/MOFs alone; GIVa: mice were given 50 μL T. spiralis larvae E/S Ags alone; and GVa mice were given 25 μL larvae E/S Ags loaded on Ca- BTC MOFs. Groups IIIa, IVa, and Va received i.p. therapy three times at 5-day intervals before AA colitis induction. The therapeutic groups (b) were then separated into five subgroups of 10 mice each: GIb (negative control group): healthy mice were given 50 μL of PBS i.p. twice, 24 and 48 hours following an intrarectal injection of 100 μL of PBS. GIIb (colitis model): mice were given 50 μL of PBS intravenously twice, 24 and 48 hours after colitis induction. GIIIb (colitis + Ca BTC): Mice were given 25 μL of Ca BTC intravenously twice, 24 and 48 hours after colitis induction. GIVb (colitis + T. spiralis larvae E/S Ags): Mice received 50 μL of E/S Ags i.p. twice following colitis induction, at 24 and 48 hours. GVb (T. spiralis larvae E/S Ags-Ca BTC MOFs): mice were given 25 μL of E/S Ags loaded on Ca BTC i.p. twice. The loading quantity of E/S Ags loaded on Ca-BTC was 271.2 mg/g and the dose concentration was 20 mg/kg mouse.
Clinical disease score
Mouse survival rates: Following colitis induction, the deaths of mice in each group were recorded daily, and survival rates were determined (Lu et al., Reference Lu, Dong, Chen, He, Peng, Wu and Li2019). Disease activity index evaluation (DAI) score: The mice were observed daily in terms of the changes in their weights, mental status, stool pattern, melena, and frequency of defecation. DAI is a score that ranges from 0 to 4 (Jeengar et al., Reference Jeengar, Thummuri, Magnusso, Naidu and Uppugunduri2017). The fecal occult blood of mice was evaluated by the FOB kit (Biodiagnostic CAT No. OB24 12). Mice serum samples were tested for kidney function (creatinine and urea) and liver function (total protein, alanine aminotransferase [ALT], and aspartate aminotransferase [AST]). We used commercial kits that we bought from Spin React Co., Spain, to measure the serum AST and ALT activity. According to Young (Reference Young2001), the test was conducted at a wavelength of 340 nm and was reported in IU/L. The commercial kits obtained from Spectrum Co., Egypt, were used. The total plasma protein content was determined colorimetrically in accordance with Tietz (Reference Tietz2015) and expressed in g/dL. A wavelength of 546 nm was used to test the absorbance. Determination of serum urea and creatinine concentrations: serum urea and creatinine concentrations (mg/dL) were estimated according to the modified Berthelot reaction (Chaney and Marbach, Reference Chaney and Marbach1962) and the Jaffe alkaline picrate method (Mazzachi et al., Reference Mazzachi, Peake and Ehrhardt2000), respectively. The test type was a colorimetric assay. The absorbance was measured at 578 and 492 nm, respectively. Liver and kidney functions were performed: serum transaminases (AST and ALT), total protein concentrations, and serum urea and creatinine levels.
Score of macroscopic inflammation
Following scarification, the colons were aseptically removed and cleaned with PBS. The colonic damage was measured macroscopically using four parameters: wall thickness, degree of mucosal edema, colonic ulcerations, and the presence of adhesions. Each parameter was assigned a score ranging from 0 (normal) to 3 (extreme). Menachem et al. (Reference Menachem, Trop, Kolker, Shibolet, Alper, Nagler and Ilan2005) defined the overall score as ranging from 0 to 12. The length of the colon was measured to determine the amount of inflammation (Moreels et al., Reference Moreels, Nieuwendijk, De Man, Winter, Herman, Van Marck and Pelckmans2004).
In terms of weight changes in mice, the day of AA induction was deemed experimental day 0. The body weights of the mice in each group were recorded using a digital weight scale at various time points from the beginning to the completion of the experiment on the first, third, fifth, and seventh days (Labib et al., Reference Labib, Shaker and Elfarouk2016).
Score of microscopic inflammation
For histopathological examination, colonic specimens were fixed in 4% paraformaldehyde. The sections were stained with hematoxylin and eosin. The slides were examined using the Fabia et al. (Reference Fabia, Willen, Ar’Rajab, Andersson, Ahren and Bengmark1992) histological scoring system.
Profiling of inflammatory cytokines and markers of oxidative stress
Following the manufacturer’s instructions, serum samples were used to evaluate the levels of IFN-γ and IL-4 using ELISA kits (Sun Red Biological Technology Co., China, Shanghai). The oxidative stress indicators (MDA and GSH-Px) were measured using a colorimetric method (Biodiagnostic Co., USA, CAT).
Analysis of Foxp3+ in colon and spleen tissue
The formalin-fixed paraffin-embedded sections of the colon and spleen were performed by using an anti-Foxp3+ rabbit polyclonal antibody kit (Servicebio Technology Co., China, Hu Bei, Wuhan). The antibodies that targeted distinct Foxp3+ epitopes produced dramatically varied staining patterns. The percentage of positive lymphocytes was calculated using 10 high-power fields, and the mean percentage was reported for each case. Following that, the instances were scored as follows:
-
0. Foxp3 has no immunoreactivity.
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1. Only around 25% of lymphocytes were stained with hot spots of inflammation.
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2. Between 25% and 50% of lymphocytes were stained.
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3. More than 50% of the lymphocytes were stained.
During scoring, the staining in the suppuration and necrotic areas was ignored. The mean score of each group was shown as the mean value (x) ± standard deviation (SD) (Ashour et al., Reference Ashour, Othman, Shareef, Gaballah and Mayah2014).
Statistical analysis
SPSS version 27.0 (IBM, 2020) was used to analyse all the data. The independent sample t-test, paired sample t-test, and ANOVA F-test were used to evaluate quantitative data, whereas the Chi-square test and Fisher’s exact test were used to analyse qualitative data. The log-rank test was performed to compare the median survival of the various groups. To make multiple comparisons between groups, the Tukey post hoc test was used. Statistical significance was defined as a P value of < 0.05. Statistical diagrams were made by Graph Pad Prism 8. 0 software.
Results
Optimizing antigen loading
The FTIR of Ca-BTC employed in this work confirms that the BTC molecule is linked to Ca ions (Figure 1a). The C=O stretching frequency of free aromatic carboxylic acid is moved to lower frequencies (Ca. 1550 cm-1). Figure (1b): The thermogravimetric analysis of Ca-BTC was used to characterise its thermal stability. The results showed three weight loss phases of Ca-BTC MOFs as the temperature increased (25–800°C). The initial weight loss was recorded at 0 to 100°C, and it was around 22%. This loss is due to the elimination of the adsorbed water. The second weight loss is 40% between 100 and 450°C, owing mostly to the degradation of the organic linker of Ca-BTC. The weight loss in step 3 (450°C–700°C) can be attributed to calcium oxide production as the temperature rises. Ca-BTC has a BET-specific surface area of 376.805 m2/g-1 and a pore size of 1.68 nm. Variations in antigen loading with changes in antigen concentrations are illustrated in Figure 1c. The loading quantity was Qm, which equaled 271.2 mg/g. Figure (1d): Variations in antigen loading as a function of stirring duration are seen. The antigen loading increased with increasing time while using 100 ppm antigen concentration and swirling at different periods. At 100 minutes, the maximum antigen loading was attained. Figure (1e): One of the most essential aspects of antigen loading was evaluating the Transmission Electron Microscopy (TEM) of the particles following antigen loading. After antigen loading, the morphology of the nanoparticle changed significantly, with the T. spiralis larvae E/S Ags appearing as white dots surrounding the Ca-BTC particles.
Weight changes and DAI scores at different times following colitis (Table 1)
Among the prophylactic groups, at different time intervals, the body weight showed no statistically significant difference in GIa, GIIa, and GIIIa, whereas a statistically significant difference was obtained in GIVa and GVa. Nevertheless, the DAI score exhibited no statistically significant difference in GIIa, a statistically significant difference in GIIIa, and a highly statistically significant difference in GIVa and GVa. Among the therapeutic groups, at different time intervals, the body weight showed no statistically significant difference in all groups except GIIb. Nevertheless, the DAI score exhibited no statistically significant difference in GIIb and GIIIb, a statistically significant difference in GIVb, and a highly statistically significant difference in GVb.
Table 1. Weight changes and DAI score at different times after colitis induction
F: one way ANOVA test, P$: repeated measure ANOVA test, NS: nonsignificant (P>0.05), *significant (P < .05), **: highly significant (P < 0.001), Tukey post hoc test a: significant versus GI, b: Significant versus GII, c, significant versus GIII, d: Significant versus GIV.
Effect of T. spiralis larvae E/ S Ags/ Ca -BTC MOFs on reducing the severity of AA- induced acute colitis
The group treated with T. spiralis larvae E/S Ags/Ca-BTC MOFs had the highest survival rates of 90.0% and 80.0% among the prophylactic and therapeutic groups, with a statistically significant difference (P < 0.05) of (Figure 1f) and (Figure 1g). Within the same groups, there were substantial drops in the colon weight/length (W/L) ratios, compared with other groups (Figure 1h).
Effect of T. spiralis larvae E/ S Ags/ Ca -BTC MOFs on macroscopic and histopathological changes of the colon
Colons of negative control groups demonstrated a normal colonic wall with no macroscopic variations (Figure 2a). Colitis models, on the other hand, had classic hemorrhagic areas, congestion, and edema (Figure 2b). In the prophylactic group, mice that were given Ca-BTC MOFs alone showed an uninterrupted large hemorrhagic area, congestion, and edema in the colonic wall (Figure 2c). Mice administered larvae E/S Ags of T. spiralis showed intestinal wall edema without congestion (Figure 2d). The colonic walls of mice that were administered T. spiralis larvae E/S Ags/Ca-BTC MOFs were normal without obvious changes (Figure 2e). Mice in the treatment group had edema, congestion in the intestinal wall, and a large hemorrhagic region when they received continuous Ca-BTC MOFs (Figure 2f). Mice that received larvae E/S Ags showed a lesser hemorrhagic area and lower congestion with edema (Figure 2g). The colons of mice that were given T. spiralis larvae E/S Ags/Ca-BTC MOFs showed less congestion and fewer edematous alterations (Figure 2h). Compared to the treatment group, the prophylactic group’s mean macroscopic colon scores were better (Figure 2i).
Figure 2. Representative photomicrographs of mouse colons at 7 days post-colitis. a: The control negative group has an apparently normal colonic wall (green arrows). b: Colitis model demonstrating characteristic large hemorrhagic area with perforation (blue arrows) of the colonic wall, edema, and congestion (yellow arrows); Prophylactic groups: c: the colon of mice that received Ca-BTC showed edema (yellow arrow) and congestion (black arrow); d: Mice that received T. spiralis larvae E/S Ags showed residual edema in the colonic wall without congestion (green and red arrows). e: Mice that received T. spiralis larvae E/S Ags Ca-BTC had a normal colonic wall (green arrow); Therapeutic groups: f: Mice that received Ca-BTC had a continuous large hemorrhagic area, edema, and congestion in the colonic wall (red arrows). g: Mice that received T. spiralis larvae E/S Ags showed a minor hemorrhagic area and lesser congestion with edema (green arrows). h: Mice that received T. spiralis larvae E/S Ags/Ca BTC (X200) showed fewer edematous changes (green arrow) with decreased congestion (black arrow). i: Macroscopic score of prophylactic and therapeutic. Results are representative as means ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001.
Seven days after the onset of colitis, histopathological assessment of the colon sections in colitis models revealed deep mucosal neutrophilic cellular infiltration, which was accompanied by a localized crypt abscess with severe mucosal, submucosal, and transmural inflammation and altered tissue architecture (Figure 3c). Mice administered Ca-BTC (Figure 3d) in the prophylactic groups showed widespread infiltration of mixed inflammatory cells in deep tissues in addition to localized erosion. T. spiralis larvae E/S Ags-treated mice showed little neutrophils and mild transmural inflammation (Figure 3e). The mice in Figure (3f), which were given T. spiralis larvae E/S Ags/Ca-BTC MOFs, had a mostly normal gut and mucosa of normal thickness with late regeneration alterations. Moreover, mice receiving Ca-BTC showed deep mucosal neutrophilic cellular infiltration of the intestinal wall with a focal crypt abscess (Figure 3g). Within the treatment groups, colons of mice treated with T. spiralis larvae E/S Ags showed considerable mucosal and submucosal aggregation of mixed inflammatory cells, together with marked surface erosion with a portion of normal mucosa and mild submucosal edema (Figure 3h). The colons of mice that received T. spiralis larvae E/S Ags/Ca-BTC MOFs (Figure 3i) showed minor interstitial edema, vascular congestion, and mucosal infiltration of mononuclear inflammatory cells.
Figure 3. Histopathological examination of the colon sections of prophylactic groups 7 days postcolitis. a: microscopic scores of the prophylactic and therapeutic groups. Results are representative as means ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001. b: Negative control group. c: Colitis models: severe mucosal, submucosal, and transmural inflammation and damaged tissue architecture. d: mice received Ca-BTC: focal erosion (black arrow), dense infiltration (yellow arrow). e: mice treated with T. spiralis larvae E/S Ags: mild transmural inflammation (black arrow). f: mice received T. spiralis larvae E/S Ags-Ca-BTC/MOFs had relatively normal intestine and late regenerative changes. Colon sections of therapeutic groups at 7 days postcolitis. g: mice received Ca-BTC: deep mucosal neutrophilic cellular infiltration of the intestinal wall (yellow arrow); focal crypt abscess (black arrow); h: mice treated with T. spiralis larvae E/S Ags: marked mucosal and submucosal aggregation of mixed inflammatory cells (blue arrow) with marked superficial erosion (black arrow); moderate submucosal edema (red arrow); i: T. spiralis larvae E/S Ags-Ca-BTC/MOFs: mild mucosal infiltration of mononuclear inflammatory cells with mild interstitial edema and vascular congestion.
Effect of T. spiralis larvae E/ S Ags/ Ca -BTC MOFs on the inflammatory cytokines
The prophylactic groups that received T. spiralis larval E/S Ags/Ca-BTC MOFs had lower levels of IFN-γ than the therapeutic groups that received the same antigen (Figure 4a). The highest up-regulation of IL-4 levels was seen in mice treated with T. spiralis larvae E/S Ags/Ca-BTC MOFs in prophylactic groups, and subsequently therapeutic groups (Figure 4b). There were highly statistically significant variations in IFN-γ and IL-4 levels between the prophylactic and therapeutic groups.
Figure 4. Inflammatory cytokines in the prophylactic and therapeutic groups: a: IFN-γ; b: IL-4. Oxidative stress parameters: c: MDA1; d: GPX. Results are representative as means ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001.
Effect of T. spiralis larvae E/ S Ags/ Ca -BTC MOFs on lipid peroxidation, antioxidant activity, and liver and kidney functions
Compared to the colitis group, mice in the prophylactic groups given T. spiralis larvae E/S Ags/Ca-BTC MOFs showed down-regulated MDA levels, followed by the therapeutic groups (Figure 4c). Within the same targeted groups, the best values of GSH-Px were found in the prophylactic and therapeutic groups, as shown in Figure 4e. The control negative group displayed negative Foxp3 expression in colonic and splenic tissues (Figure 5b, 6b), and the colitis model (Figure 5b) displayed mild Foxp3 expression with few Foxp3-positive T cells. Strong Foxp3 expression was seen in mice administered T. spiralis larvae E/S Ags/Ca-BTC MOFs and Foxp3-positive T cells that were diffusely infiltrated into the colonic and splenic organs (Fig. 5i, 6i). Within the prophylactic groups, mice administered T. spiralis larvae E/S Ags demonstrated moderate immune reactivity with Foxp3-positive T-cell infiltrations in the colon and spleen tissues (Figure 5e, 6e), whereas mild immune reactivity with a few sporadic Foxp3-positive T-cell infiltrations in the colon and spleen tissues was observed in mice administered Ca-BTC MOFs (Figure 5d, 6d). In comparison to the treatment groups, the prophylactic groups in the colon and splenic tissues exhibited a significantly higher mean percentage of Foxp3-positive Treg cell expression (Figure 5a, 6a). Along with the prophylactic and therapeutic groups, the liver function tests (ALT, AST, protein) and kidney function tests (urea, creatinine) revealed high statistically significant differences between all groups. Groups receiving T. spiralis larvae E/S Ags/Ca-BTC MOFs exhibited a significant difference with all groups except the control negative group (Table 2).
Figure 5. Immunohistochemically stained photomicrographs with anti-Foxp3+ antibody of colon tissues 7 days postcolitis among prophylactic groups. a: Fox-colon expression of the prophylactic and therapeutic. Results are representative as means ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001. b: Control the negative group. c: Colitis model immune-stained with anti-Foxp3 antibody showing positive cells. Prophylactic group: d: Mice received Ca-BTC immune-stained with anti-Foxp3 antibody showing mild expression. e: Mice were treated with E/S Ags immune-stained with anti-Foxp3 antibody showing moderate immunoreactivity of Foxp3 antibody. f: Mice were treated with T. spiralis larvae E/S Ags/Ca BTC MOFs immune-stained with anti-Foxp3 antibody showing strong immunoreactivity of Foxp3 antibody. Therapeutic group: g: mice received Ca-BTC immune-stained with anti-Foxp3 antibody, showing positive cells. h: mice were treated with T. spiralis larvae E/S Ags immune-stained with anti-Foxp3 antibody, showing mild immunoreactivity of Foxp3 antibody; i: mice were treated with T. spiralis larvae E/S Ags/Ca-BTC MOFs immune-stained with anti-Foxp3 antibody, showing moderate immunoreactivity of Foxp3 antibody.
Figure 6. Results of immunohistochemically stained photomicrographs with an anti-Foxp3+ antibody of splenic tissues 7 days postcolitis among prophylactic groups. a: Fox-spleen expression of the prophylactic and therapeutic. Results are representative as mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001. b: Control normal group showing negative immunoreactivity in all lymphoid cells of both the white and red pulp. c: Colitis model: showing positive immune-stained cells with an anti-Foxp3 antibody; Prophylactic groups: d: Mice received Ca-BTC MOFs, showing mild immunoreactivity of Foxp3 antibody; e: Mice treated with T. spiralis larvae E/S Ags showed moderate immunoreactivity of Foxp3 antibody; f: Mice treated with T. spiralis larvae E/S Ags/Ca BTC MOFs revealed marked immunoreactivity to the used marker. Therapeutic group: g: Mice received Ca-BT MOFs showing positive cells; h: Mice treated with T. spiralis larvae E/S Ags showed moderately mild immunoreactivity of the Foxp3 antibody; i: Mice treated with T. spiralis larvae E/S Ags/Ca BTC MOFs showed moderate immunoreactivity to the used marker.
Table 2. Liver and kidney function tests 7 days after AA-induced colitis
F: one way ANOVA test, NS: nonsignificant (P > 0.05), *significant (P < 0.05), **highly significant (P < 0.001), Tukey post hoc test a: significant versus GI, b: significant versus GII, c, significant versus GIII, d: significant versus GIV.
Discussion
Mice given T. spiralis larvae E/S Ags/Ca-BTC MOFs had the highest survival rates among prophylactic (90%) and therapeutic groups (80%). In the prophylactic and therapeutic groups, the colitis models demonstrated the most notable decline in survival rates (40%, 30%), respectively. This can be explained by the reduction in inflammation of AA-induced colitis offered by intraperitoneal delivery of T. spiralis-produced antigens. In mice treated with Clonorchis sinensis cysteine protease, Xie et al. (Reference Xie, Wu, Wu, Liu, Chen, Shi and Tang2022) also observed a significantly higher survival rate for dextran sulfate sodium-induced colitis.
Significant reductions in mortality, DAI score, body weight loss, colon W/L, colon weight/body weight (CW/BW) ratios, and macroscopic and microscopic colon scores were found in mice given T. spiralis larvae E/S Ags/Ca-BTC MOFs when compared to the colitis model. Following colitis induction, mice given prophylactic dosages had fewer symptoms and recovered faster than mice given therapeutic doses. Our findings could be explained by the significant inflammatory infiltration and tissue edema obtained, resulting in increased colon weight in the AA-induced colitis. Colon length decreased because of longitudinal muscle spasm, resulting in a significantly higher colon W/L ratio in the colitis group compared to the control negative group. As a result of intestinal inflammation and rectal hemorrhage, a progressive loss of body weight is mostly due to the malabsorption of several nutrients. Also, Xu et al. (Reference Xu, Liu, Yu, Wu and Lu2019) reported a substantial improvement in the DAI score in mice that received recombinant T. spiralis cysteine proteinase inhibitors in trinitrobenzene sulfonic acid (TNBS)-induced experimental colitis. Our findings agreed with those of Owusu et al. (Reference Owusu, Obiri, Ainooson, Osafo, Antwi, Duduyemi and Ansah2020) and Ansari et al. (Reference Ansari, Rehman, Karim, Soliman, Ganaie, Raish and Hamad2021), who discovered that intrarectal instillation of acetic acid in rats causes an increase in colon W/L ratio while researching the therapeutic effect of Cordia vignei leaf extract in experimental colitis. In addition, Shaaban et al. (Reference Shaaban, El-Menshawy, Salem and Othman2021) found a substantial rise in the DAI score, colon W/L, and CW/BW ratios in rats with AA-induced colitis. Yang et al. (Reference Yang, Liu, Liu, Zhang, Shi, Jia and Bai2020) also reported that T. spiralis adult E/S Ags have therapeutic potential to relieve the severity of inflammatory colitis in mice. They explained their results by immunomodulating regulatory T cells that produce regulatory and anti-inflammatory cytokines and inhibit pro-inflammatory cytokines, which alleviate TNBS-induced colitis in mice.
As regards macroscopic changes of the colon, mice that received T. spiralis larvae E/S Ags/Ca-BTC MOFs showed healing of previously existing ulcerative lesions with regeneration of superficial epithelium and a reduction in the intensity of inflammatory reactions among prophylactic and therapeutic groups (Figure 2e,h). In our investigation, the histological alterations were represented by regeneration of superficial epithelium and reactive goblet cell metaplasia in the prophylactic and therapeutic groups that received T. spiralis larvae E/S Ags/Ca-BTC MOFs (Figs. 3f,i). Positive changes in the microscopic score were more pronounced in the prophylactic groups than in the therapeutic groups (Figure 2i). This is in line with the findings of Sarazin et al. (Reference Sarazin, Dendooven, Delbeke, Gatault, Pagny, Standaert and Capron2018), who found significant improvements in macroscopic and microscopic inflammation scores in the colonic tissues of mice that had been experimentally induced with colitis and then treated with T. spiralis adult E/S products. Xu et al. (Reference Xu, Liu, Yu, Wu and Lu2019) used Schistosoma-derived protein and Trichinella-derived recombinant cysteine proteinase inhibitors and obtained the same results.
For assessment of the impact of T. spiralis larval E/S Ags/Ca-BTC MOFs on AA-induced murine colitis, the inflammatory parameters (IFN-γ and IL-4) were measured. When prophylactic and therapeutic groups receiving T. spiralis larval E/S Ags/Ca-BTC MOFs were compared to those receiving T. spiralis larval E/S Ags, the level of IFN-γ was considerably lower (Figure 4a). Besides, Yang et al. (Reference Yang, Yan, Wang, Zhan, Gu, Cheng and Zhu2014) demonstrated that T. spiralis adult E/S products alleviate the induced colitis in mice by downregulating pro-inflammatory cytokines (IFN-γ). Zhu et al. (Reference Zhu, Gu and Shen2019) reported that there was an increase in the pro-inflammatory cytokines IFN-γ via increased inflammatory cell infiltration in intestinal colitis. The prophylactic groups in our research that got T. spiralis larval E/S Ags /Ca-BTC MOFs had the highest levels of IL-4 when compared to the colitis model. The therapeutic groups that received the same antigen follow them. The up regulations of IL-4 obtained in the prophylactic and therapeutic groups (Figure 4b) were in harmony with Sun et al. (Reference Sun, Guo, Hao, Zhan, Huang and Zhu2019), who found a rise in IL-4 levels in groups that received T. spiralis muscle larvae excretory-secretory products.
Rod-shaped cationic nanoparticles, which we used in our work, are easier for endosomal absorption than other cationic nanoparticle shapes (Gratton et al., Reference Gratton, Ropp, Pohlhaus, Luft, Madden, Napier and DeSimone2008), suggesting that immune system cells may be able to recognize these NPs. That might explain why those who got T. spiralis larvae E/S Ags/Ca-BTC MOFs showed more substantial alterations than other groups. Furthermore, nanoparticles can serve as passive carriers for immunostimulatory agents such as antigens. According to Luzuriaga et al. (Reference Luzuriaga, Welch, Dharmarwardana, Benjamin, Li, Shahrivarkevishahi and Gassensmith2019), NPs enhance the stability of proteinaceous medications and regulate their release and adsorption in vivo. They can also release IBD medications into the colon and small intestine. Based on the oxidative stress markers used in this work, the preventive and therapeutic groups that received T. spiralis larval E/S Ags/Ca-BTC MOFs had the greatest reduction in blood MDA levels when compared to the colitis models (Figure 4c). On the other hand, MDA levels were higher in the ileal segments of colitis animals than in control rats, according to Bou-Fersen et al. (Reference Bou-Fersen, Anim and Khan2008). This may be explained by the fact that MDA as one of the breakdown products of lipid peroxidation, may cross-link membrane proteins, rendering them ineffective as receptors or enzymes (Alzoghaibi, Reference Alzoghaibi2013).
When compared to colitis models, the group that received prophylactic doses of T. spiralis larvae E/S Ags/Ca-BTC MOFs had the most significant blood GSH-Px levels, followed by the therapeutic group (Figure 4d). This result aligns with the research conducted by Shaaban et al. (Reference Shaaban, El-Menshawy, Salem and Othman2021), which demonstrated that the production of colitis in mice led to an oxidative stress response characterised by elevated MDA, decreased GSH-Px, and decreased superoxide dismutase activity. Moreover, Derda et al. (Reference Derda, Wandurska-Nowak and Hadaś2004) noted that the host tissues produce biochemical conditions in trichinellosis that result in the disintegration of antioxidant enzymatic systems and the manifestation of disturbance of metabolite homeostasis in the form of hyperactivation of lipid peroxidation. According to Tee et al. (Reference Tee, Ong, Bay, Ho and Leong2016), the smaller sizes of inorganic NPs may make it more feasible for them to function as antioxidants because of their high surface-to-volume ratio. This is why calcium-BTC showed a synergistic antioxidant effect against antigens in our investigation. Li et al. (Reference Li, Li, Chen, Zhang and Lu2020) stated that nanomedicines have the potential to greatly enhance the pharmacokinetic properties of antioxidant compounds while reducing their adverse effects. According to Patra et al. (Reference Patra, Mukherjee, Banik, Dutta, Begum and Basu2017), quercetin loaded on CaP in the form of nanoparticles may have antioxidant qualities. The pretreatment with these formulations may shield cells from H2O2 toxicity.
In the current study, mice treated with T. spiralis larvae E/S Ags alone or loaded on Ca-BTC MOFs had a significantly higher percentage of Foxp3+ Treg cells in their colonic and splenic tissues in both the prophylactic and therapeutic groups. This led to clinical improvement and the resolution of an inflammatory reaction on the immunohistochemically stained slides (Figure 5f,i and Figure 6f,i). In accordance with our findings, Yang et al. (Reference Yang, Yan, Wang, Zhan, Gu, Cheng and Zhu2014) stated that T. spiralis adult (E/S Ags) had the capacity to alleviate the severity of inflammatory colitis in mice by immunomodulating Treg cells to release regulatory and anti-inflammatory cytokines while inhibiting pro-inflammatory cytokines. Pang et al. (Reference Pang, Ding, Zhang, Zhang, Yang, Bai, Liu, Jin, Guo, Yang and Liu2020) also observed that T. spiralis-derived recombinant serine protease alleviated TNBS-induced colitis in mice by increasing the number of Foxp3+T-reg cells. In IBD patients, the proportion of Foxp3+ T-reg cells was lower, or their activity was compromised, with an inability to limit excessive inflammation (Negi et al., Reference Negi, Saini, Tandel, Sahu, Mishra and Tyagi2021). Furthermore, Cvetkovic et al. (Reference Cvetkovic, Ilic, Gruden-Movsesijan, Tomic, Mitic, Pinelli and Sofronic-Milosavljevic2020) proved that Trichinella antigens can enhance Treg populations in the host by engaging several pattern recognition receptors (PRRs) expressed by Tol-DCs. The NPs potentiate the immunomodulatory effects of T. spiralis components. The combination of the most potent T. spiralis products and their utmost efficient (nanomedical) approaches will be a promising strategy for the treatment of autoimmune diseases (Ilic et al., Reference Ilic, Kosanović, Gruden-Movsesijan, Glamočlija, Sofronić-Milosavljević, Čolić and Tomić2021).
Conclusion
According to what we found in our research, T. spiralis larvae E/S Ags/Ca-BTC MOFs were effectively downregulated murine colitis. The levels of the proinflammatory cytokine IFN-γ and the oxidative stress marker MDA were significantly reduced in serum samples of mice given both prophylactic and therapeutic doses of our target antigen. Moreover, there was an increase in the levels of the anti-inflammatory cytokine IL-4 and the antioxidant enzyme GSH-Px. In addition, there was a substantial increase in survival rates, a drop in colon W/L and CW/BW ratios, and an improvement in clinical symptoms, including a reduced DAI score. The targeted group showed the best improvement, reaching more or less normal values for liver and kidney functions. The prophylactic groups outperformed treatment groups in terms of macroscopic and microscopic damage ratings, as well as significantly enhanced production of Foxp3 T-reg cells in colonic and splenic tissues. With regard to antigen delivery, the Ca-BTC systems used in this study show promise. Ca-BTC MOFs, an efficient delivery system for T. spiralis antigens, offers hope for the development of new UC vaccines or potentially treatments.
Financial support
No financial support was obtained.
Competing interest
None.
Ethical standard
The Zagazig University Institutional Animal Care and Use Committee authorized the animal experimental procedure(ZU-IACUC)(Approval No. ZU-IACUC/3/F/29/2020).
