Bromelain and ficin proteolytic effects on gliadin cytotoxicity and expression of genes involved in cell-tight junctions in Caco-2 cells

Enzyme therapy for celiac disease (CeD), which digests gliadin into non-immunogenic and non-toxic peptides, can be an appropriate treatment option for CeD. Here, we have investigated the effectiveness of bromelain and ficin on gliadin digestion using in vitro, such as SDS-PAGE, HPLC, and circular dichroism (CD). Furthermore, the cytotoxicity of gliadin and 19-mer peptide before and after digestion with these enzymes was evaluated using the MTT assay in the Caco-2 cell line. Finally, we examined the effect of these treatments along with Larazotide Acetate on the expression of genes involved in cell-tight junctions, such as Occludin, Claudin 3, tight junction protein-1, and Zonulin in the Caco-2 cell line. Our study demonstrated bromelain and ficin digestion effects on the commercial and wheat-extracted gliadin by SDS-PAGE, HPLC, and CD. Also, the cytotoxicity results on Caco-2 showed that toxicity of the gliadin and synthetic 19-mer peptide was decreased by adding bromelain and ficin. Furthermore, the proteolytic effects of bromelain and ficin on gliadin indicated the expression of genes involved in cell-tight junctions was improved. This study confirms that bromelain and ficin mixture could be effective in improving the symptoms of CeD.


Introduction
Celiac disease is an autoimmune enteropathy characterized by flattened villi on the small bowel mucosa.It is induced in genetically susceptible people by ingesting gluten, the protein fraction of wheat, barley, and rye (Di Sabatino and Corazza 2009).Genetics, immunology, and environmental factors are essential in the development of CeD.The disease occurs selectively in individuals expressing HLA-DQ2 or HLA-DQ8 genes (López Casado et al. 2018).The most important environmental factor is the consumption of gluten, which triggers CeD and is known as the first stage of the pathogenesis of the disease (Alhassan et al. 2019).Gluten is a complex mixture of insoluble prolamine polypeptides found the high quantities in wheat (gliadin), barley (hordein), rye (secalin), corn (zein), sorghum (kafirin), and a low quantity in oats (avenin) and rice (orzein) (Cebolla et al. 2018).Impaired digestion of gliadin by gastrointestinal proteases produces immunogenic and toxic peptides which 13-, 19-and 33-mer peptides are more immunogenic and toxic (Chibbar and Dieleman 2019).The 19-mer peptide with binding to CXCR3 leads to the release of zonulin and tight junction (TJ) dysfunction.Due to intestinal permeability, gliadin peptides, especially 33-mer, arrive at the lamina propria where they are modified by tissue transglutaminase (tTG) enhancing their affinity to MHC II molecules and stimulating Th1 and Th2 inflammatory pathways (Alhassan et al. 2019).
CeD affects around 1% of the general population and is increasing in incidence worldwide in adults and children (Agarwal et al. 2016;Alhassan et al. 2019).Currently, the only admissible treatment for CeD is a GFD for lifelong, which is challenging, insufferable, and expensive for many patients (Tye-Din et al. 2018).In addition, the GFD alone is frequently not sufficient to control symptoms and prevent mucosal damage (Alhassan et al. 2019).Several studies in people with celiac disease have shown that despite adherence to GFD over many years, the intestinal mucosa does not improve completely (Lanzini et al. 2009;Rubio-Tapia et al. 2010;Lebwohl et al. 2014;Newnham et al. 2016).Also, many patients may be unintentionally exposed to gluten due to the contamination of food and drugs, which causes the onset of symptoms and mucosal damage (Tye-Din et al. 2018;Alhassan et al. 2019).Some patients with refractory celiac disease (RCD) do not respond to GFD (Volta and Caio 2016).However, with all the limitations mentioned, following a GFD is considered the most effective way to prevent and improve the symptoms of celiac disease.Nevertheless, developing a new non-dietary therapy such as enzyme therapy alongside a GFD to control symptoms, manage the disease, and eliminate intestinal inflammations can be useful.
Gliadin is rich in proline and glutamine, so it is resistant to digestion in the intestinal (Hausch et al. 2002;Helmerhorst et al. 2010).Gluten digestion enzymes that digest gliadin into non-immunogenic peptides can be an acceptable treatment for CeD (Yoosuf and Makharia 2019).So far, the enzymatic digestion of gluten has been studied by only a few endopeptidase enzymes derived from grains, bacteria, and fungi such as ALV003, AN-PEP, STAN-1, etc. (Yoosuf and Makharia 2019).
Bromelain and ficin are members of the papain family of cysteine proteases which are derived from pineapple and the latex of the Ficus tree, respectively (Devaraj et al. 2008;Ha et al. 2012).
Based on our knowledge of bromelain and ficin enzyme cleavage sites and their anti-inflammatory properties based on previous studies and prediction of gliadin sequence cleavage by these two enzymes, bromelain and ficin were selected in this study.Therefore, in this study, toxicity and immunogenicity of digested fragments were predicted after the simulation of enzymatic cleavage of the gliadin sequence and 33-mer and 19-mer peptides by bromelain and ficin.In the following, fragments obtained from enzymatic cleavage of the 33-mer and 19-mer peptides were subjected to simulation with transglutaminase function, and their glutamine residues were replaced with glutamic acid.These peptide fragments docked with HLA-DQ2 and HLA-DQ8, and their results were compared with intact 33-mer and 19-mer peptides.Next, we have investigated the bromelain and ficin digestion effects on the commercial and wheat-extracted gliadin by a series of in vitro assays such as SDS-PAGE, HPLC, and Circular Dichroism (CD).Also, the cytotoxicity of gliadin and 19-mer peptide before and after enzymatic digestion was investigated on the Caco-2 cell line.Finally, the effect of these treatments along with the intervention of Larazotide Acetate peptide on the expression of genes involved in cell junctions on the same cell line was evaluated.In all steps, synthetic 19-mer peptides were used as positive controls.Larazotide acetate (AT-1001) is an antizonulin that functions as a gut-tight junction regulator for the treatment of Celiac (Hoilat et al. 2022).

Peptide synthesis
The 19-mer peptide with LGQQQPFPPQQPYPQPQPF sequence was synthesized by Pepmic Co., Ltd, (Suzhou, China) using a solid-phase method applying N-9 fluorenyl methoxycarbonyl (Fmoc) chemistry and was purified to 95% using C18 reverse-phase high-performance liquid chromatography (RP-HPLC).Mass spectrometry performed on a Sciex API100 LC/MS mass spectrometer (Perkin Elm Co., Norwalk, CT, USA) in the positive-ion mode was used to determine the molecular weights of the synthesized peptide.

Simulation of enzymatic cleavage and immunoinformatics studies
The sequence of gliadin was retrieved from the NCBI database, and its two toxic peptide sequences (33-mer, and 19-mer) were determined.Also, toxic peptide sequences of gliadin based on previous experimental studies (Arendt et al. 2011;Ferretti et al. 2012;Cornell and Stelmasiak 2016;Prandi 2017;Cebolla et al. 2018) were obtained and sorted based on their origins.The cleavage sites of bromelain and ficin enzymes were determined according to previous studies, and enzymatic digestion was simulated in the complete gliadin protein, 33-mer, 19-mer, and gliadin toxic peptides.In the following, toxicity and immunogenicity of enzymedigested peptides were predicted by ToxinPred (https:// webs.iiitd.edu.in/ ragha va/ toxin pred/ algo.php) and IEDB (https:// www.iedb.org) servers, respectively.Finally, the fragments obtained from enzymatic cleavage of the 33-mer and 19-mer peptides were subjected to simulation with tissue transglutaminase function, and their glutamine residues were replaced with glutamic acid.

Molecular docking simulation
The three-dimensional structures of HLA-DQ2 (PDB ID: 1s9v) and HLA-DQ8 (PDB ID: 2nna) were obtained from the protein data bank (PDB) (https:// www.rcsb.org/).The structures were cleaned, crystallographic waters and cocrystallized molecules were deleted, and only the monomer forms of each receptor were kept.Then, these structures were prepared by the Dock prep tool in UCSF Chimera software.Also, sequences of 19-mer and 33-mer peptides and their digested derivatives were saved as separated FASTA formats.The sequence changes of all these peptides were simulated under the function of tissue transglutaminase enzyme.The GalaxyPepDock web server (http:// galaxy.seokl ab.org/ cgibin/ submit.cgi? type= PEPDO CK) was utilized to perform the protein-peptide docking between the selected human MHC class II PDB files and related peptides.The binding affinity and the dissociation constant (Kd) of peptide-protein complexes were predicted based on the PRODIGY (PROtein binDIng enerGY prediction) web server (https:// bianca.scien ce.uu.nl/ prodi gy/).The best docking result was chosen based on Prodigy (Kd), the top cluster with the lowest intermolecular energy.

Isolation and preparation of gliadins
In this study, commercial gliadin (sigma) and gliadin extracted from wheat flour were used.Gliadins were extracted from flour of Triticum aestivum according to Tatham et al. (2000).Wheat flour (0.5 g) was stirred with 5 ml of butanol-1 for 2 h at room temperature.A mixture of flour-butanol was centrifuged at 6000 rpm for 10 min and the supernatant was removed.The pellet was mixed with 5 ml of 0.5 M NaCl for 1 h at room temperature.After centrifugation, the supernatant was removed and the remaining residue was washed with water.The pellet was mixed with 5 ml of 70% ethanol and stirred for 30 min to extract gliadin.The Bradford method determined the concentration of extracted gliadin, and 0.5 mg/ml of a gliadin fraction (it) was evaluated along with commercial gliadin as a control using SDS-PAGE.For commercial gliadin, 1 mg of it was dissolved in 1 ml of ethanol 70%.

Optimization of gliadin enzymatic digestion process by bromelain and ficin
Gliadin at a concentration of 1 mg/ml was incubated with bromelain and ficin enzymes at the same concentration in V/V% 1:1 (enzyme; substrate) separately at 37 °C for 3 h.Also, in another reaction, gliadin was treated with a mix of bromelain and ficin enzymes at mentioned conditions.The enzymatic hydrolysis was stopped by boiling for 5 min.Samples were stored at − 20 °C until further chemical analysis (Cavaletti et al. 2019).
To optimize the gliadin proteolysis activity of the bromelain and ficin, four variable parameters, including gliadin and enzyme concentration, the ratio of enzyme: substrate, temperature, and incubation time were tested.Next, the best condition was selected and the test was repeated in this state.Gliadin (1 mg/ml) was incubated with bromelain and ficin enzymes separately (enzyme:substrate, 1:1, 1:10, 1:100), at room temperature, 37 °C and 60 °C for different time points (3 h, and overnight).The enzymatic hydrolysis was stopped by boiling for 5 min.

Simulation of gastric digestion of gliadin
The in vitro gastric digestion was performed by adding 1 ml of simulated gastric fluids (SGF) (0.9 mM NaH 2 PO 4 , 3 mM CaCl 2 , 0.1 M HCl, 0.15 M NaCl, 16 mM KCl, pH 2.5) to 1 mg of gliadin.After incubation at 65 °C for 2 h, 1 mg of pepsin was added and incubated at 37 °C for 3 h.The reaction was stopped by enhancing the pH to 7 with the addition of 0.5 M NaHCO 3 (Smith et al. 2015).

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
The protein concentration of the samples was measured by spectrophotometry (Eppendorf, Germany) at 595 nm, according to the Bradford assay using bovine serum albumin (BSA) as standard (Bradford 1976).
SDS-PAGE test was used for purity determination and molecular weight estimation of the purified enzyme.20 μl of eluted samples and 5 μl of prestained protein marker (BLUelf, GeneDirex) were separated by 12.5% resolving gel and 5% stacking gel which was run under 100 V for 90 min.The running buffer was Tris-glycine (pH 8.3).After electrophoresis, the gel was fixed with fixation solution for 60 min and then washed three times with UPW.Finally, the gel was stained with colloidal Coomassie brilliant blue (G-250) staining solution overnight and then de-stained with 1% v/v acetic acid solution for 1 h (Wanderley et al. 2004).

RP-HPLC for the proteolytic function of enzymes
RP-HPLC was carried out using a Knauer HPLC system (Germany).Samples were suspended in 0.1% TFA and separated by C18 column (TSKgel ODS-100V, 5 µm, 250 × 4.6 mm i.d., Tosoh Bioscience LLC, King of Prussia).The volume of injection was 20 µl.Eluent A was 0.1% TFA (v/v) in Milli-Q water, and eluent B was 0.1% TFA (v/v) in acetonitrile.The column was equilibrated at 60% B. Samples was separated by applying a linear 60-100% gradient of B over 3 min at a 2 ml/min flow rate.Chromatographic separation was performed at 37 °C, using a thermostatic column holder.The column effluent was monitored at 210 nm with a UV vs. Diode-Array (PDA) Detector (Tian et al. 2014;Cavaletti et al. 2019).

Circular dichroism (CD) studies
Changes in the secondary structure content of 19-mer peptide before and after treatment with bromelain and ficin enzymes can be another way to show the gliadin or 19-mer peptide digestion by the enzyme's proteolytic activity.In our study, to evaluate the secondary structure change in 19-mer peptide after treatment with bromelain and ficin enzyme, a CD spectroscopy measurement was performed.At first, 200 µl of 19-mer peptide aqueous solution at a concentration of 0.5 mg/ml was prepared and 100 µl of Bromelain enzyme at a concentration of 0.025 mg/ml was treated for 24 h at 37 °C.The same process was repeated for the ficin enzyme.The mean residue molar ellipticities of the mentioned reactions before (t0) and after (t24) were determined by CD spectroscopy, using a Jasco J-810 spectropolarimeter (Jasco, Japan) at 25 °C with 200 nm/min scanning speed.For this purpose, the above reactions were loaded into a 1mm quartz cell and its spectra were scanned from 190 to 240 nm (Herrera et al. 2014).

Cytotoxicity assay of gliadin and 19-mer peptide before and after enzymatic cleavage
The colorimetric MTT assay was performed (Madanchi et al. 2020) to determine the toxicity of gliadin, 19-mer peptide before and after treatment by bromelain-ficin mixture (BF), and Larazotide acetate (LA).Briefly, Caco2 cells were cultured in 96-well plates at a density of 1 × 10 5 cells/well and incubated at 37 °C, in a 5% CO 2 atmosphere with 95% relative humidity for 24 h.Then, the old media were replaced with the fresh RPMI medium containing 10% FBS and the cells were incubated for 24 h.Quintet wells were analyzed for each concentration, and column elution buffer was used as the control.A 10 μl solution of freshly prepared 5 mg/ ml MTT in PBS was added to each well and incubated for an additional 4 h at 37 °C.After removing the medium, DMSO was added at 100 μl/well.Plates were shaken gently to ensure that formed formazan crystals were entirely dissolved.Absorption values at 595 nm were measured using a microplate reader (STAT FAX 2100, BioTek, Winooski, USA).The percentages of cell toxicity and half-maximal inhibitory concentration (IC50) were calculated.All experiments were performed in triplicate.
Cell viability was calculated as follows:

Evaluating the expression of genes involved in cell junctions by qRT-PCR
To determine the gene expression of cell junction proteins, such as Occludin, Tight junction protein (TJP), or Zonula occludes-1 (ZO-1), Zonulin, and Claudin, an RT-qPCR was performed as previously described (Madanchi et al. 2022).
For this purpose, total RNA was isolated from Caco-2 cells using the blood/cultured cell Total RNA Purification Kit (Jena Bioscience, Jena, Germany) according to the manufacturer's protocol.After the final elution of extracted total RNA, the quality and quantity of total RNA extracted were detected using a NanoDrop ® spectrophotometer (Hitachi, Tokyo, Japan).cDNA synthesis performed by a SCRIPT cDNA Synthesis Kit (Jena Bioscience, Jena, Germany).qPCR was performed using an ABI system (Applied Biosystems; Thermo Fisher Scientific, Inc.) and Real Q plus 2 × Master Mix Green with high ROX (Ampliqon, Denmark).qPCR was conducted as follows: Initial denaturation at 95 °C for 15 min; 40 cycles of denaturation (at 95 °C for 20 s) and annealing (at 52-62 for 30 s); and extension at 72 °C for 30 s. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) housekeeping gene was used as an internal reference gene.Primer sequences were designed except GAPDH using AllelID software and evaluated by oligo analyzer and primer blast tools.The primers were provided by SinaClon (SinaClon, Tehran, Iran) and are shown in Table 1.

Statistical analysis
Data were evaluated using IBM SPSS Statistics version 20 (IBM Corp).Data were offered as the mean ± standard deviation.All the tests were repeated three times.Differences between groups were obtained using one-way analyses of Viability% = 100 − Toxicity%.
variance followed by Tukey's post hoc test.P < 0.05 was considered to display a significant difference.

Simulation of enzymatic digestion of gliadin, 33-mer, and 19-mer peptides
Enzyme cleavage sites with bromelain and ficin were determined in the sequence of gliadin, 33-mer, and 19-mer peptides, and other gliadin toxic peptides and enzyme cleavage was simulated in these sequences, and the resulting fragments are arranged in Tables 2, 3 and 4. Also, the toxicity and immunogenicity of obtained peptides after enzymatic cleavage of gliadin, 33-mer, and 19-mer peptides were predicted.Bromelain's enzymatic cleavage site is at the C-terminus of Tyrosine (W), Lysine (K), and Alanine (A) residues, while ficin's enzymatic cleavage site is at the C-terminus of Glycine (G), Serine (S), Tyrosine (Y), and Glutamic acid (E) residues, which are located after the hydrophobic subunits.As it is clear, bromelain and ficin digest gliadin sequences and their toxic peptides derived from gliadin, and the peptides obtained by enzymatic digestion with these two enzymes are non-toxic and non-immunogenic or have weak immunogenicity.

Simulation of HLA-DQ2 and HLA-DQ8 interaction with 33-mer and 19-mer peptides before and after digestion by bromelain and ficin
To compare the binding affinity of the peptides obtained from enzymatic cleavage of 33-mer (LELEPFPEPELPYPEPELPYPEPELPYPEP) and 19-mer (LGEEEPFPPEEPYPEPEPF) peptides by bromelain and ficin with parental peptides before digestion in interaction with HLA-DQ2 and HLA-DQ8 molecules, a molecular docking simulation was designed by the GalaxyPepdock software.As Table 2 shows, of all the peptides resulting from enzymatic cleavage of these two peptides with bromelain and ficin, only two sequences have above 11 residues and can interact with HLA-DQ.Therefore, 19-mer, 33-mer peptides and two peptides obtained from enzymatic digestion of them (PGQQQPFPPQQPY 19mer1 and LQLQPFPQPQLPY or 33mer1) were subjected to tissue transglutaminase enzyme and all their glutamines were converted to glutamate, and then, their interaction with HLA-DQ2 and HLA-DQ8 molecules was evaluated (Fig. 1).The results of molecular docking analysis with the PRODIGY software revealed that the binding affinity between 33-mer and 19-mer peptides with HLA-DQ2 and HLA-DQ8 molecules is higher than the peptides obtained after enzymatic digestion of these two peptides by bromelain and ficin (Table 5).As it is clear in Table 5, the ΔG and Kd of 19-mer and 33-mer peptides complex with HLA-DQ2 and HLA-DQ8 is much lower than when these peptides are digested with bromelain and ficin enzymes.
The smaller the value of ΔG and Kd parameters, the greater the binding affinity of the peptide with HLA-DQs, so the initiation of immune responses in digested peptides with bromelain and ficin is weaker than in their intact state.

Evaluation of extracted gliadin from flour
In the present study, gliadin was extracted from wheat flour and the fragments of gliadin (α-, β-, γ-, and ω-gliadin) were separated by SDS-PAGE results.The protein band pattern of gliadin is shown in Fig. 2. Also, a comparison of the band's pattern of extracted gliadin with commercial gliadin (sigma) is shown in Fig. 2.

Enzymatic digestion of gliadin by bromelain and ficin and optimization of this process
Enzymatic digestion of gliadin with bromelain and ficin enzymes was performed under different temperatures, treatment times, and concentration conditions and the results were analyzed with 12% SDS-PAGE.The results of SDS-PAGE gel analysis showed that in a 1:100 concentration ratio of enzyme to gliadin at all treatment times and temperatures, Bromelain enzyme was better able to break down gliadin (Fig. 3).It was also found that in the same ratio of 1-100 enzyme-gliadin in room temperature or RT conditions, the best enzyme digestion result was obtained.Also, the enzyme treatment at 37 °C temperature was better than 60 °C temperature in the same treatment ratio.All the previous articles have mentioned that the optimal temperature of Bromelain performance is 60 °C.The results of the enzymatic treatment at 3 and 16 h showed that the digestion rate of gliadin with a ratio of 1:100 enzymegliadin was better after 16 h of treatment and the bands related to gliadin became fainter.But on the whole, from treatments at room temperature and 37 °C, almost similar results were obtained.Also, in a separate experiment, three enzyme-gliadin concentration ratios were repeated at RT and 37° and for a treatment time of 16 h.The results of this test were analyzed again with 12% SDS-PAGE (Fig. 4).The results showed that in high enzyme-gliadin ratios (1:1), the enzymatic digestion rate is improved, and gliadin decomposition is better.Also, in the ratio of 1 to 1 and 1 to 10 (enzyme-gliadin) in the case of ficin, the breakdown of gliadin bands is better at RT, 16 h, and the ratios 1:100, 1:10, and 1:1, respectively.Columns 5, 6, and 7 are related to the treatment of gliadin with ficin (GF) at RT, 16 h, and the ratio 1:100, 1:10, and 1:1, respectively.Columns 8, 9, and 10 are gliadin:bromelain (Heat inactivated), and columns 18, 19, and 20 are gliadin: ficin (heat inactivated) as the control for 1:100, 1:10, and 1:1 ratio, respectively.Columns 12 to 14, GB, 37 °C, 16 h, the ratios 1:100, 1:10, and 1:1, respectively.Columns 15 to 17, GF, 37 °C, 16 h, the ratios 1:100, 1:10, and 1:1, respectively than bromelain.At the same time, in the previous test and its repetition in the same test, it was found that in the ratio of 1 to 100 treatment, bromelain has better gliadin degradation than ficin.Briefly, it can be said that based on the optimization of the conditions for the enzymatic cleavage of bromelain and ficin, the best temperature for the activity of the two enzymes in our experiment was 37 °C for 16 h.Also, Bromelain with an enzyme:substrate ratio of 1:100 showed a much better result than ficin in gliadin digestion, but in enzyme:substrate ratio of 1:1 and 1:10, ficin enzyme has performed better than Bromelain.

Digestion of gliadin by enzymes after treatment in gastric digestion condition
To simulate more and closer to the natural conditions of the body in vitro conditions, first gliadin was affected by simulated gastric fluids (SGF), and then, it was treated under the effect of bromelain and bromelain-ficin mixture (BF).Figure 5 shows the electrophoresis gel of gliadin bands before and after treatment in simulated stomach conditions and bromelain and ficin enzymes.As shown in the figure, the digestion of gliadin in SGF is incomplete and increases after treatment with bromelain and bromelain-ficin mixture.It is also clear that a mixture of bromelain-ficin had a greater effect than bromelain alone on the digestion of gliadin treated with simulated gastric fluids.

Evaluation of gliadin enzymatic digestion by HPLC
The ability of bromelain and ficin to hydrolyze the gliadin sequence was assessed by RP-HPLC analysis (Fig. 6).Gliadin treated by bromelain and ficin at time point 0 was considered a zero-point sample and was run as a reference control.As expected, a significant decrease in gliadin-related peak in chromatogram after treatment by enzymes was observed compared to the zero-point sample.Therefore, the chromatographic profile of the zero-point sample was significantly different compared to digested gliadin after the incubation period.This evidence indicated that both bromelain and ficin can proteolyze the gliadin at a certain incubation time.However, in the digestion of gliadin (at the concentration ratio 1:1 of enzyme:gliadin), ficin acts better than the bromelain enzyme.

Treatment of the synthetic gliadin 19-mer peptide by enzymes and its validation by circular dichroism (CD)
To further evaluate the enzymatic digestion of 19-mer toxic peptides derived from gliadin, we hypothesized that the secondary structure content of peptide 19 changes before and after enzymatic digestion.Therefore, we studied it with the CD method (Fig. 7).The secondary structure content (fraction ratio) of the 19-mer peptide with bromelain and ficin enzymes before (zero point) and after treatment is shown in Table 6.As Table 6 shows, after the 19-mer peptide treatment with bromelain, its alpha helix and beta sheets content decreased, and instead, the percentage of its turns and coils increased.However, ficin did not affect the amount of alpha helices but increased the beta sheets.However, the turn and coil of 19-mer peptide decreased after treatment with ficin.This change in the secondary structure content of the 19-mer peptide once again confirmed the potential of proteolytic cleavage of this peptide by these two enzymes.

Cytotoxicity results
Cytotoxicity of gliadin and 19-mer peptide alone and after (gliadin-BF and 19mer-BF) treatment with a bromelain and ficin mixture (BF) and larazotide acetate (LA) on CaCo-2 cells was determined by the MTT method (Fig. 8).Also, the toxicity of the mentioned enzymatic mixture alone was measured.The Caco2 cells were treated with different concentrations (from 15.62 to 4000 μg/ml) of each sample for 24 h.Untreated cells were used as control.After a 24-h-long exposure to different concentrations of gliadin and 19-mer peptide, the MTT assay showed a significantly decreased Fig. 5 The image shows the SDS-PAGE gel of gliadin protein bands before (column 4) and after digestion by SGF (column 3), SGF:Bromelain (column 2), and SGF:BF (column 1).Column 5 is a protein-size marker 1 3 viability of Caco-2 cells.The results showed that the toxicity of intact gliadin is significantly higher than the 19-mer peptide at the same concentrations (P < 0.05).Conversely, bromelain, ficin, and larazotide acetate did not show significant toxicity.Our results demonstrated that digestion of gliadin and 19-mer peptide with bromelain and ficin mixture has significantly reduced their toxicity (P < 0.05).

Evaluation of gene expression of tight junction proteins by a qRT-PCR after
The gene expression analysis by real-time PCR was conducted to evaluate the effects of the bromelain and ficin mixture (BF) on the gliadin and synthetic 19-mer peptide.The OCCL, CLDN, TGP (ZO-1), and ZON genes were evaluated.We also determined their effects on the expression of the desired genes along with the LA peptide.The results showed that treatment of CaCo-2 cells with gliadin alone increases the expression of all genes (OCCL, TGP, ZON, and CLDN), especially ZON, which is in response to the destruction of tight junction proteins.This increase in gene expression (especially ZON and OCCL) in the treatment of cells with 19-mer peptide was significantly higher than in the treatment with gliadin (P < 0.001).As it is known, after the treatment of gliadin and 19-mer with BF mixture, the expression of all genes involved in cell junctions, especially ZON, and OCCL, is significantly reduced compared to before their enzyme treatment (P < 0.001) (Fig. 9A).
It was also found that cells treated with LA express significantly fewer cell junction genes than when the same cells were treated with a mixture of LA and gliadin (gliadin-LA).This issue is evident in the expression of genes OCCL, TGP, and CLDN (P < 0.001).Also, after gliadin was digested with an enzyme mixture and added to the cells along with LA (gliadin-BF-LA), it was found that the expression of all genes decreased significantly compared to gliadin-LA (P < 0.05).Therefore, it was found here that the enzymatic digestion of gliadin can reduce  their toxic effects on the proteins involved in cell junctions, and again, the positive effects of LA on cell junctions appeared (Fig. 9B).

Discussion
Celiac disease (CeD) is one of the important human autoimmune diseases that is caused by extreme responses to toxic peptides resulting from incomplete digestion of gliadin, and related prolamins in genetically predisposed individuals with (HLA)-DQ2 or HLA-DQ8 haplotypes.This disease is characterized by the presence of a variable combination of gluten-dependent clinical indications, CD-specific antibodies, and small intestinal enteropathy (Gujral et al. 2012).As mentioned in the introduction, although the gluten-free diet is the only treatment for celiac disease, the presence of unwanted gluten in supplements, medicines, and other food items has reduced the effectiveness of this strategy (Alhassan et al. 2019).
Enzymes derived from plants such as amylases, invertases, papains, bromelain, ficin, lipoxygenase, etc. many uses in food preparation processes including the production of syrups, bakery products, alcoholic beverages, dairy products, etc. (Meshram et al. 2018).This study aimed to evaluate the digestive effects of bromelain and ficin enzymes on the toxicity of gliadin and toxic 19-mer peptide on CaCo2 cell lines.
In the virtual simulation of enzymatic cleavage of gliadin with bromelain and ficin, 12 peptide fragments were obtained, and all sequences obtained from enzymatic digestion by two non-toxic and non-immunogenic enzymes were predicted.In this part of the study, it was predicted that even these two enzymes digest two important toxic peptides resulting from the breakdown of gliadin (33-mer and 19-mer peptides).Also, immunoinformatic predictions showed that the fragments obtained by digestion of 19-mer and 33-mer peptides with bromelain and ficin enzymes are not immunogenic or toxic.Following these predictions, the digestive effects of these two enzymes on other 12 toxic peptides derived from gliadin were determined.It was found that bromelain and ficin separately and even mixed can also digest these toxic peptides.Also, molecular docking results show that enzymatic digestion with bromelain and ficin can, in addition to digesting toxic peptides 33-mer and 19-mer, create fragments that have a lower binding affinity to HLA-DQ2 and HLA-DQ8.Therefore, these digested fragments probably induce a weaker immune response than undigested 33-mer and 19-mer peptides.Consequently, it can be assumed that in the case of enzymatic digestion of gliadin and toxin peptides resulting from its incomplete digestion with bromelain and ficin, the pathological damages will be reduced in those prone to celiac disease.Our study is the first study that simulates enzymatic cleavage of gliadin and toxic peptides resulting from its incomplete digestion with in-silico methods and predicts the toxicity and immunogenicity of the digested fragments of gliadin.
Fig. 8 The chart displays the toxicity of gliadin and 19-mer peptide before and after treatment with bromelain and ficin mixture (BF), and larazotide acetate on the Caco-2 cell line.Error bars indicate standard deviation Also, there has been no study on the molecular docking of peptide fragments obtained from gliadin enzymatic cleavage with HLA-DQ2 and HLA-DQ8 molecules.Therefore, through these virtual studies and simulations, we designed our default for conducting in vitro studies.
Active proteases hydrolyze gliadin, and several studies showed that plant proteases are most suitable for this purpose (Jayawardana et al. 2019).Bromelain and ficin are the most broadly identified commercially accessible fruit-derived proteases that are securely used in the baking industry and can be used in protein hydrolysis (Meshram et al. 2018).Hydrolysis conditions, such as temperature and enzyme-substrate ratio, can significantly affect the efficiency of enzymatic hydrolysis (Li et al. 2016), so our study examined these conditions.For the first time, we evaluated the effect of the combination of bromelain and ficin on gliadin digestion.Also, various conditions of enzymatic digestion of gliadin and 19-mer peptides, such as temperature, enzyme-substrate ratio, and duration of treatment with these two enzymes, were optimized, and the results were observed directly by SDS-PAGE, RP-HPLC, and CD.
The temperature range for the enzyme efficacy examinations was designated based on the results of previous studies.The mentioned temperature in the earlier studies is 35-65 °C.Pang et al. reported the optimum temperature for bromelain to be 40 °C.They also stated that bromelain's activity and stability are temperature dependent, and the activity of bromelain is reduced with the increase of the incubation temperature (50-70 °C) (Pang et al. 2020).However, unlike many studies, our study showed that the most appropriate temperature for enzyme function is 37 °C (biological body temperature).The soluble protein content (SPC) and SDS-PAGE analysis of the Gliadin showed which part of this macro-polymer has been depolymerized (Thiele et al. 2004); therefore, it can be used as a method to assess the effectiveness of enzymatic digestion.Enzymatic digestion of gliadin with bromelain and ficin enzymes was performed under different conditions.The results of SDS-PAGE gel analysis showed that at a 1:100 enzyme-gliadin ratio at all times and temperatures, it could digest gliadin better than ficin according to the protein size.In the 1:100 enzyme-gliadin ratio, the best results were at room temperature (RT) and 37 °C (although all the previous research reported that the optimal temperature of Bromelain digestion is 60 °C).Buddrick et al. (2015) demonstrated that caricain from papaya latex decreases toxic gliadin content after 5 h.Li et al. reported that with enzyme-substrate ratio [E/S] (0.1/0.4%) and reaction time of 60-90 min, a very high decrease in gliadin content (up to 99.99%) for papain (Li et al. 2016).Some studies have reported the optimal temperature for ficin's performance to be 30-37 °C (Yang et al. 2017), and some other studies have reported this temperature to be 60 °C (Fadýloğlu 2001).
In the 1:1 and 1:10 ratio (enzyme-gliadin) by ficin, the digestion of the gliadin was better than bromelain.The HPLC results of enzymatic digestion of gliadin by bromelain and ficin showed that these enzymes could digest gliadin into small peptides.HPLC chromatograms demonstrated that bromelain and ficin have nearly similar effects in hydrolyzing gliadin.This confirmed the efficacy of these plant enzymes.Ficin and bromelain belong to the cysteine protease family; however, their enzymatic hydrolysis mechanism is in particular, and they cleave dissimilar protein bonds.
Therefore, we first investigated the digestion of one of the peptides, 19-mer peptide, by bromelain and ficin by studying the change in its secondary structure content.Then, we determined the cytotoxicity of gliadin and this peptide before and after enzymatic digestion with these two enzymes on the CaCo-2 cell line.Finally, we studied the effect of gliadin and 19-mer peptide before and after enzymatic digestion with bromelain and ficin on the expression of genes involved in creating cell-tight junctions.
For the first time, in an innovative approach, we hypothesized that the secondary structure content of 19-mer peptide changes after enzymatic digestion with bromelain and ficin, so we used circular dichroism (CD) to prove it.The results showed that after digesting this peptide with both bromelain and ficin enzymes, the percentage of helices, beta strands, turns, and coils changed.
MTT results of gliadin and 19-mer peptide before and after enzyme digestion on the CaCo2 cell line demonstrated that toxicity of the gliadin and synthetic 19-mer peptide was decreased after treatment of them by bromelain and ficin mixture.
Gliadin and its derived toxic peptides act on the small intestinal epithelial cells' tight junctions and increase their permeability (Yoosuf and Makharia 2019).This process increases the leakage of toxic peptides into the lamina propria and triggers more immune responses (Yoosuf and Makharia 2019).Therefore, we evaluated the effects of gliadin and 19-mer peptide before and after enzymatic digestion by a mixture of BF on the expression of some genes encoding intercellular tight junction proteins by the real-time PCR method.qRT-PCR results indicate that tight junction proteins in the treated CaCo-2 cells with gliadin, and 19-mer peptide after digested of them by BF, it is not destroyed.Therefore, CaCo-2 cells do not need to express additional genes to produce these proteins.For this reason, the gene expression of intercellular tight junction proteins decreases after the treatment of gliadin and 19-mer peptide with BF compared to intact gliadin and 19-mer peptide.In our study, the expression of the ZO-1 gene was decreased in the presence of digested gliadin by BF.The expression of these genes was increased with gliadin.The expression of ZO-1 and occludin genes is possibly the molecular origins of the abnormal structure and function of tight junction patients with CeD.The ZO-1 protein is the initial molecule to localize in the nascent close junction complex and arrangements a connecting link between the transmembranous proteins and cytoplasmic actin filament (skeleton) (Anderson et al. 1995;Fanning and Anderson 2009).Pizzuti et al. demonstrated that the expression of the ZO-1 gene decreases and the actin structure is disturbed in CeD, but after a GFD, abnormalities in ZO-1 expression and actin organization were reversed (Pizzuti et al. 2004).Claudins are the key factors for tight junction permeability.The tightness of tight junctions is determined by the composition, number, and involvement ratio of Claudins (Günzel and Yu 2013).Studies suggested that Occludin is essential for the preservation and arrangement of tight junction proteins (Saitou et al. 2000).A dysfunction of TJ proteins deteriorates intercellular bonds and stimulates intestinal permeability and inflammation (Lee 2015).Briefly, real-time PCR results in our study are interpreted in this way.Gliadin and 19-mer peptide increase the permeability of cells by increasing the expression of zonulin.Zonulin destroys other proteins, such as OCCL, TGP (ZO-1), and CLDN.Therefore, as a result of the treatment of Caco2 cells, gliadin or 12-mer peptide increases the expression of zonulin, and as a result, the expression of degraded OCCL, TGP, and CLDN also increases to compensate for their loss.Enzymatic treatment by BF mixture causes the digestion of gliadin and 19-mer peptide, so the expression of zonulin in Caco2 cells decreases compared to when they are treated with intact gliadin or 19-mer peptide, and there is no need to overcompensate OCCL, TGP, and CLDN, so their expression is also lower than when they are treated with intact gliadin.
Zonulin controls intestinal permeability by disrupting contact between the cytoplasmic anchoring protein ZO-1 and the occluding transmembrane protein (Sturgeon and Fasano 2016).The intestinal permeability is increased dependent on zonulin expression, and the management of zonulin inhibitor FZI/0 prevents an increase in intestinal permeability (Watts et al. 2005).Tammara Watts et al. demonstrated a meaningful upper serum zonulin level in type I diabetes patients compared to controls (Watts et al. 2005).Duerksen et al. showed an upper serum zonulin level in CeD patients, which normalized following a gluten-free diet (Duerksen et al. 2010).Our study showed that the expression of zonulin and occludin genes was decreased in the presence of gliadin and BF enzymes.Subsequently, these results can confirm the use of bromelain and ficin enzymes for treating CeD patients.However, this issue should be verified by in vivo and clinical trials.
We also measured the of intact gliadin and gliadin digested with these two enzymes along with LA peptide on the expression of the mentioned genes.This peptide prevents the destruction of intestinal cells in celiac patients by regulating tight junction proteins (Hoilat et al. 2022).
The results of examining the expression of genes involved in cell junctions in Caco-2 cells treated with LA showed that all the mentioned genes' expression significantly decreased compared to untreated cells.However, when gliadin is added to LA-treated cells, the expression of OCCL, CLDN, TGP, and ZON genes increases significantly.Instead, when gliadin digested with an enzyme mixture is added to the cells treated with LA, the expression of all these genes decreases, and the protective effects of LA are observed again.
Therefore, our results indicated that the enzymatic digestion of gliadin by bromelain and ficin mixture can reduce the toxic effects of gliadin on the proteins involved in cell junctions, and again, the positive effects of LA on cell junctions appeared.

Conclusion
Although consuming gluten-free foods (GFDs) is the only strategy to prevent the clinical manifestations of celiac disease, however, the unpleasant taste of gluten-free foods, the high cost of these products, and the unwanted consumption of gluten are among the problems for which a solution has not yet been proposed.Research on glutendegrading enzymes, especially plant enzymes, is growing.Our study showed that the mixture of bromelain and ficin digest gliadin and the toxic peptides derived from it well.Peptides resulting from the digestion of gliadin with bromelain and ficin enzymes have much less toxicity than intact gliadin on Caco-2 cells.Also, gene expression studies showed that digestion of gliadin and 19-mer peptide with a mixture of bromelain and ficin preserves the proteins involved in tight junctions in this cell line and strengthens the effect of larazotide acetate therapeutic peptide in maintaining tight cell junctions.Therefore, the use of bromelain and ficin, in addition to their antiinflammatory properties, can also be helpful in the digestion of toxic peptides due to the incomplete digestion of gliadin.We can hope for the therapeutic potential of these two natural enzymes in the treatment of celiac disease by conducting in vivo studies.

Fig. 1 Fig. 2
Fig.1The three-dimensional structure of the complexes was simulated with Chimera software

Fig. 6
Fig. 6 HPLC chromatograms of A bromelain, B gliadin and bromelain at zero point of enzyme treatment, C bromelain treated gliadin after the incubation period, D ficin, E gliadin and ficin at the zero point, and F gliadin after treatment by ficin

Fig. 7
Fig. 7 CD spectra of the 19-mer peptide before and after treatment with bromelain and ficin.The illustration indicates that the secondary structure of the 19-mer peptide was changed after treatment with these enzymes

Fig. 9
Fig.9Expression of genes involved in cell junctions treated with A gliadin, 19-mer peptide, and their digested derivatives with enzyme mixture (BF) and B treated with LA peptide, gliadin-larazotide acetate (gliadin-LA), and gliadin digested with BF along with LA (gliadin-BF-LA)

Table 1
Sequence of primers

Table 2
Simulation of enzymatic digestion of gliadin by bromelain and ficin

Table 4
Enzyme digestion of gliadin-derived toxic peptides by bromelain, ficin, and a mixture of these two enzymes

Table 6
Fraction ratios of secondary structure content of the 19-mer peptide with bromelain and ficin before and after treatment