Next Article in Journal
Isogenic Human-Induced Pluripotent Stem-Cell-Derived Cardiomyocytes Reveal Activation of Wnt Signaling Pathways Underlying Intrinsic Cardiac Abnormalities in Rett Syndrome
Next Article in Special Issue
Relationship between Epithelial-to-Mesenchymal Transition and Tumor-Associated Macrophages in Colorectal Liver Metastases
Previous Article in Journal
DFT Simulation-Based Design of 1T-MoS2 Cathode Hosts for Li-S Batteries and Experimental Evaluation
Previous Article in Special Issue
TGF-β Signaling in Metastatic Colorectal Cancer (mCRC): From Underlying Mechanism to Potential Applications in Clinical Development
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
IJMS
Volume 23
Issue 24
10.3390/ijms232415610
ijms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Selective Role of TNFα and IL10 in Regulation of Barrier Properties of the Colon in DMH-Induced Tumor and Healthy Rats

by
Viktoria Bekusova
1,*,
Tatiana Zudova
1,
Ilyas Fatyykhov
1,
Arina Fedorova
1,
Salah Amasheh
2 and
Alexander G. Markov
1
1
Department of General Physiology, Faculty of Biology, Saint Petersburg State University, 199034 Saint Petersburg, Russia
2
Department of Veterinary Medicine, Institute of Veterinary Physiology, Freie Universität Berlin, 14163 Berlin, Germany
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2022, 23(24), 15610; https://doi.org/10.3390/ijms232415610
Submission received: 29 October 2022 / Revised: 1 December 2022 / Accepted: 6 December 2022 / Published: 9 December 2022
(This article belongs to the Special Issue Molecular Mechanisms and Therapies of Colorectal Cancer)
Browse Figures
Review Reports Versions Notes

Abstract

:
Recently it has been reported that the tumor adjacent colon tissues of 1,2-dymethylhydrazine induced (DMH)-rats revealed a high paracellular permeability. We hypothesized that the changes might be induced by cytokines. Colorectal cancer is accompanied by an increase in tumor necrosis factor alpha (TNFα) and interleukin 10 (IL10) that exert opposite regulatory effects on barrier properties of the colon, which is characterized by morphological and functional segmental heterogeneity. The aim of this study was to analyze the level of TNFα and IL10 in the colon segments of DMH-rats and to investigate their effects on barrier properties of the proximal and distal parts of the colon in healthy rats. Enzyme immunoassay analysis showed decreased TNFα in tumors in the distal part of the colon and increased IL10 in proximal tumors and in non-tumor tissues. Four-hour intraluminal exposure of the colon of healthy rats with cytokines showed reduced colon barrier function dependent on the cytokine: TNFα decreased it mainly in the distal part of the colon, whereas IL10 decreased it only in the proximal part. Western blot analysis revealed a more pronounced influence of IL10 on tight junction (TJ) proteins expression by down-regulation of the TJ proteins claudin-1, -2 and -4, and up-regulation of occludin only in the proximal part of the colon. These data may indicate a selective role of the cytokines in regulation of the barrier properties of the colon and a prominent role of IL10 in carcinogenesis in its proximal part.

1. Introduction

The large intestine is characterized by morphological and functional segmental heterogeneity [1,2,3]. The specificity of its two main different parts—the proximal and the distal colon, is manifested in many aspects, particularly, in development of right- or left-side colorectal carcinogenesis (CRC) [4,5,6,7,8,9,10,11,12,13]. Tumors more often appears in the proximal colon in adults [14], whereas in adolescents and young adults these are predominantly located in the distal colon [15]. A precancerous lesion of CRC as serrated adenoma was predominantly found in the distal part of the colon [16].
The most adequate to human CRC model of experimental carcinogenesis is 1,2-dymethylhydrazine (DMH)-induced CRC. DMH is a pro-carcinogenic agent that is activated in the liver and transported to the intestine by bile and blood. The mechanism of action of DMH is associated primarily with DNA methylation of the stem colonocytes which are located at the base of the intestinal crypts, with the subsequent development of colon adenocarcinomas [17,18,19]. The majority of colon tumors in the DMH-rats are located in the distal colon, while are less frequent in the proximal colon [17,18,20]. The cause of this preference remains unclear.
One of the reasons for the development of CRC is considered to be an imbalance between pro- and anti-inflammatory cytokines [21]. Pre-neoplastic lesions under DMH carcinogenesis may be connected with an increase in pro-inflammatory cytokines, especially tumor necrosis factor alpha (TNFα) [22]. It can be assumed that the different incidences of the colon may be associated with heterogeneity of pro- and anti-inflammatory cytokine production. The first group includes TNFα, while the second group includes interleukin 10 (IL10). There is no data available regarding the concentration of the cytokines in intestinal tissue in DMH-induced carcinogenesis.
CRC is accompanied by destroying the interactions between neighboring epithelial cells, cell dedifferentiation, loss of polarity, and metastasis. Impairment of the barrier properties of the colon and changes in the molecular composition of tight junctions (TJs) may lead to disturbance of the epithelial integrity [23].
TJs are composed of various transmembrane proteins, which belong to the MARVEL protein family, such as occludin and tricellulin, and the claudin family, which is divided in two groups according to their contribution to paracellular permeability [24]. Whereas some claudins as claudin-1, -3, -4 reduce epithelial permeability [25], others, particularly claudin-2, form pores and increase paracellular permeability for ions, water, and macromolecules [26]. One of the functions of TJs is to combine epithelial cells into unified sheets and maintain the integrity of the epithelial layer [27]. Under conditions of possible disintegration of TJs, the expression of claudins changes [28]. Therefore, one of the features of carcinogenesis, in particular in the colon, is a change in the expression of TJ proteins [29,30,31,32]. The physiological parameter for analysis of the destroying of the epithelial barrier function, consider the change in transepithelial resistance (TEER), short-circuit current (Isc) and paracellular permeability to some macromolecules such as sodium fluorescein [33].
Previously we have shown that DMH changed the intestinal permeability and induced alteration in expression of TJ proteins in the rat colon and IPEC-J2 cells [34,35]. We hypothesized that different incidence of the colon may be due to the different effects of the cytokines in carcinogenesis. TNFα decreased barrier properties of the colon ex vivo in Ussing chambers [36] and monolayers of epithelial cells in vitro [37,38,39,40]. IL10, on the contrary, had a protective effect, preventing disruption of the intestinal barrier function [41,42] and, perhaps, IL10 prevents the development of tumors in the proximal colon. Yet, the effects of cytokines on the barrier properties of the proximal and the distal parts of the colon have not been studied before. The aim of this study was to analyze the levels of TNFα and IL10 in the different segments of rat colon in DMH-induced carcinogenesis and to investigate the barrier properties of the main parts of the colon after intraluminal incubation with the cytokines in healthy rats.

2. Results

2.1. Study of the Level of TNFα and IL10 in Segments of Different Parts of the Colon during DMH-Induced Carcinogenesis

In the colon tissues, TNFα was different significantly only in the distal part. It was lower in the tumors compared to the control and tumor-adjacent tissues (Figure 1A). In comparison, IL10 was different only in the proximal part of the colon, where it was higher in tumors and non-tumor tissues compared to the control (Figure 1B).

2.2. Study of TEER, Isc and Paracellular Permeability to Sodium Fluorescein in the Proximal and Distal Parts of the Colon

The incubation with TNFα revealed an increase in paracellular permeability with a more pronounced effect in the distal part compared to controls (from 0.6 ± 0.1 to 1.4 ± 0.3·10−4 cm/s and 1.5 ± 0.3 to 4.5 ± 0.4·10−4 cm/s accordingly) (Figure 2A). TEER did not differ significantly compared to the control. Isc increased (from 15 ± 2 to 25 ± 3 µA/cm2) only in the distal part of the colon (Figure 2C).
In the proximal colon, incubation with IL10 revealed an increase in paracellular permeability (from 0.6 ± 0.1·to 1.2 ± 0.2·10−4 cm/s) (Figure 2A) and a decrease in TEER (from 170 ± 18 to 118 ± 12 Ω·cm2) (Figure 2B), whereas Isc did not show a significant change compared to the control.

2.3. Western Blotting in the Colon Tissues

The expression of TJ proteins, namely claudin-1, -2, -3, -4, occludin and tricellulin, after incubation with the cytokines was determined by immunoblotting, revealing that the level of TJ proteins significantly changed only in the proximal part of the colon (Figure 3). There were not significant differences in the distal part (Figure 4). TNFα reduced the level of claudin-1 (55 ± 22% vs. control, n = 4), while IL10 reduced the level of claudin-1 (32 ± 16% vs. control, n = 4), claudin-2 (36 ± 13% vs. control, n = 4) and claudin-4 (47 ± 17% vs. control, n = 4). At the same time IL10 increased the level of occludin (367 ± 209% vs. control, n = 4) (Figure 3). Other TJ proteins, namely, claudin-3 and tricellulin, did not show significant differences in all studied groups.
Thus, the cytokines induced a change in TJ proteins level only in the proximal part of the colon, and IL10 showed the most pronounced effect.

3. Discussion

Previously, it was found that the tumor-adjacent tissues in DMH-rats were characterized by a high paracellular permeability [34]. We hypothesized that these changes might be induced by cytokines. Therefore, we chose pro-inflammatory TNFα and anti-inflammatory IL10, which play opposite roles in the development of CRC, as well as regarding participation in the regulation of the barrier properties of the colon [36,37,38,39,40,41,42].
The role of cytokines in the development of CRC is ambiguous. An amount of TNFα in human blood serum was negatively correlated with proliferation of tumor cells [43]; however, increased circulating TNFα was associated with poor overall and cancer-specific patient survival [44]. In contrast, the level of IL10 has been shown to be positive correlated with the intensity of tumor proliferation and apoptosis [43]. However, it decreased in colon tissues with increased tumor invasion and lesion of lymph nodes at the late stages of CRC, corresponding to poor patient survival [45].
Most authors indicate that the concentration of TNFα in human blood serum in CRC is increased compared to the control [46,47,48,49,50]. The concentration of circulating TNFα was significantly higher compared to the control at all stages of CRC and showed highest values at the last, IV stage [46]. Expression of TNFα mRNA in tumor was significantly higher than in neighboring tissues [51]. Some authors reported that the level of IL10 was increased in human blood serum in CRC [49,50,52], while others showed that its concentration remained practically unchanged [53].
We investigated the cytokines level in the segments of the colon in DMH-rats in accordance with the mapping scheme we used earlier [34]. The study of the level of the cytokines into three types of segments—tumors, adjacent to tumors, and not adjacent to tumors, allowed us to analyze their concentration in the entire organ in detail, making a note of the possible influence in the tumor and tumor microenvironment on the colon tissues.
The level of TNFα was changed only in the distal part of the colon, it was lower in tumors compared to the control and tumor-adjacent tissues. We suppose that these results are explained by the features of our experimental model. Increased production of TNFα is usually associated with inflammation, and TNFα is a key cytokine that links inflammation and carcinogenesis [54]. DMH-induced carcinogenesis is not the direct result of an inflammatory process. It is believed that in the distal colon, histogenesis follows aberrant crypt foci-adenoma-carcinoma sequences, while in the proximal colon, carcinomas arise de novo without an intermediate stage of colon carcinogenesis [17]. Taking into account the inhibitory effect of TNFα on tumor cell proliferation [43] and that its pro-tumorigenic properties may rather be invoked by low chronic TNFα production than by an intensive outburst that activates reactive oxygen species and kills malignant cells [55], reduced concentration of TNFα in the tumors should have promoted tumor growth in the distal part of the colon. In fact, our hypothesis about the potential role of TNFα in the impairment of barrier properties of the colon and the possible negative effect of the tumor on neighboring tissues through the production of TNFα in DMH-induced carcinogenesis was not confirmed. That does not exclude its possible role in the disturbance of the barrier function with other types of CRC.
In our study IL10 was higher compared to the control in tumors, tumor-adjacent and not tumor-adjacent segments in the proximal part of the colon. It was most pronounced in the proximal not tumor-adjacent segments that significantly differed from the distal not tumor-adjacent segments. Thus, IL10 was significantly increased in all the segments of the proximal part of the colon in DMH-rats with right-side carcinogenesis. The studies revealed that IL10 can be a protective factor in animal CRC models [56,57]. Based on the fact that a decrease in IL10 in cancerous tissue is an independent risk factor for poor survival [45], elevated IL10 could have a protective effect in CRC. However, given that cytokines can play a dual role in tumor development—they can either participate in the suppression of carcinogenesis, or contribute to its progress [58], increased IL10 associated with tumors in the proximal part of the colon where tumors develop only in 20–25% of cases of CRC, could stimulate their development. Some studies suggest that IL10 serum levels are lower in the control group than in the CRC patients [59]. Patients in the fourth clinical stage of CRC have a higher level of serum IL10 when compared to lower stages, while a high serum concentration of IL10 correlates with poor survival of patients with CRC [60,61]. Additionally, IL10 overexpression was positively correlated with metastasis occurrence [62].
Previously, we conducted a segmental analysis of the barrier properties of the colon [63]. It was shown that the barrier properties were more pronounced in the proximal part of the colon compared to the distal part. We assume that the heterogeneity of the barrier properties of the colon and different effects of cytokines which are elevated in pathologies may determine how pathological processes develop and contribute to different incidences of CRC in two main parts of the colon.
Based on the literature, most studies of the barrier properties of the intestinal epithelium under cytokines action were carried out in vitro on cell cultures, with stimulation mainly from the basolateral side of the epithelium [37,64,65]. We stimulated the colon in situ from the apical side of the epithelium according to the approach we successfully used in our previous study [63]. We supposed that the addition of the cytokines to the mucosal side ensured their prolonged action and prevented their rapid enzymatic degradation by tissue cells.
We have shown that the four-hour prolonged action of cytokines on the colon of healthy rats led to the changes in the parameters of its barrier properties—a decrease in TEER, an increase in Isc, and an increase in paracellular permeability of the colon. Thus, under the action of TNFα and IL10 on the apical side of the epithelium, the barrier properties of the colon were reduced.
Regarding the main hypothesis that we checked, whether cytokines have the same regulatory effect on different parts of the colon, we analyzed barrier properties of the segments taken from its proximal and distal part. It was shown that TNFα changed the barrier properties mainly of the distal part of the colon, and IL10—only of the proximal one.
TJ proteins only showed changes in the proximal colon: TNFα reduced claudin-1, while IL10 decreased claudin-1, -2, -4, and increased occludin, demonstrating the most pronounced effect. The decrease of claudin-1 and -4, which increases the barrier properties of the colon, is consistent with the data obtained in Ussing chambers—an increase in paracellular permeability under the action of TNFα and IL10 and a decrease in TEER under the action of IL10 for the proximal colon segments. At the same time, increased occludin and decreased claudin-2 indicated an increase in the barrier properties of the proximal part of the colon and indicated a different effect of IL10 on the expression of TJ proteins in this region. Changes in TJ proteins expression only in the proximal part of the colon could indicate a greater sensitivity of this region to the action of cytokines and its ability to more pronounced molecular rearrangements.
Data on low TNFα in tumors in the distal part of the colon indicated that TNFα was probably not involved in the impairment of the barrier properties of the colon in DMH-induced carcinogenesis. Increased IL10 in the proximal segments of the colon of DMH-rats with proximal tumor location indicated that IL10 could play an important role in regulating the barrier properties in this colon segment. IL10 decreased the barrier properties and contributed to the restructuring of the TJ proteins only in the proximal part of the colon. Thus, IL10 altered intercellular interactions and could influence the development of CRC in the proximal part of the colon.
Our results have provided following novel findings: (1) cytokines have selective effects on the regulation of barrier properties of the colon: TNFα decreases barrier properties mainly in the distal part of the colon, while IL10 decreases the barrier properties only in the proximal one, (2) IL10 plays a prominent role in regulation of carcinogenesis and barrier function of the proximal part of the colon and may contribute to different incidences of CRC in the proximal and distal parts of the colon.
The heterogeneity of the cytokines production during cancer development and the selective effects of the cytokines on barrier properties of the proximal and the distal parts of the colon may contribute to different involvement of the colon in CRC.

4. Materials and Methods

4.1. Animals

Male Wistar rats for Experiment 1 were obtained from the Animal Laboratory of the I.P. Pavlov Institute of Physiology (Experiment 1, below: 5.1.) and from the vivarium of St. Petersburg University (Experiment 2, below: 5.2.). They were kept under a standard light/dark cycle (12 h light:12 h dark) at 22 ± 2 °C with ad libitum access to tap water and complete pelleted feed (Delta Feeds, BioPro, Novosibirsk, Russia). The studies were carried out in accordance with the guidelines of the FELASA [66] and approved by the Ethics Committee for Animal Research of St. Petersburg State University (Conclusion No. 131-03-1 dated 2 February 2021).

4.2. Chemicals

The cytokines were obtained from Sigma-Aldrich (Taufkirchen, Germany): TNFα from rat, recombinant, expressed in E. coli, IL10 human, recombinant, expressed in HEK 293 cells, HumanKine®.

5. Experimental Design

5.1. Experiment 1

Animals weighing 120–150 g (n = 20) were randomly subdivided into 2 groups—a control group (n = 6) and an experimental group (n = 14).
The rats in the control group were not exposed to the carcinogen, whereas the rats in the experimental group were administered 5 subcutaneous injections of DMH weekly at 21 mg/kg of body weight (each dose). DMH was obtained from Sigma-Aldrich (Tokyo, Japan). Six months after the first carcinogen injection, the rats were decapitated with a guillotine (Open Science, Moscow, Russia).
Mapping of the colon segments in the experimental group of DMH-induced rats depended on tumor location and was carried out according to the previously described method [34] (Figure 5).

Study of TNFα and IL10 in Colon Tissues in DMH-Rats by Enzyme Immunoassay

The colon segments were immediately frozen at −80 °C and stored at this temperature until analysis. A total of 50–60 mg colon tissue samples were homogenized in 1 mL RIPA buffer with protease inhibitors (150 mM NaCl; 10 mM Tris-HCl, pH 7.4; 0.5% Triton X-100; 0.1% SDS), the suspension was sonicated and centrifuged (10,000× g, 5 min at 4 °C). In the supernatants, the concentration of total protein was determined. The cytokines in the samples were determined following the manufacturers’ instructions by sandwich ELISA and commercial reagent kits (High Sensitive ELISA Kit for IL10 and TNFα, Cloud-Clone Corp., Wuhan, China) using spectrophotometer SPECTROstar Nano (BMG LABTECH, Ortenberg, Germany). TNFα and IL10 concentration values were recalculated per mg of total protein.

5.2. Experiment 2

Animals weighing 280–380 g (n = 20) were randomly subdivided into four groups—one control group and three experimental groups with five animals in each group. The rats were anesthetized intraperitoneally with Zoletil 100 (Virbac, Carros, France, 100 mg/kg of the body weight). Narcosis was verified by the disappearance of the reaction to a painful stimulus (tail prick). An incision was made along the midline of the abdomen from the processus xiphoideus of the sternum in the distal direction. Two ligatures isolated the colon loop between the cecum and the anus, thus limiting the colon. Two tubes were inserted at the beginning and at the end of the loop, and the system was filled hermetically with the test solution. In the control group, the loop was luminally filled with Krebs-Ringer’s solution, while in the experimental groups it was filled with one of the cytokine solutions with 200 ng/mL TNFα and 100 ng/mL IL10. After 4 h of incubation the rats were decapitated, the large intestine was divided into proximal and distal segments, as described in our previous studies [34] and then investigated in Ussing chambers for 1 h (Figure 6).

5.2.1. Electrophysiological Assay of the Colon Segments

Isc and TEER of the large intestine wall were studied according to the previously described protocol [67]. Briefly, the segments of the large intestine were mounted in Ussing chambers filled with Krebs-Ringer solution at 37 °C, which was maintained throughout the experiment, and permanently oxygenated with a mixture of 95% oxygen and 5% carbon dioxide. The Krebs-Ringer solution was composed as follows (mM): NaCl (119), KCl (5), MgCl2∙6H2O (1.2), NaHCO3 (25), NaH2PO4∙H2O (0.4), Na2HPO4∙7H2O (1.6), CaCl2 (1.2), and D-glucose (10). The short circuit current was recorded when the voltage was set at 0 mV. To evaluate TEER, we recorded voltage fluctuations when the current was 10 μA and calculated it using Ohm’s law: R = U/I (Ω). The size of the examined tissue was calculated by using the diameter of the slotted opening between the two chamber halves (4 mm) and was equal to 0.13 cm2. With regard to the size of the examined tissue, we adjusted the obtained transepithelial voltage values for 1 cm2 of tissue (Ω·cm2).

5.2.2. Assessment of the Paracellular Permeability of the Colon Segments

To study the paracellular permeability of the intestine in the Ussing chamber, we added sodium fluorescein to the mucosal bathing solution at a final concentration of 100 μM. This concentration was determined from earlier published reports [68,69]. Thirty minutes after the experiment started, the serosal bathing solution was removed to analyze the concentration of the diffused sodium fluorescein. To assess the optical density of this solution, we used Cary Eclipse Fluorescence Spectrophotometer (Agilent Technologies, CA, USA). The excitation and emission wavelengths were 460 and 515 nm, respectively. The permeability coefficient (Papp, cm/s) was calculated using the following equitation: Papp = (dQ/dt)/(A·C0), with: dQ/dt as the concentration of sodium fluorescein in the serosal bathing solution (mol/s), A as the size of the examined tissue (cm2), and C0 as the concentration of sodium fluorescein in the mucosal bathing solution.

5.2.3. Western Blotting of Colon Tissues

TJ protein levels were analyzed in the colon segments, as described in detail earlier [70], and stain-free immunoblotting was performed, as described previously [71,72,73].
Briefly, tissues were homogenized in RIPA buffer (150 mM NaCl; 10 mM Tris-HCl, pH 7.4; 0.5% Triton X-100; 0.1% SDS) with protease inhibitor (Complete ULTRA Tablets, Mini; Roche, Mannheim, Germany), then centrifuged (15 min, 15,000× g, 4 °C), and a quantitative protein analysis using Thermo BCA assay kit (Thermo Fisher Scientific, Waltham, MA, USA) was performed by a spectrophotometer SPECTROstar Nano (BMG Labtech, Ortenberg, Germany). SDS buffer (Laemmli) was added to extracted proteins, and samples were loaded on 10% Stain-Free gels and electrophoresis was performed. Proteins from the gels were transferred to PVDF membranes with a 0.2 µm pore size (Bio-Rad, Hercules, CA, USA), which were first incubated with primary antibodies raised against claudin-1, -2, -3, -4, occludin or tricellulin and then visualized using secondary goat anti-rabbit and anti-mouse IgG antibodies and chemiluminescence reaction (Bio-Rad). The following antibodies were used: claudin-1 (#51-9000, Invitrogen, Carlsbad, CA, USA), claudin-2 (#32-5600, Invitrogen), claudin-3 (#34-1700, Invitrogen), claudin-4 (#36-4800, Invitrogen), occludin (#DF7504, Affinity Biosciences, Cincinnati, OH, USA), tricellulin (#48-8400, Invitrogen). The protein bands were detected and identified using Clarity Western ECL Substrate and the ChemiDoc XRS+ imager (Bio-Rad). Normalization of detected proteins was performed using the Image Lab 6.1 Software (Bio-Rad) to the total protein load measured in the membrane. The signal density in the control group was set as 100% (Supplementary Materials).

5.3. Statistical Analysis

Statistical analysis was performed using the Anova group analysis in GraphPad Prism 8.4.3 (Graphpad Software Inc., San Diego, CA, USA). The data were analyzed using Mann–Whitney U-test. The results of the analyses are presented as mean ± standard error (M ± SEM). Statistically reliable differences were reported with a probability value of 95% (p < 0.05).

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms232415610/s1, Figure S1: Original images.

Author Contributions

Conceptualization, A.G.M. and V.B.; methodology, A.G.M. and V.B.; investigation, V.B., T.Z., I.F. and A.F.; writing—original draft preparation, V.B. and T.Z.; writing—review and editing, S.A. and A.G.M.; supervision, A.G.M.; funding acquisition, S.A. and A.G.M. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by a grant from the Russian Foundation for Basic Research (Grant No. 20-04-01050) and the German Research Association (Grant No. AM141/11-2).

Institutional Review Board Statement

The studies were approved by the Ethics Committee for Animal Research of St. Petersburg State University (Conclusion No. 131-03-1 dated 2 February 2021).

Informed Consent Statement

Not applicable.

Data Availability Statement

Date is contained in the article. The datasets analyzed in the study are available from the corresponding author upon reasonable request.

Acknowledgments

Scientific research was performed using the Research Park of St. Petersburg State University “Center for Molecular and Cell Technologies”. We are grateful to Irina A. Lekomtseva, Department of English Philology and Translation, St Petersburg University, for help during translation of this article.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Mirakhur, M.; Diener, M. Proteinase-activated receptors regulate intestinal functions in a segment-dependent manner in rats. Eur. J. Pharmacol. 2022, 933, 175264. [Google Scholar] [CrossRef]
  2. Garg, S.; Zheng, J.; Wang, J.; Authier, S.; Pouliot, M.; Hauer-Jensen, M. Segmental differences in radiation-induced alterations of tight junction-related proteins in non-human primate jejunum, ileum and colon. Radiat. Res. 2016, 185, 50–59. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  3. Markov, A.; Veshnyakova, A.; Fromm, M.; Amasheh, M.; Amasheh, S. Segmental expression of claudin proteins correlates with tight junction barrier properties in rat intestine. J. Comp. Physiol. B-Bioch. Syst. Envir. Physiol. 2010, 180, 591–598. [Google Scholar] [CrossRef] [PubMed]
  4. Lee, M.; Menter, D.; Kopetz, S. Right Versus left colon cancer biology: Integrating the consensus molecular subtypes. J. Natl. Compr. Canc. Netw. 2017, 15, 411–419. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  5. Mendis, S.; Beck, S.; Lee, B.; Lee, M.; Wong, R.; Kosmider, S.; Shapiro, J.; Yip, D.; Steel, S.; Nott, L.; et al. Right versus left sided metastatic colorectal cancer: Teasing out clinicopathologic drivers of disparity in survival. Asia Pac. J. Clin. Oncol. 2019, 15, 136–143. [Google Scholar] [CrossRef]
  6. Testa, U.; Pelosi, E.; Castelli, G. Colorectal cancer: Genetic abnormalities, tumor progression, tumor heterogeneity, clonal evolution and tumor-initiating cells. Med. Sci. 2018, 6, 31. [Google Scholar] [CrossRef] [Green Version]
  7. Miyake, T.; Mori, H.; Yasukawa, D.; Hexun, Z.; Maehira, H.; Ueki, T.; Kojima, M.; Kaida, S.; Iida, H.; Shimizu, T.; et al. The comparison of fecal microbiota in left-side and right-side human colorectal cancer. Eur. Surg. Res. 2021, 62, 248–254. [Google Scholar] [CrossRef]
  8. Sugai, T.; Habano, W.; Jiao, Y.; Tsukahara, M.; Takeda, Y.; Otsuka, K.; Nakamura, S. Analysis of molecular alterations in left- and right-sided colorectal carcinomas reveals distinct pathways of carcinogenesis: Proposal for new molecular profile of colorectal carcinomas. J. Mol. Diagn. 2006, 8, 193–201. [Google Scholar] [CrossRef] [Green Version]
  9. Patel, M.; McSorley, S.; Park, J.; Roxburgh, C.; Edwards, J.; Horgan, P.; McMillan, D. The relationship between right-sided tumour location, tumour microenvironment, systemic inflammation, adjuvant therapy and survival in patients undergoing surgery for colon and rectal cancer. Br. J. Cancer 2018, 118, 705–712. [Google Scholar] [CrossRef] [Green Version]
  10. Natsume, S.; Yamaguchi, T.; Takao, M.; Iijima, T.; Wakaume, R.; Takahashi, K.; Matsumoto, H.; Nakano, D.; Horiguchi, S.; Koizumi, K.; et al. Clinicopathological and molecular differences between right-sided and left-sided colorectal cancer in Japanese patients. Jpn. J. Clin. Oncol. 2018, 48, 609–618. [Google Scholar] [CrossRef]
  11. Mesa, H.; Manivel, J.; Larson, W.; Dachel, S.; Reinink, A.; Jessurun, J. Immunophenotypic comparison of neoplasms of the appendix, right colon, and left colon in search of a site-specific phenotypic signature. Int. J. Surg. Pathol. 2020, 28, 20–30. [Google Scholar] [CrossRef]
  12. Kanno, H.; Miyoshi, H.; Yoshida, N.; Sudo, T.; Nakashima, K.; Takeuchi, M.; Nomura, Y.; Seto, M.; Hisaka, T.; Tanaka, H.; et al. Differences in the immunosurveillance pattern associated with DNA mismatch repair status between right-sided and left-sided colorectal cancer. Cancer Sci. 2020, 111, 3032–3044. [Google Scholar] [CrossRef]
  13. Guo, D.; Li, X.; Xie, A.; Cao, Q.; Zhang, J.; Zhang, F.; Li, W.; Chen, J. Differences in oncological outcomes and inflammatory biomarkers between right-sided and left-sided stage I-III colorectal adenocarcinoma. J. Clin. Lab. Anal. 2020, 34, e23132. [Google Scholar] [CrossRef]
  14. Siegel, R.; Miller, K.; Goding Sauer, A.; Fedewa, S.; Butterly, L.; Anderson, J.; Cercek, A.; Smith, R. Colorectal cancer statistics, 2020. CA Cancer J. Clin. 2020, 70, 145–164. [Google Scholar] [CrossRef] [Green Version]
  15. Salem, M.; Battaglin, F.; Goldberg, R.; Puccini, A.; Shields, A.; Arguello, D.; Korn, W.; Marshall, J.; Grothey, A.; Lenz, H. Molecular analyses of left- and right-sided tumors in adolescents and young adults with colorectal cancer. Oncologist 2020, 25, 404–413. [Google Scholar] [CrossRef] [Green Version]
  16. Zhao, X.; Dou, L.; Zhang, Y.; Liu, Y.; He, S.; Ke, Y.; Liu, X.; Liu, Y.; Wang, G. Clinicopathological features of the colorectal serrated adenoma and analysis on influencing factors of malignancy. Zhonghua Wei Chang Wai Ke Za Zhi 2021, 24, 75–80. [Google Scholar] [CrossRef]
  17. Perse, M.; Cerar, A. Morphological and molecular alterations in 1,2 dimethylhydrazine and azoxymethane induced colon carcinogenesis in rats. J. Biomed. Biotechnol. 2011, 2011, 473964. [Google Scholar] [CrossRef]
  18. Venkatachalam, K.; Vinayagam, R.; Anand, M.; Isa, N.; Ponnaiyan, R. Biochemical and molecular aspects of 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis: A review. Toxicol. Res. 2020, 9, 2–18. [Google Scholar] [CrossRef]
  19. De-Souza, A.; Costa-Casagrande, T. Animal models for colorectal cancer. Abcd-Arq. Bras. Cir. Dig. Braz. Arch. Dig. Surg. 2018, 31, e1369. [Google Scholar] [CrossRef]
  20. Bekusova, V.; Patsanovskii, V.; Nozdrachev, A.; Trashkov, A.; Artemenko, M.; Anisimov, V. Metformin prevents hormonal and metabolic disturbances and 1,2-dimethylhydrazine-induced colon carcinogenesis in non-diabetic rats. Cancer Biol. Med. 2017, 14, 100–107. [Google Scholar] [CrossRef]
  21. Borowczak, J.; Szczerbowski, K.; Maniewski, M.; Kowalewski, A.; Janiczek-Polewska, M.; Szylberg, A.; Marszałek, A.; Szylberg, Ł. The Role of inflammatory cytokines in the pathogenesis of colorectal carcinoma-recent findings and review. Biomedicines 2022, 10, 1670. [Google Scholar] [CrossRef]
  22. Umesalma, S.; Sudhandiran, G. Differential inhibitory effects of the polyphenol ellagic acid on inflammatory mediators NF-kappa B, iNOS, COX-2, TNF-alpha, and IL-6 in 1,2-dimethylhydrazine-induced rat colon carcinogenesis. Bas. Clinic. Pharm. Toxicol. 2010, 107, 650–655. [Google Scholar] [CrossRef]
  23. Tsukita, S.; Tanaka, H.; Tamura, A. The claudins: From tight junctions to biological systems. Trends Biochem. Sci. 2019, 44, 141–152. [Google Scholar] [CrossRef]
  24. Gunzel, D.; Fromm, M. Claudins and other tight junction proteins. Compr. Physiol. 2012, 2, 1819–1852. [Google Scholar] [CrossRef]
  25. Gunzel, D.; Yu, A. Claudins and the modulation of tight junction permeability. Physiol. Rev. 2013, 93, 525–569. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  26. Amasheh, S.; Meiri, N.; Gitter, A.; Schöneberg, T.; Mankertz, J.; Schulzke, J.; Fromm, M. Claudin-2 expression induces cation-selective channels in tight junctions of epithelial cells. J. Cell Sci. 2002, 115, 4969–4976. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  27. Markov, A.; Aschenbach, J.; Amasheh, S. The epithelial barrier and beyond: Claudins as amplifiers of physiological organ functions. Iubmb. Life 2017, 69, 290–296. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  28. Kruglova, N.; Razgovorova, I.; Amasheh, S.; Markov, A. Accumulation of milk increases the width of tight junctions in the epithelium of mouse mammary alveoli. Biol. Comm. 2020, 65, 277–280. [Google Scholar] [CrossRef]
  29. Landy, J.; Ronde, E.; English, N.; Clark, S.; Hart, A.; Knight, S.; Ciclitira, P.; Al-Hassi, H. Tight junctions in inflammatory bowel diseases and inflammatory bowel disease associated colorectal cancer. World J. Gastroenterol. 2016, 22, 3117–3126. [Google Scholar] [CrossRef]
  30. Barmeyer, C.; Fromm, M.; Schulzke, J. Active and passive involvement of claudins in the pathophysiology of intestinal inflammatory diseases. Pflug. Arch. Europ. J. Physiol. 2017, 469, 15–26. [Google Scholar] [CrossRef]
  31. Wang, X.; Tully, O.; Ngo, B.; Zitin, M.; Mullin, J. Epithelial tight junctional changes in colorectal cancer tissues. Sci. World J. 2011, 11, 826–841. [Google Scholar] [CrossRef] [Green Version]
  32. Turksen, K.; Troy, T. Junctions gone bad: Claudins and loss of the barrier in cancer. Biochim. Et Biophys. Acta-Rev. Cancer 2011, 1816, 73–79. [Google Scholar] [CrossRef]
  33. Thomson, A.; Smart, K.; Somerville, M.; Lauder, S.; Appanna, G.; Horwood, J.; Raj, L.; Srivastava, B.; Durai, D.; Scurr, M.; et al. The Ussing chamber system for measuring intestinal permeability in health and disease. BM. Gastroenterol. 2019, 19, 1–14. [Google Scholar] [CrossRef] [Green Version]
  34. Bekusova, V.; Falchuk, E.; Okorokova, L.; Kruglova, N.; Nozdrachev, A.; Markov, A. Increased paracellular permeability of tumor-adjacent areas in 1,2-dimethylhydrazine-induced colon carcinogenesis in rats. Cancer Biol. Med. 2018, 15, 251–259. [Google Scholar] [CrossRef]
  35. Bekusova, V.; Droessler, L.; Amasheh, S.; Markov, A. Effects of 1,2-dimethylhydrazine on barrier properties of rat large intestine and IPEC-J2 cells. Int. J. Mol. Sci. 2021, 22, 10278. [Google Scholar] [CrossRef]
  36. Amasheh, M.; Grotjohann, I.; Amasheh, S.; Fromm, A.; Soderholm, J.; Zeitz, M.; Fromm, M.; Schulzke, J. Regulation of mucosal structure and barrier function in rat colon exposed to tumor necrosis factor alpha and interferon gamma in vitro: A novel model for studying the pathomechanisms of inflammatory bowel disease cytokines. Scand. J. Gastroenterol. 2009, 44, 1226–1235. [Google Scholar] [CrossRef]
  37. Al-Sadi, R.; Guo, S.; Ye, D.; Ma, T. TNF-alpha modulation of intestinal epithelial tight junction barrier is regulated by ERK1/2 activation of Elk-1. Am. J. Pathol. 2013, 183, 1871–1884. [Google Scholar] [CrossRef] [Green Version]
  38. He, F.; Peng, J.; Deng, X.; Yang, L.; Camara, A.; Omran, A.; Wang, G.; Wu, L.; Zhang, C.; Yin, F. Mechanisms of tumor necrosis factor-alpha-induced leaks in intestine epithelial barrier. Cytokine 2012, 59, 264–272. [Google Scholar] [CrossRef]
  39. Khan, M.; Uwada, J.; Yazawa, T.; Islam, M.; Krug, S.; Fromm, M.; Karaki, S.; Suzuki, Y.; Kuwahara, A.; Yoshiki, H.; et al. Activation of muscarinic cholinoceptor ameliorates tumor necrosis factor-α-induced barrier dysfunction in intestinal epithelial cells. FEBS Lett. 2015, 589, 3640–3647. [Google Scholar] [CrossRef] [Green Version]
  40. Droessler, L.; Cornelius, V.; Markov, A.; Amasheh, S. Tumor necrosis factor alpha effects on the porcine intestinal epithelial barrier include enhanced expression of TNF receptor 1. Int. J. Mol. Sci. 2021, 22, 8746. [Google Scholar] [CrossRef]
  41. Sun, X.; Yang, H.; Nose, K.; Nose, S.; Haxhija, E.; Koga, H.; Feng, Y.; Teitelbaum, D. Decline in intestinal mucosal IL-10 expression and decreased intestinal barrier function in a mouse model of total parenteral nutrition. Am. J. Physiol. Gastrointest Liver Physiol. 2008, 294, G139–G147. [Google Scholar] [CrossRef] [PubMed]
  42. Al-Sadi, R.; Boivin, M.; Ma, T. Mechanism of cytokine modulation of epithelial tight junction barrier. Front. Biosci.-Landmark 2009, 14, 2765–2778. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  43. Evans, C.; Morrison, I.; Heriot, A.; Bartlett, J.; Finlayson, C.; Dalgleish, A.; Kumar, D. The correlation between colorectal cancer rates of proliferation and apoptosis and systemic cytokine levels; plus their influence upon survival. Br. J. Cancer 2006, 94, 1412–1419. [Google Scholar] [CrossRef] [PubMed]
  44. Yu, Y.; Fan, C.; Tseng, W.; Chang, P.; Kuo, H.; Pan, Y.; Yeh, K. Correlation between the Glasgow prognostic score and the serum cytokine profile in Taiwanese patients with colorectal cancer. Int. J. Biol. Markers 2021, 36, 40–49. [Google Scholar] [CrossRef] [PubMed]
  45. Toiyama, Y.; Miki, C.; Inoue, Y.; Minobe, S.; Urano, H.; Kusunoki, M. Loss of tissue expression of interleukin-10 promotes the disease progression of colorectal carcinoma. Surg. Today 2010, 40, 46–53. [Google Scholar] [CrossRef]
  46. Stanilov, N.; Miteva, L.; Dobreva, Z.; Stanilova, S. Colorectal cancer severity and survival in correlation with tumour necrosis factor-alpha. Biotech. Equip. 2014, 28, 911–917. [Google Scholar] [CrossRef]
  47. Nikiteas, N.; Tzanakis, N.; Gazouli, M.; Rallis, G.; Daniilidis, K.; Theodoropoulos, G.; Kostakis, A.; Peros, G. Serum IL-6, TNFalpha and CRP levels in Greek colorectal cancer patients: Prognostic implications. World J. Gastroenterol. 2005, 11, 1639–1643. [Google Scholar] [CrossRef]
  48. Coşkun, Ö.; Öztopuz, Ö.; Özkan, Ö. Determination of IL-6, TNF-α and VEGF levels in the serums of patients with colorectal cancer. Cell Mol. Biol. 2017, 63, 97–101. [Google Scholar] [CrossRef] [Green Version]
  49. Pengjun, Z.; Xinyu, W.; Feng, G.; Xinxin, D.; Yulan, L.; Juan, L.; Xingwang, J.; Zhennan, D.; Yaping, T. Multiplexed cytokine profiling of serum for detection of colorectal cancer. Future Oncol. 2013, 9, 1017–1027. [Google Scholar] [CrossRef]
  50. Galizia, G.; Orditura, M.; Romano, C.; Lieto, E.; Castellano, P.; Pelosio, L.; Imperatore, V.; Catalano, G.; Pignatelli, C.; De Vita, F. Prognostic significance of circulating IL-10 and IL-6 serum levels in colon cancer patients undergoing surgery. Clin. Immunol. 2002, 102, 169–178. [Google Scholar] [CrossRef]
  51. Al Obeed, O.; Alkhayal, K.; Al Sheikh, A.; Zubaidi, A.; Vaali-Mohammed, M.; Boushey, R.; Mckerrow, J.; Abdulla, M. Increased expression of tumor necrosis factor-α is associated with advanced colorectal cancer stages. World J. Gastroenterol. 2014, 20, 18390–18396. [Google Scholar] [CrossRef]
  52. Czajka-Francuz, P.; Francuz, T.; Cisoń-Jurek, S.; Czajka, A.; Fajkis, M.; Szymczak, B.; Kozaczka, M.; Malinowski, K.; Zasada, W.; Wojnar, J.; et al. Serum cytokine profile as a potential prognostic tool in colorectal cancer patients–one center study. Rep. Pract. Oncol. Radiother. 2020, 25, 867–875. [Google Scholar] [CrossRef]
  53. Szkaradkiewicz, A.; Marciniak, R.; Chudzicka-Strugała, I.; Wasilewska, A.; Drews, M.; Majewski, P.; Karpiński, T.; Zwoździak, B. Proinflammatory cytokines and IL-10 in inflammatory bowel disease and colorectal cancer patients. Arch. Immunol. Ther. Exp. 2009, 57, 291–294. [Google Scholar] [CrossRef] [Green Version]
  54. Mager, L.; Wasmer, M.; Rau, T.; Krebs, P. Cytokine-induced modulation of colorectal cancer. Front. Oncol. 2016, 6, 96. [Google Scholar] [CrossRef] [Green Version]
  55. Monteleone, G.; Pallone, F.; Stolfi, C. The dual role of inflammation in colon carcinogenesis. Int. J. Mol. Sci. 2012, 13, 11071–11084. [Google Scholar] [CrossRef] [Green Version]
  56. Gounaris, E.; Blatner, N.; Dennis, K.; Magnusson, F.; Gurish, M.; Strom, T.; Beckhove, P.; Gounari, F.; Khazaie, K. T-regulatory cells shift from a protective anti-inflammatory to a cancer-promoting proinflammatory phenotype in polyposis. Cancer Res. 2009, 69, 5490–5497. [Google Scholar] [CrossRef] [Green Version]
  57. Krause, P.; Morris, V.; Greenbaum, J.; Park, Y.; Bjoerheden, U.; Mikulski, Z.; Muffley, T.; Shui, J.; Kim, G.; Cheroutre, H.; et al. IL-10-producing intestinal macrophages prevent excessive antibacterial innate immunity by limiting IL-23 synthesis. Nat. Commun. 2015, 6, 7055. [Google Scholar] [CrossRef] [Green Version]
  58. Shrihari, T. Dual role of inflammatory mediators in cancer. Ecancermedicalscience 2017, 11, 721. [Google Scholar] [CrossRef]
  59. Abtahi, S.; Davani, F.; Mojtahedi, Z.; Hosseini, S.; Bananzadeh, A.; Ghaderi, A. Dual association of serum interleukin-10 levels with colorectal cancer. J. Cancer Res. Ther. 2017, 13, 252–256. [Google Scholar] [CrossRef]
  60. Stanilov, N.; Miteva, L.; Stankova, N.; Jovchev, J.; Deliyski, T.; Stanilova, S. Role of IL-12P40 and IL-10 in progression of colorectal cancer. Khirurgiia 2010, 4, 26–29. [Google Scholar]
  61. Miteva, L.; Stanilov, N.; Deliysky, T.; Stanilova, S. Significance of -1082A/G polymorphism of IL10 gene for progression of colorectal cancer and IL-10 expression. Tumour Biol. 2014, 35, 12655–12664. [Google Scholar] [CrossRef]
  62. Townsend, M.; Felsted, A.; Piccolo, S.; Robison, R.; O’Neill, K. Metastatic colon adenocarcinoma has a significantly elevated expression of IL-10 compared with primary colon adenocarcinoma tumors. Cancer Biol. Ther. 2018, 19, 913–920. [Google Scholar] [CrossRef] [PubMed]
  63. Bekusova, V.; Fatyykhov, I.; Amasheh, S.; Markov, A. Heterogeneity of the barrier properties of the colon in rat. Biol. Comm. 2021, 66, 160–170. [Google Scholar] [CrossRef]
  64. Ma, T.; Boivin, M.; Ye, D.; Pedram, A.; Said, H. Mechanism of TNF-alpha modulation of Caco-2 intestinal epithelial tight junction barrier: Role of myosin light-chain kinase protein expression. Am. J. Physiol. Gastrointest. Liver Physiol. 2005, 288, G422–G430. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  65. Freour, T.; Jarry, A.; Bach-Ngohou, K.; Dejoie, T.; Bou-Hanna, C.; Denis, M.; Mosnier, J.; Laboisse, C.; Masson, D. TACE inhibition amplifies TNF-alpha-mediated colonic epithelial barrier disruption. Int. J. Mol. Med. 2009, 23, 41–48. [Google Scholar] [CrossRef] [PubMed]
  66. Mahler, M.; Berard, M.; Feinstein, R.; Gallagher, A.; Illgen-Wilcke, B.; Pritchett-Corning, K.; Raspa, M. FELASA recommendations for the health monitoring of mouse, rat, hamster, guinea pig and rabbit colonies in breeding and experimental units. Lab. Anim. 2015, 49, 88. [Google Scholar] [CrossRef] [Green Version]
  67. Markov, A.; Falchuk, E.; Kruglova, N.; Radloff, J.; Amasheh, S. Claudin expression in follicle-associated epithelium of rat Peyer’s patches defines a major restriction of the paracellular pathway. Acta Physiol. 2016, 216, 112–119. [Google Scholar] [CrossRef] [Green Version]
  68. Zakelj, S.; Legen, I.; Veber, M.; Kristl, A. The influence of buffer composition on tissue integrity during permeability experiments “in vitro”. Int. J. Pharm. 2004, 272, 173–180. [Google Scholar] [CrossRef]
  69. Molenda, N.; Urbanova, K.; Weiser, N.; Kusche-Vihrog, K.; Gunzel, D.; Schillers, H. Paracellular transport through healthy and cystic fibrosis bronchial epithelial cell lines–Do we have a proper model? PLoS ONE 2014, 9, e100621. [Google Scholar] [CrossRef] [Green Version]
  70. Amasheh, S.; Milatz, S.; Krug, S.M.; Bergs, M.; Arnasheh, M.; Schulzke, J.; Fromm, M. “Na+ absorption defends from paracellular back-leakage by claudin-8 upregulation. Biochem. Biophys. Res. Comm. 2009, 378, 45–50. [Google Scholar] [CrossRef]
  71. Stein, L.; Brunner, N.; Amasheh, S. Functional analysis of gastric tight junction proteins in Xenopus laevis oocytes. Membranes 2022, 12, 731. [Google Scholar] [CrossRef]
  72. Droessler, L.; Cornelius, V.; Boehm, E.; Stein, L.; Brunner, N.; Amasheh, S. Barrier perturbation in porcine Peyer’s patches by tumor necrosis factor is associated with a dysregulation of claudins. Front. Physiol. 2022, 13, 889552. [Google Scholar] [CrossRef]
  73. Markov, A.; Fedorova, A.; Kravtsova, V.; Bikmurzina, A.; Okorokova, L.; Matchkov, V.; Cornelius, V.; Amasheh, S.; Krivoi, I. Circulating ouabain modulates expression of claudins in rat intestine and cerebral blood vessels. Int. J. Mol. Sci. 2020, 17, 5067. [Google Scholar] [CrossRef]
Ijms 23 15610 g001 550
Figure 1. Elisa kit analysis of (A) TNFα and (B) IL10 in tumor, tumor-adjacent and non- tumor segments in the proximal and distal parts of the colon in DMH-rats. * p < 0.05, ** p < 0.005, # p < 0.05, Mann–Whitney U-test. The number of symbols corresponds to the number of samples.
Figure 1. Elisa kit analysis of (A) TNFα and (B) IL10 in tumor, tumor-adjacent and non- tumor segments in the proximal and distal parts of the colon in DMH-rats. * p < 0.05, ** p < 0.005, # p < 0.05, Mann–Whitney U-test. The number of symbols corresponds to the number of samples.
Ijms 23 15610 g001
Ijms 23 15610 g002 550
Figure 2. (A) Paracellular permeability for sodium fluorescein (P), (B) TEER, (C) Isc of the proximal and distal segments of the rat colon under the action of TNFα and IL10, * p < 0.05, ** p < 0.01, *** p < 0.001, Mann–Whitney U–test. The number of symbols corresponds to the number of samples.
Figure 2. (A) Paracellular permeability for sodium fluorescein (P), (B) TEER, (C) Isc of the proximal and distal segments of the rat colon under the action of TNFα and IL10, * p < 0.05, ** p < 0.01, *** p < 0.001, Mann–Whitney U–test. The number of symbols corresponds to the number of samples.
Ijms 23 15610 g002
Ijms 23 15610 g003 550
Figure 3. Western blot analysis of TJ proteins in the proximal colon. (A) Densitometric analysis revealed decreased Cld-1 under the action of TNFα, decreased claudin (Cld)-1, -2 and -4 and increased occludin (Occl) after IL10 action, Tricellulin (Tricell), * p < 0.05, Mann–Whitney U-test, n for each protein = 4. The values were normalized to total protein amount. The number of symbols corresponds to the number of samples. (B) Representative Western blot bands.
Figure 3. Western blot analysis of TJ proteins in the proximal colon. (A) Densitometric analysis revealed decreased Cld-1 under the action of TNFα, decreased claudin (Cld)-1, -2 and -4 and increased occludin (Occl) after IL10 action, Tricellulin (Tricell), * p < 0.05, Mann–Whitney U-test, n for each protein = 4. The values were normalized to total protein amount. The number of symbols corresponds to the number of samples. (B) Representative Western blot bands.
Ijms 23 15610 g003
Ijms 23 15610 g004 550
Figure 4. Western blot analysis of TJ proteins in the distal colon. (A) Densitometric analysis did not reveal any significant differences, p < 0.05, Mann–Whitney U-test, n for each protein = 4. The values were normalized to total protein amount. The number of symbols corresponds to the number of samples. (B) Representative Western blot bands.
Figure 4. Western blot analysis of TJ proteins in the distal colon. (A) Densitometric analysis did not reveal any significant differences, p < 0.05, Mann–Whitney U-test, n for each protein = 4. The values were normalized to total protein amount. The number of symbols corresponds to the number of samples. (B) Representative Western blot bands.
Ijms 23 15610 g004
Ijms 23 15610 g005 550
Figure 5. Design of experiment 1 (scheme).
Figure 5. Design of experiment 1 (scheme).
Ijms 23 15610 g005
Ijms 23 15610 g006 550
Figure 6. Design of experiment 2 (scheme).
Figure 6. Design of experiment 2 (scheme).
Ijms 23 15610 g006
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Bekusova, V.; Zudova, T.; Fatyykhov, I.; Fedorova, A.; Amasheh, S.; Markov, A.G. Selective Role of TNFα and IL10 in Regulation of Barrier Properties of the Colon in DMH-Induced Tumor and Healthy Rats. Int. J. Mol. Sci. 2022, 23, 15610. https://doi.org/10.3390/ijms232415610

AMA Style

Bekusova V, Zudova T, Fatyykhov I, Fedorova A, Amasheh S, Markov AG. Selective Role of TNFα and IL10 in Regulation of Barrier Properties of the Colon in DMH-Induced Tumor and Healthy Rats. International Journal of Molecular Sciences. 2022; 23(24):15610. https://doi.org/10.3390/ijms232415610

Chicago/Turabian Style

Bekusova, Viktoria, Tatiana Zudova, Ilyas Fatyykhov, Arina Fedorova, Salah Amasheh, and Alexander G. Markov. 2022. "Selective Role of TNFα and IL10 in Regulation of Barrier Properties of the Colon in DMH-Induced Tumor and Healthy Rats" International Journal of Molecular Sciences 23, no. 24: 15610. https://doi.org/10.3390/ijms232415610

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop