The Combined Effect of Vitamin C and Vitamin D3 on the Intestinal Epithelial Barrier by Regulating Notch Signaling Pathway

DOI: https://doi.org/10.21203/rs.2.19153/v1

Abstract

Background: Tight junction proteins play crucial role in maintaining the intestinal mucosal barrier. Although previous studies had shown that the Notch signal is closely related to tight junction proteins, the mechanism by which it does so remains unknown. The goal of the present study was to investigate whether vitamin C combined with vitamin D3 affects intestinal mucosal barrier stability through Notch signal pathway.

Results: To assess the effect of vitamin C combined with vitamin D3 on the intestinal mucosal barrier, electron microscopic observation of ultrastructure of tight junctions was done. And tight junction proteins gene and Notch signal gene expression were analyzed by quantitative reverse-transcription polymerase chain reaction, expression of tight junction protein in SW480 cells interfered with by LPS were examined by western blot. We found that vitamin C combined with vitamin D3 had protective effect on DSS-induced ulcerative colitis in guinea pig intestinal mucosa. Electron microscopy results showed that both low dose and high dose of vitamin C combined with vitamin D3 could maintain DSS-induced ulcerative colitis in guinea pig intestinal epithelium tight junction, however, the combination of medium dose vitamin C and vitamin D3 did not have this effect; Compared with the control group, the expression level of ZO-1 mRNA in the colon tissue of high-dose vitamin C group was significantly increased. In SW480 cell experiments, compared with the control group, the cell migration and repair ability of different concentrations of vitamin C combined with vitamin D3 group were significantly improved, the protein expression of Notch-1 was increased, but the protein expression of claudin-2 was significantly decreased.

Conclusions: our results of this experiment showed that the appropriate amount of vitamin C combined with vitamin D3 might regulate the expression of claudin-2 by regulating Notch-1, slow the intestinal mucosal barrier destruction, and promote the damage repair of cell mucosal barrier.

Background

Ulcerative colitis (UC) is a kind of inflammatory bowel disease (IBD), which belongs to chronic non-specific autoimmune diseases. Its pathogenesis is not fully understood and may be related to genetic susceptibility, immunity and the other factors such as abnormal expression of cytokines, imbalance of intestinal flora, diet and infection [13]. Because the disease is prolonged and prone to recurrent episodes, it increases the risk of colon cancer [4, 5]. As one of the important interfaces between the body and the external environment, the intestinal mucosal barrier plays a key role in maintaining intestinal homeostasis. Many studies have shown that intestinal mucosal damage is the initiating factor of IBD [6], while the intestinal mucosa is destroyed resulting in increased mucosal barrier permeability, and a large amount of bacteria in the intestine are absorbed into the blood, which in turn causes an intestinal immune response [7, 8]. Therefore, protecting and repairing the intestinal mucosal barrier are the key to prevent the development of IBD.

The intestinal epithelial barrier is composed of epithelial cells, tight junction proteins and intestinal secretions [9], the tight junction acts as the most important connection between intestinal mucosal cells and plays an important role in maintaining intestinal barrier integrity. The tight junction complex mainly includes claudin, zona occludens (ZO) family, etc. The expression and distribution of claudin are closely related to the permeability of endothelial cells. ZO protein is a backbone protein linked to cells [10], and the normal expression in cells is an important condition for the stability of the cell structure, however, its abnormal expression may destroy the structure of epithelial cells and endothelial cells, resulting in impaired function. Studies have shown that the increased expression of Notch1 receptor and downstream gene Hes1 can promote the repair process of injured epithelium, suggesting that Notch1 signaling pathway may be involved in the regulation of proliferation and differentiation of epithelial cells [11].

Studies have shown that the active form of vitamin D3 (1,25 (OH) 2D3) can regulate the expression of family proteins such as claudin and ZO in the tight junction complex, which plays an important role in maintaining and repairing the integrity of the mucosal barrier [12, 13]. Vitamin C(VC) may play an important role in the activation of vitamin D3 (VD3) hydroxylation [14].

Herein, the aim of this study was to investigate whether vitamin C combined with vitamin D3 mediates the role of the Notch signaling pathway in protecting the intestinal mucosal barrier.Therefore, this study used vitamin D3 combined with vitamin C in vitro and in vivo intervention experiments on UC model is to explore whether the combination has protective effects on the intestinal mucosal barrier, and to explore the role of Notch signaling pathway in the intestinal mucosal barrier.

Results

VC+VD3 Promotes Recovery of DSS Induced Colitis in Guinea Pigs

Adaptive feeding of guinea pigs for 5 weeks, to induce acute colitis, guinea pigs were treated 2%DSS for 5 days. At the beginning of the experiment, the guinea pigs in each group had normal mental state and good health. There was no significant difference in body weight between the guinea pigs. In the low, medium and high dose VC group, the weight of the guinea pigs began to decrease on the first day after drinking 2% DSS solution. From the second day, the guinea pigs gradually showed signs of apathy, decreased mobility, loose stools, mucus, bloody stools, etc. It is aggravating, indicating that ulcerative colitis model is successful. After modeling, the body weight of each group of guinea pigs showed a downward trend, but there was no significant difference in body weight between the groups (P > 0.05)(Figure 1A).Compared with the control group, the colonic gross morphology score and histopathological score of the other groups were significantly increased (P < 0.05). Compared with the medium dose VC group, the colon length of the high dose VC group was significantly increased (P < 0.05)(Table 1).Compared with the control group, the H&E staining showed epithelial crypt damage and severe mucosal inflammation in the other group. In the low-dose VC group, a small amount of epithelial cells were exfoliated in the colon, the mucosal surface was finger-like, and the crypt opening was widened, but it was improved compared with the medium-dose VC group. The medium-dose VC group had severe epithelial cell shedding in the colon of the guinea pigs, and the mucosal surface the villous changes, the crypt shrinkage, the spacing increased, and the connective tissue in the submucosa was loose. The mucus in the colonic goblet cells of the guinea pigs in the high-dose VC group decreased, the crypts were branched and twisted, but the epithelial cell shedding was better than the medium-dose VC group (Figure 1B).

VC+VD3 Attenuate DSS-induced Epithelial Permeability through Regulates Colonic Tight Junction.

DSS model would not only affect the integrity of the mucosal barrier, but also regulate colonic tight-junctions. Damage to intestinal permeability may be due to dysregulation of the mucus or epithelial junction complex, which enhances susceptibility to colitis.

The previous reports believed that both vitamin D3 and vitamin C play a role in maintaining the integrity of the intestinal mucosal barrier and repairing the mucosal barrier [12, 13, 15].According to our results,there was no significant change in the ultrastructure of colonic epithelial cells in the control group (Figure 2A). The low, medium and high doses of the guinea pig colonic epithelial cells in the VC group showed focal reduction, shortening and sparseness in different degrees. Among them, the medium dose VC group was the most severely damaged (Figure 2C). Compared with the control group, the tight junction structure between the colonic epithelial cells of the medium-dose VC group was severely damaged, and the cell gap became larger (Figure 2C), while the tight junction gap between the colonic epithelial cells in the low-dose and high-dose VC group was slightly widened, but the tight joint structure remains essentially intact (Figure 2B, D).

VC+VD3 Regulate Notch Signaling in the Colon of DSS-treated Mice

Notch signaling pathway plays a key role in the fate of intestinal epithelial cells [16],which regulate a lot of genes including Hes-1 to promote the repair process of damaged epithelium [11].Therefore, Notch signaling pathway may play an important regulatory role in the maintenance of intestinal mucosal homeostasis by regulating the proliferation and differentiation of intestinal epithelial cells.

The results of this experiment showed that the expression levels of Notch1 and Hes1 mRNA were significantly higher than those of the control group (P<0.05), but the expression levels of the middle and high dose groups were lower than those of the low dose group (P<0.05). The expression distribution of tight junction complexes is closely related to apothecary endothelial cell permeability. The results of this experiment showed that compared with the control group, the expression level of ZO-1 mRNA in the high dose group was significantly increased (P<0.05) (Table 2). However, the mRNA expression level of claudin-2 was not significantly different among the groups.

Repair Effect of VD3 Combined VC on Tight Connection Damage between SW480 Cells Induced by Lipopolysaccharide by Notch Signal Pathway

Cell Scratch Test

The cell scratch test is a method for determining the migration and repair ability of cells. Therefore, in order to observe the effect of different concentrations of vitamin C combined with vitamin D3 on the migration of SW480 cells, the experiment was performed using a cell scratch test.

Compared with cell scratches 0h, cell migration was observed in all groups 24h after cell scratching. Among them, 0.1VC+VD3 group had the most significant cell migration compared with the control group, while 10VC+VD3 group had the least cell migration. Obviously; compared with the VD3 group, the cell migration and repair ability of the 0.1VC+VD3, 1VC+VD3, and 5VC+VD3groups was significantly improved (Figure 3).

VC+VD3 Attenuate LPS-induced Tight Junction Structure Damage in SW480 Cells

Tight junctions is essential construction for mucosal barrier, therefore, in this experiment, the LPS in vitro model was used to induce cell membrane barrier destruction, and the effects of different vitamin C and vitamin D3 on cell tight junction were observed. As marker of tight junction structure, the distribution and expression of claudin-2 and ZO-1 were detected by western blot. Compared with the control group, The expression level of claudin-2 protein was significantly decreased in LPS, 0.1VC+VD3, 1VC+VD3 and 10VC+VD3 groups (P< 0.05), and the expression level of claudin-2 protein in group VD3 was significantly higher than that in LPS group (P< 0.05), and the expression level of claudin-2 protein in the VC+VD3 group was significantly lower than that in the VD3 group (P< 0.05). Western blot analysis demonstrated decreased expression of ZO-1 in other groups vs control group (P<0.05) (Figure 4). Thus, the role of VC+VD3 in the intestinal epithelium may enhance epithelial tight junctions in SW480 cells.

Discussion

Both guinea pigs and humans lack gulono-gamma-lactone oxidase [17], and they cannot synthesize vitamin C (VC), and they must be obtained from food. Therefore, guinea pigs were selected as the study objects in this study, and VC was artificially added to study whether VC can activate vitamin D3 (VD3). Dextran sulfate sodium (DSS) is widely used in the study of inflammatory bowel disease, especially in the DSS-induced ulcerative colitis model [1820]. DSS destroys the intestinal mucosal barrier and causes intestinal substances to enter the intestinal tissue, inducing excessive activation of immune cells and triggering inflammatory reactions [2123]. In this experiment, after drinking DSS solution, guinea pigs showed different degrees of weight loss, colonic inflammatory cell infiltration, and elevated colonic score. However, before DSS modeling, guinea pigs were given a certain amount of VC, VD3 by gavage every day, which to some extent protected the damaged colon of guinea pigs.

The intestinal mucosal barrier is the key to maintaining a balance between the intestinal lumen and the mucosa, primarily through the mucous layer, intestinal epithelial cells, host innate, and adaptive immune response [24]. In order to illustrate the effect of VC and VD3 on colonic guinea pig colon in DSS, the ultrastructure of colonic colon in guinea pigs was observed. The results showed that there was no significant change in the ultrastructure of colonic epithelium in the control group. Colonic epithelium of guinea pigs in low, medium and high dose VC groups,the microvilli on the surface of the cells showed focal reduction, shortening and sparseness in different degrees. Among them, the medium-dose VC group had the most severe damage and the cell gap became larger, the other two groups had a tightly connected structure. It is suggested that VD3 can only protect the colon after DSS intervention by appropriate combination of VC.

Notch signaling pathway is a highly conserved signaling pathway involved in the proliferation and differentiation of all cells. Notch signaling is associated with many human diseases [25], and Notch signaling is also closely related to inflammatory bowel disease [26, 27], which is considered to treat cancer potential target. By detecting the genes related to colon tissue in guinea pigs, low-dose VC can highly activate the Notch/Hes-1 signaling pathway. Combined with electron microstructural ultrastructural results, it was shown that low dose of VC can play a certain protective effect on the intestinal mucosa of guinea pigs with DSS-induced colitis. Studies have found that when the intestinal epithelium is damaged, the increased expression of Notch-1 can promote the proliferation of epithelial cells, which is conducive to the repair and reconstruction of the injured site, but the persistent overexpression of Notch-1 can lead to the reduction of intestinal epithelial secretory cell lines, which is not conducive to the treatment of UC [28, 29]. And activation of the Notch signaling pathway alters the expression of tight junction proteins and affects the continuity of thier distribution, thereby reducing cell barrier permeability.Studies have shown that vitamin D3 protects the gut barrier by regulating tight junction proteins [30]. The results of this experiment show that in the high-dose VC group, although the mRNA expression level of the signal molecules in the Notch/Hes-1 pathway was significantly decreased, the expression level of tight junction protein ZO-1 was higher, and the expression level of claudin-2 mRNA was higher, and ZO-1 interacts with claudin-2 to correct the increase in permeability caused by disease [31],and high-dose VC group can significantly improve the colonic tissue shortening caused by DSS in guinea pigs. Thus, when given the same dose of VD3, high dose of VC can protect guinea pigs with DSS-induced colitis by reducing the increase of colonic mucosal permeability.

In addition, in the cell scratch experiment, VC combined VD3 had better repair effect on cell scratches than simple VD3, but with the increase of VC dose, the ability of cell migration repair did not improve significantly, indicating that VC combined VD3 was more conducive to repair of epithelial barrier damage, but not in dose-dependent reaction.

Conclusions

The effect of VC combined with VD3 on the damage repair of cell barrier was better than that of VD3 alone, indicating that VC could promote the activation of VD3 to a certain extent, and the appropriate application of VC combined with VD3 could alleviate the damage of colon mucosa and promote the damage repair of cell mucosal barrier. Although different concentrations of VC combined with VD3 have different regulatory levels of Notch signal and tight junction proteins, the combination of VC and VD3 may reduce the expression of Notch-1 and inhibit the expression of claudin-2, thereby playing a protective role in the intestinal epithelial barrier.

Methods

Animals

Animal Model and Groups

All animal procedures were approved by the Institutional Animal Care and Use Committee of Shanxi Medical University and conformed with Guide for the Care and Use of Laboratory Animals 8th Edition 2011.

Twenty-four male Dunkin-Hartley guinea pigs (360 ± 20 g; Longan experimental animal breeding center, Beijing, China) were housed in metabolic cages at 22 °C ± 1 °C with relative humidity of 55%~60% and a 12/12-h yellow light/dark cycle. Guinea pigs initially received distilled water and regular diets (Beijing Keao Xieli Feed Co, Ltd.), a standard chow for laboratory guinea pigs (GB 14924.3–2010). Adaptive feeding was carried out for 1 week, the guinea pigs were randomized to 4 groups (n = 6 for each): control group (C, 200 IU/ (kg·d) VD3 + 100 mg/ (kg·d) VC), low VC group (LC, 200 IU / (kg·d) VD3 + 10 mg/ (kg·d) VC), medium VC group (MC,200 IU/ (kg·d) VD3 + 100 mg/ (kg·d)VC) ,high VC group (HC, 200 IU/ (kg·d) VD3 + 200 mg/ (kg·d) VC). Guinea pigs in each group were fed a purified diet without vitamin D3 and vitamin C (Research Diets, America) (Table S1 of supplemental materials), and the corresponding doses of VC and VD3 were given by gavage. After 5 weeks, the control group continued drinking distilled water, and the other 3 groups were treated with 2% dextran sodium sulfate solution for 4 days. After the modeling was completed, all guinea pigs were deeply anesthetized by intraperitoneal injection with 3% pentobarbital solution, and then killed by exsanguination.

Reagents

Dextran sodium sulfate (DSS, Mr: 36,000 ~ 50,000) was obtained from MP Biochemicals (Aurora, USA). Vitamin C, Vitamin D3 were purchased from Solarbio (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China).

Colon Hematoxylin-Eosin Staining

Take the guinea pig colon tissue, after 24 hours of neutral-buffered formalin (NBF) fixation, the paraffin wrapped, sliced. And then the tissue slices were dyed after dewaxing, dyeing, dehydration and sealing, after the glue solidification, using a microscope for observation, and colon histosome pathology score. Table S2 of supplemental materials for specific scoring criteria.

Observation of Colon Electron Microscopy

For electron microscopy, 1 cm long section of the gut segments were taken from the colon 3 cm proximal to the ileo-cecal valve. The gut samples were rinsed with ice cold saline (0.9% NaCl), and then cut into square-fragments (1 cm2) which were placed in cold 2% glutaraldehyde stationary solution for 24 hours. After the samples was fixed, sent them to professional EM technicians for follow-up treatment.

RT-qPCR for Notch-1, Hes-1, Claudin-2, ZO-1

Total RNA was extracted from guinea pig tissue samples using the Eastep® Super Total RNA Extraction Kit (Promega) according to the manufacturer’s protocol. An equal amount (0.5 µg) of mRNAs was converted to cDNA using a reverse transcription kit (Promega). RT-qPCR reactions were performed using 2 µl of cDNA with a Reverse Transcription Kit (Promega). GoTaq qPCR Master Mix (Promega) was used for the qPCR analyses on the CFX96TM Real-Time System (Bio-Rad).

Guinea pig mRNA sequences were selected to design primer pairs for RT-qPCR reactions using the Oligo software. The sequence of the desired primers for guinea pigs is shown in Table S3 of supplemental data. The sequence of primers was designed using Primer3, and was obtained from Thermo Scientific (Thermo). GAPDH/β-actin was used as the endogenous loading control. Gene expression was determined using the delta-delta Ct method: 2−△△CT (△△CT= [Ct (target gene) – Ct (GAPDH/β-actin)]experiment– [Ct (target gene) –Ct (GAPDH/β-actin)] control ).

Cell culture

Cells culture, Grouping, Collection

SW480 cells (human colon cancer cells) were obtained from Shanxi Tumor Hospital. The SW480 cells were maintained in DMEM (with 100 U/ml penicillin and 100 µg/ml streptomycin) (BOSTER, Wuhan, China). The cells were cultured with heat-inactivated 10% fetal bovine serum (EVERY GREEN, Zhejiang Tianhang Biotechnology Co. Ltd, China), grown at 5% CO2 and 37 °C. Healthy cultured cells were seeded at 104 cells per well. After 24 h, except for the control group, LPS (100 ng/ml) was added in the absence or presence of VD3 (0.1 µmol/L) and VC (5 µmol/ml) for 24 h. Quantification of cell proliferation was performed using the Cell Counting Kit-8 (BOSTER, Wuhan, China).

Cell grouping: control group (without any additional intervention); LPS group (100 ng/ml LPS); VD3 group (0.1 µmol/L VD3); VC + VD3 group (0.1/1/5/10 µmol/ml VC + 0.1 µmol/L VD3).

Cell damage Repair Experiment

SW480 cells in logarithmic growth phase were divided into normal control group (C), VD3 group (0.1 µmol / L VD3), VC + VD3 group (0.1, 1, 5, 10 µmol / ml VC + 0.1 µmol / L VD3). 2 duplicate holes per group. Inoculate a 6-well culture plate at a density of 1 ~ 4 × 105 cells per well, culture for 24 hours, remove the six-well plate covered with cells, and use a 200 µl gun head perpendicular to the cell surface, and cut one end of the hole to the other end to make a cell line mark. The old medium was aspirated, and the cell surface was washed 3 times with sterile PBS. The medium containing no fetal bovine serum was added to each group. Except the normal control group, the other groups were added with the corresponding doses of VC and VD3. The trace width of the different treatment groups were measured.

Western Blot analysis for Claudin-2, Hes-1, Notch-1 and ZO-1

SW480 cells were lysed in lysis buffer containing Proteinase Inhibitor Cocktail (KeyGEN BioTECH) and Halt Phosphatase Inhibitor Cocktail (KeyGEN BioTECH). Protein concentrations were quantified by using a BCA protein kit (KeyGEN BioTECH). Samples (30 µg protein per lane) were loaded on 8%~10% SDS-PAGE gels. After electrophoresis, the proteins were transferred from gel to PVDF membranes. Then, the membranes were blocked with 5% skim milk and incubated at 4 °C overnight with the appropriate primary antibodies respectively [β-actin polyclonal antibody (Bioworld, 1:5000), Claudin-2 Antibody (Affinity,1:500), Hes-1 polyclonal antibody (Abclonal,1:500), Notch-1 polyclonal antibody (Abclonal,1:500), and ZO-1 polyclonal antibody (Abclonal,1:200)]. Immunoreactivity was detected by incubation with horseradish peroxidase-conjugated secondary antibodies (BOSTER, 1:2000) followed by chemiluminescent substrate development (Affinity). The readout was detected by using ChemiDoc XRS + with the Image Lab Software (Bio-Rad). All samples were run in parallel with three replicates.

RT-qPCR for Notch-1, Hes-1, Claudin-2, ZO-1

Total RNA was extracted from SW480 cells samples using the Eastep® Super Total RNA Extraction Kit (Promega) according to the manufacturer’s protocol, and the other steps are the same as mentioned above. The gene primer sequences required for SW480 cells are also shown in Table S3 of supplemental materials.

Statistical Analysis

Values are presented as means ± standard deviation (

±SD). The normal distribution data were compared by one-way ANOVA, LSD-t (homogeneous variance) or Dunnett’s T3 (heterogeneous variance) test. Rank sum test was used for non-normal distribution data and Spearman correlation test was used for correlation analysis. And results were considered significant at P < 0.05. Statistical tests were performed with SPSS 22.0. GraphPad Prism version 8.0 was used to generate histograms.

Declarations

Additional files

Table S1: Feed formulations that do not contain VC and VD3; Table S2: Gross morphological damage score criteria for colonic tissue by naked eye; Table S3: Polymerase chain reaction primers' gene sequences.

Ethical Approval

The animals used in this experiment were approved by the Ethics Committee of Shanxi Medical University and met the ethical requirements.

Consent to publish

Not applicable

Availability of data and materials

Data supporting the conclusions of this article are included in this article and in supplementary materials.

Conflict of Interest

The authors have declared no conflicts of interest for this article.

Funding

This work was supported by grants from the National Natural Science Foundation of China (81573156).

Authors' Contributions

FQ designed the study, performed the study, and wrote the manuscript. ZZ and YM performed the experiments, and helped prepare for the manuscript. LY provided technical support, YM and RL performed the statistical analysis. FQ helped analyze data and provided scientific advice.

Acknowledgements

We are very grateful to Shanxi Cancer Hospital for the donation of SW480 cells, which helped us successfully complete the experiment. We also thank the anonymous reviewers for their valuable advice and assistance.

Abbreviations

DSS: Dextran sodium sulfate; VC: Vitamin C; VD3: Vitamin D3; NBF: neutral-buffered formalin; LPS: lipopolysaccharide; SW480 cell: human colon cancer cells

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Tables

Table 1 Effect of VC+VD3 on DSS-induced colonic morphology in guinea pigs ( ±s, n=6)

Group

Length of colon(cm)

Colonic gross morphological score

Colonic histopathological score

Control

60.80±12.73

0.00±0.00

0.00±0.00

LVC

60.90±10.76

2.80±0.83a

3.50±0.55a

MVC

52.00±7.77

2.00±0.63a

4.00±0.71a

HVC

64.70±5.25c

2.50±0.57a

3.50±0.84a

aP indicates that compared with the control group, P<0.05;the c P expression is compared with the MVC group, P<0.05.


Table 2 Relative mRNA expression of Notch1 Hes1, claudin2, ZO-1 in colon tissue

Group

Notch-1

Hes1

Claudin-2

ZO-1

control

1.46±1.27

1.17±0.83

2.74±2.26

1.11±0.60

LVC

318.88±202.43a

9.68±6.94a

1.26±1.69

5.38±3.27

MVC

3.29±3.11

0.53±0.22b

1.49±0.40

6.42±1.21

HVC

1.12±1.53b

0.34±0.11b

1.25±1.24

8.71±4.90a

aP indicates that compared with the control group, P<0.05; the b P expression is compared with the LVC group, P<0.05.