DOI: https://doi.org/10.21203/rs.3.rs-1493817/v1
Gastroesophageal reflux disease (GERD) is the most common upper gastrointestinal (GI) disorder with a considerably high prevalence across the world. Reflux of gastric acid into the esophagus can cause irritation and inflammation of the esophagus and progress into reflux esophagitis (RE). Vitamin D has anti-inflammatory effects and plays an important regulatory role in adaptability and innate immunity. Therefore, we hypothesized that VitD3 may play a protective role in RE. A total of 70 male Sprague–Dawley rats were used to surgically induce acute RE(n = 35) or chronic RE (n = 35), and to examine effects of different doses of 1,25-dihydroxy vitamin D3(VitD3) on morphological changes and alteration of pro-inflammatory cytokine levels. Macrographic and histopathological examinations revealed various degrees of esophageal impairment in rats following surgical induction of acute or chronic RE, whereas morphologic grading of esophageal mucosa showed no significant difference between groups. The protein expression of IL-1β, IL-6, and IL-8 was significantly increased in the rats with acute or chronic RE compared with rats that received sham-surgery. Vit D3 treatment significantly reduced the levels of these pro-inflammatory cytokines in both the low-dose and high-dose VitD3 groups compared with the control group in acute RE. Although no morphologic and histopathologic changes were observed, these novel findings suggest a potential protective role of VitD3 in RE at an early stage.
Gastroesophageal reflux disease (GERD) is the most common upper gastrointestinal (GI) disorder with a considerably high prevalence across the world. Reflux of gastric acid into the esophagus can cause irritation and inflammation of the esophagus and progress into reflux esophagitis (RE). In fact, RE has long been a health concern worldwide. If left untreated or uninterrupted at an early stage, it can cause heartburn, difficulty and pain in swallowing, and other uncomfortable symptoms, and progress into more severe clinical conditions (e. g. erosive esophagitis, hemorrhagic necrosis) and even esophageal cancer in a proportion of patients. It has been noted that there is seasonal variation in the incidence of GERD, with a peak in winter [1]. In addition, higher latitude climate is associated with a greater incidence of GERD [1]. Given that vitamin D (Vit D) in the skin is synthesized from exposure to sunlight, mainly ultraviolet light B (UVB) radiation, and insufficient UVB exposure in winter and in a high latitude climate may cause Vit D deficiency, Vit D deficiency has been proposed to play a role in GERD[2–4]. However, at present, it remains to be elucidated whether Vit D is involved in RE.
Scientific evidence has shown that RE involves inflammation of the esophagus in the early stage. As such, prevention of RE could be achieved by reducing the levels of key pro-inflammatory factors, and anti-inflammation treatment could slow the progression of RE. 1,25-Dihydroxy vitamin D3 (VitD3) as a ligand can bind to its specific receptors to form a ligand-receptor complex, a critical process in the functioning of VitD3. Extensive previous studies have shown that VitD3 possesses anti-inflammatory activity[5]. For instance, combined supplementation with vitamin D and l-cysteine were effective in lowering the risk of oxidative stress and inflammation associated with type 2 diabetes or COVID-19 infection [6], and down-regulation of IL-6 and IL-β levels in those adults after ultra-marathon running with vitamin D supplementation and in activated human macrophages[7–10]. In vivo studies have indicated that Vit D supplementation can suppress the formation of reactive oxygen species (ROS), thereby enhancing the function of vascular endothelial cells [11]and reducing the mortality of cardiovascular diseases (CVD)[12]. Progress has also been made in understanding the molecular mechanisms underlying the anti-inflammatory action of VitD3, with studies suggesting that the nuclear factor-kappa B (NF-κB) signaling pathway through the Toll-like receptors 2 and 4 (TLR2/4) or Janus kinase (JAK) is likely to play an important role, at least in part, in the VitD3-modulated inflammation[13, 14]. Additionally, the anti-inflammatory activity of VitD3 can be attributed to its regulation in the NF-κB and other molecular pathways[15]. Although the anti-inflammatory activity of VitD3 has been clearly proven, the role of VitD3 in RE remains largely unclear.
Based upon these important previous findings, including those of our previous studies[16, 17], we hypothesized that VitD3 may play a protective role in RE. In the present study, we examined the assumed protective effects of VitD3 treatment for the attenuation of esophageal inflammation in rat models of acute and chronic RE.
A total of 70 male Sprague–Dawley rats, weighting 180-220g(6-7week), were purchased from the experimental animal center of Fujian Medical University [Animal License No. SCXK (Min) 2012-0001; Fuzhou, Fujian, China). All rats were kept in cages and maintained in animal rooms under standard conditions with constant temperature of 22 ± 2°C, humidity of 50 ± 5%, 12-h light-dark cycle, and AIN-93M standard pellet diet (Table 1) with drinking water. The establishment of rat models of acute and chronic RE and treatments with VitD3 are described below.
| Ingredient | AIN-93M + HVD (HVD group) | AIN-93M + VD (VD group) | AIN-93M + 10% DMSO (DMSO group and Sham group) |
|---|---|---|---|
| Protein | 14.2% | 14.2% | 14.2% |
| Carbohydrate | 73.1% | 73.1% | 73.1% |
| Fat | 4% | 4% | 4% |
| Casein | 140g | 140g | 140g |
| Corn starch | 495.7g | 495.7g | 495.7g |
| Maltodextrin 10 | 125g | 125g | 125g |
| Sucrose | 100g | 100g | 100g |
| Cellulose | 50g | 50g | 50g |
| L-Cystine | 1.8g | 1.8g | 1.8g |
| Mineral mix | 35g | 35g | 35g |
| Vitamin mix | 10g | 10g | 10g |
| Choline bitartrate | 2.5g | 2.5g | 2.5g |
| Soybean Oil | 40g | 40g | 40g |
| Vitamin D3 | 0.125 mg/d | 0.03125mg/d | 0 |
| 10%DMSO | 0.1ml/d | 0.1ml/d | 0.1ml/d |
| HVD, high concentration VitD3 supplementation; VD, low concentration VitD3 supplementation; DMSO, dimethyl sulfoxide. | |||
RE was surgically induced using the pyloric nylon loop-induced chronic acid reflux esophagitis (PNL-CARE) approach, as reported previously[18], and a schematic of the procedure is presented in Fig. 1. In brief, the experimental rats were anesthetized by intraperitoneal injection of 2% pelltobarbitalum natricum (0.2 mL/100g). The limiting ridge or transitional region between the forestomach and glandular stomach was ligated with 3 − 0 non-absorbable suture (Johnson Medical Ltd. Shanghai, China). Subsequently, the pyloric ring was occluded using home-made pyloric clips to accomplish partial obstruction of the pylorus. Pyloric rings of different sizes were created following occlusion: 4.2 mm in diameter for establishment of the acute RE model and 4.5 mm in diameter for establishment of the chronic RE model. In the sham operation group, the duodenum and the stomach were dissociated for 2 min without ligating the pylorus and the duodenum. After successful establishment of acute and chronic RE rat models, the rats were deprived of food for 48 h, but allowed free access to drinking water. The rats were then given an intraperitoneal injection of gentamicin (0.5 ml/D) to prevent infection. The occurrence and severity of acute or chronic RE were examined morphologically and pathologically.
VitD3 powder was purchased from Aladdin (Shanghai, China). To prepare injectable VitD3 solution for treatment, VitD3 powder was dissolved in dimethyl sulfoxide (DMSO). The dose of VitD3 was calculated on the basis of that for human adults (50000 IU/week)[15]. The equivalent dose for rats was 1250 IU/day. Both a lower concentration VitD3 solution (12500 IU/mL) and a higher concentration VitD3 solution (50000 IU/mL) were prepared and used for treatments.
A total of 70 male Sprague–Dawley rats were randomly divided into the acute RE and chronic RE groups (n = 35 in each group). Rats in both groups received treatment with high concentration VitD3 supplementation (HVD group, n = 10), low concentration VitD3 supplementation (VD group, n = 10), or 0.1 mL 10% dimethyl sulfoxide (DMSO) as a control (n = 10). Five rats underwent the sham procedure. Prior to model establishment in the acute RE group, the esophagi of the rats were perfused with different doses of VitD3 or 10% DMSO for 14 consecutive days. At 72 h after surgery, esophageal specimens were collected from each group. Differently, 24 h after model establishment in the chronic RE group, the esophagi of the rats were perfused with different doses of VitD3 or 10% DMSO solution as a control for 14 days. Esophageal specimens were collected for subsequent macrographic and histopathological examinations as well as western blot analysis.
Macrographical examinations and assessment of esophageal tissues were based on the Kuwahta's grading system[19]. For histophathological examinations, esophageal specimens were fixed in 10% formaldehyde solution and sectioned at 5 mm thickness. The resulting tissue sections were stained with hematoxylin-eosin (H&E) and evaluated by two independent pathologists who were blinded to the information of the experimental and control groups. The degrees of esophageal inflammation and mucosal barrier impairment (severe, moderate, and mild) were assigned in accordance with the Chinese Society of Digestive Endoscopy GERD Guidelines. Briefly, the occurrence of mucosal ulcers was defined as a severe lesion, followed by mucosal erosion as a moderate lesion, and squamous hyperplasia, and inflammatory infiltration as mild lesions[20].
Western blot analysis was performed to examine the protein expression levels of IL-1β, IL-6, and IL-8. The primary antibodies included rabbit anti-rat IL-1β antibody (Wuhan Sanying Biotechnology Co., Ltd., Wuhan, Hubei, China), rabbit anti-rat IL-6 antibody (Baaode Biotechnology Co., Ltd., China), and rabbit anti-rat IL-8 antibody (Abcam, Cambridge, MA, USA). Horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (Xiamen Lulong biotechnology, Xiamen, Fujian, China) was used as the secondary antibody. The protein expression levels were normalized to that of β-actin as a loading control.
A calcium ion test kit was purchased from Nanjing Jiancheng Bioengineering Research Institute (Nanjing, Jiangsu, China) and used for measurement of serum calcium levels. A VitD3 enzyme-linked immunosorbent assay (ELISA) kit was obtained from Nanjing Senbeijia Biotechnology (Nanjing, Jiangsu, China). Serum calcium and VitD3 levels were determined with the respective kits following the manufacturers’ instructions.
Statistical analysis was conducted using SPSS 22.0 statistical software (IBM, Chicago, IL, USA). Data with a normal distribution were expressed as mean ± standard deviation (SD). The categorical data were analyzed by chi-square test; the quantitative data were analyzed by Student's t-test. One-way analysis of variance (ANOVA) was used for comparisons among multiple groups, and the rank sum test was used for analysis of rank data. P < 0.05 was indicative of a statistically significant difference.
The various degrees of esophageal morphologic alterations are displayed in Fig. 2. Esophageal erosion, ulcer, and hemorrhagic necrosis were observed as common lesions in the mucosa of the esophagus in the RE models. The results showed that VitD3 treatment did not attenuate the esophageal lesions in rats with chronic RE or acute RE. In the HVD group of the chronic RE model, one rat showed distention or enlargement of the upper esophagus and suspected stenosis or narrowing of the esophageal ulcer of the middle and lower segments of the esophagus. In the VD group of the chronic RE model, thickening of the esophageal mucosa layer was observed in one rat. The Kruskal–Wallis H test (rank sum test) showed that there was no significant difference in the morphologic grading of esophageal mucosa between the acute and chronic RE rats (P > 0.05, Table 2). These macroscopic assessments of esophagus tissues in rats with and without VitD3 treatment indicated no significant morphologic changes in chronic or acute RE in rats (Fig. 2, Table 2).
| Groups | Acute RE | Chronic RE | |||||||
|---|---|---|---|---|---|---|---|---|---|
| - | + | ++ | +++ | - | + | ++ | +++ | ||
| HVD | 0 | 1 | 3 | 5 | 0 | 0 | 4 | 3 | |
| VD | 0 | 1 | 2 | 5 | 0 | 0 | 4 | 3 | |
| DMSO | 0 | 0 | 3 | 6 | 0 | 0 | 3 | 2 | |
| Sham | 5 | 0 | 0 | 0 | 5 | 0 | 0 | 0 | |
| HVD group, high concentration VitD3 supplementation; VD group ,low concentration VitD3 supplementation ; DMSO, dimethyl sulfoxide; Sham, sham-surgery. | |||||||||
Histopathological examinations revealed various degrees of inflammation of the esophageal tissues in rats with chronic or acute RE. As shown in Fig. 3, H&E staining of the esophagus specimens from rats with acute RE detected a considerable enhancement in neutrophil infiltration in the three layers of the submucosa, muscularis mucosae, and lamina propria. More severe lesions, such as loss of esophageal mucosa, necrotic tissues, and red blood cells, were also observed in acute RE. Compared with acute RE, there was not only a large amount of neutrophil infiltration in the three layers of submucosa, muscularis mucosae, and lamina propria, but also a few infiltrating lymphocytes and monocytes in chronic RE. The Kruskal–Wallis H test showed that there was no significant difference in HE staining-based pathological inflammatory grading of the esophageal mucosa between the acute and chronic RE rat groups (P > 0.05, Table 3, Fig. 3). Notably, the histopathological examinations of esophagus tissues from rats treated with VitD3 did not show significant differences in the histopathological changes in the chronic or acute RE rat groups (Fig. 3, Table 3).
| Groups | Acute RE | Chronic RE | |||||||
|---|---|---|---|---|---|---|---|---|---|
| - | + | ++ | +++ | - | + | ++ | +++ | ||
| HVD | 0 | 1 | 3 | 5 | 0 | 0 | 4 | 3 | |
| VD | 0 | 1 | 4 | 4 | 0 | 0 | 3 | 4 | |
| DMSO | 0 | 0 | 3 | 6 | 0 | 0 | 3 | 2 | |
| Sham | 5 | 0 | 0 | 0 | 5 | 0 | 0 | 0 | |
| HVD group, high concentration VitD3 supplementation; VD group ,low concentration VitD3 supplementation ; DMSO, dimethyl sulfoxide; Sham, sham-surgery. | |||||||||
3.3 Effects of VitD3 on IL-1β, IL6, and IL-8 protein expression in rats with acute or chronic RE
The results obtained from the measurement of inflammatory cytokine expression are presented in Figs. 4 and 5. Relative to the sham surgery group, the surgery-induced acute RE, HVD, and VD groups showed significant increasing trends in the IL-1β, IL6, and IL-8 protein expression (P < 0.05), but significant decreasing trends in comparison with the control group (DMSO) (P < 0.05). No significant difference was observed between the HVD and VD groups (P > 0.05; Figs. 4,5). For chronic RE, VitD3 treatment appeared to slightly inhibit the IL-1β, IL6, and IL-8 protein expression compared with the control group (DMSO), but the differences were not significant (P > 0.05; Figs. 4, 5).
3.4 Effects of VitD3 on serum calcium levels in the RE model rats and the association with morphologic changes of the kidney
The major consequence of over-dose VitD3-induced toxicity is hypercalcemia, which leads to various clinical symptoms (e.g., nausea, vomiting, frequent urination) and can even progress to more serious kidney problems. We therefore examined serum levels of calcium as well as performed histology of the kidney after VitD3 treatment of acute or chronic RE model rats. As shown in Table 4, the serum levels of VitD3 and calcium were significantly higher in the HVD group than in the VD group, sham-surgery group, and the control group (P < 0.05) in both the acute and chronic RE groups. Histological examination revealed no abnormal morphologic changes of the kidney in rats treated with either low- or high-dose VitD3 (Fig. 6).
| Groups | Acute RE | Chronic RE | |||
|---|---|---|---|---|---|
| Serum calcium (mmol/L) | Serum VitD3 (mmol/L) | Serum calcium (mmol/L) | Serum VitD3 (mmol/L) | ||
| HVD | 3.01 ± 0.18 | 237.00 ± 24.90 | 3.10 ± 0.24 | 237.00 ± 27.06 | |
| LVD | 2.71 ± 0.09 | 202.00 ± 19.24 | 2.81 ± 0.14 | 204.00 ± 16.73 | |
| DMSO | 2.56 ± 0.11 | 173.00 ± 18.57 | 2.52 ± 0.10 | 175.00 ± 21.79 | |
| Sham | 2.57 ± 0.03 | 168.33 ± 7.63 | 2.61 ± 0.04 | 170.00 ± 10.00 | |
| HVD group, high concentration VitD3 supplementation; VD group ,low concentration VitD3 supplementation ; DMSO, dimethyl sulfoxide; Sham, sham-surgery. | |||||
The novel finding of the present study is that of VitD3 play a potential protective role in RE at an early stage. This study of the effects of VitD3 treatment in rat models of acute and chronic RE has the following major novel findings: (1) esophageal impairments occurred in rats with surgery-induced acute and chronic RE, as evidenced by macrographic and histopathological findings; (2) no significant differences in morphologic grading of esophageal mucosa were observed after VitD3 treatment in RE rats; (3) the protein expression levels of the pro-inflammatory cytokines, including IL-1β, IL-6, and IL-8, were significantly increased in both the acute and chronic RE groups, compared with the sham-surgery group; (4) VitD3 treatment significantly reduced the levels of these pro-inflammatory cytokines in both low-dose and high-dose VitD3 groups compared with the DMSO control group; and (5) serum calcium levels increased significantly in response to VitD3 treatment, while no abnormal alterations were observed on histological examination of the kidney tissues of these rats.
RE is considered a consequence of chronic inflammation of the esophagus occurring when mainly when the tissue is irritated by reflux of gastric acid. In the present study, rat models of acute and chronic RE were surgically induced using modified procedures, namely PNL-CARE, as we reported previously[16–18]. Macrographic and histopathological findings showed characteristic esophageal impairments in rats after surgical induction of acute or chronic RE. In addition, a number of key pro-inflammatory cytokines, specifically IL-1β, IL-6, and IL-8, were significantly elevated in the rat models of acute and chronic RE, which may be attributed to the inflammatory response in the esophagus after it is irritated by gastric acid reflux[20, 21]. Furthermore, using the rat models established in this study, we subsequently tested our hypothesis that VitD3 treatment could exert a beneficial effect in the inflammatory process occurring in acute or chronic RE.
It may merit attention that VitD3 treatment, either at a low dose or high dose, significantly reduced the levels of these important pro-inflammatory cytokines or mediators of the inflammatory process, including IL-1β, IL-6, and IL-8, in comparison with the cytokine levels in the control group. Especially, with the rat model of acute RE, we found that the experimental rats in the high-dose and low-dose VitD3 groups showed significant reductions in the levels of IL-1β, IL6, and IL-8 protein expression compared with those in the control groups. However, VitD3 seemed to affect the levels of IL-1β, IL6, and IL-8 protein expression differently between acute and chronic RE. In the chronic RE rats, VitD3 treatment slightly diminished the expression levels of IL-1β, IL6, and IL-8 protein in contrast to the control groups, and no difference between groups was observed. Although VitD3 treatment significantly suppressed the expression of these pro-inflammatory factors (IL-1β, IL6, and IL-8), there was no significant difference in the morphologic grading of the esophageal mucosa between the VitD3 treatment groups and control groups of rats with acute RE. Furthermore, considering that VitD3 was given after RE occurred, these findings suggest that VitD3 alone may not improve the esophageal impairments arising from the reflux of gastric acid into the esophagus in RE. We also noted that the serum level of calcium was markedly elevated after VitD3 treatment in rats, while histological findings of the kidney tissues detected no abnormality, indicating no renal toxicity in relation to excessive serum calcium. Collectively, excessive secretion of the pro-inflammatory factors is proposed as an early event in the development of RE[22], and our findings suggest that VitD3 has the potential to slow the development of RE.
This study has a number of limitations to consider. Although we showed that VitD3 diminished the expression of pro-inflammatory cytokines (IL-1β, IL-6, and IL-8) in rats, a further in-depth mechanistic study is needed to determine the molecular pathways through which it occurs. Additionally, two doses of VitD3 were used in the present study, and more doses of VitD3 should be tested for their effects on the pro-inflammatory cytokines and to examine if the effect could be dose-dependent. These studies are currently underway in our laboratory.
In conclusion, IL-1β, IL-6, and IL-8 are markedly elevated in RE, and abnormal increases in these important pro-inflammatory cytokines are suppressed by VitD3 in rats. Although no morphologic and histopathologic changes are observed with VitD3 treatment, these novel findings suggest a potential protective role for VitD3 in RE at an early stage. VitD3 holds promise to slow down the progression of ER or prevent ER, but further studies are needed in the future.
ANOVA, analysis of variance; CVD, cardiovascular diseases; DMSO, dimethyl sulfoxide; ELISA, enzyme-linked immunosorbent assay; GERD, Gastroesophageal Reflux Disease; GI, Gastrointestinal; HVD group, high concentration VitD3 supplementation; H&E, hematoxylin-eosin; JAK, Janus kinase; NF-κB, nuclear factor-kappa B; PNL-CARE, pyloric nylon loop-induced chronic acid reflux esophagitis; RE, Reflux Esophagitis; ROS, reactive oxygen species; SD, standard deviation; TLR2/4, Toll-like receptors 2 and 4; UVB, Ultraviolet Light B; VitD3,1,25-Dihydroxy Vitamin D3; VD group ,low concentration VitD3 supplementation .
Author ContributionsZehao Zhuang contributed to the conception of the study and revised the manuscript. Jingjing Wei and Jun Zhang drafted the manuscript. Jun Zhang conducted the study and performed statistical analyses. Tingting Lian, Ying Jiang, and Peihong Zhang helped perform the analysis with constructive discussions. All authors agree to be fully accountable for ensuring the integrity and accuracy of the work, and read and approved the final manuscript.
Funding This research was supported by the Nature Science Foundation of Fujian province (Grant number: 2019J01447 and 2020J01974).
Ethical approval The study protocols involving rats were reviewed and approved by the Laboratory Animal Welfare & Ethics Committee of Fujian Medical University (Fuzhou, Fujian, China), and approval number are FJMU IACUC 2019-0050 and FJMU IACUC 2021-0335.
Conflict of interest The authors declare no conflict of interest.