Paricalcitol Ameliorated Dextran-Sulfate-Sodium-Induced Colitis in Mice through MKP-1/P38 MAPK Signaling Pathway

Research Article

Austin J Public Health Epidemiol. 2023; 10(1): 1139.

Paricalcitol Ameliorated Dextran-Sulfate-Sodium-Induced Colitis in Mice through MKP-1/P38 MAPK Signaling Pathway

Liyuan Lv1#, Guinan Liu1#, Man Yang1#, Tao Chen2, Xiang Li2, Jun Zhang2, Jie Liu2, Yi Liu2*

¹Department of Gastroenterology, The Seventh Affiliated Hospital of Sun Yat-sen University, Guangdong, China.

²Department of Gastroenterology, Huashan Hospital, Fudan University, Shanghai,China.

#Equal Contribution.

*Corresponding author: Yi LiuHuashan Hospital, Fudan University, Shanghai, China

Received: November 28, 2022; Accepted: January 09, 2023; Published: January 16, 2023


Background and Aim: Ulcerative Colitis (UC), a type of inflammatory bowel disease, of which the accurate pathogenesis is not yet well understand. Recently, the Vitamin D/VDR signaling pathway and the activated vitamin D analogues have been proved as playing important role in the pathogenesis of UC. In the present study, our objective was to evaluate the effect of Vitamin D analogues paricalcitol on dextran-sulfate-sodium-induced colitis in a mouse model.

Methods: We evaluated the effects of the activated vitamin D analogues paricalcitol on the development of Dextran-Sulfate-Sodium-(DSS)-induced colitis. Clinical symptoms were evaluated by the Disease Activity Index (DAI) and tissue samples were evaluated by Histopathological Scoring (HS). Meanwhile, the mucosal mRNA expression of cytokines, Tumor Necrosis Factoralpha (TNF-a), Interleukin-6 (IL-6), Interleukin-10 (IL-10), Interleukin-17 (IL-17) were analyzed by real-time semi quantitative reverse transcription polymerase chain reaction. The mucosal protein VDR, p38 Mitogen-Activated Protein Kinase (P38-MAPK) and Mitogen-Activated Protein Kinase Phosphatase-1 (MKP-1) expressions of the vitamin D/VDR signaling pathway were analyzed using Western blot.

Results: The results showed that the weight loss and colon length shortening of DSS-induced mice were significantly improved after paricalcitol treatment. In addition, both DAI and HS were significantly reduced. Paricalcitol down regulated the mucosal expression of messenger RNA of pro-inflammatory cytokines TNF-a, IL-6 and IL-10 and upregulated anti-inflammatory cytokines IL-17. Both VDR protein expression and MKP-1 level increased, whereas the mucosal expression of p38-MAPK was found to be decreased.

Conclusion: Activated Vitamin D analogues paricalcitol can ameliorate the development of DSS-induced colitis through the Vitamin D/VDR signaling pathway.

Keywords: Ulcerative colitis; Paricalcitol; Vitamin D Receptor (VDR); P38 MAPK; P-P38 MAPK.


Inflammatory Bowel Disease (IBD) is associated with chronic relapsing inflammation of the intestinal tract of unknown etiology. UC and Crohn’s Disease (CD) are the two major forms of IBD. Recent studies have indicated that a complex interplay of genetic, microbial, and environmental factors culminates in sustained aberrant intestinal innate immunity, which may be a central early mechanism, subsequently perpetuated by dysregulated adaptive immune responses [1].

Apart from its classical calcium-regulating effect, vitamin D serves as a potent regulator of multiple biological activities, including antimicrobial activities, the inhibition of apoptosis and immunomodulatory functions [2]. We both know that the majority of the body’s vitamin D content is derived from pho tosynthesis in the skin following UV light irradiation.1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) is the active form of vitamin D and binds with VDR. Recent studies have shown that VDR is highly expressed in Intestinal Epithelial Cells (IECs) [3], indicating that the vitamin D/VDR signaling pathway may play a key role in the process of IBD. Increasing data have suggested VDR gene polymorphisms are associated with the incidence of IBD [4,5] and VDR knockout mice have been shown to have a compromised mucosal barrier, leading to increased susceptibility to mucosal damage and an increased risk of developing IBD [6].

The vitamin D/VDR signaling pathway, commonly referred to as the P38MAPK pathway, involves a series of proteins and gytokines. The dual-specificity MKPs comprise a subfamily of 10 catalytically active proteins, including MKP-1, has been shown to regulate p38 via dephosphorylation to restrain over-activation of MAPK and to keep homeostasis. Thus, in this study, to clarify the role of the vitamin D/VDR signaling pathway in intestinal inflammation, we evaluated the effects of activated vitamin D analogues paricalcitol on the development of DSS-induced colitis. Our data demonstrated that the P38-MAPK, TNF-a, IL-6 and IL-17 expression in the mice with DSS-induced colitis were down regulated by the treatment of paricalcitol. However, the expression of IL-10, VDR and MKP-1 were upregulate.

Materials and Methods

Animals and Groups

Thirty male BALB/c mice (SPF level) purchased from an experimental animal center (Shanghai Medical College, Fudan University, China), all weighing about 25g,were randomly assigned into five groups: the normal control group (Group A ), the UC model group (group B), the low-dose (0.1ug/kg body weight) intervention group (group C), the moderate-dose (1ug/kg body weight) intervention group (group D) and the large-dose (10ug/kg body weight) intervention group (group E). All animal procedures were reviewed and approved by the Laboratory Animal Ethics Committee of Fudan University.

Main Reagents

Dilute DSS (from MP Biomedicals: M.W=36,000-50,000,60316ES25) with distilled water into 5% DSS solution.Paricalcitol Injection (from Abbott, CAS No.: 131918-61-1). RNA extraction kit (from TRIzol Reagent. Life Technologies, USA). Real-time quantitative polymerase chain reaction (Realtime qPCR) Kit (from TOYOBO ReverTra Ace® qPCR RT Kit Code No. FSQ-101, Japan). Reverse transcription polymerase chain reaction kit (from TOYOBO SYBR Green Real-time PCR Master Mix Code No.QPK-201, Japan). Anti-p38MAPK (9F12; sc-81621, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). Anti-P-P38MAPK (sc-101758; Santa Cruz Biotechnology Inc. Santa Cruz, CA, USA). Anti-MKP-1 (sc-370, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA).

Modeling of Ulcerative Colitis

All mice were fed in a standard laboratory for one week before modeling. In accordance with the classic method used by Cooper [7] Group B to Group E were given 5.0% (w/v) DSS solution as drinking water for days 1-7 to induce acute intestinal inflammation while the normal control group drank distilled water freely. Group C, D and E received paricalcitol by intragastric administration once on days 1, 4 and 7 with 0.1ug/kg paricalcitol each time for the group C, 1ug/kg paricalcitol each time for the group D,and with 10ug/kg paricalcitol each time for the group E. Group A and group B received ddH2O intragastric administration as control. All mice were killed on the eighth day and four colon specimens from each mouse were dissected from the different sections of colon for further detection (one for nucleic acid, one for cytokines, one for proteins, one for histopathology).

Evaluation of Symptoms and Physical Signs

The daily Disease Activity Index (DAI) assessment was carried out according to the classic scoring system by Cooper [7] in the process of modeling. DAI is the sum of weight loss, trait of stool, and occult blood in stool or hematochezia.

Evaluation of Histopathology Inflammation

The whole colons were harvested on day 8. The distal colon were taken and fixed in 10% formaldehyde solution overnight at 4OC, dehydrated with graded alcohol and then embedded in paraffin for histological analysis. Sections of the colon (5 um) were stained with hematoxylin and eosin. Microscopic scoring was independently by two pathologists who were blinded to the group design and the classic scoring system by Cooper [7] was carried out. The colonic damage was categorized into six segments: severity and extent of inflammation, degree of regeneration, degree of crypt damage and percentage involvement. Total scores are the sum of the scores of each individual segment.

RNA Isolation and Real-time qPCR

Total RNA was isolated by the guanidinium isothiocyanate method followed by centrifugaion. Total RNA concentration was quantified by ultraviolet spectrophotometer three times. A260nm/A280nm was 1.8-2.0.RNA was conserved at -80OC in the fridge, β-actin was selected to span an intron. Quantities of mRNA for β-actin, TNF-a, IL-6, IL-10, IL-17, VDR, p38 MAPK and MKP-1 were determined by Real-time qPCR using the sequences of the primers designed by Primer Express (Table 1). Initial denaturation at 95OC for 3min, followed by 40cycles consisting of denaturation at 95OC for 5s, primers annealing at 60OC for 30sOC and elongation at 72OC for 45s. The Fluorescent quantitation was completed by Funglyn FTC-3000 Real-time PCR System. The result of each experimental group was repeated for three times.