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Effect of ethyl gallate and propyl gallate on dextran sulfate sodium (DSS)-induced ulcerative colitis in C57BL/6 J mice: preventive and protective

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Abstract

Objective and design

Inflammatory bowel disease (IBD) is an idiopathic inflammatory condition of the digestive system marked by oxidative stress, leukocyte infiltration, and elevation of inflammatory mediators. In this study, we demonstrate the protective effect of ethyl gallate (EG), a phytochemical, and propyl gallate (PG), an anti-oxidant, given through normal drinking water (DW) and copper water (CW) in various combinations, which had a positive effect on the amelioration of DSS-induced ulcerative colitis in C57BL/6 J mice.

Materials and methods

We successfully determined the levels of proinflammatory cytokines and anti-oxidant enzymes by ELISA, tracked oxidative/nitrosative stress (RO/NS) by in vivo imaging (IVIS) using L-012 chemiluminescent probe, disease activity index (DAI), and histopathological and morphometric analysis of colon in DSS-induced colitis in a model.

Results

The results revealed that oral administration of ethyl gallate and propyl gallate at a dose of 50 mg/kg considerably reduced the severity of colitis and improved both macroscopic and microscopic clinical symptoms. The level of proinflammatory cytokines (TNF-α, IL-6, IL-1β, and IFN-γ) in colonic tissue was considerably reduced in the DSS + EG-treated and DSS + PG-treated groups, compared to the DSS alone-treated group. IVIS imaging of animals from the DSS + EG and DSS + PG-treated groups showed a highly significant decrease in RO/NS species relative to the DSS control group, with the exception of the DSS + PG/CW and DSS + EG + PG/CW-treated groups. We also observed lower levels of myeloperoxidase (MPO), nitric oxide (NO), and lipid peroxidation (LPO), and restored levels of GST and superoxide dismutase (SOD) in DSS + EG-DW/CW, DSS + PG/DW, and DSS + EG + PG/DW groups compared to DSS alone-treated group. In addition, we showed that the EG, PG, and EG + PG treatment significantly reduced the DAI score, and counteracted the body weight loss and colon shortening in mice compared to DSS alone-treated group. In this 21-day study, mice were treated daily with test substances and were challenged to DSS from day-8 to 14.

Conclusion

Our study highlights the protective effect of ethyl gallate and propyl gallate in various combinations which, in pre-clinical animals, serve as an anti-inflammatory drug against the severe form of colitis, indicating its potential for the treatment of IBD in humans. In addition, propyl gallate was investigated for the first time in this study for its anti-colitogenic effect with normal drinking water and reduced effect with copper water.

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Data availability

All the data are available in the manuscript. On reasonable request, the corresponding author will provide the datasets used or analyzed during the current work.

Abbreviations

AGEs:

Advanced glycation end products

ANOVA:

Analysis of variance

BCA:

Bicinchoninic acid

BSA:

Bovine serum albumin

BW:

Body weight

CD:

Crohn’s disease

CDNB:

1-Chloro-2: 4-dinitrobenzene

COX-2:

Cycloxygenase-2

CPCSEA:

Committee for the purpose of control and supervision of experiments on animals

Cu:

Copper

CW:

Copper water

DAI:

Disease activity index

DSS:

Dextran sulfate sodium

DTNB:

Dithiobis-nitrobenzoic acid

DW:

Drinking water

EG:

Ethyl gallate

ELISA:

Enzyme-linked immunosorbent assay

GIT:

Gastrointestinal disorder

GSH:

Reduced glutathione

GSSG:

Oxidized glutathione

GST:

Glutathione-S-transferase

H&E:

Hematoxylin and eosin staining

HO-1:

Heme oxygenase 1

IAEC:

Institutional Animal Ethics Committee

IBD:

Inflammatory bowel disease

IFN-γ:

Interferon-gamma

IL-1β:

Interleukin-1 beta

IL-6:

Interleukin 6

iNOS:

Inducible nitric oxide synthase

IκB:

Inhibitor of kappa B

LPO:

Lipid peroxidase

LD50 :

Lethal dose 50

MDA:

Malondialdehyde

MPO:

Myeloperoxidase

NFκB:

Nuclear factor-κB

NO:

Nitric oxide

Nrf2:

Nuclear factor erythroid 2-related factor 2

PBS:

Phosphate-buffered saline

PC:

Positive control (DSS alone-treated group)

PG:

Propyl gallate

RIPA:

Radioimmunoprecipitation assay

RNS:

Reactive nitrogen species

ROI:

Region of interest

RO/NS:

Reactive oxygen/nitrogen species

ROS:

Reactive oxygen species

SC:

Standard control (sulfasalazine drug)

SEM:

Standard error of the mean

SOD:

Superoxide dismutase

TBARS:

Thiobarbituric acid-reactive substances

TLR-4:

Toll-like receptor 4

TNF-α:

Tumor necrosis factor-α

UC:

Ulcerative colitis

WHO:

World Health Organization

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Acknowledgements

The authors would like to thank The Indian Council of Medical Research for funding this study [(ICMR) No. of File: 61/03/2021-IMM/BMS] and also the Director, National Institute of Nutrition for the support and motivation to work on the project. Priyanka Raju Chougule thanks Department of Science & Technology, New Delhi, India for providing the DST-INSPIRE Senior Research Fellowship (No: DST/INSPIRE Fellowship/[IF190480]), and Sangaraju Rajendra thanks ICMR, New Delhi, India for providing the ICMR-Research Associate (No. 3/1/2/300/2021-Nut). All authors thank everyone who contributed technical assistance to our research.

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Authors and Affiliations

  1. Division of Food Safety, Indian Council of Medical Research - National Institute of Nutrition, Jamai-Osmania PO, Hyderabad, 500007, India

    Priyanka Raju Chougule, Rajendra Sangaraju, Sathish Kumar Mungamuri & Sukesh Narayan Sinha

  2. Animal Facility, ICMR - National Institute of Nutrition, Jamai-Osmania PO, Hyderabad, 500007, India

    Pradeep B. Patil & S. S. Y. H. Qadri

  3. Molecular Biology Division, National Institute of Nutrition-ICMR, Jamai-Osmania, Hyderabad, 500007, India

    Virendra V. Panpatil & Sudip Ghosh

  4. Department of Biochemistry, Osmania University, Hyderabad, Telangana, 500027, India

    Priyanka Raju Chougule & Manjula Bhanoori

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  1. Priyanka Raju Chougule
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Contributions

The experiment's conception and design, data analysis, and writing the original manuscript were all done by SNS, PRC, and RS. The experiments such as IVIS, anti-oxidant assays, and ELISA were carried out by SNS, PRC, RS, PBP, SSYHQ, VVP, and MB, while the paper was reviewed by SNS, PRC, RS, PBP, SSYHQ, VVP, SG, SKM, and MB. Performers of the experiments and data analysts were SNS, PRC, RS, PBP, SSYHQ, VVP, and MB. Statistical analysis was carried out by SNS, PRC, and RS. All authors reviewed the paper.

Corresponding author

Correspondence to Sukesh Narayan Sinha.

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The authors declare no competing financial interest.

Ethics approval and consent to participate

The protocols for the use of animals were approved by the Institutional Animal Ethics Committee (IAEC) of the ICMR-NIN, and the IAEC approval number is ICMR-NIN/IAEC/02/018/2020.

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Chougule, P.R., Sangaraju, R., Patil, P.B. et al. Effect of ethyl gallate and propyl gallate on dextran sulfate sodium (DSS)-induced ulcerative colitis in C57BL/6 J mice: preventive and protective. Inflammopharmacol 31, 2103–2120 (2023). https://doi.org/10.1007/s10787-023-01254-5

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