Therapeutic effects of EGCG in experimentally induced ulcerative colitis in rats via affecting inflammation and apoptosis

Ulcerative colitis (UC) is a chronic inflammatory bowel disease that affects the colon. Epigallocatechin gallate (EGCG), a compound found in green tea with antioxidant and anti-inflammatory properties. We aimed to investigate the impact of EGCG on inflammation and apoptotic pathways in UC rats. The study involved inducing UC in rats by administering acetic acid. The UC rats were then treated with EGCG. Colon samples were collected, measured then used to evaluate gene and protein expression of various factors, including NF-κB, TNF-α, SphK, MIP-1α, BCL2, and BAX, as well as the activities of caspase-3/8/9.

Methods
Animals and treatment outlines

In this study, 36 rats belonging to the Sprague-Dawley species and weighing between 180 and 200 grams were employed. The rats were divided into three groups, each consisting of twelve rats.

Control group: Rats were subjected to mild anesthesia using ether followed by the administration of 2 ml of saline solution into the colon using a soft pediatric lubricated catheter. The rats were positioned horizontally for two minutes to prevent the saline from draining. Subsequently, they received a daily oral dose of 0.5% (w/v) methylcellulose and 2% (v/v) Tween 80, both dissolved in deionized water, for two weeks.

Ucerative Colitis group: The rats were anesthetized with ether and subsequently administered 2 ml of 4% acetic acid via a gentle pediatric catheter into their colon. Following the administration, the rats were positioned horizontally for two minutes to promote the retention of the acetic acid.

Ulcerative Colitis group treated with genistein: UC was induced utilizing the previously outlined methodology. Subsequently, the rats received an oral administration of 20 mg/kg of EGCG (Sigma-Aldrich, Inc, St. Louis, MO, United States) once daily for two weeks.

Sample collection

After the animals were euthanized, the entire colon was excised, measured, and weighed. A segment was homogenized in a ten-fold cold sodium-potassium phosphate buffer (0.01 M, pH 7.4) containing 1.15% KCl. The resulting solution was then subjected to rapid freezing at -80°C.

Estimation of caspases activity

The enzyme activities of caspase-3, -8, and -9 were evaluated using commercially available kits (Abcam, Cambridge, MA, USA). The Caspase assay protocol is based on the formation of the chromophore p-nitroaniline by cleavage from the labeled substrate DEVD-pNA. The p-NA can be quantied using a spectrophotometer or a microtiter plate reader reading absorbance at 405 nm.

Enzyme-linked immunosorbent assay (ELISA)

We utilized commercially available ELISA kits to evaluate the levels of SphK, macrophage inflammatory protein (MIP)-1α (CCL3), B-cell lymphoma 2 (BCL2), and BCL2 Associated X (BAX), as well as TNF-α under the provided instructions from the manufacturer (MyBioSource, Inc., San Diego, CA, USA).

Quantitative real-time polymerase chain reaction (RT‒PCR)

The mRNA levels of SphK, MIP-1α, BCL2, BAX, NFκB, and TNF-α in rat colon lysates were analyzed using established protocols. β-actin was used as the internal reference and housekeeping gene.