Colon MPO activity, a marker of neutrophilic infiltration, was assessed using MPO assay kit (NampoxTM, Navand Salamat Co., Iran), according to the protocol. A proximal colon sample from each mouse was excised, immediately rinsed with ice-cold saline, blotted dry and frozen at − 80 °C. Subsequently, the tissue samples were thawed, weighed and homogenized in sample buffer 1 × (pH: 6.0) containing 0.5% hexadecyl trimethyl ammonium bromide. Samples were centrifuged at 10,000 rpm for 10 min in 4 °C. A microplate reader (Lab system Multiskan EX, Helsinki, Finland) was used to measure the changes in absorbance at 450 nm in supernatant using 3,3′,5,5′-tetramethylbenzidine (TMB) and H2O2. The results were reported as the U/mg tissue [31].
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