Determination of the effects of piper betle leaf ethanolic extract on myeloperoxidase activity and mpo gene expression in neutrophils in response to staphylococcus a ureus / Yalda Modirrahmati

Piper betle leaves are consumed in traditional medicine in many Asian countries including Malaysia and it has antimicrobial and antioxidant activities. Staphylococcus aureus is a troublesome pathogen due to its ability to cause infections that withstand multiple antibiotics. Myeloperoxidase (MPO) is a heme enzyme gene encoding myeloperoxidase (mpo) with a molecular mass of 150 kDa consisting of two alpha-chains 60 kDa and two beta-chains 15 kDa. It is abundantly expressed in neutrophils (PMNs) and generates hypochlorous acid (HOC1) during respiratory burst which is utilized to kill bacteria. The effects of predetermined P. betle ethanolic leaf extract MIC (5 mg/ml) on MPO in human neutrophils that were challenged with S. aureus (ATCC 25923) were investigated. MPO expression was analyzed using SDS-PAGE and total protein concentrations in neutrophil lysates were determined using Bradford assay. SDS-PAGE analysis showed significant effects of the extract on MPO after just 1 hour treatment. Based on observed effects, the protocol was adapted for use in all proceeding assays. Myeloperoxidase activity was determined using a kit assay (MPO activity colorimetric detection kit, USA) and effect on neutrophil killing activity against S. aureus was tested. Further, mpo expression analyses were performed using quantitative real-time PCR and validated using the Livak calculation method. SDS-PAGE analyses showed that S. aureus represses expression of both the alpha and beta chains of MPO. Notably, expression of the beta chain was affected more drastically. A corresponding 19.23% reduction in protein concentrations observed in treated PMNs with S. aureus as determined by the Bradford assay. P. betle extract significantly alleviated the repressor effect by S. aureus whereby band densities of both subunits were increased. Corresponding increases in protein concentrations at 19.23% and 13.44% were seen in PMNs treated with both extract and S. aureus together and with P. betle extract only respectively. Myeloperoxidase activity analysis showed 18.33 mU/ml increased in presence of P. betle extract compared to untreated PMNs 3.33 mU/ml while PMNs treated with S. aureus showed no MPO activity. Significantly, increase of 22.5 mU/ml was observed in treated PMNs with P. betle extract even in presence of S. aureus which corresponds to increased protein profile observed in both SDS-PAGE analysis and Bradford assay. The neutrophil killing assay revealed significant effect of P. betle ethanolic extract on neutrophil killing activity. Neutrophils treated with S. aureus showed 7924% survival whereas addition of P. betle extract reduced survival to only 7.14% even in the presence of the organism, mpo expression analysis by real-time PCR revealed increased expression 2.29 fold after treatment of neutrophils by P. betle extract in the presence of S. aureus. A similar repressor effect was observed in PMNs treated with S. aureus which revealed only 0.053 fold gene expression compared to untreated neutrophils. Overall data obtained from this study confirms that S. aureus showed downregulates mpo expression resulting in reduced protein expression, enzyme activity and neutrophil killing ability. P. betle ethanolic extract overcomes these repressive effects resulting in corresponding increase in all identical parameters that were tested. This study potentially aids in discovery and development of new antimicrobials. The findings in this study elucidated mechanisms by which the ethanolic extract of P. betle significantly affects clearance of S. aureus in vitro. Future work involving in vivo studies would be beneficial to ascertain if the observable effects can be replicated. Animal model studies would reveal if the extract can be potentially developed to enhance the innate immune system, particularly involving killing potential of neutrophils and clearance of S. aureus.