Chauhan, Anil Kumar
(Department of Bioscience and Biotechnology, Konkuk University)
,
Jang, Mihee
(Department of Bioscience and Biotechnology, Konkuk University)
,
Kim, Yangmee
(Department of Bioscience and Biotechnology, Konkuk University)
Macrophages are the cells of the first-line defense system, which protect the body from foreign invaders such as bacteria. However, Gram-negative bacteria have always been the major challenge for macrophages due to the presence of lipopolysaccharides on their outer cell membrane. In the present stud...
Macrophages are the cells of the first-line defense system, which protect the body from foreign invaders such as bacteria. However, Gram-negative bacteria have always been the major challenge for macrophages due to the presence of lipopolysaccharides on their outer cell membrane. In the present study, we evaluated the effect of phloretin, a flavonoid commonly found in apple, on the protection of macrophages from Escherichia coli infection. RAW 264.7 cells infected with standard E. coli, or virulent E. coli K1 strain were treated with phloretin in a dose-dependent manner to examine its efficacy in protection of macrophages. Our results revealed that phloretin treatment reduced the production of nitric oxide (NO) and generation of reactive oxygen species along with reducing the secretion of proinflammatory cytokines induced by the E. coli and E. coli K1 strains in a concentration-dependent manner. Additionally, treatment of phloretin downregulated the expression of E. coli-induced major inflammatory markers i.e. cyclooxygenase-2 (COX-2) and hemeoxygenase-1 (HO-1), in a concentration dependent manner. Moreover, the TLR4-mediated NF-κB pathway was activated in E. coli-infected macrophages but was potentially downregulated by phloretin at the transcriptional and translational levels. Collectively, our data suggest that phloretin treatment protects macrophages from infection of virulent E. coli K1 strain by downregulating the TLR4-mediated signaling pathway and inhibiting NO and cytokine production, eventually protecting macrophages from E. coli-induced inflammation.
Macrophages are the cells of the first-line defense system, which protect the body from foreign invaders such as bacteria. However, Gram-negative bacteria have always been the major challenge for macrophages due to the presence of lipopolysaccharides on their outer cell membrane. In the present study, we evaluated the effect of phloretin, a flavonoid commonly found in apple, on the protection of macrophages from Escherichia coli infection. RAW 264.7 cells infected with standard E. coli, or virulent E. coli K1 strain were treated with phloretin in a dose-dependent manner to examine its efficacy in protection of macrophages. Our results revealed that phloretin treatment reduced the production of nitric oxide (NO) and generation of reactive oxygen species along with reducing the secretion of proinflammatory cytokines induced by the E. coli and E. coli K1 strains in a concentration-dependent manner. Additionally, treatment of phloretin downregulated the expression of E. coli-induced major inflammatory markers i.e. cyclooxygenase-2 (COX-2) and hemeoxygenase-1 (HO-1), in a concentration dependent manner. Moreover, the TLR4-mediated NF-κB pathway was activated in E. coli-infected macrophages but was potentially downregulated by phloretin at the transcriptional and translational levels. Collectively, our data suggest that phloretin treatment protects macrophages from infection of virulent E. coli K1 strain by downregulating the TLR4-mediated signaling pathway and inhibiting NO and cytokine production, eventually protecting macrophages from E. coli-induced inflammation.
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문제 정의
On the other hand, humans have a sophisticated innate immune system that deals with extraneous invaders wherein macrophages play a crucial role by eliminating bacteria by phagocytosis [4, 5]. Therefore, the proper functioning of macrophages is essential, and using this as a basis, we designed this study to investigate the effect of phloretin in the protection of macrophage from inflammation induced by standard E. coli and virulent E. coli K1.
제안 방법
Experimental results are depicted as the mean ± standard error of the mean (SEM) of three individual sets of experiments.
To examine cytokine (TNF-α and IL-6) secretion, we collected the supernatant of the non- infected, infected, and phloretintreated groups, and then TNF-α and IL-6 were examined using ELISA kits (Invivogen, USA) according to the manufacturer’s instructions.
We further evaluated the expression of TLR4, MyD88, and NF-κb at the transcriptional level by RT-PCR.
대상 데이터
coli (KCTC 1682) was purchased from the Korean Collection for Type Cultures, Korea Research Institute of Bioscience & Biotechnology (Korea). E. coli K1 strain RS218 (O18:K1:H7), was provided by Jang-Won Yoon of Kangwon National University (Korea). The cultures were maintained in Luria Bertani broth/ agar.
Phloretin (with ≥99% purity) was purchased from Sigma-Aldrich (USA).
RAW 264.7, a murine macrophage cell line, was purchased from the American Type Culture Collection (Manassas, VA) and cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Invitrogen, USA) supplemented with 10% (v/v) fetal calf serum (Invitrogen,) and 1% penicillin–streptomycin cocktail at 37℃ in a 5% CO2 incubator.
데이터처리
Statistical significance was calculated using the Student’s t-test.
성능/효과
We also examined the expression pattern of myeloid differentiation primary response 88 (MyD88), phosphorylation of transforming growth factor beta-activated kinase 1 (p-TAK1), and phosphorylation of NF-κb proteins, which are associated with the production of cytokines to induce inflammation. Results revealed that cells infected with E. coli K1 exhibited up-regulation of MyD88 and phosphorylation of TAK-1, which were significantly decreased in the phloretin-treated group in a concentration-dependent manner (Fig. 3B). Moreover, E.
Therefore, we examined the efficacy of phloretin against the production of these major pro-inflammatory cytokines IL-6 and TNF-α in our study. The results revealed that phloretin treatment decreased the secretion of both cytokines in a concentration-dependent manner. Contrastingly, between standard E.
후속연구
coli infection. However, further study using animal infection model would be needed to warrant the ex vivo efficacy of phloretin.
coli K1 strain RS218(O18:K1:H7) possesses the K1 capsular polysaccharide antigen, which is essential virulence determinant that protects the bacteria from immune attack which provide additional virulence to the bacteria [21]. Results of our study might provide new insights for exploring phloretin efficacy towards infection and modulation of innate immune response.
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