title: Therapeutic targeting of Krüppel-like factor 4 abrogates microglial activation.
abstract: BACKGROUND
  Neuroinflammation occurs as a result of microglial activation in response to invading micro-organisms or other inflammatory stimuli within the central nervous system. According to our earlier findings, Krüppel-like factor 4 (Klf4), a zinc finger transcription factor, is involved in microglial activation and subsequent release of proinflammatory cytokines, tumor necrosis factor alpha, macrophage chemoattractant protein-1 and interleukin-6 as well as proinflammatory enzymes, inducible nitric oxide synthase and cyclooxygenase-2 in lipopolysaccharide-treated microglial cells. Our current study focuses on finding the molecular mechanism of the anti-inflammatory activities of honokiol in lipopolysaccharide-treated microglia with emphasis on the regulation of Klf4.
  
  
  METHODS
  For in vitro studies, mouse microglial BV-2 cell lines as well as primary microglia were treated with 500 ng/mL lipopolysaccharide as well as 1 μM and 10 μM of honokiol. We cloned full-length Klf4 cDNA in pcDNA3.1 expression vector and transfected BV-2 cells with this construct using lipofectamine for overexpression studies. For in vivo studies, brain tissues were isolated from BALB/c mice treated with 5 mg/kg body weight of lipopolysaccharide either with or without 2.5 or 5 mg/kg body weight of honokiol. Expression of Klf4, cyclooxygenase-2, inducible nitric oxide synthase and phospho-nuclear factor-kappa B was measured using immunoblotting. We also measured the levels of cytokines, reactive oxygen species and nitric oxide in different conditions.
  
  
  RESULTS
  Our findings suggest that honokiol can substantially downregulate the production of proinflammatory cytokines and inflammatory enzymes in lipopolysaccharide-stimulated microglia. In addition, honokiol downregulates lipopolysaccharide-induced upregulation of both Klf4 and phospho-nuclear factor-kappa B in these cells. We also found that overexpression of Klf4 in BV-2 cells suppresses the anti-inflammatory action of honokiol.
  
  
  CONCLUSIONS
  Honokiol potentially reduces inflammation in activated microglia in a Klf4-dependent manner.
authors.affiliation: National Brain Research Centre, Manesar, Haryana, India.
authors.forename: Deepak Kumar
authors.initials: DK
authors.lastname: Kaushik
authors.forename: Rupanjan
authors.initials: R
authors.lastname: Mukhopadhyay
authors.forename: Kanhaiya Lal
authors.initials: KL
authors.lastname: Kumawat
authors.forename: Malvika
authors.initials: M
authors.lastname: Gupta
authors.forename: Anirban
authors.initials: A
authors.lastname: Basu
mesh_terms: D000818:Animals; D002461:Cell Line, Transformed; D051546:Cyclooxygenase 2 / Q000378:metabolism; D016207:Cytokines / Q000378:metabolism; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D007249:Inflammation / Q000139:chemically induced / Q000378:metabolism*; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D017628:Microglia / Q000187:drug effects*; D016328:NF-kappa B / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D011134:Polysaccharides / Q000009:adverse effects / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D016207:Cytokines; C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; D016328:NF-kappa B; D011134:Polysaccharides; D017382:Reactive Oxygen Species; D052247:Nitric Oxide Synthase Type II; D051546:Cyclooxygenase 2