Characterization of an in vivo model of neuroinflammation and evaluation of the anti-inflammatory and neuroprotective effects of curcumin as a potential lead compound for the development of new agents useful to treat neuroinflammatory disorders

Neuroinflammation is a complex and multifactorial response of the central nervous system (CNS) to trauma, infection and neurodegenerative diseases orchestrated by specialized immune cells (microglial and astrocytes). In particular, microglia, the main resident immune cells of the CNS, undergo rapid “activation” in response to noxious stimuli, releasing a plethora of inflammatory and potentially neurotoxic soluble factors, such as cytokines [e.g., interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α], chemokines and reactive oxygen and nitrogen species (e.g., nitric oxide). Although an efficient microglial immune response is necessary and critical for proper resolution of pathological events, it is clear that an excessive activation of these cells contributes to neuronal cell damage in neurodegenerative and psychiatric disorders. However, the molecular mechanism(s) by which these cells exert their deleterious effects on neurons remain poorly understood. Recent evidence suggests that microglial cells can be “activated” in response to a systemic inflammatory stimulus. For example, a single peripheral administration of the bacterial endotoxin lipopolysaccharide (LPS), the main component of Gram-negative bacteria walls, in adult mice, can induce microglial activation and an inflammatory state in the CNS that persists long after peripheral stimulus has decline. Identification of molecules which prevent or down-regulate microglial inflammatory responses or direct microglia towards a protective anti-inflammatory phenotype could prove efficacious in neurodegenerative diseases in which inflammation is implicated. Recently, increasing interest has focused on identifying natural compounds with potential inhibitory effects on microglial activation and subsequent inflammatory processes. Among these compounds, curcumin (diferuloylmethane), the main bioactive component isolated from the rhizome of the turmeric plant (Curcuma longa) with multiple pharmacological effects, including anti-inflammatory activities, possesses neuroprotective properties against many neurodegenerative conditions. The principal focus of this doctoral project has been the study of the possible anti-inflammatory effect of curcumin in an in vivo model of neuroinflammation based on a single systemic LPS injection. Young adult mice were intraperitoneally injected with a single dose of LPS (0.5 or 5 mg/kg) or vehicle and then tested for “sickness behavior” (e.g., changes in body weight and food intake), mRNA (real-time RT-PCR) and protein (enzyme-linked immunosorbent assay, ELISA) expression of pro-inflammatory mediators and microglia morphological changes (immune-staining with the microglial marker ionized calcium binding adaptor molecule 1) in different brain areas. Both LPS doses induced a significant decrease in food intake and body weight within the first 4 days, followed by a gradual recovery to control values; however, only 5 mg/kg LPS significantly increased TNF-α, IL-1β, IL-6, COX-2 and iNOS gene expression 2 h post-injection. Pre-treatment with 50 mg/kg curcumin (orally administered by gavage for 2 consecutive days before LPS injection) facilitated the recovery from sickness behavior (anorexia and weight loss), suppressed LPS-induced microglial morphological changes and increased of mRNA levels of TNF-α, IL-1β and COX-2 in all brain areas, while limiting expression of IL-6 and iNOS to more selected brain regions. Possible neuroprotective properties of curcumin were also investigated. In particular, mRNA expression of brain-derived neurotrophic factor (BDNF), known to play a key role in the regulation of neuronal function, as well as in learning and memory processes. Its reduced expression, described in numerous neurodegenerative disorders, contributes to the onset of structural abnormalities and functional damage in the CNS. LPS treatment reduced mRNA levels of BDNF in all brain areas analyzed up to 7 days after treatment. Pre-treatment with 50 mg/kg curcumin restored BDNF gene expression 24 hours after LPS injection, while a higher dose of curcumin (100 mg/kg) completely restored the expression of BDNF 2 hours after the inflammatory stimulus. Finally, behavioral tests demonstrated that LPS induced motor and memory impairments, that persisted even when the inflammatory process had been solved. Moreover, curcumin reversed motor deficits induced by LPS. Taken together, these data show that curcumin can prevent neuroinflammation by modulating the expression of brain pro-inflammatory mediators in vivo and also suggest a potential role of curcumin as a neuroprotective molecule able to restore BDNF levels and behavioral impairments under inflammatory conditions. In conclusion, curcumin represents a promising lead compound to discover new drug candidates, with improved therapeutic efficacy in the treatment of neurodegenerative and age-related diseases with an inflammatory etiology.

La neuroinfiammazione è una risposta complessa e multifattoriale a diversi tipi di stimoli nocivi come le infezioni virali, batteriche o traumi. é noto che in questo processo le cellule microgliali giochino in ruolo chiavenel bilancio tra la risoluzione del processo infiammatorio o la progressione di questo ferso un profilo neurodegenerativo. Nel nostro lavoro abbiamo valutato gli effetti antiinfiammatori e neuroprotettivi di curcumina in un modello in vivo di neuroinfiammazione creato mediante una singola iniezione di lipopolisaccaride (LPS) intraperitoneale ad una dose di 5 mg/Kg. L'effetto di curcumina è stato valutato sul profilo citochinico, su molecole proinfimmatorie come COX-2 e iNOS su molecole neuroprotettive come il BDNF mediante saggi di RT-PCR, immunoistochimica per evidenziare l'effetto di curcumina sull'attivazione microgliale e sulla sopravvivenza dei neuroni dopaminergici in substantia nigra, attraverso l'uso di test comportamentali ai fini di valutare l'effetto protettivo di curcumina sull'attività locomotoria e la memoria dichiarativa. In cocnlusione, la curcumina è risultata essere un valido composto con proprietà antiinfiammatorie e neuroprotettive e rappresenta una molecola chiave per lo sviuppo di nuovi composti con aumentata efficacia terapeutica nel trattamento e la prevenzione di patologie neurodegenerative su base infiammatoria.