Abstract
The inflammation is a response to some injury aimed at reestablishing
homeostasis of the affected tissue. The in vitro model using RAW 264.7
cells has been widely used, as experimental model to assess the
inflammatory response. However, there is still no consensus on which
inflammatory mediators should initially be measured to screen for
possible anti-inflammatory effects. To determine the rationality of
measuring different inflammatory mediators together with NO, we carried
out this systematic review and meta-analysis. A total of 17 studies were
included, and LPS-induced cells produced high NO levels compared to
non-LPS induced, and this production was not related to cell density.
TNF-α, IL-1β, and IL-6, also showed high levels after cells had been
stimulated with LPS; however, these effects were related to cell
density. Finally, was found that measurement of NO levels alone may be
sufficient to screen for possible anti-inflammatory action in the
context of this model.
Keywords: Lipopolysaccharide, LPS, in vitro study,
macrophages, RAW 264.7, nitric oxide, tumor necrosis factor-alpha,
interleukin-1 beta, and interleukin- 6.
Introduction
Inflammation is a physiological process that occurs in response to an
infectious agent or tissue injury, in order to restore the homeostasis
of the affected tissue. This response involves a cascade of events
characterized by vascular and cellular involvement, with activation of
modulation pathways that depend on the type of harmful agent and the
stimulus generated (Fullerton & Gilroy, 2016; Medzhitov, 2010; Nathan
& Ding, 2010; Zarrin et al., 2021). If successful, the inflammatory
response tends to progress to the post-resolution phase, through a
coordinated series of molecular and cellular events that lead to the
restoration of tissue structure, organ function, and ‘adapted
homeostasis’ (Sugimoto et al., 2016). However, uncontrolled inflammation
can lead to tissue damage that gives rise to chronic inflammatory
diseases, such as Alzheimer’s disease, atherosclerosis, cardiovascular
diseases, and cancer (Cicchitti et al., 2015; Furman et al., 2019).
In this context, there is a continuous search for new anti-inflammatory
drugs, and many experimental models have been developed and extensively
used over the Years (Kabir & Ansari, 2018; Patil et al., 2019). In
vitro models represent the beginning of this complex development, and
millions of potential compounds are proposed each year. Thus, fast and
reliable screening is required for the next level of pharmacological
research, optimizing the initial screening and reducing the use of
animals (Benam et al., 2015). For this purpose, the use of in vitro
models using RAW 264.7 cells has become widespread in research
laboratories, due to their versatility. RAW 264.7 cells are similar to
monocytes/macrophages, originating from the cell line transformed by the
Abselon Leukemia Virus derived from BALB/c mice (Taciak et al., 2018).
In vitro models using these cells are the most commonly applied models
when screening for anti-inflammatory and immunomodulatory compounds.
This is because the RAW 264.7 cell line produces a robust and well-known
inflammatory response, especially when challenged by inflammatory
stimulants such as lipopolysaccharide (LPS), a Toll-like receptor 4
(TLR4) agonist. TLR4 stimulation activates intracellular signaling
cascades, which in turn, mobilizes nuclear transcription factors such as
NF-kB, This ultimately leads to the production of inflammatory
mediators, such as Nitric Oxide (NO), Tumor Necrosis Factor-alpha
(TNF-α), Interleukin-1-beta (IL-1β), and Interleukin-6 (IL-6), among
others (Elisia et al., 2018; Lawrence, 2009; Taciak et al., 2018). In
this context, the most common inflammatory biomarker used in the
screening for possible anti-inflammatory and immunomodulatory action of
any compound or drug is nitric oxide levels (NOx). Nitric oxide is
produced after the initial action of the phlogistic agent, culminating
in nitric oxide synthase (iNOS) induction and substantial production of
this inflammatory mediator (Lind et al., 2017; Saha & Burns, 2020).
The RAW 264.7 cell line has been used in biological laboratories for
more than forty years, and the detection of NO levels is the most
commonly measured inflammatory mediator in these experiments. The
expected level of other proinflammatory mediators produced, after
stimulation of RAW cells with LPS, has not yet been explored and/or
standardized in any robust study (Taciak et al., 2018). Furthermore,
there is no consensus regarding the existence of a direct correlation
between NO production by activation of iNOS, and other important
inflammatory mediators such as TNF-α, IL-1β, and IL-6, produced after
activation of nuclear transcription factors. In order to standardize the
expected levels of NO, TNF-α, IL-1β, and IL-6 production after LPS
stimulation of RAW 264.7 cells, and to determine the rationality of
measuring numerous inflammatory biomarkers in the initial stages of the
screening process of anti-inflammatory and immunomodulatory activities,
we decided to carry out this systematic review and meta-analysis.