4. Discussion
In this systematic review and meta-analysis, we demonstrated the
efficacy of TNF-α, IL-1β, and IL-6 as in vitro inflammatory
biomarkers in the LPS-induced RAW 264.7 cell model, and showed that NO
levels still are the gold standard test to trial anti-inflammatory
agents.
As expected, NO levels increased significantly when RAW 264.7 cells were
induced by LPS compared to non-induced cells, independently of cell
density, but cytokine levels changed according to cell density. To
ensure better screening for novel drugs, the trial could be initiated by
screening for drugs showing NO levels higher than 20 µM by LPS-induced
RAW 264.7 cells with statistical significance compared to non-induced
cells. After this initial screening, it was found that all the cytokines
studied were excellent inflammatory biomarkers, since in all the studies
where NO produced by cells was > 20 µM, the cytokines were
significantly increased. Moreover, all cytokine levels presented a
positive correlation with the cell density used in the experiments.
However, significant heterogeneity was found among the studies, mainly
due to the different methodologies presented by the authors. Therefore,
it is essential to standardize the inflammation model in RAW 264.7
cells, following some specific guidelines for in vitro experimentation.
Considering our results, it is important to highlight that due to the
model chosen for the review and the characteristics of the included
studies, a meta-analysis was performed. The model applied to use LPS as
a phlogistic agent is one of the main components of gram-negative
bacteria (Romerio & Peri, 2020). This agent has the characteristic of
activating Toll-like receptors present in cells of the immune system,
including the macrophages. In Gram-negative bacterial infections, for
example, in the early stages of the sepsis process, this interaction
between LPS and cellular receptors leads to the activation of
intracellular signaling cascades activating many nuclear transcriptional
factors. This results in massive secretion of several pro-inflammatory
mediators, such as NOx, TNF-α, IL-1β and IL-6, the focus of our research
(Ciesielska et al., 2021; Poltorak et al., 1998). For this reason, the
parallel increase of NO and cytokines when culture cells are stimulated
with LPS was already expected (Dai et al., 2018). However, an important
technical detail in the cell culture must be taken into account, which
is the incubation time of the cells after LPS induction. Although the
action of TLR4 by LPS, ends with the production of many inflammatory
mediators, their release by the cells, presents a different time-course
of secretion (Hobbs et al., 2018). This fact can produce different
results in the levels of these inflammatory mediators, making comparison
between them unfeasible (Liu et al., 2017; Rahman & McFadden, 2011).
As previously commented in the introduction section, this model is
widespread among research laboratories worldwide, as it provides rich
information on the inflammatory response and helps initiate the search
for new anti-inflammatory and immunomodulatory drugs (Elisia et al.,
2018; Taciak et al., 2018). However, there is still a lack of
standardization regarding LPS doses, cell induction time, and cell
density used, among others. To resolve these shortcomings in the
standardization of this inflammatory model, we were very judicious in
the application of eligibility criteria and in the data extraction, in
order to ensure the reliability of the results obtained.
Our results show, as expected, that there is an increase in NO
production when RAW 264.7 cells are induced by LPS (Moore et al., 2019;
Ranaweera et al., 2020). Thus, considering the characteristics of the
studies included in the meta-analysis, it is plausible to affirm that
the NO measurement in the model in question is sufficient for the
purposes of initial inflammatory screening, considering that the
production of the pro-inflammatory cytokines in question accompanies the
same behavior as that observed with the determination of NO levels, at
least, when the time of cell incubation after LPS stimulation is between
12h and 24h. This data is important because, to our knowledge, there is
no standardization among researchers in regard to the choice of initial
inflammatory mediators to measure in this model, in screening for the
possible anti-inflammatory or immunomodulatory activity of some compound
or extract from a natural product. Moreover, we also found that there is
no consensus among researchers regarding the biomarkers used for
screening new anti-inflammatory drugs, cell density, LPS concentration
used, time of induction, and use of culture medium supplementation.
Thus, taking into account the information collected, we strongly advise
that the use of NO measurements be standardized when used to screen for
possible compounds with anti-inflammatory activity. Obviously, taking
into account the particularities of each laboratory is extremely
important, however, the use of LPS at a dose of 1ug
mL-1, cell density between 1 and
9.9x105, and incubation time of between 12 and 24
hours at 37°C, appear to be the minimum requirements for this
standardization (Supplementary Material S10).
Finally, regarding the risk of bias and heterogeneity of included
studies in this meta-analysis, we believe that due to this lack of
standardization among published papers, researchers have selected a
range of different mediators for their experiments. In our study, it was
demonstrated that only NO levels are sufficient to draw the initial
conclusions when seeking to determine whether some compound should be
submitted to the next levels of anti-inflammatory experimentation, when
the culture of Raw 264.7 cells induced by LPS is used as an initial
experimental platform. The use of a single marker is also economically
viable, as the technique is easy to perform, with good reproducibility
and low cost, besides being quicker than using several markers.
Regarding the strengths, in this study, all steps were performed in
pairs and validated by an expert. Also, a meta-analysis and risk of bias
were performed. No time was limited to literature search. Some
limitations were considered regarding heterogeneity between studies and
the rigorous inclusion criteria.