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.