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Inflammatory biomarkers on an LPS-induced RAW 264.7 cell model: a systematic review and meta-analysis

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Several experimental models have been designed to promote the development of new anti-inflammatory drugs. The in vitro model using RAW 264.7 cells has been widely used. 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 inflammatory mediators together with NO, such as the levels of tumor necrosis factor (TNF)-α, and interleukins (IL) 1β and 6, we carried out this systematic review (SR) and meta-analysis (MA).


We conducted this SR and MA in accordance with the Preferred Reporting of Systematic Reviews and Meta-Analysis and the Cochrane Handbook for Systematic Reviews of Intervention. This review was registered in the Open Science Framework (


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. Though with some restrictions, all studies were reliable, as the risk of bias was detected in the test compounds and systems.


Measurement of NO levels may be sufficient to screen for possible anti-inflammatory action in the context of LPS-induced RAW 264.7 cells.

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This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior [Coordination for the Improvement of Higher Education Personnel] – Brazil (CAPES) – Finance Code 001.

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BMF, GOR, ETBM and GNV performed the literature research, designed the data extraction form, and performed the data extraction and data analysis. IGD, IFK, EMD critically reviewed the analyzed the data. BMF wrote the paper. IGD, IFK, EMD critically reviewed subsequent drafts. All authors approved the final version of the manuscript for submission. All authors had full access to all of the data in the study and took responsibility for the integrity of the data and the accuracy of the data analysis.

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Correspondence to Eduardo Monguilhott Dalmarco.

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Supplementary Information

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Supplementary file1 (PDF 114 KB) Figure S1 - Forest plot of studies considered as outliers, mean differences of cytokines production between the control group and the intervention group (LPS). A) Tumor Necrosis Factor alpha (TNF-α), B) Interleukin-6 (IL-6) and C) Interleukin-1-beta (IL-1β). 95-%-CI, 95-% confidence interval; Tau2, Kendall’s Tau correlation coefficient; I2, I2 statistic; df, degrees of freedom


Supplementary file2 (PDF 140 KB) Figure S2 - Forest plot of studies considered as outliers, mean differences in cytokine production between the control group and the intervention group (LPS) of the studies by cell density subgroups (1 - 2.5 and 3 – 5 x 105 cells/mL). A) Tumor Necrosis Factor alpha (TNF-α), B) Interleukin-6 (IL-6), C) Interleukin-1-beta (IL-1β). 95-%-CI, 95-% confidence interval; Tau2, Kendall’s Tau correlation coefficient; I2, I2 statistic; df, degrees of freedom


Supplementary file3 (PDF 139 KB) Figure S3 - Forest plot of studies considered as outliers, mean differences in cytokine production between the control group and the intervention group (LPS) of the studies included by NO production subgroups (20-50µM and >50µM). A) Tumor Necrosis Factor alpha (TNF-α), B) Interleukin-6 (IL-6), C) Interleukin-1-beta (IL-1β). 95-%-CI, 95-% confidence interval; Tau2, Kendall’s Tau correlation coefficient; I2, I2 statistic; df, degrees of freedom


Supplementary file4 (PDF 71 KB) Figure S4 - Funnel plots showing the mean difference (MD) between results of the LPS-induced and the control cells, by the standard error [SE (MD)]. A) Nitric Oxide (NO); B) Tumor Necrosis Factor-alpha (TNF-α); C) Interleukin-6 (IL-6); D) Interleukin-1-beta (IL-1β)


Supplementary file5 (PDF 727 KB) Figure S5 – Checklist for use of the inflammatory model using LPS in Raw 264.7 macrophages

Supplementary file6 (PDF 93 KB) Table S1 - Checklist PRISMA

Supplementary file7 (DOCX 11 KB) Table S2 - Database search strategy

Supplementary file8 (XLSX 92 KB) Table S3 - Data Extraction

Supplementary file9 (DOCX 9 KB) Table S4 - Checklist of thirty items - Inflammation model in RAW 264.7

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Facchin, B.M., dos Reis, G.O., Vieira, G.N. et al. Inflammatory biomarkers on an LPS-induced RAW 264.7 cell model: a systematic review and meta-analysis. Inflamm. Res. 71, 741–758 (2022).

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