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. 2021 Sep 4;22(17):9612.
doi: 10.3390/ijms22179612.

Methotrexate Ameliorates Systemic Inflammation and Septic Associated-Lung Damage in a Cecal Ligation and Puncture Septic Rat Model

Josep Bringué  1   2   3 , Raquel Guillamat-Prats  1   2 , Maria Luisa Martinez  4 , Eva Torrents  5 , Marta Camprubí-Rimblas  1   2   3 , Lluís Blanch  1   2   5 , Antonio Artigas  1   2   3   5
Affiliations

Methotrexate Ameliorates Systemic Inflammation and Septic Associated-Lung Damage in a Cecal Ligation and Puncture Septic Rat Model

Josep Bringué et al. Int J Mol Sci. .

Abstract

Background: Sepsis is a serious, heterogeneous clinical entity produced by a severe and systemic host inflammatory response to infection. Methotrexate (MTX) is a folate-antagonist that induces the generation of adenosine and also inhibits JAK/STAT pathway; MTX it is widely used as an anti-inflammatory drug to control the immune system.

Objective: The aim of this study was to assess the beneficial effects of a single and low dose of MTX in the systemic response and acute lung injury (ALI) induced by sepsis. As in the clinics, we treated our animals with antibiotics and fluids and performed the source control to mimic the current clinic treatment.

Methods and main results: Sepsis was induced in rats by a cecal ligation puncture (CLP) procedure. Six hours after induction of sepsis, we proceeded to the source control; fluids and antibiotics were administered at 6 h and 24 h after CLP. MTX (2.5 mg/Kg) was administered 6 h after the first surgery in one CLP experimental group and to one Sham group. A protective effect of MTX was observed through a significant reduction of pro-inflammatory cytokines and a decrease infiltration of inflammatory cells in the lung. In addition, we found a regulation in adenosine receptor A2aR and the metalloproteinases by MTX.

Conclusion: A single, low dose of MTX attenuates sepsis lung-associated damage by decreasing pro-inflammatory response, infiltration of pro-inflammatory cells and avoiding defective tissue lung remodeling.

Keywords: acute lung injury; acute respiratory distress syndrome; methotrexate; sepsis; systemic inflammation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Body Weight and Survival. (A) Body weight measured at 0 h, 24 h and 48 h in all groups. (B) Representation of the survival rate in all groups in the curse of the experiment. Values represent group mean ± SEM. Sham and Sham + MTX n = 15, CLP and CLP + MTX n = 22.
Figure 2
Figure 2
Systemic inflammation and blood lymphocytes activation. (A) Concentration of TNF-α in plasma measured by ELISA. (B) Number of total lymphocytes from blood cells. (C) Percentage of lymphocytes expressing CD4. (D) Proportion of CD4+ lymphocytes with a Treg phenotype (CD25+FoxP3+). (E) Representative flow cytometry dot plots of all groups. Gattering of CD4+ lymphocytes and Treg phenotype lymphocytes (CD25+FoxP3+). Data is expressed as mean ± SEM. Sham and Sham + MTX n = 4; CLP and CLP + MTX n = 6. * p < 0.05 vs. Sham group; # p < 0.05 vs. CLP group. ** p < 0.01. ***/### p < 0.001.
Figure 3
Figure 3
Lung weight and bronchoalveolar lavage analysis (BAL). (A) Lung weight corrected by the body weight. (B) Concentration of protein in the bronchoalveolar lavage. (C) Number of total cells in the bronchoalveolar lavage. (D) Number of total macrophages in the bronchoalveolar lavage (E) Number of total polymorphonuclear (PMNs) cells in the the bronchoalveolar lavage (F) Number of total lymphocytes in the the bronchoalveolar lavage. Data is expressed as mean ± SEM. Sham and Sham + MTX n = 13; CLP and CLP + MTX n = 18. * p < 0.05 vs. Sham group; # p < 0.05 vs. CLP group. **/## p < 0.01; ***/### p < 0.001.
Figure 4
Figure 4
Gene expression of pro-inflammatory markers in lung tissue and total nitric oxide in bronchoalveolar lavage. (A–F): mRNA expression by qPCR. Data are expressed mean ± SEM, ∆Ct correction was applied using GAPDH as a housekeeping gene and units are relative to the expression of control group. Sham and Sham + MTX n = 10; CLP and CLP + MTX n = 15. (G) Nitric oxide measured in bronchoalveolar lavage. Sham and Sham + MTX n = 5; CLP and CLP + MTX n = 6. * p < 0.05 vs. Sham group; # p < 0.05 vs. CLP group. ** p < 0.01; ***/### p < 0.001.
Figure 5
Figure 5
Gene expression of anti-inflammatory markers, adenosine receptors and caspase 3 and caspase 3 protein in lung tissue. (A–E): mRNA expression by qPCR and (F) Caspase3 protein in lung tissue. Data are expressed mean ± SEM, ∆Ct correction was applied using GAPDH as a housekeeping gene and units are relative to the expression of control group. Sham and Sham + MTX n = 10; CLP and CLP + MTX n = 15. * p < 0.05 vs. Sham group; ## p < 0.01 vs. CLP group. ** p < 0.01; *** p < 0.001.
Figure 6
Figure 6
Gene expression of cell recruitment markers and metalloproteinases and its inhibitors in lung tissue. (A–D): mRNA expression by qPCR. Data are expressed mean ± SEM, ∆Ct correction was applied using GAPDH as a housekeeping gene and units are relative to the expression of control group. Sham and Sham + MTX n = 10; CLP and CLP + MTX n = 15. * p < 0.05 vs. Sham group; # p < 0.05 vs. CLP group. ** p < 0.01; ***/### p < 0.001.
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