Overexpression of miR‑200a‑3p promoted inflammation in sepsis‑induced brain injury through ROS‑induced NLRP3

Abstract

Sepsis, a systemic inflammatory response syndrome induced by infection, is a common complication of trauma, burns, postoperative infection and critical disease, and is characterized by an acute onset and high fatality rate. The aim of the present study was to explore the possible molecular mechanisms of microRNA‑200a‑3p (miRNA‑200a‑3p) on inflammation during sepsis. Reverse transcription‑quantitative PCR and gene microarray were used to measure the expression of miRNA‑200a‑3p. Tumor necrosis factor‑α, interleukin (IL)‑1β, IL‑6 and IL‑18 were searched by ELISA. The related proteins expression was measured using western blotting. The expression of miRNA‑200a‑3p was markedly higher in the sepsis model when compared with the normal control group. In addition, the expression of miRNA‑200a‑3p was upregulated by the miRNA‑200a‑3p plasmid in human brain microvascular endothelial cells treated with lipopolysaccharide, which further induced inflammation via the induction of NLR family pyrin domain containing 3 (NLRP3) and suppression of Kelch like ECH associated protein (Keap)‑1/nuclear factor erythroid 2 like 2 (Nrf2)/heme oxygenase (HO)‑1. The inhibition of Keap1/Nrf2/HO‑1 attenuated the effects of anti‑miRNA‑200a‑3p on inflammation. However, the inhibition of NLRP3 attenuated the effects of miRNA‑200a‑3p on inflammation. In conclusion, to the best of our knowledge, the results of the present study demonstrated for the first time that overexpression of miRNA‑200a‑3p promoted inflammation in sepsis‑induced brain injury through reactive oxygen species‑induced NLRP3.

Figure 1

Expression of miRNA-200a-3p in sepsis model. (A) TNF-α, (B) IL-6, (C) IL-1β and (D) IL-18 levels were determined by ELISA. The (E) escape latency, (F) tracking maps, (G) the time in the target quadrant and (H) the number of crossings were analyzed via behavior assessments. (I) Evaluation of the number of neuronal cells in hippocampal tissues (magnification, ×100), (J) gene chip analysis and (K) reverse transcription-quantitative PCR for miRNA-200a-3p expression were also conducted. Data are presented as the mean ± standard error of the mean. ^##P<0.01 vs. sham group. Sham, sham group; Sepsis, sepsis model group; TNF-α, tumor necrosis factor-α; IL, interleukin; miRNA/miR, microRNA.

Figure 2

miRNA-200a-3p regulates NLRP3 and keap1 expression in vitro model. (A) miRNA-200a-3p expression in cells transfected with negative and miR-200a-3p plasmids. (B) Gene chip analysis for NLRP3. (C) The miRNA-200a-3p binding site in the 3'-UTR of NLRP3 mRNA, and (D) luciferase assay activity levels. (E) The miRNA-200a-3p binding site in the 3'-UTR of Keap1 mRNA, and (F) the luciferase assay activity levels. (G) The network signaling path. Data are presented as the mean ± standard error of the mean. ^##P<0.01 vs. negative group. Negative, negative plasmid group; miR-200a-3p, miR-200a-3p overexpression group; miRNA/miR, microRNA; NLRP3, NLR family pyrin domain containing 3; Keap1, Kelch like ECH associated protein-1; UTR, untranslated region.

Figure 3

Effect of miRNA-200a-3p overexpression on inflammation in vitro. (A) Keap1, (B) Nrf2, (C) HO-1, (D) NLRP3 and (E) caspase-1 protein expressions were determined by (F) western blotting analysis. (G and H) ROS levels (magnification, ×200), and (I) IL-18 and (J) IL-1β levels were also assessed. Data are presented as the mean ± standard error of the mean. ^##P<0.01 vs. negative group. Negative, negative group; miR-200a-3p, miR-200a-3p overexpression group; miRNA/miR, microRNA; NLRP3, NLR family pyrin domain containing 3; Keap1, Kelch like ECH associated protein-1; Nrf2, nuclear factor erythroid 2 like 2; HO-1, heme oxygenase; ROS, reactive oxygen species; IL, interleukin.

Figure 4

Effect of miRNA-200a-3p downregulation on inflammation in vitro. (A) miRNA-200a-3p expression following anti-miRNA-200a-3p plasmid trans-fection. (B) Keap1, (C) Nrf2, (D) HO-1, (E) caspase-1 and (F) NLRP3 protein expressions were determined by (G) western blotting analysis. (H and I) ROS levels (magnification, ×200), and (J) IL-1β and (K) IL-18 levels were also assessed. Data are presented as the mean ± standard error of the mean. ^##P<0.01 vs. negative group. Negative, negative group; anti-200a-3p, miR-200a-3p downregulation group; miRNA/miR, microRNA; NLRP3, NLR family pyrin domain containing 3; Keap1, Kelch like ECH associated protein-1; Nrf2, nuclear factor erythroid 2 like 2; HO-1, heme oxygenase; ROS, reactive oxygen species; IL, interleukin.

Figure 5

Inhibition of Keap1 reduces the effects of anti-miRNA-200a-3p on inflammation in vitro. (A) Keap1, (B) Nrf2, (C) HO-1, (D) NLRP3 and (E) caspase-1 protein expressions were determined by (F) western blotting analysis. The levels of (G and H) ROS (magnification, x200), (I) IL-1β and (J) IL-18 were also assessed. Data are presented as the mean ± standard error of the mean. ^##P<0.01 vs. negative group; ^**P<0.01 vs. miR-200a-3p downregulation group. Negative, negative group; anti-200a-3p, miR-200a-3p downregulation group; si-Keap1, downregulation of miR-200a-3p and si-Keap1 group; si-, small interfering RNA; miRNA/miR, microRNA; NLRP3, NLR family pyrin domain containing 3; Keap1, Kelch like ECH associated protein-1; Nrf2, nuclear factor erythroid 2 like 2; HO-1, heme oxygenase; ROS, reactive oxygen species; IL, interleukin.

Figure 6

Inhibition of Nrf2 reduces the effects of anti-miRNA-200a-3p on inflammation in vitro. (A) Nrf2, (B) HO-1, (C) NLRP3 and (D) caspase-1 protein expressions were determined by (E) western blot analysis. The levels of (F and G) ROS (magnification, x200), (H) IL-1β and (I) IL-18 were also assessed. Data are presented as the mean ± standard error of the mean. ^##P<0.01 vs. negative group; ^**P<0.01 vs. miR-200a-3p downregulation group. Negative, negative group; anti-200a-3p, miR-200a-3p downregulation group; si-Nrf2, downregulation of miR-200a-3p and si-Nrf2 group; si-, small interfering RNA; miRNA/miR, microRNA; NLRP3, NLR family pyrin domain containing 3; Keap1, Kelch like ECH associated protein-1; Nrf2, nuclear factor erythroid 2 like 2; HO-1, heme oxygenase; ROS, reactive oxygen species; IL, interleukin.

Figure 7

Inhibition of HO-1 reduces the effects of anti-miRNA-200a-3p on inflammation in vitro. (A) HO-1, (B) caspase-1 and (C) NLRP3 protein expressions were determined by (D) western blot analysis. The levels of (E and F) ROS (magnification, x200), (G) IL-1β and (H) IL-18 were also assessed. Data are presented as the mean ± standard error of the mean. ^##P<0.01 vs. negative group; ^**P<0.01 vs. miR-200a-3p downregulation group. Negative, negative group; anti-200a-3p, miR-200a-3p downregulation group; si-HO-1, downregulation of miR-200a-3p and si-HO-1 group; si-, small interfering RNA; miRNA/miR, microRNA; NLRP3, NLR family pyrin domain containing 3; Keap1, Kelch like ECH associated protein-1; Nrf2, nuclear factor erythroid 2 like 2; HO-1, heme oxygenase; ROS, reactive oxygen species; IL, interleukin.

Figure 8

ROS participates in the effect of miRNA-200a-3p on inflammation in vitro. (A and B) ROS levels were assessed in cells treated with miR-200a-3p plasmid and ROS inhibitor (magnification, ×200). (C) Caspase-1 and (D) NLRP3 protein expressions were determined by (E) western blotting analysis. (F) IL-1β and (G) IL-18 levels were also determined. Data are presented as the mean ± standard error of the mean. ^##P<0.01 vs. negative group; ^**P<0.01 vs. miR-200a-3p overexpression group. Negative, negative group; miR-200a-3p, miR-200a-3p overexpression group; ROS inhibitor, overexpression of miR-200a-3p and ROS inhibitor group; miR, microRNA; NLRP3, NLR family pyrin domain containing 3; Keap1, Kelch like ECH associated protein-1; Nrf2, nuclear factor erythroid 2 like 2; HO-1, heme oxygenase; ROS, reactive oxygen species; IL, interleukin.

Figure 9

Inhibition of NLRP3 reduces the effects of miRNA-200a-3p on inflammation in vitro. (A) NLRP3 and (B) caspase-1 protein expressions were determined by (C) western blotting analysis. (D) IL-1β and (E) IL-18 expression levels were also evaluated. Data are presented as the mean ± standard error of the mean. ^##P<0.01 vs. negative group; ^**P<0.01 vs. miR-200a-3p overexpression group. Negative, negative group; miR-200a-3p, miR-200a-3p overexpression group; si-NLRP3, overexpression of miR-200a-3p and si-NLRP3 group; si-, small interfering RNA; miRNA/miR, microRNA; NLRP3, NLR family pyrin domain containing 3; IL, interleukin.

Figure 10

Overexpression of miR-200a-3p promotes inflammation in sepsis-induced brain injury through Keap1/Nrf2/HO-1/ROS-induced NLRP3. miR, microRNA; NLRP3, NLR family pyrin domain containing 3; Keap1, Kelch like ECH associated protein-1; Nrf2, nuclear factor erythroid 2 like 2; HO-1, heme oxygenase; ROS, reactive oxygen species; IL, interleukin.