MicroRNA-146a improves sepsis-induced cardiomyopathy by regulating the TLR-4/NF-κB signaling pathway

Abstract

The aim of the current study was to investigate the regulatory effect of miR-146a on the toll-like receptor 4 (TLR-4)/NF-κB pathway and therefore inflammation in septic cardiomyopathy. A total of 60 healthy male Sprague Dawley rats were equally divided into a control, LPS, miR-146a agonist and miR-146a inhibitor group. Blood samples were collected from rats 24 h after intraperitoneal lipopolysaccharide injection and myocardial tissues were subsequently collected. After hematoxylin and eosin staining of rat myocardial tissues, the degree of inflammatory cell infiltration and myocardial damage was observed. The content of certain myocardial injury markers were also observed, including cardiac troponin I (cTnI), B-type natriuretic peptide (BNP), creatine kinase myocardial bound (CK-MB) and myoglobin (Mb). Western blot analysis was performed to detect the expression of NF-κB/TLR-4, tumor necrosis factor (TNF-α) and intercellular adhesion molecule-1 (ICAM-1) in myocardial tissues. Reverse transcription-quantitative (RT-q) PCR was used to detect the expression of miR-146a, TNF-α, interleukin (IL)-1α and IL-1β mRNA in myocardial tissues. In the LPS group, myocardial interstitial tissue edema occurred, with enlarged and loosely arranged cardiomyocytes. Compared with the sepsis model group, myocardial interstitial tissue edema was relieved in the miR-146a agonist group, but was aggravated in the miR-146a inhibition group. The serum levels of cTnI, BNP, CK-MB, Mb, NF-κB, TLR-4, TNF-α and ICAM-1 in the sepsis model group were higher than those in the control group. In the miR-146a agonist group, levels of myocardial injury markers were lower than those in the sepsis model group, but were higher in the miR-146a inhibition group. The results of RT-qPCR demonstrated that the expression of miR-146a, TNF-α, IL-1α and IL-1β in the sepsis model group were upregulated compared with the control group. In addition, miR-146a expression in the miR-146a agonist group and the miR-146a inhibition group was increased, but TNF-α, IL-1α and IL-1β mRNA was downregulated. miR-146a may regulate the TLR-4/NF-κB signaling pathway via negative feedback mechanisms, leading to the improvement of the inflammatory response and cardiac dysfunction in sepsis-induced cardiomyopathy.

Keywords: lipopolysaccharide; microRNA-146a; sepsis; toll-like receptor 4/nuclear factor-κB pathway.

Figure 1.

Morphological changes of myocardial tissue in four groups of rats. Morphological changes of rat myocardial tissues in the (A) control, (B) LPS, (C) miR-146a agonist and (D) miR-146a inhibitor group. miR, microRNA; LPS, lipopolysaccaride.

Figure 2.

Detection of cardiomyocyte apoptosis in four groups of rats. Cardiomyocyte apoptosis of rat myocardial tissue in the (A) control, (B) model (C) agonist and (D) miR-146a inhibitor group. miR, microRNA; LPS, lipopolysaccaride. (E) Quantitative analysis of apoptotic index. *P<0.05 vs. the control group; #P<0.05 vs. the LPS group.

Figure 3.

Levels of myocardial injury markers in four groups of rats. Comparison of serum (A) cTnI, (B) BNP, (C) CK-MB and (D) Mb levels in the indicated groups. *P<0.05 vs. the control group; #P<0.05 vs. the LPS group. cTnI, cardiac troponin I; BNP, B-type natriuretic peptide; CK-MB, creatine kinase myocardial bound; Mb, myoglobin; LPS, lipopolysaccaride.

Figure 4.

TLR-4/NF-κB-associated protein expression in the myocardial tissue of the four treatment groups. (A) Western blot analysis and (B) subsequent quantification of NF-κB and TLR-4 expression in the myocardial tissue of four groups of rats. (C) Westen blot analysis and (D) subsequent quantification of TNF-α and ICAM-1 expression in the myocardial tissue of rats. *P<0.05 vs. the control group; #P<0.05 vs. the model group. TLR-4, toll-like receptor-4; NF-κB, nuclear factor-κB; TNF-α, tumor necrosis factor-α; ICAM-1, intercellular adhesion molecule-1; LPS, lipopolysaccaride.

Figure 5.

Expression of myocardial tissue-associated mRNA in four groups of rats. (A) miRNA-146a, (B) TNF-α, (C) IL-1α and (D) IL-1β mRNA expression was determined in the myocardial tissue of the four treatment groups. miR, microRNA; TNF-α, tumor necrosis factor-α; IL-1α, interleukin-1α; IL-1β, interleukin-1β; LPS, lipopolysaccaride. *P<0.05 vs. the control group; #P<0.05 vs. the LPS group.