miR-143-3p impacts on pulmonary inflammatory factors and cell apoptosis in mice with mycoplasmal pneumonia by regulating TLR4/MyD88/NF-κB pathway

Abstract

miR-143-3p is correlated with inflammatory pain responses, such as hsa-miR-143-3p expression reduction in fibromyalgia. The present study aimed to explore the effects of miR-143-3p and Toll-like receptor (TLR) 4/myeloid differentiation factor 88 (MyD88)/NF-κB signaling pathway on pulmonary inflammatory factors levels and alveolar epithelial cell apoptosis in mycoplasmal pneumonia mice. Twenty mice were selected as normal group. The 120 successfully modeled Mycoplasma pneumoniae (MP) infection mice were randomly divided into model group (without any treatment), negative control (NC) group (injected with NC mimic), miR-143-3p mimic group (injected with miR-143-3p mimic), miR-143-3p inhibitor group (injected with miR-143-3p inhibitor), TAK-242 group (treatment with TAK-242), and miR-143-3p inhibitor + TAK-242 group (treatment with miR-143-3p inhibitor + TAK-242). Compared with model group, model mice had up-regulated miR-143-3p expression and decreased MyD88 and p-NF-κB p50 protein expressions (all P<0.05); Model mice treated with miR-143-3p mimic and TAK-242 had reduced interleukin (IL)-2 and tumor necrosis factor (TNF)-α contents and protein expressions of MyD88, p-NF-κB p50, increased IL-10 content, fewer alveolar epithelial cell apoptosis, lower Bax expression and higher Bcl-2 expression (all P<0.05); however, mice with miR-143-3p inhibitor treatment showed opposite trends in terms of above indicators. The exacerbation of mycoplasmal pneumonia caused by miR-143-3p inhibitor was partly improved by miR-143-3p inhibitor + TAK-242 combination treatment (all P<0.05). Therefore, up-regulation of miR-143-3p expression may ameliorate pulmonary inflammatory factors levels and reduce alveolar epithelial cell apoptosis in mycoplasmal pneumonia mice by inhibiting TLR4/MyD88/NF-κB signaling pathway.

Keywords: TLR4/MyD88/NF-κB signaling pathway; apoptosis; inflammation; miR-143-3p; mycoplasmal pneumonia.

Figure 1. Pathologic changes of the lung tissue (400×)

Figure 2. Content of IL-2, IL-10 and TNF-α in the serum

Compared with normal group, *P<0.05; compared with model group, ^#P<0.05; compared with NC group, ^%P<0.05; compared with miR-143-3p mimic group, ^&P<0.05; compared with miR-143-3p inhibitor group, ^$P<0.05; compared with TAK-242 group, ^@P<0.05.

Figure 3. Apoptosis in the lung tissue

(A) Apoptosis in the lung tissue detected by TUNEL staining (200×). (B) Number of TUNEL-positive cells in the lung tissue. Compared with normal group, *P<0.05; compared with model group, ^#P<0.05; compared with NC group, ^%P<0.05; compared with miR-143-3p mimic group, ^&P<0.05; compared with miR-143-3p inhibitor group, ^$P<0.05; compared with TAK-242 group, ^@P<0.05.

Figure 4. Bax and Bcl-2 mRNA and protein expressions in the lung tissue

(A) Bax and Bcl-2 mRNA expressions, (B) Bax and Bcl-2 protein bands, (C) Bax and Bcl-2 protein expressions. Compared with normal group, *P<0.05; compared with model group, ^#P<0.05; compared with NC group, ^%P<0.05; compared with miR-143-3p mimic group, ^&P<0.05; compared with miR-143-3p inhibitor group, ^$P<0.05; compared with TAK-242 group, ^@P<0.05.

Figure 5. miR-143-3p inhibited the expression of TLR4/MyD88/NF-κB signaling pathway

(A) Sequence of 3′-UTR region in which miR-143-3p bound with MYD88. (B) Dual-luciferase reporter system assay verified the target relationship between miR-143-3p and MYD88. Compared with NC mimic, ^ΦP<0.05. (C) Expressions of miR-143-3p as well as TLR4, MyD88 and NF-κB p50 mRNA, (D) protein bands of TLR4, MyD88, NF-κB p50 and p-NF-κB p50, (E) protein expressions of TLR4, MyD88, NF-κB p50 and p-NF-κB p50. Compared with normal group, *P<0.05; compared with model group, ^#P<0.05; compared with NC group, ^%P<0.05; compared with miR-143-3p mimic group, ^&P<0.05; compared with miR-143-3p inhibitor group, ^$P<0.05; compared with TAK-242 group, ^@P<0.05.