Transplantation of endothelial progenitor cells attenuated paraquat-induced acute lung injury via miR-141-3p-Notch-Nrf2 axis

Abstract

Background: Paraquat (PQ) presents with high toxicity for humans and animals, and the lungs become the main target organ by the poisoning of PQ leading to acute lung injury. Endothelial progenitor cells (EPCs) were proved to have the repair function on acute lung injury (ALI). We aimed to invatigate the underlying mechanism of EPCs in PQ-induced ALI involving miR-141-3p.

Methods: Endothelial progenitor cells were isolated from peripheral blood of C57BL/6J mice and identified by flow cytometry. Lung wet-to-dry (W/D) weight ratios, lung injury score and the number of total leukocyte and the number of neutrophils in BALF were used to analyze the degree of lung injury. The transfection was performed with Lipofectamine 2000. The levels of miRNA and mRNA were determined by qRT-PCR, and the protein levels were detected by Western blot assay.

Results: Endothelial progenitor cells alleviated lung wet-to-dry (W/D) weight ratios, lung injury score and the number of total leukocyte and the number of neutrophils in BALF in PQ-induced ALI mice. EPCs inhibited miR-141-3p expression, and enhanced the levels of Notch-Nrf2 axis in PQ-induced ALI mice. MiR-141-3p knockdown reversed the PQ induced-inhibition on Notch-1 and Hesr1 expression. MiR-141-3p over-expression could inhibit the expression of Notch-1 pathway significantly in the pulmonary epithelial cell line MLE-12. Both miR-141-3p over-expression and si-Notch-1 abolished the protection effect of EPCs on lung injury induced by PQ in vivo.

Conclusions: Endothelial progenitor cells could provide therapeutic effect on PQ-induced ALI via miR-141-3p-Notch-Nrf2 Axis.

Keywords: ALI; EPC; Notch-Nrf2 axis; PQ; miR-141-3p.

Fig. 1

Characterization of endothelial progenitor cells (EPCs). a The morphology of EPCs on day 7 of the culture. b EPCs were identified by flow cytometry

Fig. 2

EPCs alleviated the lung injury induced by paraquat (PQ) in vivo. C57BL/6J mice were randomly divided into four groups (n = 10 per group). The control group, mice received PBS by through tail vein injection; the EPCs group, mice were injected with EPCs (approximately 5 × 10⁶ in 50 μL PBS through tail vein injection); the 10 × 10⁶ EPCs group, mice were injected with approximately 10 × 10⁶ EPCs in 50 μL PBS through tail vein; the PQ group, mice were received the intraperitoneal injection of PQ 50 mg/kg; the PQ + 1.25 × 10⁶ EPCs group, mice were received the injection of PQ, and then received EPCs injection after 6 h; the PQ + 2.5 × 10⁶ EPCs group, mice were received the injection of PQ, and then received EPCs injection after 6 h; the PQ + 5 × 10⁶ EPCs group, mice were received the injection of PQ, and then received EPCs injection after 6 h; the PQ + 10 × 10⁶ EPCs group, mice were received the injection of PQ, and then received EPCs injection after 6 h. a The lung W/D ratio, b lung injury score and c the number of total leukocyte and the number of neutrophils in BALF of the mice were detected. **P < 0.01 vs. control; ^##P < 0.01 vs. PQ

Fig. 3

The expression profile of miRNAs in damaged lung tissues induced by PQ. a The expressions of miR-200a-3p, miR-21, miR-141-3p and miR-153 in lung tissues were determined using qRT-PCR. b, c The expressions of Notch-1 and Hesr1 in lung tissues were detected at both the mRNA and protein levels. **P < 0.01 vs. control; ^##P < 0.01 vs. PQ

Fig. 4

The expression profile of miRNAs in damaged lung tissues induced by PQ. a, b The expressions of Nrf2, Hmox1 and Txnrd1 in lung tissues were detected at both the mRNA and protein levels. **P < 0.01 vs. control; ^##P < 0.01 vs. PQ

Fig. 5

The interaction between miR-141-3p and Notch-1 in pulmonary epithelial cells. a The pulmonary epithelial cell line MLE-12 was transfected with miR-141-3p mimic or its control (pre-NC), and b the mRNA and c protein expressions of Notch-1 and Hesr1 were then determined. d MLE-12 cells were transfected with miR-141-3p inhibitor or its control (NC) and then treated by PQ. The expressions of Notch-1 and Hesr1 protein were detected using western blot. e MLE-12 cells were divided into six groups, which were received diverse treatment: MLE-12 cells cultured in normal condition; MLE-12 cells treated by PQ; MLE-12 cells transfected with miR-141-3p inhibitor or its control (NC) and then treated by PQ. MLE-12 cells co-transfected with miR-141-3p inhibitor and si-Notch-1 or si-control and then treated by PQ. The expressions of Nrf2, Hmox1 and Txnrd1 protein were detected using western blot. **P < 0.01 vs. pre-NC

Fig. 6

MiR-141-3p over-expression abolished the protection effect of EPCs on lung injury induced by PQ in vivo. C57BL/6J mice were randomly divided into four groups (n = 10 per group), the PQ group; the PQ + EPCs group; the EPCs group; the PQ + EPCs + pre-NC group; the PQ + EPCs + miR-141-3p mimic group. a The lung W/D ratio and b lung injury score of the mice were detected. c The expressions of Notch-1 and Hesr1 protein in lung tissues were detected using western blot. **P < 0.01 vs. PQ; ^##P < 0.01 vs. PQ + EPCs + pre-NC

Fig. 7

Notch-1 silencing abolished the protection effect of EPCs on lung injury induced by PQ in vivo. C57BL/6J mice were randomly divided into four groups (n = 10 per group), the PQ group; the PQ + EPCs group; the EPCs group; the PQ + EPCs + si-control group; the PQ + EPCs + si-Notch-1 group. a The lung W/D ratio and b lung injury score of the mice were detected. c The expressions of Notch-1, Nrf2, Hmox1 and Txnrd1 protein in lung tissues were detected using western blot. The representing section images of the injured lung tissues are shown in (d). **P < 0.01 vs. PQ; ^##P < 0.01 vs. PQ + EPCs + si-control