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. 2019 Jun 15;11(6):3707-3721.
eCollection 2019.

The protective effects of bone mesenchymal stem cells on paraquat-induced acute lung injury via the muc5b and ERK/MAPK signaling pathways

Lichun Zhang  1 , Qiuhe Li  1 , Zhenning Liu  1 , Yu Wang  1 , Min Zhao  1
Affiliations

The protective effects of bone mesenchymal stem cells on paraquat-induced acute lung injury via the muc5b and ERK/MAPK signaling pathways

Lichun Zhang et al. Am J Transl Res. .

Abstract

Objective: To evaluate the protective effect of bone mesenchymal stem cells (BMSCs) on paraquat (PQ)-induced acute lung injury (ALI) and investigate the possible underlying mechanisms.

Methods: Male Sprague Dawley rats were treated with BMSCs (3 × 106) 1 h after intraperitoneal injection of PQ. The cell apoptosis rate and mitochondrial membrane potential in rat pulmonary alveolar type II epithelial (ATII) cells were quantitated by flow cytometry. IL-17, IL-6, and MUC5B levels in bronchoalveolar lavage fluid (BALF) and ATII culture medium were measured. Lung tissues were collected to determine the wet-to-dry (W/D) ratios and lung injury scores, in addition to the protein and mRNA expression levels of ERK1/2, Bcl-2, Bax, and muc5b.

Results: BMSCs had decreased mRNA expression of Muc5b in lung tissue of rats with PQ-induced ALI as shown by RNA-seq. Treatment with BMSCs also alleviated the PQ-induced increases in protein expression in the BALF and reduced the concentration of IL-17, IL-6, and Muc5b in both the BALF and ATII culture medium. In addition, the ATII cell apoptosis rate and mitochondrial membrane potential, as well as the W/D ratios, were decreased by BMSC treatment. Moreover, BMSCs ameliorated the expression levels of Bax mRNA and active caspase-3 proteins and increased Bcl-2 mRNA expression. Furthermore, BMSCs attenuated ERK1/2 activation upon PQ-induced ALI in lung tissue.

Conclusion: BMSC therapy can protect against PQ-induced ALI in rats. A possible mechanism is the suppression of the muc5b and ERK/MAPK signaling pathways, resulting in an improvement in the endothelial permeability and a decrease in inflammation and cell apoptosis.

Keywords: Bone marrow derived mesenchymal stem cells; Muc5b and ERK/MAPK signaling pathways; acute lung injury; paraquat poisoning.

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Conflict of interest statement

None.

Figures

Figure 1
Figure 1
Isolation and characterization of BMSCs. BMSCs carrying GFP were observed by fluorescence microscopy. The transfection efficiency was over 80% (A, B). 24 hours after BMSC administration, the distribution of BMSC fluorescence was observeded throughout the distal lungs. (C) Normal control group; (D) PQ group.
Figure 2
Figure 2
BMSCs ameliorated the histopathological changes in lung tissues of rats with PQ-induced ALI. Lung tissues were fixed in 10% formalin, sectioned, and stained with H&E (200 ×). Photomicrograph of lung tissue from the control group (A). Photomicrograph of lung tissue from the PQ group (B). Photomicrograph of lung tissue from the PQ+BMSC group (C). Histopathological scoring of lung injury in the three groups (D). BMSCs reduced the lung W/D ratio in rats with PQ-induced ALI (E). Data are presented as the mean ± SD, n = 10. *P < 0.05 as compared with control rats; #P < 0.05 as compared with rats with PQ.
Figure 3
Figure 3
BMSCs decreased the mRNA expression of muc5b in the lung tissue of rats with PQ-induced ALI as shown by whole transcriptome analysis (A). Data are presented as the mean ± SD, n = 3. *P < 0.05 as compared with control rats; #P < 0.05 as compared with rats with PQ. BMSCs reduced the mRNA expression of muc5b in the lung tissue of rats with PQ-induced ALI as shown by RT-qPCR (B). BMSCs inhibited the concentration of Muc5b in the BALF of rats with PQ-induced ALI using ELISA (C). Effect of PQ on ATII cell viability as examined by the MTT assay (D). BMSCs decreased the concentration of Muc5b in ATII cell culture medium following the administration of PQ by ELISA (E). Immunohistochemistry staining of Muc5b in lung tissue from the control group, the PQ group, and the PQ+BMSC group. 3,3’-diaminobenzidine 4-HCl was used as a chromogen to stain the cells of interest (brown), and a hematoxylin counterstain (blue) was used for background staining. (F) Muc5b immunostaining at 200 × magnification, (G) Changes in OD values in lung with positive expression. Data are presented as the mean ± SD, n = 10. *P < 0.05 as compared with control rats; #P < 0.05 as compared with rats with PQ.
Figure 4
Figure 4
BMSCs decreased the cell apoptosis rate of ATII cells following PQ administration by flowcytometry (A). BMSCs decreased the mitochondrial membrane potential in ATII cells following PQ administration by flowcytometry (B). BMSCs increased Bcl-2 mRNA expression in the lung tissue of rats with PQ-induced ALI by RT-qPCR (C). BMSCs decreased Bax mRNA expression in the lung tissue of rats with PQ-induced ALI by RT-qPCR (D). BMSCs decreased the percentage of caspase-3 activity in rats with PQ-induced ALI using the Caspase-3 Colorimetric Assay kit (E). Data are presented as the mean ± SD, n = 10. *P < 0.05 as compared with control rats; #P < 0.05 as compared with rats with PQ.
Figure 5
Figure 5
BMSCs decreased the content of protein in the BALF from rats with PQ-induced ALI using ELISA (A). BMSCs inhibited the concentration of IL-17 and IL-6 in the BALF from rats and ATII cell culture medium following the administration of PQ by ELISA (B-E). Data are presented as the mean ± SD, n = 10. *P < 0.05 as compared with control rats; #P < 0.05 as compared with rats with PQ.
Figure 6
Figure 6
BMSCs attenuated ERK1/2 activation in PQ-induced ALI tissue by western blotting. (A) Expression of ERK1/2 and p-ERK1/2 proteins in lung tissue; (B) p-ERK1/2/total ERK1/2 ratio. Immunohistochemistry staining of ERK1/2 in lung tissue from the control group, the PQ group, and the PQ+BMSC group. 3,3’-diaminobenzidine 4-HCl was used as a chromogen to stain the cells of interest (brown), and a hematoxylin counterstain (blue) was used for background staining. (C) ERK1/2 immunostaining at 200 × magnification, (D) Changes in OD values in lung with positive expression. Data are presented as the mean ± SD, n = 10. *P < 0.05 as compared with control rats; #P < 0.05 as compared with rats with PQ.
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