HMSCs exosome-derived miR-199a-5p attenuates sulfur mustard-associated oxidative stress via the CAV1/NRF2 signalling pathway

Abstract

Sulfur mustard (SM) is a blister-producing chemical warfare agent which could lead to a cascade of systemic damage, especially severe acute lung injury. Oxidative stress is considered to be vital processes for the SM toxicity mechanism. We previously proved the therapeutic effect of exosomes derived from bone marrow mesenchymal stromal cells in promoting the repair of alveolar epithelial barrier and inhibiting apoptosis. However, the key functional components in exosomes and the underlying mechanisms have not been fully elaborated. This research shed light on the function of the key components of human umbilical cord mesenchymal stem cell-derived exosomes (HMSCs-Ex). We noted that HMSCs-Ex-derived miR-199a-5p played a vital role in reducing pneumonocyte oxidative stress and apoptosis by reducing reactive oxygen species, lipid peroxidation products and increasing the activities of antioxidant enzymes in BEAS-2B cells and mouse models after exposure to SM for 24 h. Furthermore, we demonstrated that the overexpression of miR-199a-5p in HMSCs-Ex treatment induced a further decrease of Caveolin1 and the activation of the mRNA and protein level of NRF2, HO1 and NQO1, compared with HMSCs-Ex administration. In summary, miR-199a-5p was one of the key molecules in HMSCs-Ex that attenuated SM-associated oxidative stress via regulating CAV1/NRF2 signalling pathway.

Keywords: acute lung injury; exosomes; human umbilical cord mesenchymal stem cells; miRNA-199a-5p; oxidative stress; sulfur mustard.

FIGURE 1

HMSCs‐Ex rescued SM‐induced lung injury in vivo. (A) Survival curves of SM‐injured mice treated with HMSCs‐Ex (3 × 10⁸ particles, resuspended in 150 μL of PBS) by tail vein injection. HMSCs‐Ex administration protected recipient animals from lung injury (n = 8; **p < 0.01). (B) Representative images of H&E‐stained lung tissues from SM‐injured mice after PBS, HFLs‐Ex or HMSCs‐Ex treatment. Original magnification: ×400. The HMSCs‐Ex group revealed conspicuous protection against SM‐injured lung damage, as showed by the relatively normal alveolar cavity, mucosal epithelium, and airways as well as minimal inflammatory cell infiltration and septal thickening. Comparison of pathological lung injury scores in SM‐exposed mice (n = 3; *p < 0.05; **p < 0.01; ***p < 0.001 compared with the SM group). (C and D) BALF protein levels (C) and wet‐to‐dry lung weight ratios (D) are reduced in HMSCs‐Ex administration group (n = 3; *p < 0.05; **p < 0.01; and ***p < 0.001). CTRL, control; HFLs‐Ex, human lung fibroblasts‐derived; HMSCs‐Ex, HMSCs‐derived exosomes; NAC, N‐acetylcysteine, PBS, phosphate‐buffered saline; SM, sulfur mustard.

FIGURE 2

HMSCs‐Ex ameliorates SM‐induced lung oxidative injury. (A) DHE‐stained lung tissue slides from SM‐injured mice after HMSCs‐Ex or NAC treatment. Original magnification: ×400. Data showed the relative DHE fluorescence intensity in lung tissues from different groups. Values are the mean ± SEM (n = 3; *p < 0.05; and ***p < 0.001). (B) Immunohistochemical staining of mouse lung tissue slides; reduced oxidative DNA damage marker 8‐OHdG expression was found in HMSCs‐Ex group (n = 3; *p < 0.05; **p < 0.01; and ***p < 0.001). Original magnification: × 400. (C) Lipid peroxidation injury marker MDA content was detected as oxidative stress parameters. Data show that HMSCs‐Ex treatment markedly reduced MDA content (n = 3; *p < 0.05; **p < 0.01; and ***p < 0.001). (D and E) SOD activity (D) and GHS/GSSG ratio (E) in mouse lungs were measured. HMSCs‐Ex treatment significantly increased antioxidant enzyme content (n = 3; *p < 0.05; **p < 0.01; and ***p < 0.001). (F) TUNEL staining of mouse lung tissue slides; reduced apoptosis‐positive cells was found in HMSCs‐Ex group after treatment (n = 3; *p < 0.05; **p < 0.01; and ***p < 0.001). Original magnification: × 400. CTRL, control; HFLs‐Ex, human lung fibroblasts‐derived; HMSCs‐Ex, HMSCs‐derived exosomes; NAC, N‐acetylcysteine, PBS, phosphate‐buffered saline; SOD, superoxide dismutase; SM, sulfur mustard.

FIGURE 3

HMSCs‐Ex suppressed SM‐induced oxidative stress via regulating the NRF2 signalling pathway in pneumonocyte. (A) The effect of HMSCs‐Ex on pneumocytes survival was assessed by CCK‐8 assays. Data indicated that significant increases in the cell viability were observed in the HMSCs‐Ex treatment. (B–D) Intracellular MDA (B), SOD (C) and GSH/GSSG (D) content were measured, and HMSCs‐Ex treatment reduced the MDA release and increased the level of GSH and SOD activity (n = 3; *p < 0.05; **p < 0.01; and ***p < 0.001). (E) Hoechst 33342 staining of apoptotic SM‐injured BEAS‐2B cells cultured with PBS, NAC, HFLs‐Ex or HMSCs‐Ex for 24 h. HMSCs‐Ex treatment reduced BEAS‐2B cell apoptosis compared with PBS and HFLs‐Ex groups (n = 3; *p < 0.05; **p < 0.01). Original magnification: ×200. (F) Mitochondrial membrane potentials in PBS, HFLs‐Ex, and HMSCs‐Ex groups. Increases in green fluorescence indicate perturbed membrane potentials (n = 3; **p < 0.01). HMSCs‐Ex blocked the increase ratio of green to red fluorescence by SM treatment. Original magnification: ×400. (G) Expression of oxidative stress‐related mRNA in SM‐exposed BEAS‐2B cells was measured with qRT‐PCR 24 h after PBS, NAC, HFLs‐Ex or HMSCs‐Ex treatment. NRF2, HO1 and NQO1 mRNA levels were increased in the HMSCs‐Ex group. (H) Western blot analysis of oxidative stress‐related proteins in SM‐exposed BEAS‐2B cells treated with PBS, NAC, HFLs‐Ex or HMSCs‐Ex for 24 h. The levels of total NRF2, HO1, NQO1 and nuclear NRF2 were quantitated by densitometric analysis using ImageJ software and normalized to Tubulin shown as numbers under individual blots (n = 3; *p < 0.05; **p < 0.01; and ***p < 0.001). CTRL, control; HFLs‐Ex, human lung fibroblasts‐derived; HMSCs‐Ex, HMSCs‐derived exosomes; NAC, N‐acetylcysteine, PBS, phosphate‐buffered saline; SOD, superoxide dismutase; SM, sulfur mustard.

FIGURE 4

HMSCs‐Ex suppressed the oxidative stress by transferring MiR‐199a‐5p. (A) Quantitative analysis of relative miR‐199a‐5p levels in BEAS‐2B cells after the indicated treatment for 24 h (n = 3; ***p < 0.001). (B) ROS production in SM‐injured BEAS‐2B cells detected by DCF probe staining after treatment with PBS, HMSCs‐Ex, miR‐NC‐HMSCs‐Ex or miR‐199a‐HMSCs‐Ex. Representative images of DCF fluorescence in SM‐injured BEAS‐2B cells and relative DCF fluorescent values showed reduced ROS production in miR‐199a‐HMSCs‐Ex treated BEAS‐2B cells (n = 3; *p < 0.05; ***p < 0.001). (C–E) Intracellular MDA, SOD and GSH content were measured, and miR‐199a‐HMSCs‐Ex group treatment reduced the MDA release and increased the level of GSH and SOD activity compared with HMSCs‐Ex and miR‐NC‐ HMSCs‐Ex groups (n = 3; *p < 0.05; **p < 0.01; and ***p < 0.001). (F) The induction of apoptosis in BEAS‐2B cells was determined by Annexin V/PI double staining and flow cytometry. MiR‐199a‐Ex incubation decreased SM‐induced BEAS‐2B cell apoptosis compared with HMSCs‐Ex and miR‐NC‐HMSCs‐Ex groups (n = 3; *p < 0.05; **p < 0.01; and ***p < 0.001). The quantification of apoptotic cells is presented as the percent of total cells. CTRL, control; GSH, glutathione; HFLs‐Ex, human lung fibroblasts‐derived; HMSCs‐Ex, HMSCs‐derived exosomes; MDA, malondialdehyde; NAC, N‐acetylcysteine, PBS, phosphate‐buffered saline; ROS, reactive oxygen species; SOD, superoxide dismutase; SM, sulfur mustard.

FIGURE 5

HMSCs‐Ex‐derived miR‐199a‐5p activated the NRF2 signalling pathway by targeting CAV1. (A) Expression of oxidative stress‐related mRNA in SM‐exposed BEAS‐2B cells was measured with qRT‐PCR 24 h after PBS, HMSCs‐Ex, miR‐NC‐HMSCs‐Ex or miR‐199a‐HMSCs‐Ex treatment. NRF2, HO1 and NQO1 mRNA levels were increased in the miR‐199a‐HMSCs‐Ex group. The miR‐199a‐HMSCs‐Ex treatment led to a marked increasement in HO1, NQO1 and NRF2 mRNA levels compared with miR‐NC‐HMSCs‐Ex. (n = 3; *p < 0.05; **p < 0.01; and ***p < 0.001). (B) Western blot analysis showed total NRF2, HO1, NQO1 and nuclear NRF2 protein expression changed accordingly with the corresponding mRNA levels. Quantitative analysis for relative total NRF2, HO1, NQO1 and nuclear NRF2 levels after the indicated treatment for 24 h (n = 3; *p < 0.05; **p < 0.01; and ***p < 0.001). (C) The binding site between miR‐199a‐5p and CAV1 3′UTR was analysed by TargetScan website. (D) The relationship between miR‐199a‐5p and CAV1 was validated by dual‐luciferase reporter assay. (E) Quantitative analysis of relative CAV1 mRNA expression in BEAS‐2B cells with qRT‐PCR 24 h after treated with PBS, HMSCs‐Ex, miR‐NC‐HMSCs‐Ex or miR‐199a‐HMSCs‐Ex (n = 3; *p < 0.05; **p < 0.01; and ***p < 0.001). CAV1 expression decreased in miR‐199a‐HMSCs‐Ex treated BEAS‐2B cells compared with HMSCs‐Ex and miR‐NC‐HMSCs‐Ex groups. (F) Western blot quantification of CAV1 expression in SM, HMSCs‐Ex, miR‐NC‐HMSCs‐Ex or miR‐199a‐HMSCs‐Ex. CAV1 protein was highly expressed in SM groups compared with HMSCs‐Ex, miR‐NC‐HMSCs‐Ex and miR‐199a‐HMSCs‐Ex groups. (G) Confocal laser scanning immunofluorescence analysis to detect the interaction between CAV1 and NRF2 expression in BEAS‐2B cells after the indicated treatment for 24 h. Original magnification: ×400 (n = 3; *p < 0.05; **p < 0.01; and ***p < 0.001). (H) Western blot quantification of CAV1, NRF2, HO1 and NQO1 expression in wild‐BEAS‐2B cells and CAV1‐overexpressed BEAS‐2B cells. CAV1 overexpression weakened the miR‐199a‐HMSCs‐Ex mediated promotion of the nuclear translocation of NRF2 and the expression of HO1 and NQO1. Data are expressed as relative ratios of specific proteins to Tubulin shown as numbers under individual blots (n = 3; *p < 0.05). CTRL, control; HFLs‐Ex, human lung fibroblasts‐derived; HMSCs‐Ex, HMSCs‐derived exosomes; NAC, N‐acetylcysteine, PBS, phosphate‐buffered saline; SM, sulfur mustard.

FIGURE 6

HMSCs‐Ex‐derived miR‐199a‐5p attenuated SM‐induced oxidative stress by regulating CAV1/NRF2 signal pathway in vivo. (A) Quantitative analysis of relative miR‐199a‐5p levels in lung sections of SM‐injured mice after treated with PBS, HMSCs‐Ex, miR‐NC‐HMSCs‐Ex or miR‐199a‐HMSCs‐Ex (n = 3; *p < 0.05; **p < 0.01; and ***p < 0.001). (B) Representative histological micrograph analysis of H&E staining of lung slides after the indicated treatment for 24 h. In addition, the quantitative assay is done using Image J software. Original magnification: ×400 (n = 3; *p < 0.05, **p < 0.01). (C) Immunohistochemical detection of positive NRF2 and CAV1 staining in the lung of SM‐expose mice 24 h after treated with PBS, HMSCs‐Ex, miR‐NC‐HMSCs‐Ex or miR‐199a‐HMSCs‐Ex (n = 3; *p < 0.05; **p < 0.01; and ***p < 0.001). (D) Quantitative analysis of relative CAV1, NRF2, HO1 and NQO1 in lung tissues was determined by qRT‐PCR (n = 3; *p < 0.05; **p < 0.01). (E) Protein levels of CAV1, total NRF2, HO1, NQO1 and nuclear NRF2 in lung tissues were determined by western blot analysis. Quantification of the protein expression in SM‐injured mice 24 h after indicated treatment (n = 3; *p < 0.05, **p < 0.01).