Melatonin augments apoptotic adipose-derived mesenchymal stem cell treatment against sepsis-induced acute lung injury

Abstract

This study investigated whether combining melatonin and apoptotic adipose-derived mesenchymal stem cells (A-ADMSC) was superior to ADMSC alone in ameliorating sepsis-induced acute lung injury. Adult male Sprague-Dawley rats (n=50) were randomized equally into five groups: sham controls (SC), sepsis induced by cecal-ligation and puncture (CLP), CLP-melatonin, CLP-A-ADMSC, and CLP-melatonin-A-ADMSC. Circulating interleukin (IL)-6 at 6, 18, and 72 hrs, were highest in CLP and lowest in SC groups, higher in CLP-melatonin than CLP-A-ADMSC and CLP-melatonin-A-ADMSC groups, higher in CLP-A-ADMSC than CLP-melatonin-A-ADMSC groups (all p<0.001). Immune reactivity (indicated by circulating cytotoxic-, and regulatory-T cells) and WBC count at 72 h exhibited the same pattern as that of circulating IL-6 (all p<0.001). Changes in histological scoring of lung parenchyma and the number of CD68+ and CD14+ cells showed a similar pattern compared to that of IL-6 level in all groups (all p<0.001). Changes in protein expressions of inflammatory (oxidative stress, RANTES, TNF-α, NF-κB, MMP-9, MIP-1, IL-1β), apoptotic (cleaved caspase 3 and PARP, mitochondrial Bax), fibrotic (Smad3, TGF-β) markers and those of reactive-oxygen-species (NOX-1, NOX-2) displayed an identical pattern compared to that of circulating IL-6 in all groups (all p<0.001). Anti-oxidative capacities (GR+, GPx+, HO-1, NQO-1+) and angiogenesis marker (CXCR4+ cells) were lowest in SC group but highest in CLP-melatonin-A-ADMSC group, lower in CLP than CLP-melatonin and CLP-A-ADMSC groups, and lower in CLP-melatonin than CLP-A-ADMSC groups (all p<0.001). In conclusion, combined melatonin and A-ADMSC were superior to A-ADMSC alone in protecting the lung from sepsis-induced injury.

Keywords: Sepsis-induced organ injury; adipose-derived mesenchymal stem cells; and melatonin; inflammation; oxidative stress; reactive oxygen species.

Figure 1

Microscopic findings for the incidence of A-ADMSC after starvation-hypoxia stress and flow cytomtric Analysis of circulating Immune Cells (n=6). (A and B) Immunofluorescent (IF) microscopic findings (400 x) of cellular apoptosis of apoptotic adipose-derived mesenchymal stem cells (A-ADMSCs). (C) Cellular apoptosis was significantly higher in A-ADMSC than in healthy ADMSC. * vs. normal control, p<0.0001. Blue color indicates DAPI stain for nuclei. (D) Flow cytometric analysis of CD3+/CD4+ cells. * vs. other groups with different symbols (*, †, ‡), p<0.001. (E) Flow cytometric analysis of CD3+/CD8+ cells. * vs. other groups with different symbols (*, †, ‡), p<0.001. (F) Flow cytometric analysis of Treg+ cells. * vs. other groups with different symbols (*, †, ‡), p<0.001. Statistical analysis in (F) using one-way ANOVA, followed by Bonferroni multiple comparison post hoc test. Symbols (*, †, ‡) indicate significance (at 0.05 level). SC=sham control; CLP=cecal-ligation and puncture; Mel=melatonin.

Figure 2

Histopathological findings in lung parenchyma at 72 hours after CLP procedure (n=6). (A to F) Illustrations of microscopic findings (100 x) of H & E stain in lung sections. (G) Analytic results of number of alveolar sacs, * vs. other groups with different symbols (*, †, ‡, §, ¶), p<0.001. (H) Analytic results of crowded score, * vs. other groups with different symbols (*, †, ‡, §, ¶), p<0.0001. The scale bars in right lower corner represent 100 μm. Statistical analysis in (G) and (H) using one-way ANOVA, followed by Bonferroni multiple comparison post hoc test. Symbols (*, †, ‡, §, ¶) indicate significance (at 0.05 level). HPF=high-power field; SC=sham control; CLP=cecal-ligation and puncture; Mel=melatonin; A-ADMSC=apoptotic adipose-derived mesenchymal stem cell.

Figure 3

Inflammatory cells infiltrated in lung parenchyma at 72 hours after CLP procedure (n=6). (A to E) Microscopic findings (200 x) of immunohistochemical (IHC) stain for CD14+ cells (brown color). (F) Analytic results of CD14+ cells, * vs. other groups with different symbols (*, †, ‡, §, ¶), p<0.0001. The scale bars in right lower corner represent 50 μm. (G to K) Microscopic findings (200 x) of immunofluorescent (IF) stain for CD68+ cells (green color of DAPI+ cells). Abundant Dil dye stained A-ADMSCs (white arrows) were found in (J) and (K) indicated that the transplanted A-ADMSCs were trapped in lung parenchyma. (L) Analytic results of CD68+ cells, * vs. other groups with different symbols (*, †, ‡, §, ¶), p<0.0001. The scale bars in right lower corner represent 50 μm. Statistical analysis in (F) and (L) using one-way ANOVA, followed by Bonferroni multiple comparison post hoc test. Symbols (*, †, ‡, §, ¶) indicate significance (at 0.05 level). HPF=high-power field; SC=sham control; CLP=cecal-ligation and puncture; Mel=melatonin; A-ADMSC=apoptotic adipose-derived mesenchymal stem cell.

Figure 4

Expressions of GR and GPx anti-oxidant cells in lung parenchyma 72 hours after CLP procedure (n=6). (A to E) Microscopic findings (200 x) of IHC stain for glutathione reductase (GR)+ cells (brown color) in lung parenchyma. (F) Analytic results of GR+ cells, * vs. other groups with different symbols (*, †, ‡, §, ¶), p<0.0001. The scale bars in right lower corner represent 50 μm. (G to K) Microscopic findings (200 x) of IHC stain for glutathione peroxidase (GPx)+ cells (brown color) in lung parenchyma. (L) Analytic results of GPx+ cells, * vs. other groups with different symbols (*, †, ‡, §, ¶), p<0.0001. The scale bars in right lower corner represent 50 μm. Statistical analysis in (F) and (L) using one-way ANOVA, followed by Bonferroni multiple comparison post hoc test. Symbols (*, †, ‡, §, ¶) indicate significance (at 0.05 level). SC=sham control; CLP=cecal-ligation and puncture; Mel=melatonin; A-ADMSC=apoptotic adipose-derived mesenchymal stem cell.

Figure 5

Expressions of NQO 1 and HO-1 anti-oxidant cells in lung parenchyma 72 hours after CLP procedure (n=6). (A to E) Microscopic findings (200 x) of IHC stain for NAD(P)H quinone oxidoreductase (NQO) 1+ cells (brown color) in lung parenchyma. (F) Analytic results of NQO 1+ cells, * vs. other groups with different symbols (*, †, ‡, §, ¶), p<0.0001. The scale bars in right lower corner represent 50 μm. (G to K) Microscopic findings (200 x) of IF stain for heme oxygenase (HO)-1+ cells (green color of DAPI+ cells) in lung parenchyma. Abundant Dil dye stained A-ADMSCs (white arrows) were found in (J) and (K) indicated that the transplanted A-ADMSCs were trapped in lung parenchyma. Additionally, some transplanted A-ADMSCs showing positive stain for OH-1 (yellow arrows). L) Analytic results of HO-1+ cells, * vs. other groups with different symbols (*, †, ‡, §, ¶), p<0.0001. The scale bars in right lower corner represent 50 μm. Statistical analysis in (F) and (L) using one-way ANOVA, followed by Bonferroni multiple comparison post hoc test. Symbols (*, †, ‡, §, ¶) indicate significance (at 0.05 level). SC=sham control; CLP=cecal-ligation and puncture; Mel=melatonin; A-ADMSC=apoptotic adipose-derived mesenchymal stem cell.

Figure 6

Protein expressions of oxidative stress and ROS in lung parenchyma 72 hours after CLP procedure (n=6). A. Protein expression of NOX-1 in lung parenchyma, * vs. other groups with different symbols (*, †, ‡, §), p<0.001. B. Protein expression of NOX-2 in lung parenchyma, * vs. other groups with different symbols (*, †, ‡, §, ¶), p<0.001. C. Protein expression of oxidative index (protein carbonyls) in lung parenchyma, * vs. other groups with different symbols (*, †, ‡, §, ¶), p<0.0001. (Note: Right lane and left lane shown on the upper panel represent control oxidized molecular protein standard and protein molecular weight marker, respectively). DNP=1-3 dinitrophenylhydrazone. All statistical analyses were with one-way ANOVA followed by Bonferroni multiple comparison post hoc test. Symbols (*, †, ‡, §, ¶) indicate significance (at 0.05 level). SC=sham control; CLP=cecal-ligation and puncture; Mel=melatonin; A-ADMSC=apoptotic adipose-derived mesenchymal stem cell.

Figure 7

Protein expressions of inflammatory biomarkers in lung parenchyma 72 hours after CLP procedure. A. Protein expressions of interleukin (IL)-1 in lung parenchyma, * vs. other groups with different symbols (*, †, ‡, §, ¶), p<0.001. B. Protein expressions of tumor necrosis factor (TNF)-α in lung parenchyma, * vs. other groups with different symbols (*, †, ‡, §, ¶), p<0.001. C. Protein expressions of tumor nuclear factor (NF)-κB in lung parenchyma, * vs. other groups with different symbols (*, †, ‡, §, ¶), p<0.001. D. Protein expressions of matrix metalloproteinase (MMP)-9 in lung parenchyma, * vs. other groups with different symbols (*, †, ‡, §, ¶), p<0.005. E. Protein expressions of macrophage inflammatory protein (MIP)-1α in lung parenchyma, * vs. other groups with different symbols (*, †, ‡, §, ¶), p<0.005. All statistical analyses were with one-way ANOVA followed by Bonferroni multiple comparison post hoc test. Symbols (*, †, ‡, §, ¶) indicate significance (at 0.05 level). SC=sham control; CLP=cecal-ligation and puncture; Mel=melatonin; A-ADMSC=apoptotic adipose-derived mesenchymal stem cell.

Figure 8

Protein expressions of DNA damage, apoptotic and anti-inflammatory biomarkers. A. Protein expression of γ-H2AX in lung parenchyma, * vs. other groups with different symbols (*, †, ‡, §, ¶), p<0.0001. B. Protein expression of mitochondrial Bax in lung parenchyma, * vs. other groups with different symbols (*, †, ‡, §, ¶), p<0.001. C. Protein expression of cleaved caspase (c-Csp) 3 I lung parenchyma, * vs. other groups with different symbols (*, †, ‡, §, ¶), p<0.0001. D. Protein expression of cleaved poly (ADP-ribose) polymerase (PARP) in lung parenchyma, * vs. other groups with different symbols (*, †, ‡, §, ¶), p<0.001. E. Protein expression of Bcl-2 in lung parenchyma, * vs. other groups with different symbols (*, †, ‡, §, ¶), p<0.008. All statistical analyses were with one-way ANOVA followed by Bonferroni multiple comparison post hoc test. Symbols (*, †, ‡, §, ¶) indicate significance (at 0.05 level). SC=sham control; CLP=cecal-ligation and puncture; Mel=melatonin; A-ADMSC=apoptotic adipose-derived mesenchymal stem cell.

Figure 9

Protein expressions of Fibrotic, anti-Fibrotic and anti-oxidant biomarkers. A. Protein expression of phosphorylated (p)-Smad3 in lung parenchyma, * vs. other groups with different symbols (*, †, ‡, §, ¶), p<0.0001. B. Protein expression of transforming growth factor (TGF)-β, * vs. other groups with different symbols (*, †, ‡, §, ¶), p<0.0001. C. Protein expression of p-Smad1/5 in lung parenchyma, * vs. other groups with different symbols (*, †, ‡, §, ¶), p<0.001. D. Protein expression of Bone morphogenic protein (BMP)-2 in lung parenchyma, * vs. other groups with different symbols (*, †, ‡, §, ¶), p<0.001. E. Protein expression of HO-1 in lung parenchyma, * vs. other groups with different symbols (*, †, ‡, §, ¶), p<0.0001. F. rotein expression of NQ0 1 in lung parenchyma, * vs. other groups with different symbols (*, †, ‡, §, ¶), p<0.0001. All statistical analyses were with one-way ANOVA followed by Bonferroni multiple comparison post hoc test. Symbols (*, †, ‡, §, ¶) indicate significance (at 0.05 level). SC=sham control; CLP=cecal-ligation and puncture; Mel=melatonin; A-ADMSC=apoptotic adipose-derived mesenchymal stem cell.

Figure 10

IF stain for identifying surface makers of endothelial cell (EC). (A to E) IF microscopic findings (200 x) for identification of positively-stained CD31 cells (white arrows). (D and E) Abundant transplanted A-ADMSCs with positively Dil-dye stain (red color) were found to be present in the lung parenchyma. Merge of double stain (i.e. Dil dye + CD31 staining) showing some of A-ADMSCs (D, E) exhibited EC surface marker of CD31 (yellow arrows). Some of transfused A-ADMSCs were identified to engraft into lung parenchyma (E) (asterisk marker). (F) Analytic results of CD31+ cells, * vs. other groups with different symbols (*, †, ‡, §), p<0.0001. The scale bars in right lower corner represent 50 μm. Symbols (*, †, ‡, §, ¶) indicate significance (at 0.05 level). SC=sham control; CLP=cecal-ligation and puncture; Mel=melatonin; A-ADMSC=apoptotic adipose-derived mesenchymal stem cell.

Figure 11

IF stain for identifying surface makers of endothelial progenitor cells. (A to E & G to H) IF microscopic findings (400 x) for identification of positively-stained CXCR4 cells (white arrows). (D & E and G & H) Numerous transplanted A-ADMSCs with positively Dil-dye stain (red color) were found to be present in the lung parenchyma. (D & E) Merge of double stain (i.e. Dil dye + CXCR4 staining) showing some of A-ADMSCs exhibited EPC surface marker of CXCR4 (yellow arrows). Some of transfused A-ADMSCs were identified to engraft into the pulmonary arteries (G, H) (pink arrow) and lung parenchyma (E, G) (asterisk marker). (F) Analytic results of CXCR4+ cells, * vs. other groups with different symbols (*, †, ‡, §, ¶), p<0.0001. The scale bars in right lower corner represent 20 μm. All statistical analyses were with one-way ANOVA followed by Bonferroni multiple comparison post hoc test. Symbols (*, †, ‡, §, ¶) indicate significance (at 0.05 level). SC=sham control; CLP=cecal-ligation and puncture; Mel=melatonin; A-ADMSC=apoptotic adipose-derived mesenchymal stem cell.

Figure 12

Proposed mechanisms underlying the effects of melatonin-A-ADMSC therapy on CLP-induced lung parenchymal injury in a rodent model based on findings of the present study. ADMSC=adipose-derived mesenchymal stem cell; BMP=bone morphogenetic protein; GPx=glutathione peroxidase; GR=glutathione reductase; HO=heme oxygense; IL-1β=interleukin (IL)-1β; MMP-9=matrix metalloproteinase-9; MIP=macrophage inflammatory protein; NQO 1=NAD(P)H quinone oxidoreductase; NOX=nicotinamide adenine dinucleotide phosphate (NADPH) oxidase; PARP=poly (ADP-ribose) polymerase; TNF=tumor necrotic factor; TGF=transforming growth factor.