Conditioned medium of mesenchymal stem cells pretreated with H2O2 promotes intestinal mucosal repair in acute experimental colitis

Abstract

Mesenchymal stem cells (MSCs) are a new therapeutic strategy for inflammatory bowel disease (IBD), and their efficacy has been widely recognized. However, there are still some challenges in cell therapy, including stable cell passage, laboratory conditions for cell culture, high-cost burden, and poor transplantation. The conditioned medium (CM) of MSCs is considered be an excellent alternative to cell transplantation, but the paracrine group in MSC-CM is limited in variety and low in concentration, which cannot meet the therapeutic needs of injured tissues and needs to be optimized. Pretreatment with low concentration of hydrogen peroxide (H₂O₂) can not only protect cells from oxidative damage, but also play a role similar to growth factors and regulate the physiological function of stem cells, to obtain an improved conditioned medium. To determine the optimal protocol for pretreatment of MSCs with H₂O₂, and to study the efficacy and potential mechanism of MSC-CM pretreated with H₂O₂ on Dextran Sulfate Sodium (DSS)-induced acute experimental colitis. MSCs were exposed to different concentrations of H₂O₂, and the optimal H₂O₂ pretreatment conditions were determined by evaluating their critical cell functional properties. H₂O₂-pretreated MSC-CM was transplanted into experimental mouse colitis by enema at 2, 4, and 6 days in modeling, and the changes of colonic tissue structure, the levels of inflammation and oxidative stress, the molecular changes of Nrf2/Keap1/ARE axis, and the related indicators of apoptosis in colonic epithelial cells were observed in each group. In vitro, Pretreated MSCs with 25 μM H₂O₂ significantly enhanced cell proliferation, migration, and survival, but had no effect on apoptosis. In vivo, MSC-CM treatment decreased apoptosis and extracellular matrix deposition, and maintained the mechanical barrier and permeability of colonic epithelial cells in experimental mouse colitis. Mechanistically, H₂O₂-pretreated MSC-CM against reactive oxygen species (ROS) production and MDA generation, accompanied by increases in components of the enzymatic antioxidant system includes SOD, CAT, GSH-PX, and T-AOC, which is through the up-regulation of the Nrf2, HO-1, and NQO-1 antioxidant genes. Our data confirmed that 25 μM H₂O₂ pretreated MSC-CM treatment could effectively improve intestinal mucosal repair in experimental colitis, which may be achieved by activating Nrf2/Keap1/ARE pathway.

Figure 1

Optimization of H₂O₂ concentrations for preconditioning MSCs. (A) Cell viability was analyzed by ATP assay. (B) The effect of H₂O₂ on MSCs proliferation was determined by the CCK-8 assay. (C) Crystal violet staining assessed the cell density are shown, quantitative analysis of cell density. Magnification: × 40. Scale bar, 200 μm. (D) The apoptosis of MSCs treated with different concentrations of H₂O₂ for 24 h was analyzed by flow cytometry. The bottom right quadrant represents annexin V-FITC-stained cells (early-phase apoptotic cells), and the top right quadrant represents PI- and annexin V-FITC-dual-stained cells (late-phase apoptotic/necrotic cells). Apoptotic cells are represented as the percentage. (E) Western blot analysis of Bcl-2, Bax, Caspase 3, and Cleaved Caspase3 in MSCs exposed to 25–400 μM H₂O₂ for 24 h, and semi-quantitative analysis of the related proteins. Data are expressed as the mean ± SD (n = 3). *P < 0.05, **P < 0.01, and ***P < 0.001; ns no significance.

Figure 2

25 μM H₂O₂ pretreatment of MSCs can reduce apoptosis caused by ROS generation by activating the Nrf2/Keap1/ARE signaling pathway. (A) Immunoblotting assay determined the abundance of Nrf2, Keap1, HO-1, and NQO-1 proteins, and semi-quantitative analysis of the related proteins. (B) Immunoblotting assay determined the abundance of Bax, Bcl-2, Caspase 3, and Cleaved Caspase3 proteins, and semi-quantitative analysis of the related proteins. (C) Representative images of flow cytometric analysis by annexin V-FITC/PI dual staining. Apoptotic cells are represented as the percentage. (D) Crystal violet staining assessed the cell density are shown, quantitative analysis of cell density. Magnification: × 40. Scale bar, 200 μm. (E) The quantitative evaluation of oxidative stress in MSCs including O₂⁻, OH⁻, and MDA. The data are expressed as the mean ± SD (n = 3). *P < 0.05, **P < 0.01, and ***P < 0.001; ns no significance.

Figure 3

Therapeutic effects of H₂O₂ pretreated MSC-CM in DSS-induced colitis mice. (A) Experimental protocol for DSS-colitis model. Mice received drinking water with 2% DSS for 7 days, and treatment groups were given an enema administration of vehicle control, 25 μM H₂O₂ pretreated MSC-CM (200 µl), MSC-CM (200 µl), or ML385 (30 mg/kg). (B) Macroscopic images of representative colons at sacrifice. (C) Disease activity score is based on the loss of body weight (a), stool condition (b), and fecal occult blood (c) at sacrifice. (D) Disease-related shortening of the colon and quantified in a bar graph. (E) Disease-related lightening of the spleen and quantified in a bar graph. The data are expressed as the mean ± SD. n = 6 animals per group per time point. *P < 0.05, **P < 0.01, and ***P < 0.001, significantly different between groups; ns = no significance. ^#P < 0.1, ^##P < 0.01, ^###P < 0.001, significantly different within groups.

Figure 4

H₂O₂ pretreated MSC-CM decreases the inflammatory cell in colon tissue. (A) Distal colons were removed and sectioned followed by H&E staining. Representative sections are displayed. Inflammation scores were evaluated in bar graph. (B) IHC analysis for the macrophage specific marker CD68 was performed, and subsequently positive cells were evaluated. Magnification: × 100. Scale bar, 100 μm. (C) IHC analysis for the neutrophil specific marker MPO was performed, and subsequently positive cells were evaluated. The insets are magnified images of the indicated rectangles. Magnification: × 100. Scale bar, 100 μm. Data are expressed as the mean ± SD. n = 6 animals per group per time point. *P < 0.05, **P < 0.01, and ***P < 0.001; ns no significance.

Figure 5

H₂O₂ pretreated MSC-CM regulates the inflammatory cytokines in colon tissue. (A) The levels of inflammatory cytokines including TNF-α (a), IL-6 (b), IL-1β (c), and IL10 (d) in colon tissue homogenate were detected by ELISA. Data are expressed as the mean ± SD. n = 6 animals per group per time point. *P < 0.05, **P < 0.01, and ***P < 0.001; ns no significance. (B) Sections of colonic tissues were immunostained for revealing NF-κB p65 proteins (red) and Foxp3 proteins (red) as indicated. The slides were counterstained with DAPI (blue), and the images were captured using an inverted fluorescence microscope. Magnification: × 400.

Figure 6

H₂O₂ pretreated MSC-CM reduces intestinal mucosal barrier injury in colon tissue. (A) Masson staining assessed the collagen deposition in the colonic interstitium, the collagen area ratio was evaluated in bar graph. The insets are magnified images of the indicated rectangles. Magnification: × 100. Scale bar, 100 μm. Values are the means ± SD. n = 6 animals per group per time point. *P < 0.05, **P < 0.01, and ***P < 0.001; ns no significance. (B) Sections of colonic tissues were immunostained for revealing ZO-1 proteins (red) as indicated. The slides were counterstained with DAPI (blue), and the images were captured using an inverted fluorescence microscope. Magnification: × 400. (C) Immunoblotting assay determined the abundance of ZO-1 proteins. Semiquantitative analysis of proteins of interest by densitometry assay. The data are expressed as the mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001; ns no significance.

Figure 7

H₂O₂ pretreated MSC-CM exhibits anti-oxidant and anti-apoptosis activities in colon tissue. (A) The quantitative evaluation of oxidative stress in colon homogenate including O₂⁻ (a), OH⁻ (b), and MDA (c). The indexes of the enzyme antioxidant system including SOD (d), CAT (e), GSH-Px (f), and T-AOC (g). (B) Immunoblotting assay determined the abundance of Bax, Bcl-2, Caspase 3, Cleaved Caspase3, Nrf2, Keap1, HO-1, and NQO-1 proteins. Semiquantitative analysis of proteins of interest by densitometry assay. The data are expressed as the mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001; ns no significance.