Gene expression analysis reveals inhibition of radiation-induced TGFβ-signaling by hyperbaric oxygen therapy in mouse salivary glands

Abstract

A side effect of radiation therapy in the head and neck region is injury to surrounding healthy tissues such as irreversible impaired function of the salivary glands. Hyperbaric oxygen therapy (HBOT) is clinically used to treat radiation-induced damage but its mechanism of action is largely unknown. In this study, we investigated the molecular pathways that are affected by HBOT in mouse salivary glands two weeks after radiation therapy by microarray analysis. Interestingly, HBOT led to significant attenuation of the radiation-induced expression of a set of genes and upstream regulators that are involved in processes such as fibrosis and tissue regeneration. Our data suggest that the TGFβ-pathway, which is involved in radiation-induced fibrosis and chronic loss of function after radiation therapy, is affected by HBOT. On the longer term, HBOT reduced the expression of the fibrosis-associated factor α-smooth muscle actin in irradiated salivary glands. This study highlights the potential of HBOT to inhibit the TGFβ-pathway in irradiated salivary glands and to restrain consequential radiation induced tissue injury.

Figure 1

Summary of microarray data by PCA and Venn diagrams. (A) PCA-mapped scatter plot. The global gene expression profiles of the submandibular glands for different treatment groups and control analyzed by PCA. The figure represents the first three principal components of microarray analysis data (PC1, PC2 and PC3) in x, y and z axes, respectively. (B) Venn diagram that represents the number of differentially expressed Affymetrix probe sets in RT versus control (red circle), HBOT versus control (green circle) and RT + HBOT versus RT (black circle), with the number overlapping probe sets inside the circles.

Figure 2

Differentially expressed probe sets between the RT− and RT + HBOT group. OmniViz treescape showing the hierarchical clustering of differentially expressed Affymetrix probe sets between the submandibular glands of the RT and RT + HBOT group (middle groups). Expression of these probe sets for the control and HBOT-group is shown on the outside. Red indicates upregulated probe sets compared with the geometric mean and blue indicates downregulated probe sets compared with the geometric mean. The color intensity correlates with the degree of change. Rectangle shows probe sets that are upregulated in the RT group, while downregulated in the RT + HBOT group. Genes within this rectangle are summarized.

Figure 3

qPCR validation of genes of interest. qPCR validation of microarray results for the expression of genes of interest at 2 wks after RT in the submandibular glands. y Axis shows mean fold change relative to controls. Lines above bars represent statistically significant differences (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 4

Influence of HBOT on the TGFβ-pathway. Differential expression of genes involved in the TGFβ-pathway in irradiated submandibular glands compared with control (A) and in irradiated glands that received HBOT compared with irradiated glands (B), by Ingenuity Pathway Analysis of microarray data. Colors show up- (red) and downregulated (green) genes (≥ 1.2-fold change, FDR 0.05). Notice the reverse expression of genes when HBOT is applied to irradiated glands. qPCR validation (C) and immunohistochemical staining (D) of Tgfβ1 and Serpine1 at 2 wks after RT. Scale bars left pictures 200 μm, right pictures 50 μm.

Figure 4

Influence of HBOT on the TGFβ-pathway. Differential expression of genes involved in the TGFβ-pathway in irradiated submandibular glands compared with control (A) and in irradiated glands that received HBOT compared with irradiated glands (B), by Ingenuity Pathway Analysis of microarray data. Colors show up- (red) and downregulated (green) genes (≥ 1.2-fold change, FDR 0.05). Notice the reverse expression of genes when HBOT is applied to irradiated glands. qPCR validation (C) and immunohistochemical staining (D) of Tgfβ1 and Serpine1 at 2 wks after RT. Scale bars left pictures 200 μm, right pictures 50 μm.

Figure 4

Influence of HBOT on the TGFβ-pathway. Differential expression of genes involved in the TGFβ-pathway in irradiated submandibular glands compared with control (A) and in irradiated glands that received HBOT compared with irradiated glands (B), by Ingenuity Pathway Analysis of microarray data. Colors show up- (red) and downregulated (green) genes (≥ 1.2-fold change, FDR 0.05). Notice the reverse expression of genes when HBOT is applied to irradiated glands. qPCR validation (C) and immunohistochemical staining (D) of Tgfβ1 and Serpine1 at 2 wks after RT. Scale bars left pictures 200 μm, right pictures 50 μm.

Figure 5

Expression of α smooth muscle actin. Relative expression (mean fold change relative to controls) of α smooth muscle actin (α-Sma) at 2, 10 and 24 wks after RT in the submandibular glands (A). Line above bars represents statistically significant difference (*P < 0.05). Immunohistochemical staining of α-SMA in submandibular glands of control and irradiated mice, either with hyperbaric oxygen therapy (RT + HBOT) or without (RT). Graph shows the percentage of positive α-SMA staining for the different groups (B). Line above bars represents statistically significant difference (**P < 0.01; P = 0.053 for RT versus control). Scale bars upper pictures 200 μm, lower pictures 50 μm.