Fig 1.
Experimental scheme of the protective effects of CA on abdominal IR-induced radiation enteropathy.
Abdominal IR was performed using X-Rad320, as shown in the photo. CA (10 mg/kg per dose) was intraperitoneally administered to C3H mice at 24 h and 1 h before and 24, 48 and 72 h after IR. The mice were euthanized at 6 h after IR for apoptosis detection and at 3.5 days after IR for crypt survival assays (n = 6/group). To assess sub-acute radiation enteropathy, 6 mice from the control and CA-only groups and 15 mice from the IR and CA and IR combination groups were sacrificed at 30 days after abdominal IR.
Fig 2.
CA treatment inhibits apoptotic epithelial and endothelial cell death after IR.
A. Apoptosis in the jejunum at 6 h after 12.5 Gy abdominal IR. Apoptotic FITC-TUNEL (green) and endothelial rhodamine-PECAM1 (red) are shown counterstained with DAPI (blue). Scale bar = 30 μm. B. The distribution of apoptotic cells in crypts and lamina propria and PECAM1 in a villus unit of the jejunum after 12.5 Gy IR. The values represent the mean ± SEM (*p < 0.05, n = 6).
Fig 3.
CA treatment rescues jejunal crypt survival after high-dose abdominal IR.
A. Hematoxylin and eosin (H&E)-stained jejunal sections harvested from vehicle- or CA-treated mice at 3.5 days after 12.5 Gy abdominal IR. The inserted squares depict whole cross sections of the jejunum. Original magnification, 5x. The arrows indicate crypts that survived following IR. Scale bar = 50 μm. B. Quantitative analysis of morphological changes in the jejunum following CA and IR. Data are presented as the mean ± SEM (*p < 0.05, n = 6). C. Immunohistochemical analysis of Ki-67, a proliferation marker, in a section of the jejunum at 3.5 days after IR. Ki-67 was evaluated using diaminobenzidine (DAB, brown stain) and hematoxylin counterstaining (blue). The arrows indicate crypts, representing intestinal stem cell proliferation.
Fig 4.
CA treatment ameliorates radiation-induced enteritis.
A. A H&E-stained section from mice treated with CA at 30 days after 12.5 Gy abdominal IR. Scale bar = 50 μm. Inserted squares show whole cross sections of the jejunum. Original magnification, 5x. B. Quantitative analysis of morphological changes in the jejunum following IR. Numbers of crypts, villi heights, and numbers of villi are shown. The data are presented as the mean ± SEM (*p < 0.05, n = 6). C. Immunohistochemical analysis of PECAM1 in jejunal sections. PECAM1 was detected using DAB (brown) and hematoxylin counterstaining (blue). Scale bar = 25 μm. The arrows indicate PECAM1-positive cells.
Fig 5.
CA confers protection against radiation-induced cell death by inducing HSF1 and HSP70 expression.
A. HSF1 and HSP70 expression in IEC6 cells and HUVECs was detected by Western blotting after treatment with 0–10 μM of CA at the time points indicated. B. Apoptotic cell death was measured by Annexin V/PI assay and flow cytometry at 48 h after 15 Gy IR. C. IEC6 cells and HUVECs were transfected with si-con, si-HSF1 or siHSP70 and treated with 5 μM of CA and 15 Gy of IR. Western blotting was conducted to detect cleaved caspase-3 and cleaved PARP at 48 h after 15 Gy IR in IEC6 cells and HUVECs. Actin was used as a loading control. Three different experiments were performed.
Fig 6.
CA promotes endothelial cell function via HSF1.
A. HUVECs were incubated in the presence of cycloheximide (CHX, 10 μg/ml) with or without 5 μM of CA and were analyzed by Western blotting (*p < 0.05 vs. control cell). B. HUVECs were transfected with si-con and si-HSF1, and the expression of HSF1 and eNOS was analyzed after 48 h of CA treatment and IR. C. A tube formation assay in Matrigel was performed using HUVECs. HUVECs were treated with 5 μM CA for 1 h before exposure to 10 Gy IR. The cells were detached from a 100-mm dish, and 4.5×104 cells were seeded on Matrigel in a 96-well plate at 24 h after IR. After 16 h of incubation, the tubes on the Matrigel were counted, and the results are depicted as a graph (**p < 0.01, n = 3).
Fig 7.
CA treatment did not protect tumors against IR.
A. Colony formation assays were performed with human cancer cell lines. Cells were seeded and cultured 7 to 10 days following CA treatment (0 and 2.5μM) and IR (0–6 Gy). B. HUVECs were transfected with si-con or si-HSF1 and treated with 5 μM of CA and 15 Gy of IR. Western blotting was conducted to detect HSF1 and eNOS at 48 h after IR. Actin was used as a loading control. Three different experiments were performed. C. The effect of CA on tumor growth was measured in CT26 allografts in BALB/c mice. Five CA doses (10 mg/kg, i.p.) were administered to tumor-bearing mice before and after 8 or 12.5 Gy IR. Tumor volumes were measured three times per week for 21 days. The data are presented as the mean ± SEM (*p < 0.05, n = 5).