Figure Legend
Figure 1: LPA attenuates radiation-induced disruption of tight junctions in the intestinal epithelial monolayers .
Caco-2 and m-ICC12 cell monolayers on Transwell inserts were irradiated (2-15 Gy) with or without LPA (10 µM) administered 30 min prior to irradiation. One hour after irradiation, the tight junction integrity was examined. A & B : Caco-2 cell monolayers exposed to radiation with or without LPA were fixed and stained for occludin (green) and ZO-1 (red). Confocal images (A) and fluorescence density at the junctions (B) are presented. Values are Mean ± SEM (n = 4). The corresponding p-values (on top of the bars) indicate the significant differences between groups. NS indicates that the p-value is greater than 0.05. C : Caco-2 cell monolayers exposed to radiation (10 Gy) with or without LPA were stained for F-actin (green) and cofilinpS3 (red). D : Caco-2 cell monolayers were pretreated with toxin-B (TB) 30 min prior to LPA treatment. Monolayers were irradiated (10 Gy) 30 min after LPA treatment and stained for occludin (green), ZO-1 (red), and nucleus (blue).E : Confocal images for ZO-1 in m-ICC12 cell monolayers that were treated with LPA or vehicle prior to irradiation.
Figure 2: Effect of LPA2 receptor deficiency on the radiation-induced disruption of tight junction and adherens junction .
Wild type (WT) and LPA2 receptor knockout (Lpar2-/- ) mice were subjected to TBI (9.5 Gy). Two hours after irradiation, colonic sections were stained for tight junction and adherens junction proteins. A : Merged fluorescence images for occludin (green), ZO-1 (red), and nucleus (blue). B : Fluorescence images for E-Cadherin (green), β-Catenin (red), and nucleus (blue). Representative images from 4 mice per each irradiated group of mice and 2 mice for control groups are presented.
Figure 3: RP1 blocks TBI-induced disruption of tight junction and adherens junction, barrier dysfunction, and endotoxemia .
Wild type mice were injected with RP1 (0.5 mg/kg) or vehicle (Veh) 30 min prior to TBI (9.5 Gy) (IR and IR+RP1); the control group was sham-treated. At 2 hours post-irradiation, colonic sections were co-stained for occludin, ZO-1, and nucleus (A) or E-cadherin, β-catenin, and nucleus (B). Confocal images were captured, and fluorescence densities at the junctions were measured. Fluorescence densities for ZO-1 (C) and E-cadherin (D) are presented. At 2 and 4 hours post-irradiation, mucosal permeability in vivo in the colon (E) and ileum (F) and the plasma LPS levels (G) were measured. Values are Mean ± SEM (n = 4). The corresponding p-values (on top of the bars) indicate the significant differences between groups. NS indicates that the p-value is greater than 0.05. Experiment was repeated with similar results.
Figure 4: RP1 mitigates TBI-induced disruption of AJC, mucosal barrier dysfunction, and endotoxemia .
Adult wild type mice were subjected to TBI (9.5 Gy). At 24 hours after irradiation, mice were injected with RP1 (0.5 mg/kg daily) or vehicle (Veh); the control group was sham-treated. At 24 and 48 hours after RP1 treatment, junctional integrity (A-F), gut permeability in vivo(G & H), and endotoxemia (I) were evaluated. Sections of the colon were co-stained for occludin, ZO-1, and nucleus (A), E-cadherin, β-catenin, and nucleus (B) or claudin-3, Claudin-2, and nucleus (C). Confocal fluorescence images were captured, and fluorescence densities at the junctions were measured. Fluorescence densities for ZO-1 (D) and β-catenin (E) and Claudin-3 (F) are presented. Mucosal permeabilityin vivo in the colon (G) and ileum (H) and the plasma LPS levels (I) were measured. Values are Mean ± SEM (n = 4). The corresponding p-values (on top of the bars) indicate the significant differences between groups. NS indicates that the p-value is greater than 0.05. Experiment was repeated with similar results. Similar results were also produced in a similar experiment analyzed at 7 days post-irradiation.
Figure 5: RP1 alleviates TBI-induced downregulation of antioxidant gene expression .
Wild type (WT) mice were subjected to TBI (9.5 Gy). At 24 hours after irradiation, mice were injected with RP1 (0.5 mg/kg daily) or vehicle (Veh); the control group was sham-treated. At 24 hours after RP1 treatment, RNA extracted from colonic mucosa was analyzed for mRNA forGpx1 (A), SOD1 (B), CAT (C), Prdx1 (D), andNrf2 (E) by RT-qPCR. Values are Mean ± SEM (n = 4). The corresponding p-values (on top of the bars) indicate the significant differences between groups. NS indicates that the p-value is greater than 0.05.
Figure 6: RP1 blocks PBI-induced disruption of AJC, barrier dysfunction, and endotoxemia .
Adult wild type mice were injected with RP1 (3 mg/kg) or vehicle (Veh) daily starting 1 day after partial body irradiation (PBI; 15.6 Gy); the control group was sham-treated. At varying times after irradiation, colonic sections were co-stained for occludin, ZO-1, and nucleus (A) or E-cadherin, β-catenin, and nucleus (B). Confocal fluorescence images were captured, and the fluorescence densities of ZO-1 (C) and E-cadherin (D) at the junctions were measured. Values are Mean ± SEM (n = 4). The corresponding p-values (on top of the bars) indicate the significant differences between groups. NS indicates that the p-value is greater than 0.05. Similar results were produced in similar experiment when examined at 72 and 96 hours post-irradiation.
Figure 7: RP1 mitigates PBI-induced oxidative stress .
Adult mice were injected with RP1 (3 mg/kg) or vehicle (Veh) daily starting one day after partial body irradiation (PBI-BM5; 15.6 Gy); the control group was sham-treated. At 48 hours after irradiation (or 24 hours after RP1), colonic sections were stained for reduced-protein thiols, oxidized-protein thiols, and NRF2. Antioxidant gene expression was analyzed by RT-qPCR. A & B : Confocal images for protein thiols were captured (A), and the fluorescence densities were measured (B). C & D : Colonic sections were co-stained for F-actin (green), NRF2 (red) and nucleus (blue) (C). The protein extracts were immunoblotted for NRF2, and the band densities were measured.E-J : RNA isolated from colonic mucosa was analyzed for mRNA forNrf2 (E), SOD1 (F), Gpx1 (G), CAT (H),Trx1 (I), and Prdx1 (J) by RT-qPCR. Values are Mean ± SEM (n = 4). The corresponding p-values (on top of the bars) indicate the significant differences between groups. NS indicates that the p-value is greater than 0.05. Similar results were produced in similar experiment when examined at 72 and 96 hours post-irradiation.
Figure 8: RP1 mitigates PBI-induced F-actin remodeling and its association with apical junctional proteins .
Adult mice were injected with RP1 (3 mg/kg) or vehicle (Veh) daily starting one day after partial body irradiation (PBI-BM5; 15.6 Gy); the control group was sham-treated. A-E: At 48 hours after irradiation (or 24 hours after RP1), detergent-insoluble fractions of colonic mucosa were immunoblotted for tight junction and adherens junction proteins as well as NRF2 and β-actin (A). Band densities for E-cadherin (B), β-catenin (C), Cldn-3 (D), ZO-1 (E), and β-actin (F) were measured. G: Cryosections of the colon were fixed and stained for F-actin (red) and nucleus (blue). H & I: Colonic sections were co-stained for cofilinpS3 (red), F-actin (green) and nucleus (blue) (H). Mucosal extracts were immunoblotted for cofilinpS3 and β-actin (I). Band densities were measured, and the cofilinpS3 band densities presented by values normalized to corresponding actin band densities (I). In all graphs, values are Mean ± SEM (n = 4). The corresponding p-values (on top of the bars) indicate the significant differences between groups. NS indicates that the p-value is greater than 0.05.