in vivo
The LPA2 receptor plays a crucial role in many intestinal mucosal protective functions of LPA, including inhibition of cholera toxin-induced diarrhea (Li et al., 2005), intestinal ion transport (Lin et al., 2010; Singla et al., 2010; Thompson et al., 2018), prevention of radiation-induced intestinal mucosal atrophy and stem cell ablation (Kuo et al., 2018). In this study, we examined the effect of LPA2 deficiency on the severity of radiation-induced disruption of colonic epithelial tight junctionsin vivo at 2 hours after irradiation. Immunofluorescence confocal images show that redistribution of occludin and ZO-1 from the epithelial junctions was more severe in Lpar2-/- mice compared to that present in WT mouse colons (Fig. 2A). Similarly, radiation-induced redistribution of adherens junction proteins, E-cadherin, and β-catenin from the epithelial junctions was more severe in the colon of Lpar2-/- mice, compared to that of WT mice (Fig. 2B).
RP1 blocks radiation-induced disruption of apical junctional complexes, barrier dysfunction, and endotoxemia
RP1, a stable non-lipid LPA2-specific agonist (Patil et al., 2014), protects the intestinal mucosa from diarrhea and mucosal atrophy by various types of insults (Kuo et al., 2018; Patil et al., 2015; Thompson et al., 2018). In the current study, we evaluated the effect of RP1 on the radiation-induced disruption of intestinal epithelial tight junctions and adherens junctions, the increase in mucosal permeability, and endotoxemia. Prophylactic administration of RP1 (0.5 mg/kg; s.c.) at 30 min pre-irradiation blocked the radiation-induced loss of junctional distribution of occludin and ZO-1 when examined at 2 hours post-irradiation (Fig. 3A). Similarly, RP1 administration blocked radiation-induced redistribution of E-cadherin and β-catenin from the epithelial junctions (Fig. 3B). Densitometric analysis of ZO-1 (Fig. 3C) and E-cadherin (Fig. 3D) fluorescence at the junctions indicated that RP1 completely attenuated radiation-induced redistribution of these proteins from the junctions. Mucosal permeability in the colon and ileum in vivo was measured at 2 hours and 4 hours after irradiation. RP1 significantly reduced radiation-induced mucosal permeability in the colon (Fig. 3E). RP1 showed no significant effect in the ileum at these time points (Fig. 3F). Prevention of radiation-induced colonic mucosal permeability by RP1 at 4 hours post-irradiation was associated with a significant (p = 0.04) reduction of the radiation-induced increase in plasma LPS levels (Fig. 3G).
RP1 mitigates radiation-induced disruption of apical junctional complexes, barrier dysfunction, and endotoxemia
In this study, RP1 was administered 3 hours after irradiation (0.5 mg/kg daily; s.c.). The post-irradiation treatment with RP1 reversed the radiation-induced loss of junctional occludin and ZO-1 when examined at 24 hours post-irradiation (Fig. 4A). Similarly, RP1 reversed radiation-induced redistribution of E-cadherin and β-catenin from the epithelial junctions (Fig. 4B). Radiation also induced redistribution of claudin-3, another transmembrane protein of tight junction in mouse colons (Fig. 4C). Post-irradiation treatment with RP1 reversed this effect of radiation on the colonic epithelial distribution of claudin-3. Densitometric analysis of ZO-1 (Fig. 4D), β-catenin (Fig. 4E), and claudin-3 (Fig. 4F) at the junctions confirmed that RP1 completely attenuated radiation-induced redistribution of these proteins from the junctions. Mucosal permeability in colon and ileum in vivo was measured at 24 and 48 hours post-irradiation. RP1 significantly alleviated radiation-induced mucosal permeability in the colon (Fig. 4G) and ileum (4H). Restoration of radiation-induced loss of intestinal mucosal permeability by RP1 was associated with a significant (p = 0.015 at 24 hours & 0.0001 at 48 hours) reduction of radiation-induced elevation of plasma LPS (Fig. 4I).
RP1 mitigates radiation-induced suppression of antioxidant gene expression
Our previous study demonstrated that radiation induces oxidative stress and the antioxidant N-acetyl-L-cysteine blocks as well as mitigates radiation-induced disruption of tight junctions and barrier dysfunction in mouse colons (Shukla et al., 2016). In this study, we evaluated the effect of RP1 on radiation-induced effects on antioxidant gene expression. RP1 (0.5 mg/kg daily; s.c.), administered at 24 hours post-irradiation, reversed the radiation-induced reduction of mRNA forGpx1 (Fig. 5A), Sod1 (Fig. 5B), and Prdx1 (Fig. 5C). However, RP1 failed to reverse radiation-induced reduction ofCAT (catalase) mRNA (Fig. 5D). The mRNA levels for Nrf2 , the transcription factor involved in antioxidant gene expression, was reduced by radiation; however, it was reversed by RP1 treatment (Fig. 5E).
Effects of RP1 on tight junction and adherens junction integrity in the PBI-BM5 model
The impact of PBI-BM5 on the intestinal epithelial tight junction integrity and its prevention by RP1 treatment were evaluated next. Immunofluorescence confocal microscopy showed that PBI-BM5 induced a redistribution of occludin and ZO-1 from the colonic epithelial junctions when examined at 52 hours post-irradiation, and RP1 (3 mg/kg daily; s.c.; beginning 24 hours after irradiation) treatment alleviated this effect of RP1 (Fig. 6A). Similarly, PBI induced redistribution of adherens junction proteins, E-cadherin, and β-catenin from the epithelial junctions and RP1 alleviated this effect of PBI (Fig. 6B). Densitometric analysis for ZO-1 fluorescence at the junctions indicated that PBI-BM5 did not affect tight junction integrity at 28 hours post-irradiation but induced a severe disruption of tight junctions at 52 and 76 hours (Fig. 6C). RP1 treatment completely blocked PBI-induced ZO-1 redistribution at both 52 and 76 hours post-irradiation. RP1 also prevented PBI-induced reduction of junctional E-cadherin levels (Fig. 6D).
RP1 attenuates PBI-BM5-induced oxidative stress in colonic mucosa
Protein thiol oxidation was examined by fluorescence staining of reduced-protein thiols and oxidized-protein thiols at 52 hours post-irradiation. Fluorescence images (Fig. 7A) and densitometric analyses (Fig. 7B) indicated that PBI-BM5 depleted reduced protein thiols with a corresponding increase in oxidized protein thiols and that RP1 treatment (3 mg/kg daily; s.c.; beginning 24 hours after irradiation) reversed PBI-induced protein thiol oxidation. The expression of Nrf2 was examined by immunofluorescence staining (Fig. 7C), immunoblot analysis (Fig. 7D), and Nrf2 -specific mRNA measurement (Fig. 7E). All these analyses indicated that PBI significantly reduced Nrf2 expression, while RP1 blocked this effect of PBI. PBI significantly reduced the levels of mRNA forSOD1 (Fig. 7F), Gpx1 (Fig. 7G), CAT (catalase) (Fig. 7H), Trx1 (Fig. 7I), and Prdx1 (Fig. 7J), and RP1 attenuated these effects of PBI.
RP1 attenuates PBI-BM5-induced F-actin remodeling and its association of apical junctional complexes
The association of tight junction and adherens junction proteins with the actin cytoskeleton was assessed by Western blot analysis of the actin-rich, detergent-insoluble fraction for tight junction and adherens junction proteins. Densitometric analysis of immunoblot bands (Fig. 8A) for E-cadherin (Fig. 8B), β-catenin (Fig. 8C), Claudin-3 (Fig. 8D), and ZO-1 (Fig. 8E) indicated that PBI-BM5 significantly reduced the detergent-insoluble fraction of E-cadherin, β-catenin, and claudin-3, which was blocked by RP-1 treatment (3 mg/kg daily; s.c.; beginning 24 hours after irradiation). Although PBI-BM5 did not reduce ZO-1 levels, nevertheless RP1 significantly increased ZO-1 density. Immunofluorescence analysis of F-actin showed that PBI reduced F-actin levels, and RP1 treatment attenuated this effect of PBI-BM5 (Fig. 8F). Immunofluorescence staining (Fig. 8G) and immunoblot analysis (Fig. 8H) indicated that PBI-BM5 reduced the levels of cofilinpS3 in the colonic mucosa, which was blocked by RP1 treatment.