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.