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. 2018 May 9:2018:2481418.
doi: 10.1155/2018/2481418. eCollection 2018.

VSports - Deoxycholic Acid-Mediated Sphingosine-1-Phosphate Receptor 2 Signaling Exacerbates DSS-Induced Colitis through Promoting Cathepsin B Release

Shengnan Zhao  1   2   3 , Zizhen Gong  1   2   3 , Xixi Du  1   2   3 , Chunyan Tian  4   5 , Lingyu Wang  1   2   3 , Jiefei Zhou  1   2   3 , Congfeng Xu  6 , Yingwei Chen  2   3 , Wei Cai  1   2   3 , Jin Wu  1   2   3
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V体育安卓版 - Deoxycholic Acid-Mediated Sphingosine-1-Phosphate Receptor 2 Signaling Exacerbates DSS-Induced Colitis through Promoting Cathepsin B Release

VSports最新版本 - Shengnan Zhao et al. J Immunol Res. .

V体育安卓版 - Abstract

We recently have proved that excessive fecal DCA caused by high-fat diet may serve as an endogenous danger-associated molecular pattern to activate NLRP3 inflammasome and thus contributes to the development of inflammatory bowel disease (IBD). Moreover, the effect of DCA on inflammasome activation is mainly mediated through bile acid receptor sphingosine-1-phosphate receptor 2 (S1PR2); however, the intermediate process remains unclear. Here, we sought to explore the detailed molecular mechanism involved and examine the effect of S1PR2 blockage in a colitis mouse model. In this study, we found that DCA could dose dependently upregulate S1PR2 expression. Meanwhile, DCA-induced NLRP3 inflammasome activation is at least partially achieved through stimulating extracellular regulated protein kinases (ERK) signaling pathway downstream of S1PR2 followed by promoting of lysosomal cathepsin B release VSports手机版. DCA enema significantly aggravated DSS-induced colitis in mice and S1PR2 inhibitor as well as inflammasome inhibition by cathepsin B antagonist substantially reducing the mature IL-1β production and alleviated colonic inflammation superimposed by DCA. Therefore, our findings suggest that S1PR2/ERK1/2/cathepsin B signaling plays a critical role in triggering inflammasome activation by DCA and S1PR2 may represent a new potential therapeutic target for the management of intestinal inflammation in individuals on a high-fat diet. .

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Figures

Figure 1
Figure 1
DCA induces S1PR2 expression in macrophages. (a) J774A.1 macrophages were pretreated with or without LPS and then stimulated with different dosage of DCA (0, 50, 100, and 200 μM) for 4 h. Relative S1PR2 mRNA expression level was analyzed by real-time PCR. (b) J774A.1 macrophages were pretreated with or without LPS and then stimulated with DCA (100 μM) for different time courses. Relative S1PR2 mRNA expression level was analyzed by real-time PCR. (c) J774A.1 macrophages were pretreated with or without LPS and then stimulated with different dosage of DCA (0, 50, 100, and 200 μM) for 24 h. S1PR2 protein level was detected by Western blotting. (d) Real-time PCR determinant of relative S1PR2 mRNA expression in murine peritoneal macrophages treated with different dosage of DCA. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. Error bars indicate s.e.m. Representative data from 3 independent experiments giving similar results are shown.
Figure 2
Figure 2
DCA activates ERK signaling and promotes cathepsin B release. (a) Immunoblot analysis of phospho-ERK, total ERK, phospho-AKT, and total AKT of LPS-primed J774A.1 macrophages treated with or without DCA (100 μM) for different time courses. β-Actin was regarded as a loading control. The relative intensity of the bands was quantitated. (b) LPS-primed J774A.1 macrophages were treated with DCA (100 μM) in the presence or absence of JTE-013 or U0126. The cells were incubated with cathepsin B substrate which emits a red signal upon cleavage by cathepsin B, followed by Hoechst staining (blue). (c–d) LPS-primed J774A.1 macrophages were treated with DCA (100 μM) in the presence or absence of JTE-013 or U0126, and cytosolic protein was extracted for the immunoblot analysis of mature cathepsin B (c) as well as fluorometric assay of cathepsin B activity (d). ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. Error bars indicate s.e.m. The data shown are from 3 independent experiments.
Figure 3
Figure 3
Blockage of ERK signaling prevents ASC speck formation and mature IL-1β secretion. (a) LPS-primed J774A.1 macrophages were treated with DCA in the presence or absence of JTE-013 or U0126. The cells were then stained with ASC antibody and DAPI. Representative immunofluorescent images of ASC specks were shown. (b) LPS-primed J774A.1 macrophages were treated with DCA in the presence or absence of JTE-013 or U0126 for 24 h. Immunoblot was performed to determine mature IL-1β (mIL-1β, 17kD) in the culture supernatants (SN) and precursors of IL-1β (pro-IL-1β) in cell lysates, and (c) secreted IL-1β in supernatants was also analyzed by ELISA. ∗∗ p < 0.01 and ∗∗∗ p < 0.001. Error bars indicate s.e.m. The data shown are representative of 3 independent experiments.
Figure 4
Figure 4
DCA administration exacerbates DSS-induced colitis and inhibition of cathepsin B release reverses the intestinal inflammation. Colitis was induced in mice with 2.5% DSS and animals were divided into control, DSS-treated, DSS-treated plus DCA enema, DSS-treated plus DCA enema, and cathepsin B inhibitor (Ca-074Me)-injection groups. (a) Loss of basal body weight, (b) colon length, (c) hematochezia score, and (d) MPO activity in colon tissue were detected. (e) Immunoblot analysis of mature IL-1β (17kD) in colonic homogenates. (f) HE staining of distal colon sections of differently treated mice. ∗ p < 0.05 and ∗∗ p < 0.01 compared to the DSS-treated alone mice. # # p < 0.01 and # # # p < 0.001 compared to the DSS-treated plus DCA enema mice. Error bars indicate s.e.m. The data shown are from 3 individual experiments.
Figure 5
Figure 5
S1PR2 antagonist abrogates the exacerbating role of DCA in the DSS-induced colitis. (a) Loss of basal body weight, (b) colon length, (c) hematochezia score, and (d) MPO activity in colon tissue of control, DSS-treated, DSS-CL, DSS-treated plus DCA enema, and DSS-treatedplus DCA enema, and S1PR2 antagonist (JTE-013)-injection mice. (e) Immunoblot analysis of mature IL-1β (17kD) and mature cathepsin B in colon tissue. (f) HE staining and of distal colon sections of differently treated mice as described above. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 compared to the DSS-treated alone mice. # p < 0.05, # # p < 0.01, and # # # p < 0.001 compared to the DSS-treated plus DCA enema mice. Error bars indicate s.e.m. Representative data from 3 individual experiments are shown.
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