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. 2017 Aug 15;7(14):3446-3460.
doi: 10.7150/thno.20359. eCollection 2017.

rSj16 Protects Against DSS-Induced Colitis by Inhibiting the PPAR-α Signaling Pathway

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Free PMC article

rSj16 Protects Against DSS-Induced Colitis by Inhibiting the PPAR-α Signaling Pathway

Lifu Wang et al. Theranostics. .
Free PMC article

Abstract

Background: Epidemiologic studies and animal model experiments have shown that parasites have significant modulatory effects on autoimmune disorders, including inflammatory bowel disease (IBD). Recombinant Sj16 (rSj16), a 16-kDa secreted protein of Schistosoma japonicum (S.japonicum) produced by Escherichia coli (E. coli), has been shown to have immunoregulatory effects in vivo and in vitro. In this study, we aimed to determine the effects of rSj16 on dextran sulfate sodium (DSS)-induced colitis. Methods: DSS-induced colitis mice were treated with rSj16. Body weight loss, disease activity index (DAI), myeloperoxidase (MPO) activity levels, colon lengths, macroscopic scores, histopathology findings, inflammatory cytokine levels and regulatory T cell (Treg) subset levels were examined. Moreover, the differential genes expression after treated with rSj16 were sequenced, analyzed and identified. Results: rSj16 attenuated clinical activity of DSS-induced colitis mice, diminished pro-inflammatory cytokine production, up-regulated immunoregulatory cytokine production and increased Treg percentages in DSS-induced colitis mice. Moreover, DSS-induced colitis mice treated with rSj16 displayed changes in the expression levels of specific genes in the colon and show the crucial role of peroxisome proliferator activated receptor α (PPAR-α) signaling pathway. PPAR-α activation diminished the therapeutic effects of rSj16 in DSS-induced colitis mice, indicating that the PPAR-α signaling pathway plays a crucial role in DSS-induced colitis development. Conclusions: rSj16 has protective effects on DSS-induced colitis, effects mediated mainly by PPAR-α signaling pathway inhibition. The findings of this study suggest that rSj16 may be useful as a therapeutic agent and that PPAR-α may be a new therapeutic target in the treatment of IBD.

Keywords: PPAR-α.; inflammatory bowel disease; parasites; protective effects; rSj16.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
rSj16 treatment attenuates clinical activity in DSS-induced colitis mice. The following groups of mice were used in the study: Water+PBS (n=5), Water+rSj16 (n=5), DSS+PBS (n=5), DSS+rSj16 (n=5), DSS+Sj (n=5), DSS+GST (n=5), DSS+DXM (n=5), and each group was used for three independent experiments. The mean values ± SEMs are represented by bars. (A) The daily mean weight change in each group (*P <0.05, DSS+rSj16 versus DSS+PBS). (B) The changes in DAI, scored from diarrhea, bleeding and body weight loss (*P <0.05, DSS+rSj16 versus DSS+PBS). (C) Colonic MPO activity. (D) On day 6, the mice were sacrificed, their colons were removed, and the lengths of their colons were measured and recorded. (E) Macroscopic appearance of the colon, as represented by the colon with the mean colon length and typical injury findings. (F) Mean colon macroscopic scores in each group. (G) The histopathological changes in the colon tissue samples were examined by H&E staining (5×, 10×, 20×, 40×). (H) Histopathological scores were determined for the colon tissue samples. Statistical analysis was performed with one-way ANOVA followed by Dunnett's multiple comparison test versus DSS+PBS. *P <0.05, **P <0.01, ***P <0.001.
Figure 2
Figure 2
rSj16 regulates pro-inflammatory cytokine and immunoregulatory cytokine production in the colons of DSS-induced colitis mice. (A) TNF-α, IFN-γ, IL-17A, Chil3, TGF-β, and IL-10 protein expression in colon tissue was detected by immunohistochemistry. Positive immunoreactivity for TNF-α, IFN-γ, IL-17A, Chil3, TGF-β, and IL-10 protein expression is indicated by the red-brown color. The slides were counterstained with hematoxylin (blue). In addition, the sum of IOD was analyzed. (B) The relative mRNA expression levels of the inflammatory cytokines TNF-α, IFN-γ, IL-12, IL-17A, IL-22, Chil3, Chil1, IL-6, and IL-1β in colon tissue were determined by RT-PCR, and the housekeeping gene GAPDH was used as an internal reference. The bars indicate the fold changes in gene expression, which were determined by quantitative RT-PCR. The mean values ± SEMs are represented by bars. Statistical analysis was performed by one-way ANOVA followed by Dunnett's multiple comparison test versus the DSS+PBS. * P <0.05, ** P <0.01, *** P <0.001.
Figure 3
Figure 3
rSj16 expands the Treg population in experimental colitis in mice. Mice were sacrificed, and their spleens and MLNs were isolated. To detect Tregs, we stained the spleens (A) and MLNs (B) with anti-mouse CD4, CD25, and Foxp3 mAbs, as described in the Materials and Methods. Treg percentages were analyzed by FACS (left), and the results of the statistical analysis are shown (right). Data are shown as the means ± SEM (three independent experiments) of each group (n=5). Statistical analysis was performed by one-way ANOVA followed by Dunnett's multiple comparison test versus the DSS+PBS. * P <0.05, ** P <0.01, *** P <0.001
Figure 4
Figure 4
The expression profiles of the Water+PBS (n=3), DSS+PBS (n=3) and DSS+rSj16 (n=3) groups. (A) Venn diagram showing the genes that were unique to each group and shared among the three groups. The cluster number for each component is listed. (B) Unsupervised hierarchical clustering was performed on the gene expression profiles of the three groups (Water+PBS, DSS+PBS and DSS+rSj16). A heat map of the differentially expressed genes, as determined by the clustering analysis, is shown in the figure. Each column represents a specimen, and each row represents a gene. The red and blue colors indicate the genes that were up-regulated and down-regulated, respectively.
Figure 5
Figure 5
The crucial role of the PPAR-α signaling pathway in DSS-induced colitis mice treated with rSj16, as determined by sequencing. We screened the top 100 genes that were up-regulated in DSS+rSj16 group compared with the DSS+PBS group, as well as the genes that were down-regulated in the DSS+PBS group compared with the Water+PBS group. We also screened the top 100 genes that were down-regulated in the DSS+rSj16 group compared with the DSS+PBS group, as well as those that were up-regulated in the DSS+PBS group compared with the Water+PBS group. (A) The 200 genes that were most significantly differentially expressed were analyzed by KEGG enrichment analysis, which showed that the genes in question were enriched in the PPAR signaling pathway. (B) The most significantly differentially expressed genes (DSS+rSj16 versus DSS+PBS) were those associated with the PPAR-α pathway. The down-regulated genes are boxed in red, and the up-regulated genes are boxed in blue. (C) PPAR-α protein expression in colon tissue was detected by immunohistochemistry and the sum of IOD was analyzed. (D) Western blotting analysis of PPAR-α protein expression in colon tissue. (E) The differentially expressed genes (DSS+rSj16 versus DSS+PBS) associated with the PPAR-α signaling pathway were identified by quantitative RT-PCR, and the housekeeping gene GAPDH was used as a reference. The bars indicate the fold changes in gene expression, as determined by quantitative RT-PCR results. The mean values ± SEMs are represented by bars. Statistical analysis was performed by one-way ANOVA followed by Dunnett's multiple comparison test against the DSS+PBS. *P <0.05, ** P <0.01, *** P <0.001.
Figure 6
Figure 6
Activating PPAR-α diminished the therapeutic effects of rSj16 on DSS-induced colitis mice. The following groups of mice were used in the study: Water+PBS (n=5), DSS+PBS (n=5), DSS+rSj16 (n=5), DSS+PBS+PPAR-α(+) (n=5), DSS+rSj16+PPAR-α(+) (n=5), DSS+PBS+PPAR-α(-) (n=5), and DSS+rSj16+PPAR-α(-) (n=5). (A) Changes in body weight. (*P <0.05, DSS+rSj16 versus DSS+PBS; # P <0.05, DSS+rSj16+PPAR-α (+) versus DSS+PBS; &P <0.05, DSS+rSj16+PPAR-α (-) versus DSS+PBS) (B) DAI. (*P <0.05, DSS+rSj16 versus DSS+PBS; &P <0.05, DSS+rSj16+PPAR-α (-) versus DSS+PBS) (C) Colonic MPO activity. (D) Mean colon length. (E) Macroscopic appearance of the colon. (F) Mean colon macroscopic scores. (G) The histopathological changes in the colon tissue samples were examined by H&E staining. (H) Colon tissue histopathological scores. Mean values ± SEMs are represented by bars. Statistical analysis was performed by one-way ANOVA followed by Dunnett's multiple comparison test against the DSS+PBS. * P <0.05, ** P <0.01, *** P <0.001.

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