Fig 1: Transcriptome alterations in the sciatic nerve induced by myeloid-cell-specific TGR5 knockdown 7 days after pSNL. (A) PCA of the sciatic nerve in the shControl and shGPBAR1 groups at POD7. (B) Volcano plots of DEGs in the shGPBAR1 group compared with those in the shControl group, which included 77 upregulated and 5 downregulated genes, respectively. GO-enriched biological processes (C) and KEGG-enriched pathways (D) of 77 upregulated genes. (E) Heat map of DEGs involved in the production of cytokines. The gene expression of Ccl3 (F), Ccl4 (G), Tnfα (H), Il1b (I), and Nlrp3 (J) were validated through RT-qPCR. n = 6 per group. *P < 0.05; unpaired t test. Data are presented as the mean ± SEM. DEG, differentially expressed gene; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; PCA, principal component analysis; RT-qPCR, real-time quantitative PCR.
Fig 2: Effects of myeloid-cell-specific TGR5 knockdown on the expression of inflammatory mediators 7 days after pSNL. Protein levels of CCL3 (A), CCL2 (B), TNF-α (C), and IL-6 (D) in the sciatic nerve at POD7 were measured using cytometric bead array analysis. n = 6 to 8 per group. *P < 0.05; unpaired t test. Representative Western blotting bands (E) and densitometric quantification of NLRP3 (F), ASC (G), pro-caspase-1 (H), caspase-1 p20 (I), and IL-1β p17 (J) in the sciatic nerve of Lyz2-Cre mice treated with shControl virus or shGPBAR1 virus. n = 4 per group. *P < 0.05, **P < 0.01, ***P < 0.001; unpaired t test. Data are presented as the mean ± SEM. (K) Representative microphotographs showing immunofluorescence staining of F4/80 (green), NLRP3 (red), and ASC (cyan) in the sciatic nerve after pSNL. Nuclei were stained with DAPI (blue). Scale bar: 100 μm. (L) Representative microphotographs showing immunofluorescence staining of F4/80 (green) and IL-1β (red) in the sciatic nerve after pSNL. Nuclei were stained with DAPI (blue). Scale bar: 100 μm. DAPI, 4′,6-diamidino-2-phenylindole; POD, postoperative day; pSNL, partial sciatic nerve ligation.
Fig 3: Effects of INT-777 administration on alterations in inflammatory mediators and macrophages induced by pSNL at POD7. Protein levels of CCL3 (F (2, 21) = 18.27) (A), CXCL9 (F (2, 21) = 4.893) (B), TNF-α (F (2, 21) = 8.638) (C), and IL-6 (F (2, 21) = 21.10) (D) in the sciatic nerve at POD7 were measured using cytometric bead array analysis. n = 4 to 5 per group. The displayed data originated from 2 independent repeated tests. *P < 0.05, **P < 0.01, ****P < 0.0001; 1-way ANOVA, Tukey post hoc test. (A) (E) Representative flow cytometry pseudocolor dot plots of myeloid cell populations in the sciatic nerve at POD7 in mice treated with DMSO or INT-777. Percentages of CD11b+ cells (F), F4/80+ cells (G), and CD11b+F4/80+ cells (H) among single cells, defined by FS-A and FS-W. Percentages of CD86+CD206− cells (I) and CD86-CD206+ cells (J) among CD11b+F4/80+ cells. (K) The ratio of CD86+CD206− macrophages to CD86-CD206+ macrophages. n = 5 per group. *P < 0.05; unpaired t test. Data are presented as the mean ± SEM. ANOVA, analysis of variance; DMSO, dimethylsulfoxide; POD, postoperative day; pSNL, partial sciatic nerve ligation.
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