Fig 2: Bmi-1 inhibitor PTC209 attenuates the protective effects of TLR4 KO against senescence and activation of NLRP3 pathway in mouse gingival fibro-blasts after LPS stimulation. (A) Western blot analysis of the effect of PTC209 on the expression of Bmi-1, NLRP3, ASC and Caspase-1. (B) Relative protein levels of Bmi-1, NLRP3, ASC and Caspase-1 normalized to GAPDH. (C) Activity of ß-gal determined via flow cytometry. (D) Western blot analysis of p16, p19, p21 and p53 protein levels following treatment with PTC209. (E) Western blot analysis of IL-1ß, IL-6 and TNF-a protein levels following treatment with PTC209. (F) Histograms showing relative ß-gal levels. (G) Relative protein levels of p16, p19, p21 and p53 normalized to GAPDH. (H) Relative protein levels of IL-1ß, IL-6 and TNF-a normalized to GAPDH. Data are presented as the mean ± SD (n=3/group). #P<0.05, ##P<0.01, ###P<0.001 vs. control; *P<0.05, **P<0.01, ***P<0.001 vs. WT; @P<0.05, @@P<0.01, @@@P<0.001 vs. TLR4 KO. TLR4, Toll-like receptor 4; WT, wild-type; KO, knockout; Bmi-1, B cell-specific Moloney murine leukemia virus integration site 1; NLRP3, nucleotide-binding and oligomerization domain-like receptor 3; ASC, apoptosis-associated speck-like protein containing a CARD domain; ß-gal, ß-galactosidase; IL, interleukin; TNF, tumor necrosis factor; FDG, fluorodeoxyglucose.

Fig 3: P16 deletion improved TNF-a–dependent epithelial barrier destruction in Bmi-1–/– mice. The experiments were carried out on the 7-week-old Bmi-1–/–, Bmi-1–/–p16–/–, and WT mice. (A) Representative micrographs showing immunofluorescence for TNF-a and F4/80, with DAPI staining the nuclei. (B) Percentage of TNF-a–positive cells. (C) Percentage of TNF-a and F4/80 double-positive cells. (D) Serous TNF-a protein level (pg/mL) was detected with ELISA assay. (E) TNF-a protein levels in jejunum, ileum, and colon (pg/mL) were detected with ELISA assay. (F) Representative micrographs showing immunofluorescence for NF-?B–p65 and F4/80, with DAPI staining the nuclei. (G,H) Percentage of NF-?B–p65 and F4/80 double-positive cells or areas in villus and crypt. Six mice per group were used for experiments. Statistical analysis was performed with one-way ANOVA test. Values are mean ± SEM from six determinations per group, *p < 0.05, **p < 0.01, ***p < 0.001 compared with the WT group; #p < 0.05, ##p < 0.01, ###p < 0.001 compared with the Bmi-1–/– group.

Fig 4: P16 deletion improved intestinal microbial dysbiosis in Bmi-1–/– mice. High-throughput sequencing of V4 region amplicon of 16S rRNA from intestinal microbiota was conducted using the fecal of 7-week-old WT, Bmi-1–/– (KO), and Bmi-1–/–p16–/– (DKO) mice. (A) Principal components at the phylum level in the intestinal microbiota of WT, Bmi-1–/–, and Bmi-1–/–p16–/– mice. Relative contribution of the top 10 phylum of each sample was shown; “others” represents the sum of abundance except the top 10 phylum, n = 8. (B) Proportion of intestinal microbiota in 7-week-old WT, KO, and DKO mice at the phylum level. Circle represents a taxonomy at the level of phylum; the size of the circle represents the abundance of the microbiota at the phylum level. (C) Abundance display curve of intestinal microbiota in WT, KO, and DKO mice. Abscissa represents sequencing depth; ordinate represents the number of species found. (D) Box plot showing abundance dilution. The end of the curve is close to gentle, indicating that the sample size of sequencing meets the requirements of diversity. (E) The observed species number and a-diversity index of intestinal bacteria were examined. Significant differences are indicated: Wilcoxon rank sum test, n = 8 per group, *p < 0.05, compared with the WT group. (F) Venn diagrams demonstrate the number of species shared among WT, KO, and DKO groups. (G) Heat map showing distribution of intestinal microbiota at genus level from 7-week-old WT, Bmi-1–/–, and Bmi-1–/–p16–/– mice; the phylum of each genus is listed in the note, n = 8. The heat map is colored based on row Z scores. The mice with the highest and lowest bacterial levels are in red and blue, respectively.

Fig 5: A schematic diagram illustrating the proposed mechanism. Orange arrows indicate the endogenous status; diabetic EPCs have an impaired Shh/Bmi1/p53 axis, which induces EPC apoptosis and dysfunction, results in decreased capillary system, increased myocardial fibrosis and worse cardiac function in diabetic myocardial infarction. Green arrows indicate the potential therapeutic role of the Shh protein, which reverses the impaired Shh/Bmi1/p53 in diabetic EPCs and eventually ameliorates the status of diabetic myocardial infarction. EPCs, endothelial progenitor cells; Shh, sonic hedgehog; Bmi1, BMI1 proto-oncogene.