Fig 1: RNA-seq and bioinformatics analysis reveal the potential effects of Rux treatment on mouse hearts. (A) PCA identified the mice as two groups, namely the SCM group and the Rux group. N = 4 in each group. (B) DEGs between the SCM group and the Rux treatment group were shown in the volcano map. (C) KEGG analysis identified changes in the cardiac pathways of mice with SCM following Rux therapy. (D) The heat map indicated that senescence markers, specifically P53 and P21, were downregulated in mice hearts with SCM after Rux treatment. N = 4 in each group. (E) The relative levels of SASP-related genes were displayed in the heatmap between the SCM and the Rux groups, including CXCL1, CXCL5, MMP3, IL-1α, TGFβ2, CXCL14, IL-6, CXCL10, GDL15, and IL-1β. N = 4 in each group. (F) GSEA analysis demonstrated that the JAK-STAT pathway was up-regulated in SCM-induced mouse hearts and down-regulated after treatment with Rux. (G) GO analysis identified changes in the cardiac biological processes of SCM mice following Rux therapy. (H) The heatmap demonstrated that the relative levels of MRC complex-related genes were upregulated post Rux treatment in SCM-induced mice hearts. N = 4 in each group. (I) The heatmap demonstrated that the relative levels of oxidative stress-related genes were up-regulated post Rux treatment in SCM-induced mice hearts. N = 4 in each group. Data are presented as mean ± SD. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001. KEGG, Kyoto Encyclopedia of Genes and Genomes; GSEA, Gene Set Enrichment Analysis; MRC, mitochondrial respiratory chain; SASP, senescence-associated secretory phenotype; GO, Gene Ontology; Rux, ruxolitinib; SCM, septic cardiomyopathy; DEGs, differentially expressed genes.
Fig 2: Senomorphic therapy with Rux exerts an anti-SASP effect and attenuates cellular senescence in mouse models with SCM. (A) The mRNA levels of SASP-related genes were detected using qRT-PCR in mouse hearts treated with 30 mg/kg and 75 mg/kg of Rux or saline post LPS-injection compared with the Sham group, including CXCL1, CXCL3, CCL5, TGFβ2 and GDF15. N = 4. (B,C) Representative Western blot bands and quantitative analysis of P16, P21, and P53 in mouse hearts treated with 30 mg/kg and 75 mg/kg of Rux or saline post LPS injection compared with the Sham group. N = 4. (D) The mRNA levels of P16, P21, and P53 were detected using qRT-PCR in mouse hearts treated with 30 mg/kg and 75 mg/kg of Rux or saline post LPS injection compared with the Sham group. N = 4. (E,F) Representative fluorescence images and quantitative analysis of P21 with cTnI and DAPI in mouse hearts treated with 30 mg/kg and 75 mg/kg of Rux or saline post LPS injection compared with the Sham group. Scale bar = 20 μm, N = 3. (G,H) Representative Western blot bands and quantitative analysis of γ-H2AX in mouse hearts treated with 30 mg/kg and 75 mg/kg of Rux or saline post LPS injection compared with the Sham group. N = 4. (I) The mRNA levels of SASP-related genes were detected using qRT-PCR in mouse hearts treated with 30 mg/kg and 75 mg/kg Rux or saline post CLP-surgery compared with the Sham group, including IL-1β, IL-6, TNF-α, CXCL1, CXCL3, CXCL10, CCL2, TGFβ2, and GDF15. N = 4. (J,K) Representative Western blot bands and quantitative analysis of P16 and P21 in mouse hearts treated with 30 mg/kg and 75 mg/kg of Rux or saline post CLP-surgery compared with the Sham group. N = 4. (L) The mRNA levels of P16 and P21 were detected using qRT-PCR in mouse hearts treated with 30 mg/kg and 75 mg/kg of Rux or saline post CLP-surgery compared with the Sham group. N = 4. (M-O) Representative fluorescence images and quantitative analysis of P21 and γ-H2AX with cTnI and DAPI in mouse hearts treated with 30 mg/kg and 75 mg/kg of Rux or saline post CLP-surgery compared with the Sham group. Scale bar = 20 μm, N = 3. (P) Representative images of SA-β-gal staining in mouse hearts treated with 30 mg/kg and 75 mg/kg of Rux or saline post CLP-surgery compared with the Sham group. Scale bar = 20 μm. N=3. Data are presented as mean ± SD. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001. SA-ß-gal, senescence-associated β-galactosidase; LPS, Lipopolysaccharide; CLP, cecal ligation and puncture; SASP, senescence-associated secretory phenotype; Rux, ruxolitinib.
Fig 3: Low-dose Rux and ABT263 demonstrated comparable efficacy in ameliorating SCM. (A,B) Cardiac function indices were measured by echocardiography in mice treated with Rux or ABT263 post LPS injection compared with the Sham group. N = 5. (C,D) Representative Western blot bands and quantitative analysis of P16 and P21 in LPS-induced NRCMs treated with ABT263 or Rux. N = 4. (E) The mRNA levels of P16 and P21 were detected using qRT-PCR in NRCMs treated with ABT263 or Rux. N = 5. (F) Representative fluorescence images and quantitative analysis of P16 with cTnI and DAPI in mouse hearts treated with Rux or ABT263 post LPS injection compared with the Sham group. Scale bar = 20 μm, N = 3. (G) The mRNA levels of SASP-related genes were detected using qRT-PCR in mouse hearts treated with Rux or ABT263 post LPS injection compared with the Sham group, including CXCL1, CXCL3, CXCL10, CCL2, IL-6, and GDF15. N = 5. (H) Representative images of SA-β-gal staining in mouse hearts treated with Rux or ABT263 post LPS injection compared with the Sham group. Scale bar = 50 μm. Data are presented as mean ± SD. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001. Lipopolysaccharide; Rux, ruxolitinib; SA-ß-gal, senescence-associated β-galactosidase
Fig 4: Cellular senescence is induced in SCM: evidence from human, animal, and cell models. (A,B) Representative Western blot bands and quantitative analysis of P16, P21, P53, and γ-H2AX in NRCMs between the control and LPS-stimulation groups. N = 4. (C) The mRNA levels of P16, P21 and P53 in NRCMs between the control and LPS-stimulation groups were detected using qRT-PCR. N = 4. (D,E) Representative fluorescence images and quantitative analysis of P16 with DAPI in NRCMs between the control and LPS-stimulation groups. Scale bar = 20 μm, N = 3. (F,G) Representative images and quantitative analysis of SA-β-gal staining in NRCMs. Scale bar = 20 μm, N = 3. (H) The mRNA levels of SASP-related genes were detected using qRT-PCR post LPS-stimulation in NRCMs compared to the control group, including IL-1β, IL-6, TNF-α, CXCL1, CXCL3, CXCL10, CCL2, CCL5 and GDF15. N = 3 - 4. (I,J) Representative Western blot bands and quantitative analysis of P16, P21, P53 and γ-H2AX in LPS-induced mice models compared with the Sham group. N = 6. (K) The mRNA levels of P16, P21, P53 in LPS-induced mice models compared with the Sham group were detected using qRT-PCR. N = 6. (L,M) Representative fluorescence images and quantitative analysis of P16 with cTnI and DAPI in LPS-induced mice models compared with the Sham group. Scale bar = 100 μm, N=3. (N) The mRNA levels of SASP-related genes were detected using qRT-PCR in LPS-induced mice models compared with the Sham group, including IL-1β, IL-6, TNF-α, CXCL1, CXCL3, CCL2, CCL5, GDF15 and EDN3. N = 6. (O,P) Representative images and quantitative analysis of SA-β-gal staining in LPS-induced mice models compared with the Sham group. Scale bar = 20 μm, N = 3. (Q,R) Representative Western blot bands and quantitative analysis of P16, P21, P53, and γ-H2AX in CLP-induced mice models compared with the Sham group. N = 6. (S) The mRNA levels of P16, P21, P53 in CLP-induced mouse models compared with the Sham group were detected using qRT-PCR. N = 4. (T) The mRNA levels of SASP-related genes were detected using qRT-PCR in CLP-induced mouse models compared with the Sham group, including IL-1β, IL-6, TNF-α, CXCL1, CXCL3, CCL2, CCL5, GDF15, and TGFβ2. N = 4. (U,V) Representative fluorescence images and quantitative analysis of P21 with cTnI and DAPI in CLP-induced mice models compared with the Sham group. Scale bar = 20 μm, N = 3. Data are presented as mean ± SD. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001. SA-ß-gal, senescence-associated β-galactosidase; LPS, Lipopolysaccharide; CLP, cecal ligation and puncture; NRCMs, neonatal rat cardiomyocytes; SASP, senescence-associated secretory phenotype.
Fig 5: HA-induced inflammation stimulated protein O-GlcNAcylation in hearts. A, Immunoblot analysis of IL-1β and IL-6 in the Control and HA hearts (n = 9 per group). B, Immunoblot analysis of IL-1β and IL-6 in the H9c2 cells (IL-1β: n = 7 per group; IL-6: n = 8 per group). C-D, The linear regression analysis between the expression of IL-1β (C) or IL-6 (D) and protein O-GlcNAcylation level. E-F, Representative blots of protein O-GlcNAc modification in H9c2 cells treated with increasing concentration of IL-1β (E) or IL-6 (F). G, The glucose uptake rate measure by 2-NBDG in H9c2 cells. H, Immunoblot analysis of protein O-GlcNAc modification in H9c2 cells after HA and neutralizing antibody treatments (n = 4 per group). I, Immunoblot analysis of protein O-GlcNAc modification in H9c2 cells with indicated treatments (n = 7 per group). Data are shown as mean ± SEM. *P < 0.05, **P < 0.01 for indicated comparisons.
Supplier Page from MedChemExpress for IL-6 Protein, Rat (HEK293)