Fig 1: 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 2: 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 3: 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 4: Senomorphic therapy with Rux exerts an anti-SASP effect and attenuates LPS-induced cellular senescence in NRCMs. (A) Schematic diagram of in vitro experiments in NRCMs. (B) The mRNA levels of SASP-related genes were detected using qRT-PCR in NRCMs treated with Rux or not post LPS-stimulation, including IL-1β, IL-6, TNF-α, CXCL1, CXCL3, CXCL10, CCL2, CCL5 and GDF15. N = 4. (C,D) Representative Western blot bands and quantitative analysis of P16 and P21 in NRCMs treated with Rux or not post LPS stimulation compared with the control group. N = 4. (E) The mRNA levels of P16, P21, and P53 were detected using qRT-PCR in NRCMs treated with Rux or not post LPS stimulation compared with the control group. N = 4. (F-I) Representative fluorescence images and quantitative analysis of P16 and P21 with DAPI in NRCMs treated with Rux or not post LPS stimulation. Scale bar = 20 μm, N = 3. (J,K) Representative images and quantitative analysis of SA-β-gal staining in NRCMs treated with Rux or not post LPS stimulation. Scale bar = 20 μm, N = 3. (L,M) Representative Western blot bands and quantitative analysis of γ-H2AX in NRCMs treated with Rux or not post LPS stimulation. N = 4. Data are presented as mean ± SD. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001. Rux, ruxolitinib; Con, control; LPS, Lipopolysaccharide; SASP, senescence-associated secretory phenotype; NRCMs, neonatal rat cardiomyocytes.
Fig 5: Inhibition of the JAK2-STAT3 pathway is critical for the protective effects of Rux in alleviating cellular senescence and SASP in SCM. (A) The mRNA levels of SASP-related genes were detected using qRT-PCR after reactivating the JAK2-STAT3 pathway using r-IL6 in NRCMs, including IL-1β, IL-6, TNF-α, CXCL3, CXCL10, CCL2 and GDF15. N = 4. (B,C) The mRNA levels of P16 and P21 were detected using qRT-PCR after reactivating the JAK2-STAT3 pathway using r-IL6 in NRCMs. N = 4. (D,E) Representative Western blot bands and quantitative analysis of P16 and P21 after reactivating the JAK2-STAT3 pathway using r-IL6 in NRCMs. N = 4. (F,G) Representative fluorescence images and quantitative analysis of γ-H2AX after reactivating the JAK2-STAT3 pathway using r-IL6 in NRCMs. N = 3. (H,I) Representative images and quantitative analysis of SA-β-gal staining after reactivating the JAK2-STAT3 pathway using r-IL6 in NRCMs. Scale bar = 20 μm, N = 3. Data are presented as mean ± SD. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001. LPS, Lipopolysaccharide; Rux, ruxolitinib; Con, control; r-IL6, recombinant IL-6; SA-ß-gal, senescence-associated β-galactosidase; NRCMs, neonatal rat cardiomyocytes.
Supplier Page from MedChemExpress for IL-6 Protein, Rat (HEK293)