Inhibition of mir-155-5p alleviates cardiomyocyte pyroptosis induced by hypoxia/reoxygenation via targeting SIRT1-mediated activation of the NLRP3 inflammasome

Fig. 1. H/R induced NLRP3-mediated pyroptosis in H9c2 cells. The H9c2 cells in the H/R group were subjected to hypoxia for 6 h and reoxygenation for 4 h, while normal cultured H9c2 cells were designated as the Control group. (A) The proliferative activity of H9c2 cells was assessed using the CCK-8 assay. (B) The level of LDH released by H9c2 cells was quantified using colorimetric analysis. (C) The death of H9c2 cells was observed by Calcein-AM/PI staining. The live cells were stained green by Calcein-AM, whereas the dead cells were stained red by PI. Scale bar = 50 μm. The levels of IL-1β (D) and IL-18 (E) in the supernatant of H9c2 cell were detected by ELISA. (F) The protein expression levels of NLRP3, GSDMD-N, and C-caspase-1 in H9c2 cells were assessed using Western blot. n = 3. **P < 0.01

Fig. 2. Inhibition of miR-155-5p attenuates NLRP3 inflammasome mediated pyroptosis in H/R-exposed H9c2 cells. (A) The expression level of miR-155-5p in H9c2 cells was quantified using qRT-PCR. The miR-155-5p inhibitor and the inhibitor-NC were separately transfected into H9c2 cells, followed by exposure to H/R. (B) The expression of miR-155-5p in H9c2 cells was assessed by qRT-PCR following transfection with a miR-155-5p inhibitor and inhibitor-NC. (C) CCK-8 assay was used to detect the proliferative activity of H9c2 cells. (D) Colorimetric analysis was used to quantify LDH activity in the supernatant of H9c2 cells. (E) Calcein-AM /PI staining was used to observe the death of H9c2 cells. The viable cells were stained green by Calcein-AM, whereas the dead cells were stained red by PI. Scale bar = 50 μm. (F-G) ELISA was used to determine the levels of IL-1β and IL-18 in the supernatant of H9c2 cells. (H) Western blot was used to assess the protein expression levels of NLRP3, GSDMD-N, and C-caspase-1 in H9c2 cells. n = 3. *P < 0.05, **P < 0.01

Fig. 3. SIRT1 overexpression inhibits the activation of NLRP3 inflammasome in H/R-induced H9c2 cells. (A) The mRNA expression level of SIRT1 in H9c2 cells was detected by qRT-PCR. (B) The protein expression level of SIRT1 in H9c2 cells was assessed by Western blot. The SIRT1 overexpression plasmid (oe-SIRT1) and empty carrier plasmid (Vector) were separately transfected into H9c2 cells, followed by exposure to H/R. The expression levels of SIRT1 mRNA (C) and protein (D) in H9c2 cells were quantified using qRT-PCR and Western blot. (E) The proliferative activity of H9c2 cells was assessed using the CCK-8 assay. (F) The LDH activity in the supernatant of H9c2 cells was quantified using colorimetric analysis. (G) The death of H9c2 cells was observed by Calcein-AM /PI staining. The viable cells were stained green by Calcein-AM, whereas the dead cells were stained red by PI. Scale bar = 50 μm. (H-I) The levels of IL-1β and IL-18 in the supernatant of H9c2 cells were detected by ELISA. (J) The protein expression levels of NLRP3, GSDMD-N, and C-caspase-1 in H9c2 cells were assessed using Western blot. n = 3. *P < 0.05, **P < 0.01

Fig. 4. miR-155-5p directly targets SIRT1 in H9c2 cells. (A) The binding sites between miR-155-5p and SIRT1 were predicted. (B) The relative fluorescence activity of SIRT1-WT and SIRT1-MUT in H9c2 cells was assessed using a dual luciferase assay. The H9c2 cells were transfected with the miR-155-5p inhibitor and the inhibitor-NC, respectively. (C) The expression level of SIRT1 mRNA in H9c2 cells was determined by qRT-PCR. (D) The protein expression level of SIRT1 in H9ce cells was detected by Western blot. n = 3. **P < 0.01

Fig. 5. SIRT1 silencing partially reversed the inhibition of H/R-induced H9c2 cell pyroptosis by miR-155-5p inhibitor. The miR-155-5p inhibitor and SIRT1 siRNA interference plasmid (si-SIRT1) or negative control plasmid (si-NC) were transfected into H9c2 cells, followed by exposure to H/R. (A) Western blot was used to detect the protein expression level of SIRT1 in H9c2 cells. (B) CCK-8 assay was used to assess the proliferative activity of H9c2 cells. (C) Colorimetric analysis was used to quantify LDH activity in the supernatant of H9c2 cells. (D) Calcein-AM /PI staining was used to observe the death of H9c2 cells. The viable cells were stained green by Calcein-AM, whereas the dead cells were stained red by PI. Scale bar = 50 μm. (E-F) ELISA was used to determine the levels of IL-1β and IL-18 in the supernatant of H9c2 cells. (G) Western blot was used to assess the protein expression levels of NLRP3, GSDMD-N, and C-caspase-1 in H9c2 cells. n = 3. *P < 0.05, **P < 0.01