Fu Fang Zhen Zhu Tiao Zhi Capsules Protect against Myocardial Ischemia by Inhibiting Cardiomyocyte Pyroptosis

Figures

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  Figure 1. FTZ improves dysregulation in the cardiac function and structure in mice with MI. (a–c) Echocardiograms and EF%, FS%, LVIDd, and LVIDs in the sham, MI, and MI + FTZ groups. ∗p < 0.05, ∗∗p < 0.01 vs. sham; #p < 0.05 and ##p < 0.01 vs. MI; n = 8 for EF%, FS%, LVIDd, and LVIDs. (d–e) Quantitative data analysis of TTC-stainedand H&E staining. (f) Representative TTC-stained transverse sections; n = 3. (g) H&E staining of the heart tissue after FTZ treatment. Black arrows indicate positions of inflammation infiltrate. Scale bar indicates 100 μm; n = 3.

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  Figure 2. FTZ inhibits pyroptosis in ischemic hearts. (a and b) Double-fl;uorescence staining with DAPI (blue) and TUNEL (green) and quantitative TUNEL data. Scale bar indicates 100 μm (right);n = 3. (c and d) Representative western blots (3 independent experiments) (left) and quantiflcation of western blots. β-actin was used as an internal control. (e) Quantitative RT-PCR for NLRP3, GSDMD, ASC, C-Caspase-1, 1L-1β, and 1L-18. (f and g) Serum IL-18 and IL-1β levels were measured using ELISA. ∗p < 0.05, ∗∗p < 0.01 vs. sham; #p < 0.05, ##p < 0.01 vs. MI. (h) NLRP3 representative IHC images. Black arrows indicate the positions of NLRP3. Scale bar indicates 100 μm; n = 3.

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  Figure 3. FTZ inhibits pyroptosis in vitro. (a and b) Double-fl;uorescence staining with DAPI (blue) and TUNEL (green) and quantitative TUNEL data. Scale bar indicates 100 μm (right); n = 3. (c and d) Representative western blots (3 independent experiments) (left) and quantiflcation of western blots in NMCMs. β-actin was used as an internal control. (e) Quantitative RT-PCR for NLRP3, GSDMD, ASC, C-Caspase-1, and GSDMD-N. (f) The degree of cell insult was assessed using % LDH release. ∗p < 0.05 and ∗∗p < 0.01 vs. CTRL;#p < 0.05 and ##p < 0.01 vs. H2O2. (g) NLRP3 representative immunofluorescence images. Scale bar indicates 100 μm; n = 3.

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  Figure 4. NLRP3 is a target of FTZ. (a and b) Double-fluorescence staining with DAPI (blue) and TUNEL (green) and quantitative TUNEL data. Scale bar indicates 100 μm (right); n = 3. (c and d) Representative western blots (3 independent experiments) (left) and quantiflcation of western blots in NMCMs. β-actin was used as an internal control. (e) Quantitative RT-PCR for NLRP3, GSDMD, ASC, C-Caspase-1, and GSDMD-N. (f) Degree of cell insult was assessed using % LDH release. ∗p < 0.05 and ∗∗p < 0.01 vs. NC; #p < 0.05 and ##p < 0.01 vs. H2O2 + NC; and p < 0.05 and p < 0.01 vs. H2O2 + FTZ + NC.