Inhibition of P2X7 Purinergic Receptor Ameliorates Cardiac Fibrosis by Suppressing NLRP3/IL-1β Pathway

Figures

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  Figure 1. P2X7R was upregulated in the mice heart after TAC surgery. (a) Cardiac images and HW/BW (n = 8). (b) HE-, Sirius Red-, and Masson's trichrome-stained sections of the hearts following four weeks of TAC treatment (n = 6). (c) Representative images of an echocardiographic assessment of mice after TAC (4 weeks). Cardiac function indicators measured by echocardiography (LVEF (%), FS (%), LVPWd, IVSd, and LVIDd) in TAC- and sham-operated mice. (d) Expression of CTGF, periostin, and α-SMA mRNAs and proteins in heart cells (n = 6). (e) Gene expression of P2X receptors in TAC- and sham-operated mice (n = 6) at 4 weeks and gene expression of P2X7R in heart cells at 3 days, 1 week, 2 weeks, and 4 weeks after TAC- or sham-operated treatment (n = 6). (f) Expression of P2X7R protein in mouse heart cells (n = 6). Results are presented as means ± standard error of the mean. ∗ indicates P < 0.05, ∗∗ indicates P < 0.01, and ∗∗∗ indicates P < 0.001 versus sham.

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  Figure 2. TGF-β1-treatment upregulated P2X7R in CFs. (a) Immunofluorescence images showing α-SMA expression in CFs (α-SMA (green) and nuclei DAPI (blue), n = 300). (b) Rate of cell proliferation (EdU-positive cells (red) and nuclei DAPI (blue), n = 120). (c) Expression of TGF-β, α-SMA, CTGF, and periostin mRNAs (n = 3). (d) Expression of CTGF, α-SMA, periostin, and TGF-β proteins (n = 3). (e) Western blot results showing P2X7R expression in CFs (n = 3). (f) Immunofluorescence images showing P2RX7 expression in CFs (P2X7R (red) and nuclei DAPI (blue), n = 60). Results are presented as means ± standard error of the mean; ∗ indicates P < 0.05, ∗∗ indicates P < 0.01, and ∗∗∗ indicates P < 0.001 versus control.

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  Figure 3. Knockdown of P2X7R alleviates the effects of TGF-β1 on CF activation. (a) Expression of P2X7R mRNA after siP2X7R transfection (n = 6). (b) Expression of collagen I, CTGF, α-SMA, and TGF-β mRNAs after transfection of CFs with siP2X7R (n = 6). (c, d) Expression of periostin, α-SMA, CTGF, P2X7R, NLRP3, IL-1β, and caspase-1 proteins after transfection of CFs with siP2X7R (n = 6). (e) Expression of α-SMA in CFs by immunofluorescence staining (α-SMA (green) and nuclei DAPI (blue), n = 300). (f) Changes in CF proliferation (EdU-positive cells (red) and nuclei DAPI (blue), n = 120). (g) Expression of collagen I, CTGF, α-SMA, and TGF-β mRNAs after treatment of CFs with BBG (n = 6). (h) Expression of P2X7R protein after BBG treatment (n = 6). (i) Expression of periostin, α-SMA, CTGF, NLRP3, IL-1β, and caspase-1 proteins after BBG treatment (n = 6). (j) Expression of periostin, α-SMA, TGF-β and CTGF proteins in activated CFs after BzATP treatment (n = 6). Results are presented as means ± standard error of the mean; ∗ indicates P < 0.05, ∗∗ indicates P < 0.01, and ∗∗∗ indicates P < 0.001 versus control or siNC; # indicates P < 0.05, ## indicates P < 0.01, and ### indicates P < 0.001.

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  Figure 4. Inhibition of P2X7Rs suppresses cardiac fibrosis induced by pressure overload. (a–d) Effects of BBG on TAC-triggered fibrosis (a, b), histological (c, d), echocardiographic analyses (e). (a) Schematic diagram outlining the in vivo experiments. Mice were treated with BBG every two days at three days after TAC. (b) HW/BW and cardiac imaging results (n = 6). (c) Masson's trichrome staining (n = 6). (d) Periostin protein levels in ventricular tissues (periostin (brown) and nucleus (blue). (e) Representative echocardiographic images of mice after TAC (4 weeks); the cardiac function indicators evaluated by echocardiography (LVEF (%), FS (%), LVPWd, IVSd, and LVIDd) in TAC- and sham-operated mice. (f) Expression of periostin, α-SMA, CTGF, NLRP3, IL-1β, and caspase-1 proteins (n = 6). Results are presented as means ± standard error of the mean; ∗ indicates P < 0.05, ∗∗ indicates P < 0.01, and ∗∗∗ indicates P < 0.001 versus sham+saline. # indicates P < 0.05, ## indicates P < 0.01, and ### indicates P < 0.001.

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  Figure 5. A schematic illustration of the mechanism of P2X7R in pressure overload-induced cardiac fibrosis and TGF-β1-induced CF activation. In TAC-induced injury, pathological stress, such as pressure overload and TGF-β1, triggers the release of ATP. Elevated ATP activates P2X7 receptors, contributing to P2X7-mediated NLRP3 inflammasome (NLRP3, ASC, and caspase-1) activation. This induces the release of IL-1β, causing severe inflammation that precipitates cardiac fibrosis and aggravates TGF-β1-induced CF activation. Moreover, inhibition of P2X7R with BBG inhibitor in TAC mice or CFs primed with TGF-β1 reduces fibrotic extracellular matrix (ECM) gene expression and cardiac fibrosis.