P2Y1 Receptor Agonist Attenuates Cardiac Fibroblasts Activation Triggered by TGF-β1

FIGURE 1. Expression pattern of P2Y receptor subtypes in TAC-induced cardiac fibrosis mice. (A) Representative image of an echocardiographic detection of mice left ventricle after TAC operation (4 weeks). (B) Representative image of HE-, Sirius Red-, and Masson’s trichrome-stained sections of mice after TAC operation. (C) Expression of mRNA of fibrosis markers (CTGF, POSTN, and α-SMA) in heart tissues of mice after TAC operation (n = 6). (D) Expression of protein of fibrosis markers (CTGF, POSTN and α-SMA) in heart tissues of mice after TAC operation (n = 6). (E) Gene expression of P2Y receptors subtypes in heart tissues of mice after TAC operation (n = 6). (F) Protein expression of P2Y1R in heart tissues of mice after TAC operation (n = 6). Results are presented as means ± standard deviation. ∗ indicates p < 0.05, ∗∗ indicates p < 0.01, and ∗∗∗ indicates p < 0.001.

FIGURE 2. Expression pattern of P2Y receptor family in TGF-β1 induced activation of CFs. (A) Expression of CTGF, POSTN and α-SMA mRNAs in TGF-β1-induced activation CFs (n = 6). (B) Expression of CTGF, POSTN and α-SMA proteins in TGF-β1-induced activation CFs (n = 6). (C) Representative images of immunofluorescence staining of α-SMA (green) in CFs and nuclei DAPI (blue) (n = 6). (D) EdU staining images showing rate of cell proliferation. Proliferation cells stained with EdU (red) and nuclei stained with DAPI (blue) (n = 6). (E) Gene expression of P2Y receptors subtypes in TGF-β1-induced activation CFs (n = 6). Results are presented as means ± standard deviation. ∗ indicates p < 0.05, ∗∗ indicates p < 0.01, and ∗∗∗ indicates p < 0.001.

FIGURE 3. Activation of P2Y1R alleviates TGF- β1-stimulated CFs activation. (A) Expression of CTGF, POSTN and α-SMA mRNAs after CFs treated with TGF-β1 and MRS2365 (n = 6). (B) Protein levels of COL-1, POSTN, CTGF, and TGF-β after CFs treated with TGF-β1 and MRS2365 (n = 6). (C) Representative images of immunofluorescence staining of α-SMA (green) in CFs and nuclei DAPI (blue) (n = 6). (D) EdU staining images showing rate of cell proliferation. Proliferation cells stained with EdU (red) and nuclei stained with DAPI (blue) (n = 6). (E) Protein levels of p-P38 MAPK and p-ERK1/2 after CFs treated with TGF-β1 and MRS2365 (n = 6). Results are presented as means ± standard deviation. ∗ indicates p < 0.05, ∗∗ indicates p < 0.01, and ∗∗∗ indicates p < 0.001.

FIGURE 4. Inhibition of P2Y1R accelerates TGF- β1-induced CFs activation. (A) Expression of P2Y1R mRNA after si-RNA transfection (n = 3). (B) Expression of CTGF, POSTN and α-SMA mRNAs after CFs transfected with siRNA and treated with TGF-β1 (n = 6). (C) Protein levels of COL-1, POSTN, CTGF, TGF-β after CFs transfected with siRNA and treated with TGF-β1 (n = 6). (D) Representative images of immunofluorescence staining of α-SMA (green) in CFs and nuclei DAPI (blue) (n = 6). (E) EdU staining images showing rate of cell proliferation. Proliferation cells stained with EdU (red) and nuclei stained with DAPI (blue) (n = 6). (F) Protein levels of p-P38 MAPK and p-ERK1/2 after CFs transfected with siRNA and treated with TGF-β1 (n = 6). (G) Expression of CTGF, POSTN and α-SMA mRNAs after CFs treated with TGF-β1 and BPTU (n = 6). (H) Protein levels of COL-1, POSTN, CTGF, TGF-β after CFs treated with TGF-β1 and BPTU (n = 6). I Protein levels of p-P38 MAPK and p-ERK1/2 after CFs treated with TGF-β1 and BPTU (n = 6). Results are presented as means ± standard deviation. ∗ indicates p < 0.05, ∗∗ indicates p < 0.01, and ∗∗∗ indicates p < 0.001.