Silencing of TLR4 Inhibits Atrial Fibrosis and Susceptibility to Atrial Fibrillation via Downregulation of NLRP3-TGF-β in Spontaneously Hypertensive Rats

Figures

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  Figure 1. Systolic blood pressure, heart rate, and body weight of rats. Systolic blood pressure (a), heart rate (b), and body weight (c) before (14 weeks) and after (22 weeks) intervention. n = 6 in each group. Data are expressed as the mean ± SEM. Statistical analyses: P values were evaluated by ANOVA, followed by a post hoc Tukey's test. ∗Compared with control; P < 0.001 indicates a significant difference.

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  Figure 2. Silencing of TLR4 alleviated susceptibility to atrial fibrillation (AF) in spontaneously hypertensive rats. (a) Typical surface electrogram lead II recordings of sinus rhythm and induced atrial fibrillation after burst pacing. (b–d) Statistically significant results of P wave maximum duration, total time of atrial fibrillation episodes, and probability of induced AF in each group (n = 6 for each). Data are expressed as mean ± SEM. Statistical analyses: P values were evaluated by ANOVA, followed by a post hoc Tukey's test. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 versus the control; #P < 0.05, ##P < 0.01, and ###P < 0.001 versus the SHR group; &P < 0.05 versus the SHR+vehicle group; significantly different as indicated.

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  Figure 3. Silencing of TLR4 alleviated atrial fibrosis. (a) Representative hematoxylin-eosin (HE) stain of rat left atrial tissue sections (200x). (b) Representative Masson's trichrome stain of rat left atrial tissue sections (200x). (c) Expression of collagen I protein was detected by immunohistochemical (IHC) staining in left atrial tissue; scale bar = 50 μm. (d) IHC staining for collagen III in magnified views; scale bar = 50 μm. (e) Volume fraction of collagen was quantified by Masson stain; data are representative of six samples per group. (f) Expression of collagen I protein was quantified by immunohistochemistry; data are representative of six samples per group. (g) Expression of collagen III protein was quantified by immunohistochemistry; data are representative of six samples per group. All the aforementioned data are represented as mean ± SEM. Statistical analyses: P values were evaluated by ANOVA, followed by a post hoc Tukey's test. ∗P < 0.001 versus the control group; #P < 0.001 versus the SHR group; &P < 0.001 versus the SHR+vehicle group; significantly different as indicated.

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  Figure 4. Silencing of TLR4 reduced mRNA expression of NLRP3 and TGF-β in vivo. Relative mRNA expression of TLR4, NLRP3, TGF-β, and collagen I normalized to GAPDH in left atrial tissue (a–d). All aforementioned data are represented as mean ± SEM (n = 3). Statistical analyses: the P values were evaluated by ANOVA, followed by a post hoc Tukey's test. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 versus the control; #P < 0.05, ##P < 0.01, and ###P < 0.001 versus the SHR group; &P < 0.05, &&P < 0.01, and &&&P < 0.001 versus the SHR+vehicle group; significantly different as indicated.

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  Figure 5. Silencing of TLR4 reduced expression of NLRP3 and TGF-β proteins in vivo. Protein expression of TLR4, NLRP3, pro-caspase-1, caspase-1-p20, TGF-β, IL-1β, and IL-18 detected by western blots in left atrial tissue. (a) Representative western blots show expression of TLR4, NLRP3, pro-caspase-1, caspase-1-p20, TGF-β, IL-1β, and IL-18. (b–h) Relative protein expression of TLR4, NLRP3, pro-caspase-1, caspase-1-p20, TGF-β, IL-1β, and IL-18 normalized to GAPDH in left atrial tissue. All the aforementioned data are represented as mean ± SEM (n = 3). Statistical analyses: P values were evaluated by ANOVA, followed by a post hoc Tukey's test. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 versus the control; #P < 0.05, ##P < 0.01, and ###P < 0.001 versus the SHR group; &P < 0.05, &&P < 0.01, and &&&P < 0.001 versus the SHR+vehicle group; significantly different as indicated.

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  Figure 6. Silencing of TLR4 inhibited proliferation of cardiac fibroblasts (CFs). (a) CF morphology was observed by transmission light microscopy (100x) and immunofluorescence was used to identify vimentin expressed in CFs (100x). (b) CFs were treated with different concentrations of Ang II, and cell proliferation was evaluated using a CCK-8 assay kit (n = 3). (c) The effect of TLR4-shRNA on proliferation of cardiac fibroblasts measured by CCK-8 (n = 3). (d) The concentration of IL-1β in cell culture supernatants was detected by enzyme-linked immunosorbent assay (ELISA) (n = 6). (e) The concentration of IL-18 in cell culture supernatants was detected by ELISA (n = 6). Data are expressed as mean ± SEM. Statistical analyses: P values were evaluated by ANOVA, followed by a post hoc Tukey's test. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 versus the control; #P < 0.05, ##P < 0.01, and ###P < 0.001 versus the SHR group; &P < 0.05, &&P < 0.01, and &&&P < 0.001 versus the SHR+vehicle group; significantly different as indicated.

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  Figure 7. Silencing of TLR4 inhibited mRNA expression of NLRP3 and TGF-β in vitro. Relative mRNA expression of TLR4, NLRP3, TGF-β, and collagen I normalized to GAPDH in CFs (a–d). All the aforementioned data are represented as mean ± SEM (n = 3). Statistical analyses: P values were evaluated by ANOVA, followed by a post hoc Tukey's test. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 versus the control; #P < 0.05, ##P < 0.01, and ###P < 0.001 versus the Ang II group; &P < 0.05, &&P < 0.01, and &&&P < 0.001 versus the Ang II+vehicle group; significantly different as indicated.

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  Figure 8. Silencing of TLR4 inhibited expression of NLRP3 and TGF-β proteins in vitro. Protein expression of TLR4, NLRP3, caspase-1, TGF-β, and collagen I detected by western blots in CFs. (a) Representative western blots show expression of TLR4, NLRP3, pro-caspase-1, caspase-1-p20, TGF-β, and collagen I. (b–g) Relative protein expression of TLR4, NLRP3, pro-caspase-1, caspase-1-p20, TGF-β, and collagen I normalized to GAPDH in CFs. All the aforementioned data are represented as mean ± SEM (n = 3). Statistical analyses: P values were evaluated by ANOVA, followed by a post hoc Tukey's test. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 versus the control; #P < 0.05, ##P < 0.01, and ###P < 0.001 versus the Ang II group; &P < 0.05, &&P < 0.01, and &&&P < 0.001 versus the Ang II+vehicle group; significantly different as indicated.

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  Figure 9. Diagram of the mechanisms investigated in the present study.