Dual Role of Triptolide in Interrupting the NLRP3 Inflammasome Pathway to Attenuate Cardiac Fibrosis

Abstract

In a previous paper, we reported that triptolide (TP), a commonly used immunomodulator, could attenuate cardiac hypertrophy. This present study aimed to further explore the inhibition of cardiac fibrosis by TP and the possible mechanism from the perspective of the NOD-like receptor protein 3 (NLRP3) inflammasome. Hematoxylin-eosin and Masson's staining, immunohistochemistry, and immunofluorescence were performed to observe cardiac fibrotic changes in mice and mouse cardiac fibroblasts (CFs). The Western blot, colocalization, and immunoprecipitation were applied to detect protein expression and interactions. Results suggested that TP dose-dependently inhibited cardiac fibrosis induced by isoproterenol and collagen production of CFs induced by angiotensin II. TP exhibited an antifibrotic effect via inhibiting activation of the NLRP3 inflammasome, which sequentially decreased IL-1β maturation, myeloid differentiation factor 88 (MyD88)-related phosphorylation of c-Jun N-terminal kinase (JNK), extracellular regulated protein kinase 1/2 (ERK1/2), and TGF-β1/Smad signaling, and ultimately resulted in less collagen production. Moreover, TP showed no antifibrotic effect in Nlrp3-knockout CFs. Notably, TP inhibited the expression of NLRP3 and apoptosis-associated speck-like proteins containing a caspase recruitment domain (ASC) as well as inflammasome assembly, by interrupting the NLRP3-ASC interaction to inhibit inflammasome activation. Finally, TP indeed inhibited the NLRP3-TGFβ1-Smad pathway in vivo. Conclusively, TP was found to play a dual role in interrupting the activation of the NLRP3 inflammasome to attenuate cardiac fibrosis.

Keywords: NOD-like receptor protein 3; apoptosis-associated speck-like protein containing a CARD; cardiac fibrosis; inflammasome; triptolide.

Figure 1

Triptolide attenuates cardiac fibrosis in mice. Cardiac fibrosis was induced by continuous Iso infusion (40 mg/kg/day for 14 days; s.c.) in mice (n = 5). Mice were treated with normal saline (control), Iso and/or triptolide (10, 30, 100 μg/kg) for 14 days, respectively. Cross-sections of the mid-ventricle were used for subsequent assays. (A) Hematoxylin-eosin (HE) staining. (B) Masson staining. (C–D) Immunohistochemistry (IHC) assays of collagen I/III (Col-I/III). Bar = 50 μm. (E) Fibrosis score. (F) Expression of Col-I/III in the mid-ventricle. (G) Western blot analysis of Col-I/III expression in mid-ventricle tissue lysate. Histograms represent protein ratios normalized to GAPDH (n = 3). * p < 0.01 vs. control; # p < 0.01 vs. Iso.

Figure 2

Triptolide inhibits collagen production in vitro. Cardiac fibroblasts (CFs) grown in plates were treated with AngII (1 μM) and/or TP (1, 3, 10 μg/L) for 24 h. (A) Cytoskeleton staining by rhodamine-phalloidin (acts on F-actin). (B–C) Immunofluorescence staining of Col-I/III. (D) Cell size (n = 50). (E–F) Expression of Col-I and Col-III in CFs (n = 50). (G) Western blot analysis of the expression of Col-I, Col-III, and α-smooth muscle actin (α-SMA) in CFs. Histograms represent the protein ratio normalized to GAPDH (n = 3). Bar = 10 μm. * p < 0.01 vs. medium; # p < 0.01 vs. AngII.

Figure 3

The TGF-β1/Smad pathway is crucial for the triptolide-mediated inhibition of collagen production in cardiac fibroblasts. (A) Cells were treated as described in Figure 2. The cell lysate was analyzed via Western blot with antibodies against TGF-β1, Smad2/3, and phospho-Smad2^Ser465/467/3^Ser423/425 (p-Smad2/3). (B) Cells grown in dishes were treated with AngII (1 μM) and TP (10 μg/mL), alone or in combination and with or without TGF-β1 (10 μg/mL). The cell lysate was analyzed by Western blot with antibodies against Col-I, Smad2/3, and p-Smad2/3. Histograms represent the protein ratio normalized to GAPDH (n = 3). * p < 0.01 vs. medium; # p < 0.01 vs. AngII.

Figure 4

Triptolide inhibits ERK1/2 and JNK activation in cardiac fibroblasts. The cells were treated as described in Figure 2 and analyzed by Western blot with antibodies against (A) JNK and p-JNK, (B) ERK1/2 and p-ERK1/2, (C) p38 and p-p38, and (D) MyD88. Histograms represent the protein ratio, normalized to total JNK, total ERK, total p38, or GAPDH (n = 3), respectively. * p < 0.01 vs. medium; # p < 0.01 vs. AngII.

Figure 5

Triptolide downregulates NLRP3 inflammasome expression in cardiac fibroblasts (CFs). (A) Cells were treated as presented in Figure 2. Western blot was performed to detect the expression of NLRP3, apoptosis-associated speck-like proteins containing a caspase recruitment domain (ASC) and pro-caspase-1 in total cell lysate, and the release of caspase-1 and IL-1β in the supernatant. (B) CFs isolated from wildtype C57 mice or Nlrp3-knockout C57 mice (Nlrp3^−/−) were treated with AngII (1 μM) and/or triptolide (TP; 10 μg/mL) for 24 h. Cell lysate was used to detect the expression of NLRP3, Col-I, and p-Smad2/3 by Western blot. Histograms represent the protein ratio normalized to GAPDH (n = 3). * p < 0.01 vs. medium; # p < 0.01 vs. AngII. n.d., not detected.

Figure 6

Triptolide inhibits NLRP3 inflammasome assembly in cardiac fibroblasts (CFs). (A) CFs were placed on glass slides and treated with AngII (1 μM) and/or triptolide (TP; 10 μg/mL) for 24 h. Cells were then stained for NLRP3 (Alexa Fluor 488; green) and ASC (Alexa Fluor 555; red) and observed under a laser confocal microscope; bar = 5 μm. (B) Experiments were performed as described in A, and the cells were stained for NLRP3 (Alexa Fluor 488; green) and caspase-1 (Alexa Fluor 555; red); bar = 5 μm. (C) Colocalization of NLRP3 with ASC or caspase-1, calculated using Pearson’s correlation coefficient (Rr) via ImageJ (n = 50). (D–E) Cells were transfected with a Flag-Nlrp3 construct and treated as in A. Total lysate was used for immunoprecipitation (IP) using an anti-Flag antibody, and the associated pro-caspase-1 and ASC were detected via immunoblotting (IB). Histograms represent the protein ratio normalized to Flag (n = 3) * p < 0.01 vs. medium; # p < 0.01 vs. AngII.

Figure 7

Triptolide inhibits NLRP3-TGFβ1-Smad signaling in the fibrotic ventricle of mice. Experiments were performed as presented in Figure 1. (A–C) IHC for NLRP3, TGF-β1 and p-Smad2/3; bar = 50 μm. (D–F) Fibrosis score. * p < 0.01 vs. control; # p < 0.01 vs. Iso.