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Myocardial fibrosis functions as an independent risk factor involved in various cardiac diseases such as hypertension, left ventricular diastolic dysfunction and myocardial infarction. Our previous study indicated triptolide (TP), a diterpenoid isolated from Tripterygium wilfordii, attenuated cardiac hypertrophy and myocardial fibrosis in vivo. Herein, the present study was designed to assess the protective effect of TP against cardiac fibrosis and the underlying mechanisms in vivo and in vitro.
Myocardial fibrosis of mice was induced by continuous infusion of isoproterenol (ISO) using osmotic mini-pump at 40 mg/kg/d for 14 days. Collagen deposition of primary mouse cardiac fibroblasts (CFs) was induced by angiotensin II (AngII, 1 μmol/L). Collagen was respectively assessed with MASSON staining in vivo, or immunofluorescence staining and western blot in vitro. Cytoskeleton of CFs was probed by rhodamine-phalloidine and observed using confocal imaging. Protein expression such as NOD-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC), Caspase-1, IL-1β, myeloid differentiation factor 88 (MyD88), MAPKs including Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38, transforming growth factor β1 (TGF-β1) and phopho-Smad2/3 (p-Smad2/3) was detected by western blot in vitro or immunohistochemistry in vivo.
TP (10, 30, 100 μg/kg) dose-dependently decreased total collagen deposition as well as collagen I/III expression induced by ISO in left ventricle. In addition, TP (1, 3, 10 μg/mL) also concentration-dependently inhibited cell hypertrophy and collagen I/III expression induced by AngII in CFs. Critically, even low-dose of TP (10 μg/kg or 1 μg/mL) showed significant anti-fibrotic effect in vivo and in vitro. TGF-β1 and p-Smad2/3 were increased by ISO in fibrotic area of left ventricle or AngII in CFs, but significantly decreased by TP treatments, suggesting the anti-fibrotic effect of TP is related to TGF-β1/Smad pathway which is considered crucial for collagen production and fibrosis. Moreover, TP decreased expression of IL-1β and MyD88, as well as phosphorylation of JNK and ERK, but not p38 MARK, induced by AngII. However, the inhibition of TP on TGF-β1 mediated collagen production completely lost in IL-1β receptor knock-out CFs, indicating IL-1β/MAPK/Smad signaling is critical for TP's effect. NLRP3 inflammasome plays a critical role in IL-1β production and fibrotic pathogenesis. Herein, TP also dose-dependently decreased the expression of NLRP3 and ASC, not pro-caspase-1, in fibrotic left ventricle as well as in AngII-treated CFs. Not surprisingly, the inhibition of TP on collagen production completely lost in NLRP3 knock-out CFs, indicating the inhibition of TP on cardiac fibrosis was related to decrease NLRP3 inflammasome activation.
Triptolide significantly inhibits cardiac fibrosis in vitro and in vivo via inhibiting NLRP3 inflammasome mediated MAPK/TGF-β1/Smad signaling and may therefore be useful in preventing fibrotic process in heart diseases.