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

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-1ß 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-ß1/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-TGFß1-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.

Cyclin-Dependent Kinase Inhibitor p21WAF1/CIP1 Facilitates the Development of Cardiac Hypertrophy.

BACKGROUND/AIMS: Adult cardiomyocytes can re-enter cell cycle as stimulated by prohypertrophic factors although they withdraw from cell cycle soon after birth. p21WAF1/CIP1, a cyclin-dependent kinase inhibitor, has been implicated in cardiac hypertrophy, however, its precise contribution to this process remains largely unclear. METHODS: The gene expression profile in left ventricle (LV) of spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats was determined using quantitative PCR array and verified by real-time PCR and Western blotting. Hypertrophic response of H9c2 cells and neonatal rat ventricular myocytes (NRVM) were induced by angiotensin II (1 µmol/L). Cardiac hypertrophy of mice was elicited by isoproterenol (ISO) infusion (40 mg/kg per day for 14 days). p21-adenovirus and p21-siRNA were employed to transfect NRVM, and sterigmatocystin (STE, 3 mg/kg, ip, qd) was used to inhibit p21 activity. mRNA and protein expression levels of α- and ß-myosin heavy chain (MHC), p21WAF1/CIP1, calcineurin (CaN) and atrial natriuretic peptide (ANP) were assayed by realtime PCR and WB, respectively. RESULTS: Sixteen genes showed two-fold or greater changes between SHR and WKY rats, in which the expression of p21WAF1/CIP1 was upregulated by 4.15-fold (P=0.002) and reversed by losartan. Surface area, protein content, mRNA and protein expressions of ß-MHC, ANP and p21WAF1/CIP1 in H9c2 cells treated with AngII elevated significantly compared with control group. p21-Ad transfection markedly increased the surface area and ß-MHC mRNA expression of normal NRVMs, and p21-siRNA transfection decreased them in AngII-treated NRVMs. STE treatment decreased HW/BW and cross-sectional area, expression levels of ß-MHC, ANP and p21 significantly in ISO-treated mice. CONCLUSION: Our findings suggest that p21 facilitates the development of cardiac hypertrophy, and regulating the expression of p21 may be an approach to attenuate hypertrophic growth of cardiomyocytes.

Effect of Triptolide on Temporal Expression of Cell Cycle Regulators During Cardiac Hypertrophy.

Adult mammalian cardiomyocytes may reenter the cell cycle and cause cardiac hypertrophy. Triptolide (TP) can regulate the expressions of various cell cycle regulators in cancer cells. However, its effects on cell cycle regulators during myocardial hypertrophy and mechanism are unclear. This study was designed to explore the profile of cell cycle of cardiomyocytes and the temporal expression of their regulators during cardiac hypertrophy, as well as the effects of TP. The hypertrophy models employed were neonatal rat ventricular myocytes (NRVMs) stimulated with angiotensin II (Ang II) for scheduled times (from 5 min to 48 h) in vitro and mice treated with isoprenaline (Iso) for from 1 to 21 days, respectively. TP was used in vitro at 1 µg/L and in vivo at 10 µg/kg. NRVMs were analyzed using flow cytometry to detect the cell cycle, and the expression levels of mRNA and protein of various cell cycle regulators were determined using real-time PCR and Western blot. It was found NRVM numbers in phases S and G2 increased, while that in the G1 phase decreased significantly after Ang II stimulation. The mRNA expression levels of p21 and p27 increased soon after stimulation, and thereafter, mRNA expression levels of all cell cycle factors showed a decreasing trend and reached their lowest levels in 1-3 h, except for cyclin-dependent kinase 1 (CDK1) and CDK4 mRNA. The mRNA expression levels of CDK1, p21, and p27 increased markedly after stimulation with Ang II for 24-48 h. In myocardium tissue, CDK and cyclin expression levels peaked in 3-7 days, followed by a decreasing trend, while those of p21 and p27 mRNA remained at a high level on day 21. Expression levels of all protein were consistent with the results of mRNA in NRVMs or mice. The influence of Ang II or Iso on protein expression was more obvious than that on mRNA. TP treatment effectively prevented the imbalance in the expression of cell cycle regulators in the hypertrophy model group. In Conclusion, an imbalance in the expression of cell cycle regulators occurs during cardiac hypertrophy, and triptolide corrects these abnormal expression levels and attenuates cardiac hypertrophy.

Triptolide Upregulates Myocardial Forkhead Helix Transcription Factor p3 Expression and Attenuates Cardiac Hypertrophy.

The forkhead/winged helix transcription factor (Fox) p3 can regulate the expression of various genes, and it has been reported that the transfer of Foxp3-positive T cells could ameliorate cardiac hypertrophy and fibrosis. Triptolide (TP) can elevate the expression of Foxp3, but its effects on cardiac hypertrophy remain unclear. In the present study, neonatal rat ventricular myocytes (NRVM) were isolated and stimulated with angiotensin II (1 µmol/L) to induce hypertrophic response. The expression of Foxp3 in NRVM was observed by using immunofluorescence assay. Fifty mice were randomly divided into five groups and received vehicle (control), isoproterenol (Iso, 5 mg/kg, s.c.), one of three doses of TP (10, 30, or 90 µg/kg, i.p.) for 14 days, respectively. The pathological morphology changes were observed after Hematoxylin and eosin, lectin and Masson's trichrome staining. The levels of serum brain natriuretic peptide (BNP) and troponin I were determined by enzyme-linked immunosorbent assay and chemiluminescence, respectively. The mRNA and protein expressions of α- myosin heavy chain (MHC), ß-MHC and Foxp3 were determined using real-time PCR and immunohistochemistry, respectively. It was shown that TP (1, 3, 10 µg/L) treatment significantly decreased cell size, mRNA and protein expression of ß-MHC, and upregulated Foxp3 expression in NRVM. TP also decreased heart weight index, left ventricular weight index and, improved myocardial injury and fibrosis; and decreased the cross-scetional area of the myocardium, serum cardiac troponin and BNP. Additionally, TP markedly reduced the mRNA and protein expression of myocardial ß-MHC and elevated the mRNA and protein expression of α-MHC and Foxp3 in a dose-dependent manner. In conclusion, TP can effectively ameliorate myocardial damage and inhibit cardiac hypertrophy, which is at least partly related to the elevation of Foxp3 expression in cardiomyocytes.

Gαq protein carboxyl terminus imitation polypeptide GCIP-27 improves cardiac function in chronic heart failure rats.

BACKGROUND: Gαq protein carboxyl terminus imitation polypeptide (GCIP)-27 has been shown to alleviate pathological cardiomyocyte hypertrophy induced by various factors. Pathological cardiac hypertrophy increases the morbidity and mortality of cardiovascular diseases while it compensates for poor heart function. This study was designed to investigate the effects of GCIP-27 on heart function in rats with heart failure induced by doxorubicin. METHODS AND RESULTS: Forty-eight rats were randomly divided into the following six groups receiving vehicle (control), doxorubicin (Dox), losartan (6 mg/kg, i.g.) and three doses of GCIP-27 (10, 30, 90 µg/kg; i.p., bid), respectively. Heart failure was induced by Dox, which was administered at a 20 mg/kg cumulative dose. After 10 weeks of treatment, we observed that GCIP-27 (30, 90 µg/kg) significantly increased ejection fraction, fraction shortening, stroke volume and sarcoplasmic reticulum Ca2+ ATPase activity of Dox-treated hearts. Additionally, GCIP-27 decreased myocardial injury, heart weight index and left ventricular weight index, fibrosis and serum cardiac troponin-I concentration in Dox-treated mice. Immunohistochemistry, western blotting and real-time PCR experiments indicated that GCIP-27 (10-90 µg/kg) could markedly upregulate the protein expression of myocardial α-myosin heavy chain (MHC), Bcl-2, protein kinase C (PKC) ε and phosphorylated extracellular signal-regulated kinase (p-ERK) 1/2 as well as the mRNA expression of α-MHC, but downregulated the expression of ß-MHC, Bax and PKC ßII, and the mRNA expression levels of ß-MHC in Dox-treated mice. It was also found that GCIP-27 (30, 90 µg/L) decreased cell size and protein content of cardiomyocytes significantly in vitro by comparison of Dox group. CONCLUSIONS: GCIP-27 could effectively ameliorate heart failure development induced by Dox. PKC-ERK1/2 signaling might represent the underlying mechanism of the beneficial effects of GCIP-27.