Cantharidin induces senescence via inhibition of AMP-activated protein kinase and activation of NLRP3 inflammasome in H9c2 cardiomyocytes

Cantharidin is a natural compound with cardiotoxicity. Cellular senescence and senescence-associated secretory phenotype (SASP) are implicated in chemotherapy-associated cardiotoxicity. We here investigated how cantharidin induced cardiomyocyte senescence. H9c2 cells were treated with cantharidin. Senescence, mitochondrial functions, SASP, NOD-like receptor thermal protein domain associated protein 3 (NLRP3) signaling and AMP-activated protein kinase (AMPK) phosphorylation were examined. Cantharidin inhibited viability and increased expression of senescence-associated β--galactosidase (SA-β-Gal), p16 and p21 in H9c2 cells, suggesting occurrence of senescence. Cantharidin impaired mitochondrial functions evidenced by reduction in basal respiration, ATP levels and spare respiratory capacity. Cantharidin also decreased mitochondrial DNA copy number and down-regulated mRNA levels of cytochrome c oxidase-I, -II and -III. Moreover, cantharidin suppressed activity of mitochondria complex-I and -II. Examinations of SASP showed that cantharidin promoted expression and secretion of SASP cytokines interleukin-1β-, -6 and -8 and tumor necrosis factor-α, associated with activation of NLRP3/caspase-1 pathway. Finally, cantharidin suppressed AMPK phosphorylation. AMPK activator GSK621 abrogated the up-regulation of SA-β--Gal, p16 and p21 and counteracted the activation of NLRP3 and caspase-1 in cantharidin-challenged H9c2 cells. In conclusion, cantharidin stimulated senescence and SASP in cardiomyocytes through activation of NLRP3 inflammasome and inhibition of AMPK, providing novel molecular insights into cantharidin-induced cardiotoxicity.

 

Comments:

The study you described investigated the molecular mechanisms behind cantharidin-induced cardiomyocyte senescence. Here's a summary of the findings:

**1. Senescence Induction:**
   - Cantharidin treatment reduced cell viability and increased the expression of senescence markers, including senescence-associated β-galactosidase (SA-β-Gal), p16, and p21 in H9c2 cells, indicating the occurrence of cellular senescence.

**2. Mitochondrial Dysfunction:**
   - Cantharidin impaired mitochondrial functions, evidenced by a decrease in basal respiration, ATP levels, and spare respiratory capacity.
   - It reduced mitochondrial DNA copy number and down-regulated the expression of cytochrome c oxidase-I, -II, and -III.
   - Cantharidin suppressed the activity of mitochondrial complexes I and II.

**3. Senescence-Associated Secretory Phenotype (SASP) Activation:**
   - Cantharidin promoted the expression and secretion of SASP cytokines, including interleukin-1β, -6, and -8, and tumor necrosis factor-α.
   - Activation of the NLRP3/caspase-1 pathway was associated with the increased expression of SASP cytokines.

**4. Inhibition of AMPK Pathway:**
   - Cantharidin suppressed AMP-activated protein kinase (AMPK) phosphorylation, a key regulator of cellular energy balance and senescence.
   - Treatment with the AMPK activator GSK621 counteracted the up-regulation of senescence markers (SA-β-Gal, p16, and p21) and the activation of the NLRP3/caspase-1 pathway in cantharidin-treated H9c2 cells.

**Conclusion:**
   Cantharidin induced senescence and SASP in cardiomyocytes by activating the NLRP3 inflammasome and inhibiting the AMPK pathway. These findings provide new insights into the molecular mechanisms underlying cantharidin-induced cardiotoxicity. AMPK activators like GSK621 showed potential in mitigating the senescence and inflammatory responses triggered by cantharidin in cardiomyocytes.