ABSTRACT
Cardiac toxicity has been the major concern when using doxorubicin (DOX) in cancer therapy. Thrombopoietin (TPO) protects cardiac cells from DOX-induced cell damage; however, its molecular mechanism remains exclusive. The anti-autophagic and anti-apoptotic effects of TPO upon DOX treatment were studied in the cardiac H9C2 cell line, with bafilomycin A1 treatment as a positive control for autophagy inhibition. Cell viability was measured by Cell Counting Kit-8 assay in different treatment groups. The mRNA and/or protein levels of apoptotic markers and autophagy-associated factors were detected. The mean number of microtubule-associated protein 1A/1B-light chain 3 (LC3) puncta per cell was quantified to indicate autophagosomes and autolysosomes, of which the ones co-stained with lysosomal-associated membrane protein 1 were considered as autolysosomes. DOX treatment (5 µg/ml, 24 h) significantly impaired H9C2 cell viability compared with the control, while TPO pretreatment (10 ng/ml, 36 h) improved cell viability upon DOX treatment. DOX exposure markedly increased LC3 puncta in H9C2 cells, and TPO pretreatment reduced the number of autophagosomes, but showed no significant inhibitory effect on autolysosome formation. The autophagy inhibition by TPO upon DOX treatment was confirmed according to protein quantification of LC3-II and nucleoporin 62. TPO also suppressed autophagy-promoting protein Beclin-1, and elevated the anti-autophagic factors GATA-binding protein-4 and B cell lymphoma-2. Furthermore, TPO reduced DOX-induced apoptosis in H9C2 cells, as reflected by the amount changes of caspase-3. Taken together, these results revealed that TPO has a protective role in H9C2 cells from DOX-induced autophagy as well as apoptosis, and indicated that TPO may act as a cardioprotective drug in DOX-treated patients.
ABSTRACT
AIMS: The molecular events leading from cardiomyocyte ischaemia to inflammatory cytokine production are not well understood. We previously found that heat shock protein 60 (HSP60) appeared in extracellular space after cardiomyocyte ischaemia. This study examined the activation and regulation of toll-like receptors (TLRs) by HSP60 in cardiomyocytes. METHODS AND RESULTS: Cytokine production and TLRs regulation mediated by TLRs signalling were examined in response to exogenous HSP60 (exHSP60) and endogenous HSP60 (enHSP60) released extracellularly under ischaemia. The results showed that exHSP60 induced inflammatory cytokine production in adult rat cardiomyocytes and H9c2 cells (a standard cardiac cell line derived from embryonic cells), through a pathway dependent on TLR4, myeloid differentiation factor 88 (MyD88), p38, and nuclear factor-κB (NF-κB). Further study revealed up-regulated expression of both TLR2 and TLR4 by exHSP60, which was dependent on the activation of TLR4, MyD88, c-Jun NH2-terminal kinase (JNK), and NF-κB, but not on p38. In myocytes exposed to ischaemia, enHSP60 was released into the media, and triggered cytokine production and TLR2/4 overexpression, through the same pathways as exHSP60. In rats subjected to LAD ligation, the released enHSP60 contributed to cytokine production and TLR2/4 overexpression in the ischaemic myocardium. CONCLUSION: Extracellular HSP60 induces cytokine production via TLR4-MyD88-p38/NF-κB pathway, and up-regulates TLR2/4 expression via TLR4-MyD88-JNK/NF-κB pathway. Both pathways contribute to myocardial inflammation induced by ischaemia.