| Summary: | 博士 === 國立陽明大學 === 藥理學研究所 === 100 === Our laboratory has previously demonstrated that sodium fluoride (NaF) caused a rapid increase of free calcium concentration and resulted in apoptosis by increasing NADPH oxidase-dependent reactive oxygen species production in H9c2 cardiomyocytes. From pharmaceutical aspect, this study was designed to uncover the endogenous mechanisms and/or pharmacological manipulations for providing effective protection against the cellular damages caused by NaF in H9c2 cardiomyocyte.
Akt has been demonstrated to play protective roles for cardiomyocytes under a variety of insult conditions. The first series of experiments was carried out to clarify the possible role of Akt in NaF-induced apoptosis of H9c2 cardiomyocytes. The results showed that Akt was significantly activated after 1-2 hours exposure to NaF. In addition, NaF-induced apoptosis of H9c2 cells was aggravated by API-2 (an Akt inhibitor). As was expected, NaF-induced apoptosis of H9c2 cardiomyocytes was also inhibited by wortmannin, a PI3K inhibitor, suggesting the involvement of PI3K/Akt signaling pathway. These results suggest a protective role for Akt against apoptotic damage of H9c2 cardiomyocytes produced by NaF intoxication. Akt has been shown to activate CREB, a stimulus-induced transcription factor, and then reduce apoptosis by increasing the expression of Bcl-2. Furthermore, based on the finding that the anti-apoptotic effect of Akt was more significant at 48 hours after NaF exposure, this study thus proposed that Akt may reduce apoptosis via activation of CREB/Bcl-2 signaling. The results revealed that CREB phosphorylation could be upregulated by NaF and demonstrated a pro-apoptotic effect, while pretreatment with API-2 or CREB inhibitor (KG-501) was failed to affect the activation of CREB.
Adrenomedullin (ADM) has been demonstrated to exhibit anti-apoptotic effect on cardiomyocytes after ischemia/reperfusion injury. Thus, the second series of experiments of this study was aimed to verify whether ADM can abrogate NaF-induced apoptosis of cardiomyocytes. Following pretreatment with 0.1 pM ADM, significant decreases in the caspase-3activity and the percentage of sub G0/G1 phase were observed in NaF-treated H9c2 cells. It is suggested that pharmacological application of ADM may provide beneficial effect on the NaF-induced apoptosis of cardiomyocytes. Our laboratory has previously found that treatment with NaF could decrease the activity of autophagy and in turn cause cell death in H9c2 cardiomyocytes. In this study, ADM was further found to reverse the NaF-elicited inhibition of autophagy.
In conclusion, the results from this investigation suggest that NaF-induced apoptosis of H9c2 cardiomyocytes could be balanced off by, on the one hand, an endogenous protection mechanism through PI3K/Akt signaling pathway activated by NaF-derived stress. On the other hand, ADM may pharmacologically provide beneficial effect on NaF-induced apoptosis of H9c2 cardiomyocytes via restoring their autophagic activity.
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