Yanqing Wu, Xilin Qiao, Shuaiyu Chen, Yalan Lu, Bingyan Mao, Mengdie Zhu, Xinzhi Chen, Nipi Chen, Zhishan Ding, Bo Jin
The results showed that miR-29b-3p was downregulated in OGD/R-treated HUVECs, an effect that was reversed by PCA and the ROS inhibitor N-acetylcysteine (NAC). The miR-29b-3p mimic enhanced the promoting effects of PCA and NAC on miR-29b-3p expression, whereas miR-29b-3p inhibitor reversed these effects. Similarly, PCA-induced autophagy and inhibition of apoptosis were enhanced by the miR-29b-3p mimic and reversed by the inhibitor. To determine whether SIRT1 acts downstream of miR-29b-3p, we used EX527 (a SIRT1 inhibitor) and found that it attenuated the PCA-mediated upregulation of miR-29b-3p. The miR-29b-3p mimic enhanced PCA-induced SIRT1 activity and protein expression, while the inhibitor had the opposite effect. Additional experiments revealed that NAC suppressed ROS by upregulating miR-29b-3p, leading to increased SIRT1 expression and reduced apoptosis.
INTRODUCTION: MicroRNA-29b-3p (miR-29b-3p) plays a critical role in regulating endothelial apoptosis and autophagy. We previously demonstrated that protocatechualdehyde (PCA), a water-soluble phenolic acid derived from Salvia miltiorrhiza Bge., protects human umbilical vein endothelial cells (HUVECs) against oxygen-glucose deprivation/reoxygenation (OGD/R)-induced injury. However, whether miR-29b-3p mediates this protective effect remains unclear.
METHODS: Using the OGD/R in vitro model of ischemia-reperfusion injury, we investigated the molecular mechanisms underlying ischemic damage at the cellular level. RT-qPCR was used to measure miR-29b-3p expression, Western blotting to assess autophagy- and apoptosis-related proteins, and MDC staining and flow cytometry to evaluate autophagy and apoptosis, respectively.
RESULTS: The results showed that miR-29b-3p was downregulated in OGD/R-treated HUVECs, an effect that was reversed by PCA and the ROS inhibitor N-acetylcysteine (NAC). The miR-29b-3p mimic enhanced the promoting effects of PCA and NAC on miR-29b-3p expression, whereas miR-29b-3p inhibitor reversed these effects. Similarly, PCA-induced autophagy and inhibition of apoptosis were enhanced by the miR-29b-3p mimic and reversed by the inhibitor. To determine whether SIRT1 acts downstream of miR-29b-3p, we used EX527 (a SIRT1 inhibitor) and found that it attenuated the PCA-mediated upregulation of miR-29b-3p. The miR-29b-3p mimic enhanced PCA-induced SIRT1 activity and protein expression, while the inhibitor had the opposite effect. Additional experiments revealed that NAC suppressed ROS by upregulating miR-29b-3p, leading to increased SIRT1 expression and reduced apoptosis.
DISSCUSSION: Collectively, these findings demonstrate that PCA ameliorates OGD/R-induced endothelial injury by promoting autophagy and inhibiting apoptosis via the ROS/miR-29b-3p/SIRT1 regulatory axis.