Effect of Shuangdan Mingmu capsule, a Chinese herbal formula, on oxidative stress-induced apoptosis of pericytes through PARP/GAPDH pathway.

BACKGROUND: Diabetic retinopathy (DR) has become a worldwide concern because of the rising prevalence rate of diabetes mellitus (DM). Despite much energy has been committed to DR research, it remains a difficulty for diabetic patients all over the world. Since apoptosis of retinal microvascular pericytes (RMPs) is the early characteristic of DR, this study aimed to reveal the mechanism of Shuangdan Mingmu (SDMM) capsule, a Chinese patent medicine, on oxidative stress-induced apoptosis of pericytes implicated with poly (ADP-ribose) polymerase (PARP) / glyceraldehyde 3-phosphate dehydrogenase (GAPDH) pathway. METHODS: Network pharmacology approach was performed to predict biofunction of components of SDMM capsule dissolved in plasma on DR. Both PARP1 and GAPDH were found involved in the hub network of protein-protein interaction (PPI) of potential targets and were found to take part in many bioprocesses, including responding to the regulation of reactive oxygen species (ROS) metabolic process, apoptotic signaling pathway, and response to oxygen levels through enrichment analysis. Therefore, in vitro research was carried out to validate the prediction. Human RMPs cultured with media containing 0.5 mM hydrogen oxide (H2O2) for 4 h was performed as an oxidative-damage model. Different concentrations of SDMM capsule, PARP1 inhibitor, PARP1 activation, and GAPDH inhibitor were used to intervene the oxidative-damage model with N-Acetyl-L-cysteine (NAC) as a contrast. Flow cytometry was performed to determine the apoptosis rate of cells and the expression of ROS. Cell counting kit 8 (CCK8) was used to determine the activity of pericytes. Moreover, nitric oxide (NO) concentration of cells supernatant and expression of endothelial nitric oxide synthase (eNOS), superoxide dismutase (SOD), B cell lymphoma 2 (BCL2), vascular endothelial growth factor (VEGF), endothelin 1 (ET1), PARP1, and GAPDH were tested through RT-qPCR, western blot (WB), or immunocytochemistry (ICC). RESULTS: Overproduction of ROS, high apoptotic rate, and attenuated activity of pericytes were observed after cells were incubated with media containing 0.5 mM H2O2. Moreover, downregulation of SOD, NO, BCL2, and GAPDH, and upregulation of VEGFA, ET1, and PARP1 were discovered after cells were exposed to 0.5 mM H2O2 in this study, which could be improved by PARP1 inhibitor and SDMM capsule in a dose-dependent way, whereas worsened by PARP1 activation and GAPDH inhibitor. CONCLUSIONS: SDMM capsule may attenuate oxidative stress-induced apoptosis of pericytes through downregulating PARP expression and upregulating GAPDH expression.

MiR-340/iASPP axis affects UVB-mediated retinal pigment epithelium (RPE) cell damage.

Long-term exposure to ultraviolet B (UVB) light increases the risk of UVB damage due to increased UVB absorption by the retina and may further lead to age-related eye diseases. The retinal pigment epithelium (RPE) cell is a main target of UVB reaching the retina; its degeneration is an essential event in UVB-mediated age-related macular degeneration (AMD). Herein, we first evaluated the expression and effect of iASPP, an inhibitory regulator of apoptosis, in UVB-induced RPE cell damage. Through the mechanism of RNA interference at the post-transcriptional level, miRNA affects a variety of cellular processes, including UVB-mediated cell damage. We next screened for upstream candidate miRNAs that may regulate iASPP expression. Among 8 candidate miRNAs, UVB significantly increased miR-340 levels. We also confirmed the direct binding of miR-340 to the 3'UTR of iASPP, and assessed the combined effect of miR-340 and iASPP on UVB-induced RPE cell damage. Taken together, we demonstrated the possible mechanisms involved in UVB-induced retinal damage. In RPE cells, UVB irradiation inhibits iASPP expression through inducing miR-340 expression, thereby promoting RPE cell apoptosis and suppressing cell viability via affecting p53, p21 and caspase-3 protein expression. Targeting miR-340 to rescue iASPP expression in RPE cells may help treat UVB-mediated retinal damage.