P38-DAPK1 axis regulated LC3-associated phagocytosis (LAP) of microglia in an in vitro subarachnoid hemorrhage model.

BACKGROUND: The phagocytosis and homeostasis of microglia play an important role in promoting blood clearance and improving prognosis after subarachnoid hemorrhage (SAH). LC3-assocaited phagocytosis (LAP) contributes to the microglial phagocytosis and homeostasis via autophagy-related components. With RNA-seq sequencing, we found potential signal pathways and genes which were important for the LAP of microglia. METHODS: We used an in vitro model of oxyhemoglobin exposure as SAH model in the study. RNA-seq sequencing was performed to seek critical signal pathways and genes in regulating LAP. Bioparticles were used to access the phagocytic ability of microglia. Western blot (WB), immunoprecipitation, quantitative polymerase chain reaction (qPCR) and immunofluorescence were performed to detect the expression change of LAP-related components and investigate the potential mechanisms. RESULTS: In vitro SAH model, there were increased inflammation and decreased phagocytosis in microglia. At the same time, we found that the LAP of microglia was inhibited in all stages. RNA-seq sequencing revealed the importance of P38 MAPK signal pathway and DAPK1 in regulating microglial LAP. P38 was found to regulate the expression of DAPK1, and P38-DAPK1 axis was identified to regulate the LAP and homeostasis of microglia after SAH. Finally, we found that P38-DAPK1 axis regulated expression of BECN1, which indicated the potential mechanism of P38-DAPK1 axis regulating microglial LAP. CONCLUSION: P38-DAPK1 axis regulated the LAP of microglia via BECN1, affecting the phagocytosis and homeostasis of microglia in vitro SAH model. Video Abstract.

PDK4 Decrease Neuronal Apoptosis via Inhibiting ROS-ASK1/P38 Pathway in Early Brain Injury After Subarachnoid Hemorrhage.

Aims: Metabolic disorders may play key roles in oxidative stress and neuronal apoptosis in response to early brain injury (EBI) after subarachnoid hemorrhage (SAH). Pyruvate dehydrogenase (PDH) is related to oxidative stress in EBI, and its activity obviously decreases after SAH. We discovered that only pyruvate dehydrogenase kinase 4 (PDK4) expression was obviously increased among the four PDK isozymes after SAH in preliminary experiments. Therefore, we attempted to investigate the effects and corresponding mechanisms of PDK4 on oxidative stress after SAH. Results: First, we confirmed that PDK4 overexpression promoted PDH phosphorylation, inhibited PDH activity, and changed cell metabolism after SAH. A small interfering RNA (siRNA) targeting PDK4, a lentiviral PDK4 overexpression vector, and dichloroacetic acid (DCA) were used to regulate the expression and activity of PDK4. The siRNA decreased PDH phosphorylation, promoted reactive oxygen species (ROS) production, activated the apoptosis signal-regulating kinase 1 (ASK1)/P38 pathway, and induced neuronal apoptosis. The lentivirus further attenuated PDH activity, oxidative stress, and neuronal apoptosis. DCA inhibited the activity of PDK4, but increased the expression of PDK4 due to a feedback mechanism. Inactivated PDK4 did not effectively suppress PDH activity, which increased ROS production, activated the ASK1/P38 pathway, and led to neuronal apoptosis. Innovation: This study provides new insights into the potential antioxidant and antiapoptotic effects of the PDK4-PDH axis on EBI after SAH. Conclusions: The early overexpression of PDK4 after SAH may attenuate neuronal apoptosis by reducing oxidative stress via the ROS/ASK1/P38 pathway. PDK4 may be a new potential therapeutic target to ameliorate EBI after SAH. Antioxid. Redox Signal. 36, 505-524.

Transcriptome-Wide m6A Analysis Provides Novel Insights Into Testicular Development and Spermatogenesis in Xia-Nan Cattle.

Testis is the primary organ of the male reproductive tract in mammals that plays a substantial role in spermatogenesis. Improvement of our knowledge regarding the molecular mechanisms in testicular development and spermatogenesis will be reflected in producing spermatozoa of superior fertility. Evidence showed that N6-Methyladenosine (m6A) plays a dynamic role in post-transcription gene expression regulation and is strongly associated with production traits. However, the role of m6A in bovine testis has not been investigated yet. In this study, we conducted MeRIP-Seq analysis to explore the expression profiles of the m6A and its potential mechanism underlying spermatogenesis in nine bovine testes at three developmental stages (prepuberty, puberty and postpuberty). The experimental animals with triplicate in each stage were chosen based on their semen volume and sperm motility except for the prepuberty bulls and used for testes collection. By applying MeRIP-Seq analysis, a total of 8,774 m6A peaks and 6,206 m6A genes among the studied groups were identified. All the detected peaks were found to be mainly enriched in the coding region and 3'- untranslated regions. The cross-analysis of m6A and mRNA expression exhibited 502 genes with concomitant changes in the mRNA expression and m6A modification. Notably, 30 candidate genes were located in the largest network of protein-protein interactions. Interestingly, four key node genes (PLK4, PTEN, EGR1, and PSME4) were associated with the regulation of mammal testis development and spermatogenesis. This study is the first to present a map of RNA m6A modification in bovine testes at distinct ages, and provides new insights into m6A topology and related molecular mechanisms underlying bovine spermatogenesis, and establishes a basis for further studies on spermatogenesis in mammals.

Notch4 inhibition reduces migration and invasion and enhances sensitivity to docetaxel by inhibiting Akt/fascin in pancreatic cancer cells.

Overexpression of Notch4 is associated with a variety of tumor types. Only sparse information exists on Notch4 expression in pancreatic cancer (PC). The present study demonstrated that Notch4 expression was significantly upregulated in PC cell lines compared with a non-transformed pancreatic epithelial cell line, HPDE6c-7. To investigate the possible role of Notch4 in PC cells, an RNA interference approach was used to silence Notch4 expression. The results revealed that small interfering RNA (siRNA) targeting Notch4 significantly impeded the viability, migration and invasion abilities of PC cells in vitro. Downregulation of Notch4 with siRNA sensitized cells to the action of docetaxel. Furthermore, Notch4 downregulation enhanced the inhibition of Akt activation and the fascin expression induced by docetaxel in PC cells. Together, these data provide insight into the function of Notch4 and suggest that Notch4 may represent a new potential target for gene therapy in PC.