ABSTRACT
BACKGROUND: Local brain tissue can suffer from ischaemia/reperfusion (I/R) injury, which lead to vascular endothelial damage. The peptide δ opioid receptor (δOR) agonist [D-ala2, D-leu5]-Enkephalin (DADLE) can reduce apoptosis caused by acute I/R injury in brain microvascular endothelial cells (BMECs). OBJECTIVE: This study aims to explore the mechanism by which DADLE enhances the level of mitophagy in BMECs by upregulating the expression of transient receptor potential vanilloid subtype 4 (TRPV4). METHODS: BMECs were extracted and made to undergo oxygen-glucose deprivation/reoxygenation (OGD/R) accompanied by DADLE. RNA-seq analysis revealed that DADLE induced increased TRPV4 expression. The CCK-8 method was used to assess the cellular viability; quantitative PCR (qPCR) was used to determine the mRNA expression of Drp1; western blot was used to determine the expression of TRPV4 and autophagy-related proteins; and calcium imaging was used to detect the calcium influx. Autophagosomes in in the cells' mitochondria were observed by using transmission electron microscopy. ELISA was used to measure ATP content, and a JC-1 fluorescent probe was used to detect mitochondrial membrane potential. RESULTS: When compared with the OGD/R group, OGD/R+DADLE group showed significantly enhanced cellular viability; increased expression of TRPV4, Beclin-1, LC3-II/I, PINK1 and Parkin; decreased p62 expression; a marked rise in calcium influx; further increases in mitophagy, an increase in ATP synthesis and an elevation of mitochondrial membrane potential. These protective effects of DADLE can be blocked by a TRPV4 inhibitor HC067047 or RNAi of TRPV4. CONCLUSION: DADLE can promote mitophagy in BMECs through TRPV4, improving mitochondrial function and relieving I/R injury.
ABSTRACT
Background: Diabetic foot is a major complication of diabetes. The bone transverse transport method could be applied in clinics for treatment, which could improve the metabolism of the tissues via lasting distraction forces. However, the process' specific regulating mechanism is still unknown. Methods: Based on the notion that the healing of bones involves the recruitment of calcium ions, in this study, we established the model of tibial cortex transverse transport (TTT) on rats and then used tissue immunologic detection, such as the double fluorescent staining to explore the expression of the calcium channels' calcium release-activated calcium modulator 1 (Orai1)/stromal interaction molecule 1 (STIM1), which belong to the store-operated calcium entry (SOCE) signaling pathways on the tissues around the bone transport area. By using the laser capture microdissection (LCM) tool, we acquired samples of tissues around the bone and endeavored to identify pivotal protein molecules. Subsequently, we validated the functions of key protein molecules through in vitro and in vivo experiments. Results: After protein profile analysis, we found the differentially expressed key protein osteopontin (OPN). The in vitro experiments verified that, being stimulated by OPN, the migration, proliferation, and angiogenesis of human umbilical vein endothelial cells (HUVEC) were observed to be enhanced. The activation of Orai1/STIM1 might increase the activity of endothelial nitric oxide synthase (eNOS) and its effect on releasing nitric oxide (NO). Subsequently, the migration and proliferation of the HUVECs are improved, which ultimately accelerates wound healing. These signaling pathway was also observed in the OPN-stimulated healing process of the skin wound surface of diabetic mice. Conclusion: This study identifies the molecular biological mechanism of OPN-benefited the migration and proliferation of the HUVECs and provides ideas for searching for new therapeutic targets for drugs that repair diabetes-induced wounds to replace invasive treatment methods. The translational potential of this article: The OPN is highly expressed in the tissues surrounding the TTT bone transfer area, which may possibly stimulate the activation of eNOS to increase NO release through the SOCE pathway mediated by Orai1/STIM1. This mechanism may play a significant role in the angiogenesis of diabetic foot's wounds promoted by TTT, providing new therapeutic strategies for the non-surgical treatment for this disease.
ABSTRACT
Stroke is one of the leading causes of death. (D-ala2, D-leu5) enkephalin (DADLE) is a synthetic peptide and highly selective delta opioid receptor (δOR) agonist that has exhibited protective properties in ischemia. However, the specific target and mechanism are still unclear. The present study explores the expression of δOR on brain microvascular endothelial cells (BMECs) and whether DADLE could relieve I/R-induced injury by reducing apoptosis. A lateral ventricular injection of DADLE for pretreatment, the neurofunctional behavior score, and TTC staining, were used to evaluate the protective effect of DADLE. Immunofluorescence technology was used to label different types of cells with apoptosis-positive signals to test co-localization status. Primary cultured BMECs were separated and treated with DADLE, accompanied by OGD/R. The CCK-8 test was conducted to evaluate cell viability and TdT-mediated dUTP Nick-end Labelling (TUNEL) staining to test apoptosis levels. The levels of apoptosis-related proteins were analyzed by Western blotting. The co-localization results showed that BMECs, but not astrocytes, microglia, or neurons, presented mostly TUNEL-positive signals, especially in the Dentate gyrus (DG) area of the hippocampus. Either activation of δORs on rats' brains or primary BMECs mainly reduce cellular apoptosis and relieve the injury. Interference with the expression δOR could block this effect. DADLE also significantly increased levels of Bcl-2 and reduced levels of Bax. δOR's expressions can be detected on the BMECs, but not on the HEK293 cells, by Western blotting and IFC. Therefore, DADLE exerts a cytoprotective effect, primarily under hypoxia-ischemic injury/reperfusion conditions, by targeting BMECs to inhibit apoptosis.