Suppression of circular RNA serum and glucocorticoid-induced kinase 1 elevates antioxidant molecules and angiogenesis in trophoblast cells to attenuate preeclampsia via microRNA-508-3p to target and restrain PUM homolog 1.

AIM: Preeclampsia (PE) is a pregnancy-specific syndrome characterized by hypertension and proteinuria. Recently, multiple circular RNAs (circRNAs) were considered latent clinical diagnostic markers or therapeutic targets. This study was to explore the impact of circRNA serum and glucocorticoid-induced kinase 1 (SGK1) on PE via influencing the microRNA (miR)-508-3p/PUM homolog 1 (PUM1) axis. METHODS: Placental tissues of 34 pregnant women with PE and 34 normal pregnant women were collected to measure circRNA SGK1 (circSGK1), miR-508-3p, and PUM1. Human placental trophoblasts HTR-8/SVneo were transfected with plasmids, thereafter to observe proliferation, migration, invasion, and apoptosis, analyze antioxidant molecules Troxerutin (TXN), Glutamate-cysteine ligase catalytic subunit (GCLC), NAD (P) H-quinone oxidoreductase 1 (NQO1), and determine angiogenesis. After the construction of the PE rat model, antioxidant molecules TXN, GCLC, and NQO1, vascular-associated factor vascular endothelial growth factor A (VEGF-A), and histopathological conditions were tested. Examination of the binding of circSGK1 and miR-508-3p with PUM1 was performed. RESULTS: Our data showed that circSGK1 expression was elevated in the placenta of patients with PE. Silenced circSGK1 or elevated miR-508-3p promoted the growth and antioxidant molecules and angiogenesis in trophoblast cells; CircSGK1 combined with miR-508-3p, and miR-508-3p targeted PUM1. CONCLUSIONS: In summary, suppression of circSGK1 augments antioxidant molecules and angiogenesis in trophoblast cells to attenuate PE via miR-508-3p to target PUM1.

The anti-inflammatory and analgesic effects of intraperitoneal melatonin after spinal nerve ligation are mediated by inhibition of the NF-κB/NLRP3 inflammasome signaling pathway.

OBJECTIVE: To explore the potential analgesic effect of melatonin and its underlying molecular mechanisms in a neuropathic pain model induced by spinal nerve ligation (SNL). METHODS: The experimental animals were divided into different groups including sham, vehicle, melatonin (MT) treatment, caspase-1 inhibitor (VX-765) treatment and MT2 antagonist (4P-PDOT) treatment. On the first three successive postoperative days, rats were intraperitoneally administered with MT, VX-765 or combination of MT and 4P-PDOT. Hyperalgesic behavior after SNL was evaluated using the paw withdrawal threshold (PWT). We then assessed expression of tumor necrosis factor-α (TNF-α), IL-18, interleukin-1ß (IL-1ß), NLRP3 inflammasome components, and nuclear factor-κB (NF-κB) activation using enzyme-linked immunosorbent assay kits (ELISA), real-time PCR, immunohistochemistry, and western blot, respectively, in spinal cord horn tissues extracted on postoperative day 7. RESULTS: The results showed that melatonin treatment alleviated SNL-induced allodynia. We observed an SNL-induced upregulation of TNF-α, IL-18, IL-1ß, NLRP3, ASC, cleaved caspase-1, and NF-κB in the lumbar spinal cord horn of rats, which was significantly attenuated by intraperitoneal injection of melatonin or VX-765. Additionally, co-treatment of melatonin and 4P-PDOT abrogated the analgesic and anti-inflammatory effect of melatonin. CONCLUSION: Melatonin had potent analgesic and anti-inflammatory effects in SNL-induced neuropathic pain via NF-κB/NLRP3 inflammasome signaling pathway. Our results therefore suggested that this pathway could represent a novel therapeutic target for the management of neuropathic pain.

The Protective Effects of Juglanin in Cerebral Ischemia Reduce Blood-Brain Barrier Permeability via Inhibition of VEGF/VEGFR2 Signaling.

INTRODUCTION: Ischemic brain injury due to stroke or other pathologies is a major contributor to disability and mortality worldwide. Upon the occurrence of stroke, neuronal cells undergo apoptosis due to the deprivation of oxygen and nutrients and failure of the blood-brain barrier (BBB). In the moments immediately following a stroke, widespread perfusion resulting from hyperpermeability is accompanied by an acute inflammatory response, which induces neovascularization and often permanent neurological injury. Vascular endothelial growth factor (VEGF) and its receptor VEGF receptor 2 (VEGFR2) have been targeted to suppress cerebral ischemia. Recently, natural products including flavonoids, such as juglanin, have been receiving increasing attention for their impressive physiological effects. METHODS: Twenty mg/kg body weight juglanin was administrated for 3 weeks before inducing middle cerebral artery occlusion (MCAO) in mice. The animal brain infarction volume, neurological deficit score, blood-brain barrier permeability, and the expression of tight junction proteins were evaluated. Endothelial permeability and tight junction protein expression were also assessed in brain microvascular endothelial cells (HMBVECs) exposed to oxygen-glucose deprivation/reperfusion (OGD/R). RESULTS: Juglanin significantly reduced occlusion-induced infarct volume and improved neurological score by suppressing BBB hyperpermeability. Juglanin inhibited both the mRNA and protein expression of VEGF and VEGFR2 and restored the normal expression of occludin and zonula occludens-1 (ZO-1), two important tight junction proteins, in MCAO mice. Meanwhile, the results of in vitro experiments show that the protective effects of juglanin against increased BBB permeability and reduced tight junction functionality are dependent on the VEGF/VEGFR2 signaling pathway, as evidenced by the capacity of exogenous VEGF-A to abolish the effects of juglanin. CONCLUSION: Our findings indicate a potent ability of juglanin to prevent neuronal injury resulting from cerebral ischemia by modulating the VEGF/VEGFR2 signaling pathway. Further research will help elucidate the exact mechanisms behind the protective effects of juglanin.

MicroRNA-665 mediates propofol-induced cell apoptosis in human stem cell-derived neurons.

We aimed to evaluate the neurotoxicity and mechanisms of anesthetics propofol in hESC-derived neurons. Cell apoptosis in hESC-derived neurons' exposure to 4, 10 and 20 µg/mL propofol for 6 h was assessed using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate in situ nick end labeling (TUNEL) staining and microRNA-665 (miR-665) expression was assessed using quantitative reverse transcription polymerase chain reaction (qRT-PCR). miR-665 was overexpressed and knocked down using a miR-665 mimic and anti-665 transfection, respectively. The results showed that hESCs exposed to propofol showed a dose-dependent cell apoptosis, followed by the upregulation of miR-665 expression. Overexpression of miR-665 increased propofol-induced apoptosis in hESC cells. And targeting miR-665 decreased propofol-induced cell apoptosis in hESC cells. These data suggest that propofol induces cell death in hESC-derived neurons and the propofol-induced cell apoptosis may occur via miR-665-dependent mechanism.

SC79, the AKT Activator Protects Cerebral Ischemia in a Rat Model of Ischemia/Reperfusion Injury.

BACKGROUND Activation of AKT pathway attenuates brain damage and neuronal apoptosis during cerebral ischemia/reperfusion (I/R) injury. SC79 is a novel, selective and highly-efficient Akt activator. This study aimed to investigate the neuroprotective effect of SC79 against cerebral I/R injury in a rat model, and to explore the possible underlying mechanisms. MATERIAL AND METHODS Male Sprague-Dawley rats received cerebral ischemia for 1 hour, followed by brain reperfusion for 0.5-24 hours. The cerebral I/R injury animal model were treated with SC79 alone or SC79 in combination with LY294002. Western blots were used to detect the levels of expression of phosphatidylinositol AKT (p-Akt), Bax, and bcl-2. Twenty-four hours after cerebral I/R, the degree of brain injury was evaluated by detecting the neurological deficit score (NDS). The infarct rate of brain tissue was observed by TTC (2, 3, 5-triphenyltetrazolium chloride) staining. TUNEL (terminal deoxynucleotidyl transferase-mediated UTP nick end labeling) staining was used to detect cell apoptosis. RESULTS p-Akt was activated during early cerebral I/R at 0.5 hours, and reached the highest levels at 4 hours, then gradually decreased from 6 hours, and reached and maintained the lowest levels at 12-24 hours. Bax expression was gradually increased from 6 hours and reached the highest level at 24 hours. However, bcl-2 expression was gradually increased and reached the highest levels at 4 hours, then gradually decreased from 6 hours, and reached the lowest levels at 24 hours. Administration of SC79 decreased infarct volumes and improved neurological function significantly. LY294002 in combination with SC79 lost the capability of SC79 to resist the cerebral I/R injury. SC79 treatment alone activated p-Akt and promoted anti-apoptotic bcl-2 and inhibited anti-apoptotic Bax expression in middle cerebral artery occlusion (MCAO) mice. However, combined SC79 and LY294002 treatment abolished SC79-induced p-Akt activity, inhibited anti-apoptotic bcl-2 and promoted anti-apoptotic Bax expression in MCAO mice. Furthermore, SC79 treatment alone attenuated apoptotic neuronal cell death, but abolished this effect in SC79 in combination with LY294002 treated groups. CONCLUSIONS SC79 significantly increased Akt activation and reduced infarct volume and subsequently improved neurological function in ischemic brain after cerebral I/R injury in rats. These findings suggested that SC79 may be as a neuroprotective drug to be potentially used in the clinic.

Clusterin Reduces Cold Ischemia-Reperfusion Injury in Heart Transplantation Through Regulation of NF-kB Signaling and Bax/Bcl-xL Expression.

BACKGROUND/AIMS: Ischemia-reperfusion (I/R) injury is an unavoidable event occurring during heart transplantation and is a key factor in graft failure and the long-term survival rate of recipients. Therefore, there is an urgent need for the development of new therapies to prevent I/R injury. Clusterin is a hetero-dimeric glycoprotein with an antiapoptotic function. In this study, we investigated whether clusterin was cardioprotective in heart transplantation against I/R injury using an in vivo rat model and an in vitro cell culture system, and examined the underlying mechanisms of I/R injury. METHODS: Heart grafts from wild-type C57BL/6 mice were preserved in UW solution (control) or UW solution containing recombinant human apolipoprotein-J (hr clusterin) for 24 h. The preserved hearts were implanted into recipient mice of the same strain as the donors for 72 h, and the heart grafts were then taken for histopathological and gene expression analyses. An in vitro ischemia reperfusion model using H9C2 cells or H9C2/clusterin cDNA cells was constructed. The expression of clusterin, p65, Bax, Bcl-xL, IL-1ß, and TNF-α protein and mRNA in heart tissue and H9C2 cells was detected by western blot, reverse transcription-polymerase chain reaction (RT-PCR), and quantitative RT-PCR assays; IL-1ß and TNF-α protein was detected by enzyme-linked immunosorbent assays; NF-kB activity was detected by an electrophoretic mobility shift assay; cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and flow cytometric analyses. RESULTS: Cold I/R caused severe morphologic myocardial injury to heart grafts from wild-type C57BL/6 mice, whereas grafts from hr clusterin preservation showed less damage, as demonstrated by decreased cell apoptosis/death, decreased neutrophil infiltration, and the preservation of the normal structure of the heart. Clusterin reduced the expression of p65, pre-inflammatory IL-1ß, and TNF-α, and the pro-apoptotic gene Bax, while it enhanced the expression of the anti-apoptotic gene Bcl-xL in vitro and in vivo. Clusterin inhibited cell apoptosis/death and reduced pre-inflammatory. CONCLUSION: Clusterin is a promising target for preventing cold I/R injury in heart transplantation. This study also shows that the resultant protective effects of clusterin are mediated by NF-κB signaling and Bax/Bcl-xL expression.

Protective effects of the AKT activator SC79 on renal ischemia-reperfusion injury.

BACKGROUND AND AIMS: SC79, an AKT activator, has been reported to protect experimental ischemia-elicited neuronal death, brain injury, and myocardiocyte hypoxia/reoxygenation (H/R) injury. However, the protection of SC79 from renal ischemia-reperfusion (I/R) injury and the precise mechanisms involved are unknown. Here, we investigated the effects of SC79 in renal tubular epithelial cells in vitro and in mouse kidney in vivo following hypoxia-reoxygenation (H/R) and renal I/R injury. METHODS: The kidneys of Sprague-Dawley rats were subjected to 30 min of warm ischemia followed by 24 h of reperfusion. Murine renal tubular epithelial NRK-52E cells were subjected to hypoxia for 6 h and reoxygenation for 24 h. The NRK-52E cells and the renal I/R injury model were treated with SC79 and/or LY294002 at different times and concentrations. Serum creatinine (Cr) concentration, renal histology, cellular viability, and cell apoptosis were assessed. Levels of phospho-Akt, bad, Bim, bax, bcl-2, and bcl-XL in NRK-52E cells and renal tissues were determined by western blotting. RESULTS: SC79 improved viability and inhibited apoptosis in NRK-52E cells following H/R. SC79 decreased serum Cr and markedly improved pathology and decreased cell apoptosis in kidneys following I/R. In addition, SC79 promoted the expression of phospho-Akt, bcl-2, and bcl-XL, and decreased the expression levels of bid, bax, and bim. PI3K inhibitor (LY294002) pre-treatment completely abolished these effects of SC79. CONCLUSIONS: The protective role of SC79 against H/R of NRK-52E cells or renal I/R injury is related to activation of phosphorylation of AKT, resulting in a decrease in the pro-apoptotic proteins bim, bax, and bad and an increase in the anti-apoptotic proteins bcl-2 and bcl-XL induced by cell H/R and renal I/R injury.

Effects of sevoflurane on rats with ischemic brain injury and the role of the TREK-1 channel.

The purpose of this investigation was to determine the effects of sevoflurane on rats with ischemic brain injury and to determine the potential role of the TREK-1 channel in this process. Normal rats were randomly divided into three groups: Sham operation, sevoflurane anesthesia or chloral hydrate anesthesia group, an additional group of TREK-1 knockout rats were also studied. Semi-quantitative PCR and western blot analysis confirmed the lack of TREK-1 expression in the brain of TREK-1 knockout rats. The thread-tie method was used to establish middle cerebral artery occlusion (MCAO) model to induce cerebral ischemic brain injury. All rates were treated for 4 days prior to ischemia (for 2 h) followed by a 24 h reperfusion period. Physiological indexes of rats in each group both prior to and after surgery showed no statistical difference (P>0.05). Neurological function was scored both before (no statistical difference) and after surgery where it was found to be significantly better (lower score) in the sevoflurane anesthesia group than in chloral hydrate anesthesia and TREK-1 knockout groups (P<0.01). The area of cerebral infarction was measured by triphenyl tetrazolium chloride staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay to detect the apoptosis of brain cells. TTC staining showed different degrees of cerebral infarction in the various groups; the area of cerebral infarction in sevoflurane anesthesia group was significantly lower than that in chloral hydrate anesthesia and TREK-1 knockout groups (P<0.01). TUNEL assay showed that the number of TUNEL-positive cells was significantly lower in sevoflurane anesthesia group than in TREK-1 knockout and chloral hydrate anesthesia groups (P<0.01). In conclusion, results from this investigation showed that sevoflurane can protect the nerve function of rats with cerebral ischemic brain injury possibly by affecting the expression of proteins involved in the TREK-1 signaling pathway.