Morphine Induced Neuroprotection in Ischemic Stroke by Activating Autophagy Via mTOR-Independent Activation of the JNK1/2 Pathway.

Morphine (Mor) has exhibited efficacy in safeguarding neurons against ischemic injuries by simulating ischemic/hypoxic preconditioning (I/HPC). Concurrently, autophagy plays a pivotal role in neuronal survival during IPC against ischemic stroke. However, the involvement of autophagy in Mor-induced neuroprotection and the potential mechanisms remain elusive. Our experiments further confirmed the effect of Mor in cellular and animal models of ischemic stroke and explored its potential mechanism. The findings revealed that Mor enhanced cell viability in a dose-dependent manner by augmenting autophagy levels and autophagic flux in neurons subjected to oxygen-glucose deprivation/reoxygenation (OGD/R). Pretreatment of Mor improved neurological outcome and reduced infarct size in mice with middle cerebral artery occlusion/reperfusion (MCAO/R) at 1, 7 and 14 days. Moreover, the use of autophagy inhibitors nullified the protective effects of Mor, leading to reactive oxygen species (ROS) accumulation, increased loss of mitochondrial membrane potential (MMP) and neuronal apoptosis in OGD/R neurons. Results further demonstrated that Mor-induced autophagy activation was regulated by mTOR-independent activation of the c-Jun NH2- terminal kinase (JNK)1/2 Pathway, both in vitro and in vivo. Overall, these findings suggested Mor-induced neuroprotection by activating autophagy, which were regulated by JNK1/2 pathway in ischemic stroke.

Morphine Preconditioning Alleviates Ischemia/Reperfusion-induced Caspase-8-dependent Neuronal Apoptosis through cPKCγ-NF-κB-cFLIPL Pathway.

BACKGROUND: Perioperative cerebral ischemia/reperfusion injury is a major contributor to postoperative death and cognitive dysfunction in patients. It was reported that morphine preconditioning (MP) can mimic ischemia/hypoxia preconditioning to protect against ischemia/reperfusion injury. However, the mechanism of MP on the ischemia/reperfusion-induced neuronal apoptosis has not been fully clarified. METHODS: The middle cerebral artery occlusion/reperfusion (MCAO/R) model of mice and the oxygen-glucose deprivation/reoxygenation (OGD/R) model in primary cortical neurons were used to mimic ischemic stroke. In vivo, the infarct size was measured by using TTC staining; NDSS, Longa score system, and beam balance test were performed to evaluate the neurological deficits of mice; the expression of the protein was detected by using a western blot. In vitro, the viability of neurons was determined by using CCK-8 assay; the expression of protein and mRNA were assessed by using western blot, RT-qPCR, and immunofluorescent staining; the level of apoptosis was detected by using TUNEL staining. RESULTS: MP can improve the neurological functions of mice following MCAO/R (P<0.001, n=10 per group). MP can decrease the infarct size (P<0.001, n=10 per group) and the level of cleaved-caspase-3 of mice following MCAO/R (P<0.01 or 0.001, n=6 per group). MP can increase the levels of cPKCγ membrane translocation, p-p65, and cFLIPL, and decrease the levels of cleaved-caspase-8, 3 in neurons after OGD/R or MCAO/R 1 d (P<0.05, 0.01 or 0.001, n=6 per group). In addition, MP could alleviate OGD/R-induced cell apoptosis (P<0.001, n=6 per group). CONCLUSION: MP alleviates ischemia/reperfusion-induced Caspase 8-dependent neuronal apoptosis through the cPKCγ-NF-κB-cFLIPL pathway.

Study on the combination of remazolam besylate and sufentanil in elderly patients with percutaneous vertebroplasty.

This study analyzed the application effect of remimazolam besylate combined with sufentanil on percutaneous vertebroplasty (PVP). Forty elderly patients with osteoporotic vertebral compression fractures (OVCF) were randomly divided into sufentanil (A group) and remimazolam besylate + sufentanil groups (B group). A group was given sufentanil anesthesia, B group was given remimazolam besylate combined with sufentanil anesthesia. Heart rate (HR), mean arterial pressure (MAP), saturation of pulse oxygen (SpO2) and Ramsay sedation score were recorded at different time points, including T0 (before administration), T1 (beginning of surgery), T2 (perfusion of bone cement) and T3 (end of surgery). HR, MAP, SpO2 were continuously monitored by electrocardiogram monitor. The visual analogue scale (VAS) pain scores of the two groups were recorded at before anesthesia and immediately after surgery. The adverse reactions (AR) were observed, including respiratory depression, nausea and vomiting, and restlessness during the recovery period. Compared with group A, MAP was increased at T2 and T3 time points in group B. Ramsay sedation scores at T2 time points in group B were higher than that in group A. The VAS score of immediately after operation and incidence of AR of group B was lower than that of group A. In elderly patients undergoing PVP, remimazolam besylate combined with sufentanil has no obvious effect on patients' respiratory parameters, with clear analgesic and sedative effects and low incidence of AR, which is worthy of promotion.

[Effect of Electroacupuncture Intervention on ß-endorphin Levels of Hypothalamus and Spinal Cord Tissues in Rats with Pelmatic Incisional Pain].

OBJECTIVE: To observe the effect of electroacupuncture (EA) intervention on pain thresholds (PT) and contents of ß-endorphin (EP) in the hypothalamus and spinal cord, and the expression of 5-HT in the dorsal raphe nucleus(DRN)in rats with pelmatic incisional pain, so as to investigate the underlying mechanisms of acupuncture in reducing post-operative pain. METHODS: Wistar rats were randomized into normal control, model, EA and non-acupoint groups (n=8/group). The pelmatic pain model was induced by making an incision (about 1 cm in length, to the fascia and muscle layers) from the heel towards the toes. EA (2 Hz, 1.5-2 V) was applied to "Zusanli" (ST 36) and "Kunlun" (BL 60) or non-acupoint (about 3 mm beside the ST 36 and BL 60) on the affected side for 20 min, once daily for three days. The thermal PT and mechanical PT were measured before and after operation and after EA. The contents of ß-EP in hypothalamus and L3-S4 spinal cord were detected using enzyme linked immunosorbent assay (ELISA) and the expressions of ß-EP in hypothalamus and 5-HT in DRN were measured with immunohistochemistry. RESULTS: After EA intervention, the markedly decreased mechanical and thermal pain thresholds on day 1 and 3 after paw incision were significantly increased in the EA group (P<0.05), but not in the non-acupoint group (P>0.05). The hypothalamic ß-EP content was significantly higher in the model group than in the normal group (P<0.05), and further up-regulated in the EA group (not the non-acupoint group) than in the model group (P<0.05). In addition, the hypothalamic ß-EP immunoreactive (IR)-positive cell number and 5-HT immunoactivity level in DRN were also considerably up-regulated in the EA group (P<0.05) but not in the non-acupoint group (P>0.05). No significant changes were found in the lumbar spinal ß-EP contents in the model, EA and non-acupoint groups (P>0.05). CONCLUSIONS: EA stimulation of "Zusanli"(ST 36) and "Kunlun" (BL 60) has an analgesic effect in pelmatic incision pain rats, which may be related to its effects in raising the level of hypothalamic ß-EP and the expression of 5-HT in DRN.

Morphine Preconditioning Downregulates MicroRNA-134 Expression Against Oxygen-Glucose Deprivation Injuries in Cultured Neurons of Mice.

BACKGROUND: Brain protection by narcotics such as morphine is clinically relevant due to the extensive use of narcotics in the perioperative period. Morphine preconditioning induces neuroprotection in neurons, but it remains uncertain whether microRNA-134 (miR-134) is involved in morphine preconditioning against oxygen-glucose deprivation-induced injuries in primary cortical neurons of mice. The present study examined this issue. MATERIALS AND METHODS: After cortical neurons of mice were cultured in vitro for 6 days, the neurons were transfected by respective virus vector, such as lentiviral vector (LV)-miR-control-GFP, LV-pre-miR-134-GFP, LV-pre-miR-134-inhibitor-GFP for 24 hours; after being normally cultured for 3 days again, morphine preconditioning was performed by incubating the transfected primary neurons with morphine (3 µM) for 1 hour, and then neuronal cells were exposed to oxygen-glucose deprivation (OGD) for 1 hour and oxygen-glucose recovery for 12 hours. The neuronal cells survival rate and the amount of apoptotic neurons were determined by MTT assay or TUNEL staining at designated time; and the expression levels of miR-134 were detected using real-time reverse transcription polymerase chain reaction at the same time. RESULTS: The neuronal cell survival rate was significantly higher, and the amount of apoptotic neurons was significantly decreased in neurons preconditioned with morphine before OGD than that of OGD alone. The neuroprotection induced by morphine preconditioning was partially blocked by upregulating miR-134 expression, and was enhanced by downregulating miR-134 expression. The expression of miR-134 was significantly decreased in morphine-preconditioned neurons alone without transfection. CONCLUSIONS: By downregulating miR-134 expression, morphine preconditioning protects primary cortical neurons of mice against injuries induced by OGD.

Impact of microRNA-134 on neural cell survival against ischemic injury in primary cultured neuronal cells and mouse brain with ischemic stroke by targeting HSPA12B.

As a newly discovered member of the HSP70 family, heat shock protein A12B (HSPA12B) is involved in brain ischemic injury. According to our previous study, microRNA-134 (miR-134) could target HSPA12B by binding to its 3'-untranslated region (UTR). However, the regulation of miR-134 on HSPA12B and their role in protecting neuronal cells from ischemic injury are unclear. In this study, the miR-134 expression level was manipulated, and the HSPA12B protein levels were also determined in oxygen-glucose deprivation (OGD)-treated primary cultured neuronal cells in vitro and mouse brain after middle cerebral artery occlusion (MCAO)-induced ischemic stroke in vivo. The results showed that miR-134 expression levels increased in primary cultured neuronal cells and mouse brain from 12h to 7 day reoxygenation/reperfusion after 1h OGD or 1h MCAO treatment. miR-134 overexpression promoted neuronal cell death and apoptosis by decreasing HSPA12B protein levels. Conversely, downregulating miR-134 reduced neuronal cell death and apoptosis by enhancing HSPA12B protein levels. Also, HSPA12B siRNA could block miR-134 inhibitor-mediated neuroprotection against OGD-induced neuronal cell injury in vitro. Taken together, miR-134 might influence neuronal cell survival against ischemic injury in primary cultured neuronal cells and mouse brain with ischemic stroke by negatively modulating HSPA12B protein expression in a posttranscriptional manner.

A retrospective review of the prognostic value of ALDH-1, Bmi-1 and Nanog stem cell markers in esophageal squamous cell carcinoma.

Stem cell markers are upregulated in various cancers and have potential as prognostic indicators. The objective of this study was to determine the expression of three stem cell markers, aldehyde dehydrogenase 1 (ALDH-1), B cell-specific Moloney murine leukemia virus integration site 1 (Bmi-1), and Nanog, in esophageal squamous cell carcinoma (ESCC) tissues. Immunohistochemistry was used to measure the expression of ALDH-1, Bmi-1, and Nanog in ESCC tissues from 41 patients who received pre-operative chemoradiation. We evaluated the relationship between expression of these markers, and clinicopathological features, tumor regression grade (TRG), and 5-year overall survival (OS). There were no significant associations of ALDH-1 or Bmi-1 expression with age, gender, clinical stage, and treatments (p>0.05). However, patients with Nanog-positive tumors were significantly older than those whose tumors were Nanog-negative (p = 0.033). TRG after treatment was significantly associated with expression of ALDH-1 (p = 0.001), Bmi-1 (p = 0.004), and Nanog (p<0.001). Although OS was significantly better in patients with low TRGs (p = 0.001), there were no significant correlations between ALDH-1, Bmi-1, or Nanog with OS. Expression of ALDH-1, Bmi-1, and Nanog correlated with TRG, but not OS. Further large studies are necessary to fully elucidate the prognostic value of these stem cell markers for ESCC patients.