Herkinorin negatively regulates NLRP3 inflammasome to alleviate neuronal ischemic injury through activating Mu opioid receptor and inhibiting the NF-κB pathway.

Herkinorin is a novel opioid receptor agonist. Activation of opioid receptors, a member of G protein coupled receptors (GPCRs), may play an important role in Herkinorin neuroprotection. GPCRs may modulate NOD-like receptor protein 3 (NLRP3)-mediated inflammatory responses in the mechanisms of inflammation-associated disease and pathological processes. In this study, we investigated the effects of Herkinorin on NLRP3 and the underlying receptor and molecular mechanisms in oxygen-glucose deprivation/reperfusion (OGD/R)-treated rat cortex neurons. First, Western blot analysis showed that Herkinorin can inhibit the activation of NLRP3 and Caspase-1, decrease the expression of interleukin (IL)-1ß, and decrease the secretion of IL-6 and tumour necrosis factor α detected by enzyme-linked immunosorbent assay in OGD/R-treated neurons. Then we found that Herkinorin downregulated NLRP3 levels by inhibiting the activation of nuclear factor kappa B (NF-κB) pathway, reducing the phosphorylation level of p65 and IκBα in OGD/R-treated neurons (p < .05 or .01, n = 3 per group). Instead, both the mu opioid receptor (MOR) inhibitor, ß-funaltrexamine, and MOR knockdown reversed the effects of Herkinorin on NLRP3 (p < .05 or .01, n = 3 per group). Further, we found that the level of ß-arrestin2 decreased in the cell membrane and increased in the cytoplasm after Herkinorin pretreatment in OGD/R-treated neurons. In co-immunoprecipitation experiments, Herkinorin increased the binding of IκBα with ß-arrestin2, decreased the ubiquitination level of IκBα, and ß-arrestin2 knockdown reversed the effects of Herkinorin on IκBα in OGD/R-treated neurons (p < .05 or .01, n = 3 per group). Our data demonstrated that Herkinorin negatively regulated NLRP3 inflammasome to alleviate neuronal ischemic injury through inhibiting NF-κB pathway mediated primarily by MOR activation. Inhibition of the NF-κB pathway by Herkinorin may be achieved by decreasing the ubiquitination level of IκBα, in which ß-arrestin2 may play an important role.

Validation of manufacturers' laryngeal mask airway size selection standard: a large retrospective study.

BACKGROUND: Laryngeal mask airway (LMA) is a prominent supraglottic airway device, widely used especially in difficult airway management. However, the LMA sizes recommended by the manufacturers are not always well matched in clinical practice, which leads to complications. To date, there are rare models to validate whether the manufacturers' standard is suitable for use in clinical practice. METHODS: A total of 58,956 patients undergoing general anesthesia using LMA device were included in the study between January 1, 2011 and December 31, 2018, to validate the adherence rate of LMA sizes according to the manufacturers' recommendations. A logistic regression analysis was performed based on the actual LMA size used in clinical practice to establish separately size selection guidelines with gender, weight, and age as variables in adults, adolescents, and children. RESULTS: LMA insertions were analyzed in 50,776 (86.1%) adults, 3,548 (6%) adolescents, and 4,632 (7.9%) children. Suitability of manufacturers' recommendations was higher in children [male: 86.02%; female: 85.09%] than adults [male: 72.75%; female: 78.13%] or adolescents [male: 73.4%; female: 70.79%]. For adults and adolescents, LMA size was better predicted using the regression model rather than the manufacturers' recommendations [male adults: 82.4% (81.16-83.57%) vs. 73.21% (71.79-74.59%), P<0.05; female adults: 87.82% (86.65-88.9%) vs. 77.07% (75.6-78.48%), P<0.05; male adolescents: 79.45% (74.86-83.4%) vs. 72.05% (67.09-76.53%), P<0.05; female adolescents: 78.4% (71.11-84.31%) vs. 72.22% (64.54-78.82%), P<0.05]. For children, there was equal performance suitability using the regression model and the manufacturers' recommendations. CONCLUSIONS: The model-based guidelines may provide more accurate directions for LMA size selection for adolescents and adults than the manufacturers' weight-based recommendations, whereas the manufacturers' recommendation in children is consistent with clinical practice.

Neurons derived from human-induced pluripotent stem cells express mu and kappa opioid receptors.

Neuroprotection studies have shown that induced pluripotent stem (iPS) cells have the possibility to transform neuroprotection research. In the present study, iPS cells were generated from human renal epithelial cells and were then differentiated into neurons. Cells in the iPS-cell group were maintained in stem cell medium. In contrast, cells in the iPS-neuron group were first maintained in neural induction medium and expansion medium containing ROCK inhibitors, and then cultivated in neuronal differentiation medium and neuronal maturation medium to induce the neural stem cells to differentiate into neurons. The expression of relevant markers was compared at different stages of differentiation. Immunofluorescence staining revealed that cells in the iPS-neuron group expressed the neural stem cell markers SOX1 and nestin on day 11 of induction, and neuronal markers TUBB3 and NeuN on day 21 of induction. Polymerase chain reaction results demonstrated that, compared with the iPS-cell group, TUBB3 gene expression in the iPS-neuron group was increased 15.6-fold. Further research revealed that, compared with the iPS-cell group, the gene expression and immunoreactivity of mu opioid receptor in the iPS-neuron group were significantly increased (38.3-fold and 5.7-fold, respectively), but those of kappa opioid receptor had only a slight change (1.33-fold and 1.57-fold increases, respectively). Together, these data indicate that human iPS cells can be induced into mu opioid receptor- and kappa opioid receptor-expressing neurons, and that they may be useful to simulate human opioid receptor function in vitro and explore the underlying mechanisms of human conditions.

Effects of pumpless extracorporeal lung assist on hemodynamics, gas exchange and inflammatory cascade response during experimental lung injury.

Pumpless extracorporeal lung assist (pECLA) has been reported to efficiently remove the systemic CO2 production and provide mild to moderate oxygenation, thereby allowing for ventilator settings and modes prioritizing oxygenation and lung protection. However, an adequate bypass flow, the capacity to provide respiratory support and the effect on the inflammatory cascade response and tissue perfusion require further study to be determined. After induction of acute lung injury (ALI) by oleic acid injection, pECLA was implemented in 12 anaesthetized and mechanically ventilated dogs for 48 h. Improved oxygenation [partial oxygen pressure (PaO2) and oxygen saturation (SaO2) was measured by arterial blood gas analysis, and increased by 29 and 18%, respectively] and CO2 elimination (partial CO2 pressure decreased by 43.35%) were obtained after pECLA implementation. A maximum arterio-venous shunt flow of up to 25% of the foundational CO resulted in stable hemodynamics. The pECLA procedure did not elicit any further increase in the concentration of tumor necrosis factor-α, interleukin (IL)-6, IL-8 and endothelin-1 compared with that in the group subjected to oleic acid injection only. In addition, the pECLA procedure had no effect on lactate levels and urine production. In conclusion, pECLA is an efficient and promising strategy for providing a mild to moderate oxygenation and adequate decarboxylation, while avoiding excessive inflammatory cascade response and tissue hypoperfusion in an experimental ALI model.

Decellularized Rat Lung Scaffolds Using Sodium Lauryl Ether Sulfate for Tissue Engineering.

Perfusion decellularization with detergents is effective to maintain the architecture and proteins of extracellular matrix (ECM) for use in the field of lung tissue engineering (LTE). However, it is unclear which detergent is ideal to produce an acellular lung scaffold. In this study, we obtained two decellularized rat lung scaffolds using a novel detergent sodium lauryl ether sulfate (SLES) and a conventional detergent sodium dodecyl sulfate (SDS). Both decellularized lung scaffolds were assessed by histology, immunohistochemistry, scanning electron microscopy, DNA quantification, sulfated glycosaminoglycans (GAGs) quantification and western blot. Subsequently, the scaffolds were implanted subcutaneously in rats for 6 weeks and were evaluated via hematoxylin and eosin staining and Masson staining. Results indicated that SLES was effective to remove cells; moreover, lungs decellularized with SLES showed better preservation of sulfated GAGs, lung architecture, and ECM proteins than SDS. After 6 weeks, SLES scaffolds demonstrated a significantly greater potential for cell infiltration and blood vessel formation compared with SDS scaffolds. Taken together, we conclude that SLES is a promising detergent to produce an acellular scaffold using LTE for eventual transplantation.

Exosomes from iPSCs Delivering siRNA Attenuate Intracellular Adhesion Molecule-1 Expression and Neutrophils Adhesion in Pulmonary Microvascular Endothelial Cells.

The pro-inflammatory activation of pulmonary microvascular endothelial cells resulting in continuous expression of cellular adhesion molecules, and subsequently recruiting primed neutrophils to form a firm neutrophils-endothelium (PMN-EC) adhesion, has been examined and found to play a vital role in acute lung injury (ALI). RNA interference (RNAi) is a cellular process through harnessing a natural pathway silencing target gene based on recognition and subsequent degradation of specific mRNA sequences. It opens a promising approach for precision medicine. However, this application was hampered by many obstacles, such as immunogenicity, instability, toxicity problems, and difficulty in across the biological membrane. In this study, we reprogrammed urine exfoliated renal epithelial cells into human induced pluripotent stem cells (huiPSCs) and purified the exosomes (Exo) from huiPSCs as RNAi delivery system. Through choosing the episomal system to deliver transcription factors, we obtained a non-integrating huiPSCs. Experiments in both vitro and vivo demonstrated that these huiPSCs possess the pluripotent properties. The exosomes of huiPSCs isolated by differential centrifugation were visualized by transmission electron microscopy (TEM) showing a typical exosomal appearance with an average diameter of 122 nm. Immunoblotting confirmed the presence of the typical exosomal markers, including CD63, TSG 101, and Alix. Co-cultured PKH26-labeled exosomes with human primary pulmonary microvascular endothelial cells (HMVECs) confirmed that they could be internalized by recipient cells at a time-dependent manner. Then, electroporation was used to introduce siRNA against intercellular adhesion molecule-1 (ICAM-1) into exosomes to form an Exo/siRNA compound. The Exo/siRNA compound efficiently delivered the target siRNA into HMVECs causing selective gene silencing, inhibiting the ICAM-1 protein expression, and PMN-EC adhesion induced by lipopolysaccharide (LPS). These data suggest that huiPSCs exosomes could be used as a natural gene delivery vector to transport therapeutic siRNAs for alleviating inflammatory responses in recipient cells.

Differentiation of Urine-Derived Human Induced Pluripotent Stem Cells to Alveolar Type II Epithelial Cells.

Human alveolar type II (AT II) epithelial cells are valuable for the cellular therapy of lung disease. Human induced pluripotent stem cells (iPSCs) have the ability to generate AT II cells that can be used in modeling and treatment of lung disease caused by dysfunction of AT II cells. In this study, we present a simple, effective, and noninvasive way of obtaining human iPSCs from exfoliated renal epithelial cells, which exist in urine. Alkaline phosphatase (AP) staining, immunofluorescence staining, karyotyping, and teratoma experiments have proved that these iPSCs are pluripotent. Urinary iPSCs (UiPSCs) can differentiate into AT II cells with our four-step induction protocol. These cells have phenotypic properties similar to mature human AT II cells, such as outstretched and epithelium-like morphology and the specific expression markers of AT II cells (surfactant proteins A, B, and C). This study indicates that AT II cells can be generated from UiPSCs and these cells may be useful for the study of human lung development and regenerative medicine.