Dexmedetomidine promotes colorectal cancer progression via Piwil2 signaling.

PURPOSE: α2-adrenoceptor agonist dexmedetomidine (DEX) has been reported to promote tumorigenesis. Stem-cell protein Piwil2 is associated with cancer progression. Whether Piwil2 plays a role in tumor-promoting effects of DEX is unknown. METHODS: We examined the expression of Piwil2 in human colorectal cancer (CRC) cell lines with/without DEX treatment. We also studied the roles of Piwil2 in proliferation, invasion, migration, as well as expressions of epithelial-mesenchymal transition (EMT)-related proteins in DEX-treated in vitro and in vivo CRC models. And the experiments with genetic and pharmacological treatments were conducted to investigate the underlying molecular mechanism. RESULTS: RNA-sequencing (RNA-seq) analysis found Piwil2 is one of most upregulated genes upon DEX treatment in CRC cells. Furthermore, Piwil2 protein levels significantly increased in DEX-treated CRC cancer cells, which promoted proliferation, invasion, and migration in both CRC cell lines and human tumor xenografts model. Mechanistically, DEX increased nuclear factor E2-related factor 2 (Nrf2) expression, which enhanced Piwil2 transcription via binding to its promoter. Furthermore, in vitro experiments with Piwil2 knockdown or Siah2 inhibition indicated that DEX promoted EMT process and tumorigenesis through Siah2/PHD3/HIF1α pathway. The experiments with another α2-adrenoceptor agonist Brimonidine and antagonists yohimbine and atipamezole also suggested the role of Piwil2 signaling in tumor-promoting effects via an α2 adrenoceptor-dependent manner. CONCLUSION: DEX promotes CRC progression may via activating α2 adrenoceptor-dependent Nrf2/Piwil2/Siah2 pathway and thus EMT process. Our work provides a novel insight into the mechanism underlying tumor-promoting effects of α2-adrenoceptor agonists.

Parecoxib sodium attenuates acute lung injury following burns by regulating M1/M2 macrophage polarization through the TLR4/NF-κB pathway.

High temperature-induced burn injury often leads to an excessive inflammatory cascade resulting in multiple organ dysfunction syndrome, such as acute lung injury (ALI), in addition to skin tissue damage. As a specific COX2 inhibitor, parecoxib sodium suppresses the inflammatory response during burn injury. The effect of parecoxib sodium on ALI induced by burn injury and the associated molecular mechanism still need to be investigated. The role of parecoxib sodium in burn injury-induced ALI through the TLR4/NF-κB pathway was explored in the present study. A burn-induced ALI mouse model was constructed, and M1/M2 macrophages in lung tissue and markers involved in the TLR4/NF-κB signalling pathway were evaluated in bronchoalveolar lavage fluid (BALF) and MH-S mouse alveolar macrophages in vitro. The results indicated that parecoxib sodium attenuated lung injury after burn injury, decreased iNOS and TNF-α expression, increased IL-10 expression in BALF, and regulated the CD86-and CD206-mediated polarization of M1/M2 macrophages in lung tissue along with MH-S mouse alveolar macrophages. The effect of parecoxib sodium might be reversed by a TLR4 agonist. Overall, the results suggested that parecoxib sodium can regulate the polarization of M1/M2 macrophages through the TLR4/NF-κB pathway to attenuate ALI induced by skin burns.

Propofol modulates neural dynamics of thalamo-cortical system associated with anesthetic levels in rats.

The thalamocortical system plays an important role in consciousness. How anesthesia modulates the thalamocortical interactions is not completely known.  We simultaneously recorded local field potentials(LFPs) in thalamic reticular nucleus(TRN) and ventroposteromedial thalamic nucleus(VPM), and electrocorticographic(ECoG) activities in frontal and occipital cortices in freely moving rats (n = 11). We analyzed the changes in thalamic and cortical local spectral power and connectivities, which were measured with phase-amplitude coupling (PAC), coherence and multivariate Granger causality, at the states of baseline, intravenous infusion of propofol 20, 40, 80 mg/kg/h and after recovery of righting reflex. We found that propofol-induced burst-suppression results in a synchronous decrease of spectral power in thalamus and cortex (p < 0.001 for all frequency bands). The cross-frequency PAC increased by propofol, characterized by gradually stronger 'trough-max' pattern in TRN and stronger 'peak-max' pattern in cortex. The cross-region PAC increased in the phase of TRN modulating the amplitude of cortex. The functional connectivity (FC) between TRN and cortex for α/ß bands also significantly increased (p < 0.040), with increased directional connectivity from TRN to cortex under propofol anesthesia. In contrast, the corticocortical FC significantly decreased (p < 0.047), with decreased directional connectivity from frontal cortex to occipital cortex. However, the thalamothalamic functional and directional connectivities remained largely unchanged by propofol anesthesia.  The spectral powers and connectivities are differentially modulated with the changes of propofol doses, suggesting the changes in neural dynamics in thalamocortical system could be used for distinguishing different vigilance levels caused by propofol. Supplementary Information: The online version contains supplementary material available at 10.1007/s11571-022-09912-0.

Methylene blue targets PHD3 expression in murine microglia to mitigate lipopolysaccharide-induced neuroinflammation and neurocognitive impairments.

Methylene blue (MB) has anti-inflammatory properties, however, its underlying molecular mechanism remains elusive. This study aimed to investigate whether and how MB could attenuate lipopolysaccharide (LPS)-induced microglial activation, neuroinflammation, and neurobehavioral deficits. We measured the expression of pro-inflammatory factors and performed three neurobehavioral tests to assess the effect of MB on neuroinflammation and neurocognitive dysfunction in LPS-treated adult C57BL/6N male mice or LPS-stimulated microglia cells. In vitro and in vivo experiments were further performed to investigate the molecular mechanism underlying MB inhibition of neuroinflammation using various experimental methods, including western blot, RT-qPCR, immunofluorescence, seahorse measurement, positron emission tomography (PET) scan, and flow cytometry analyses. Our results demonstrated that microglial activation and M1 polarization were induced by LPS exposure, resulting in an inflammatory response and neuronal apoptosis. Furthermore, LPS induced metabolic reprogramming in microglial cells. However, MB treatment substantially inhibited LPS-induced elevated levels of pro-inflammatory factors and reversed metabolic activation in vivo, which eventually led to the resolution of neuroinflammation and neurobehavioral improvement. Mechanistically, MB specifically inhibited the LPS-induced overexpression of PHD3 in vitro and in vivo. The pharmacological and genetic manipulations unveiled that the Siah2/Morg1/PHD3 signaling pathway may mediate MB protection against LPS-induced neuroinflammation and neurotoxicity. Therefore MB inhibited PHD3-dependent neuroinflammation may via Siah2/Morg1/PHD3 pathway, and that PHD3 expressed in microglia may be a drug target for the treatment of neuroinflammation-related brain disorders.

Efficient oral delivery of water-soluble CT contrast agent using an W1/O/W2 alginate hydrogel matrix.

HYPOTHESIS: Iohexol (IOH) is a commonly used second-generation nonionic iodinated contrast agent. However, low gastrointestinal mucosal adherence and high downstream speed limit its application in intestinal Computed tomography (CT) imaging. We hypothesize that oral IOH delivery carriers composed of environmentally responsive materials enable the intestinal targeted delivery and prolong the intestinal residence time of IOH, enhancing the intestinal disease detection efficiency. EXPERIMENTS: An emulsion-filled alginate hydrogel system was developed as the intestinal targeting vehicle for IOH. The formulation optimization was determined by response surface analysis. After a thorough study of the physicochemical properties of this hydrogel matrix, the pH sensitivity and the ability to control release were investigated, followed by a vitro cell experiment evaluating its bioactivity and CT imaging capability. FINDINGS: This alginate hydrogel matrix was sparsely structured and rapidly released IOH at pH 7.4. Meanwhile the swelling degree was 4.4 times higher than that at pH 1.2, indicating a good selective responsiveness to the gastrointestinal simulated environment. It improved the CT visual contrast of A549 cells without affecting cell morphology, suggesting that it would be an effective oral administration for water-soluble nonionic contrast agents and a potential candidate for intestinal disease detection tools.

Predictive Value of Eosinophil Count on COVID-19 Disease Progression and Outcomes, a Retrospective Study of Leishenshan Hospital in Wuhan, China.

BACKGROUND: The potential protective role of eosinophils in the COVID-19 pandemic has aroused great interest, given their potential virus clearance function and the infection resistance of asthma patients to this coronavirus. However, it is unknown whether eosinophil counts could serve as a predictor of the severity of COVID-19. METHODS: A total of 1004 patients with confirmed COVID-19 who were admitted to Leishenshan Hospital in Wuhan, China, were enrolled in this study, including 905 patients in the general ward and 99 patients in the intensive care unit (ICU). We reviewed their medical data to analyze the association between eosinophils and ICU admission and death. RESULTS: Of our 1004 patients with COVID-19, low eosinophil counts/ratios were observed in severe cases. After adjusting for confounders that could have affected the outcome, we found that eosinophil counts might not be a predictor of ICU admission. In 99 ICU patients, 58 of whom survived and 41 of whom died, low eosinophil level was an indicator of death in severe COVID-19 patients with a cutoff value of 0.04 × 109/L, which had an area under the curve of 0.665 (95% CI = 1.089-17.839; P = .045) with sensitivity and specificity of 0.569 and 0.7317, respectively. CONCLUSION: Our research revealed that a low eosinophil level is a predictor of death in ICU patients rather than a cause of ICU admission.

Inhibition or Deletion of Hydroxylases-Prolyl-4-Hydroxyases 3 Alleviates Lipopolysaccharide-induced Neuroinflammation and Neurobehavioral Deficiency.

It is well known that neuroinflammation plays a key role in neurodegenerative diseases. Hypoxia-inducible factor (HIF) and its hydroxylases-Prolyl-4-hydroxyases (PHDs) have been found to modulate the inflammatory processes. Here, the effects of PHDs enzyme onlipopolysaccharide-induced neuroinflammation and neurocognitive deficits were investigated. BV2 microglia cells were stimulated by LPS (1 µg/ml) as neuroinflammation model in vitro. Dimethyloxalylglycine (DMOG, 100 µM) and PHD3-siRNA were used to suppress the expression of PHD3. In vivo, mice received consecutive intraperitoneal injection of LPS (500 µg/kg) for 7 days, and intraperitoneal injection of DMOG (100 mg/kg) was applied 1 h before LPS at the same days. Several neurobehavioral tests (Open field, Novel object recognition and Morris water maze) were used to measure cognitive function. RT-qPCR and Western blotting were used to investigate the expression of inflammatory cytokines, HIF-PHDs protein. Metabolic reprogramming was measured by seahorse method. The results revealed that LPS induced neuroinflammation and PHD3 expression in vivo and vitro. DMOG and PHD3knockout decreased expression of inflammatory cytokines and improved the metabolic reprogramming caused by LPS treatment. Furthermore, pretreatment of DMOG reversed learning and memory deficits in systemic LPS-exposed mice through anti-neuroinflammation, which is independent of DMOG angiogenesis. These findings suggested that PHD3 may mediate LPS-induced microglial activation and neuroinflammation-associated neurobehavioral deficits.

Discovery of 4-aminopyrimidine analogs as highly potent dual P70S6K/Akt inhibitors.

Activation of the PI3K/Akt/mTOR kinase pathway is associated with human cancers. A dual p70S6K/Akt inhibitor is sufficient to inhibit strong tumor growth and to block negative impact of the compensatory Akt feedback loop activation. A scaffold docking strategy based on an existing quinazoline carboxamide series identified 4-aminopyrimidine analog 6, which showed a single-digit nanomolar and a micromolar potencies in p70S6K and Akt enzymatic assays. SAR optimization improved Akt enzymatic and p70S6K cellular potencies, reduced hERG liability, and ultimately discovered the promising candidate 37, which exhibited with a single digit nanomolar value in both p70S6K and Akt biochemical assays, and hERG activities (IC50 = 17.4 µM). This agent demonstrated dose-dependent efficacy in inhibiting mice breast cancer tumor growth and covered more than 90% pS6 inhibition up to 24 h at a dose of 200 mg/kg po.

Orthogonal Regulation of Nucleophilic and Electrophilic Sites in Pd-Catalyzed Regiodivergent Couplings between Indazoles and Isoprene.

Depending on the reactant property and reaction mechanism, one major regioisomer can be favored in a reaction that involves multiple active sites. Herein, an orthogonal regulation of nucleophilic and electrophilic sites in the regiodivergent hydroamination of isoprene with indazoles is demonstrated. Under Pd-hydride catalysis, the 1,2- or 4,3-insertion pathway with respect to the electrophilic sites on isoprene could be controlled by the choice of ligands. In terms of the nucleophilic sites on indazoles, the reaction occurs at either the N1 - or N2 -position of indazoles is governed by the acid co-catalysts. Preliminary experimental studies have been performed to rationalize the mechanism and regioselectivity. This study not only contributes a practical tool for selective functionalization of isoprene, but also provides a guide to manipulate the regioselectivity for the N-functionalization of indazoles.

Preoperative Chronic and Acute Pain Affects Postoperative Cognitive Function Mediated by Neurotransmitters.

The effective prevention of postoperative cognitive dysfunction (POCD) needs to be explored, and the effect of preoperative pain on POCD remains unclear. We established a chronic pain model induced by chronic constriction injury (CCI) and models of acute pain and anxiety without pain in mice that were subsequently subjected to partial hepatectomy surgery. Morris water maze (MWM) tests were performed to evaluate the learning and memory abilities of the mice. ELISA was used to measure IL-1ß, IL-6, and TNF-α in serum, and HPLC-MS was used to detect neurotransmitters in the prefrontal cortices and hippocampi of the mice. The results indicated that chronic pain induced by CCI might have significantly impaired the learning and memory abilities of mice, while acute pain and anxiety without pain only affected the memory abilities of mice. Perioperative acute pain increased the level of IL-1ß in serum, and CCI might have increased the level of IL-6. CCI and acute pain increased dopamine (DA) levels in the cortex, similar to anxiety. Like anxiety, CCI increased 5-hydroxytryptamine (5-HT) levels in the prefrontal cortex and hippocampus. Acute pain led to a decrease in the acetylcholine (ACH) level in the hippocampus. Our results suggest that acute pain and CCI-induced chronic pain might aggravate postoperative cognitive dysfunction via neurotransmitters and by changing the levels of inflammatory factors such as IL-1ß and IL-6.

Preoperative Acute Pain Is Associated with Postoperative Delirium.

BACKGROUND: Studies have provided some evidence that pain is a risk factor for postoperative delirium (POD). Therefore, we investigated the relationship between preoperative pain and POD after noncardiac surgery. METHODS: POD was assessed with the Montreal Cognitive Assessment, and preoperative cognition was assessed with the Mini-Mental State Examination. Plasma C-reactive protein (CRP) was detected by enzyme-linked immunosorbent assay before surgery. Preoperative pain was classified by its duration before surgery as chronic pain (lasting more than 1 month), acute pain (lasting less than 1 month), or no pain (no obvious pain). Multiple linear regression was used to adjust for confounding. RESULTS: From October 15, 2018, through August 12, 2019, a total of 67 patients were randomized; 7 were excluded because they were discharged before the seventh postoperative day. The prevalence of POD was significantly higher in the acute pain group (13 of 20; 65%) than in the chronic pain group (5 of 20; 25%) or the no pain group (6 of 20; 30%) (P = 0.019), indicating that delirium is associated with preoperative acute pain. The plasma level of preoperative CRP was also higher in the acute pain group than in the other two groups (mean [interquartile range]: 10.7 [3.3, 29.3] vs 1 [0.5, 3.8]mg/l; P < 0.001), suggesting that elevated preoperative plasma levels of CRP were associated with delirium. CONCLUSIONS: Preoperative acute pain was associated with POD, and increased plasma levels of CRP provide a marker. In addition, we found that illiteracy and advanced age were risk factors for POD.

Multiple Anesthesia/Surgery Cannot Impair Reference Memory in Adult Mice.

Postoperative cognitive dysfunction increases mortality and morbidity in perioperative patients. Numerous studies have demonstrated that multiple surgery/anesthesia during the neurodevelopmental period affects cognitive function, whereas a single anesthesia/surgery rarely causes cognitive dysfunction in adults. However, whether adults who undergo multiple anesthesia/surgery over a short period will experience cognitive dysfunction remains unclear. In this study, central nervous system inflammation and changes in cholinergic markers were investigated in adult mice subjected to multiple laparotomy procedures over a short period of time. The results showed that despite the increased expression of IL-6 and TNF-α in the hippocampus after multiple operations and the activation of microglia, multiple anesthesia/surgery did not cause a decline in cognitive function in adult mice. There were no changes in the cholinergic markers after multiple anesthesia/surgery.

Discovery of novel Cyclophilin D inhibitors starting from three dimensional fragments with millimolar potencies.

Fragment-based screening by SPR enabled the discovery of chemical diverse fragment hits with millimolar binding affinities to the peptidyl-prolyl isomerase Cyclophilin D (CypD). The CypD protein crystal structures of 6 fragment hits provided the basis for subsequent medicinal chemistry optimization by fragment merging and linking yielding three different chemical series with either urea, oxalyl or amide linkers connecting millimolar fragments in the S1' and S2 pockets. We successfully improved the in vitro CypD potencies in the biochemical FP and PPIase assays and in the biophysical SPR binding assay from millimolar towards the low micromolar and submicromolar range by >1000-fold for some fragment derivatives. The initial SAR together with the protein crystal structures of our novel CypD inhibitors provide a suitable basis for further hit-to-lead optimization.

Central cholinergic neuronal degeneration promotes the development of postoperative cognitive dysfunction.

Postoperative cognitive dysfunction (POCD) is consistently associated with increased morbidity and mortality. However, its mechanism remains poorly understood. We hypothesized that central cholinergic neuronal degeneration facilitates the development of POCD. The impact of anesthesia/surgery (appendectomy) on learning and memory and the levels of choline acetyltransferase (ChAT), acetylcholinesterase (AChE), vesicular acetylcholine transporter (VAChT), and choline transporter (CHT) in adult and aged mice were measured. Separate cohorts were analyzed after pretreatment with donepezil, an AChE inhibitor, in aged mice or with murine-p75-saporin (mu-p75-sap), a cholinergic-specific immunotoxin, in adult mice. Morris Water Maze was used to measure the learning and memory changes after anesthesia/surgery. Western blot was used to measure the changes in the protein levels of the biomarkers of the central cholinergic system. We found that anesthesia/surgery-induced memory decline and attenuation of central cholinergic biomarkers (ChAT and VAChT) in aged mice but not in adult mice. Donepezil pretreatment reduced central cholinergic impairment in the aged mice and prevented learning and memory declines after anesthesia/surgery. In contrast, when central cholinergic neurons were pre-injured with mu-p75-sap, cognitive dysfunction developed in the adult mice after anesthesia/surgery. These data suggest that central cholinergic neuronal degeneration facilitates the development of POCD.

Central cholinergic system mediates working memory deficit induced by anesthesia/surgery in adult mice.

Background: Postoperative cognitive dysfunction (POCD) is consistently associated with increased morbidity and mortality, which has become a major concern of patients and caregivers. Although POCD occurs mainly in aged patients, it happens at any age. Previous studies demonstrated that anesthesia/surgery had no effects on reference memory of adult mice. However, whether it impairs working memory remains unclear. Working memory deficit would result in many deficits of executive function. We hypothesized that anesthesia/surgery impaired the working memory of adult mice and the central cholinergic system was involved. Method: Tibial fracture internal fixation under the anesthesia of isoflurane was performed in two-month-old C57BL/6 mice. Two days later, the spatial reference memory and working memory were measured by a Morris Water Maze (MWM). Donepezil, an inhibitor of acetylcholinesterase (AChE), was administered in another cohort mice for 4 weeks. Then, the working memory was measured by MWM 2 days after anesthesia/surgery. Western blot was used to detect the protein levels of acetylcholine transferase (ChAT), AChE, vesicular acetylcholine transporter (VAChT), and choline transporter (ChT) in the prefrontal cortex (PFC). Results: We found that anesthesia/surgery had no effects on the reference memory, but it impaired the working memory in adult mice. Meanwhile, we also found that the protein level of ChAT in PFC decreased significantly compared with that in control group. Donepezil pretreatment prevented working memory impairment and the decrease of the protein levels of ChAT induced by anesthesia/surgery. Conclusion: These results suggest that anesthesia/surgery leads to working memory deficits in adult mice and central cholinergic system impairment is involved.

A loop region of BAFF controls B cell survival and regulates recognition by different inhibitors.

The B cell survival factor (TNFSF13B/BAFF) is often elevated in autoimmune diseases and is targeted in the clinic for the treatment of systemic lupus erythematosus. BAFF contains a loop region designated the flap, which is dispensable for receptor binding. Here we show that the flap of BAFF has two functions. In addition to facilitating the formation of a highly active BAFF 60-mer as shown previously, it also converts binding of BAFF to TNFRSF13C (BAFFR) into a signaling event via oligomerization of individual BAFF-BAFFR complexes. Binding and activation of BAFFR can therefore be targeted independently to inhibit or activate the function of BAFF. Moreover, structural analyses suggest that the flap of BAFF 60-mer temporarily prevents binding of an anti-BAFF antibody (belimumab) but not of a decoy receptor (atacicept). The observed differences in profiles of BAFF inhibition may confer distinct biological and clinical efficacies to these therapeutically relevant inhibitors.

Data for the crystal structure of APRIL-BAFF-BAFF heterotrimer.

The TNF family ligands B cell activation factor (BAFF) and a proliferation-inducing ligand (APRIL) modulate B cell function by forming homotrimers and heterotrimers. To determine the structure of a heterotrimer of BAFF and APRIL, these ligands were expressed as a single chain protein in HEK 293 cells, purified by affinity and size exclusion chromatographies, and crystallized. Crystals belonging to the orthorhombic crystal system with a space group of C2221 diffracted to 2.43 Å. Initial structural solution was obtained by the molecular replacement method, and the structure was further refined to an R factor of 0.179 and free R factor of 0.234. The atomic coordinates and structure factors have been deposited into the Protein Data Bank (accession code 4ZCH).