Quercetin, Main Active Ingredient of Moutan Cortex, Alleviates Chronic Orofacial Pain via Block of Voltage-Gated Sodium Channel.

BACKGROUND: Chronic orofacial pain (COP) therapy is challenging, as current medical treatments are extremely lacking. Moutan Cortex (MC) is a traditional Chinese medicine herb widely used for chronic inflammatory diseases. However, the mechanism behind MC in COP therapy has not been well-established. The purpose of this study was to identify the active ingredients of MC and their specific underlying mechanisms in COP treatment. METHODS: In this study, the main active ingredients and compound-target network of MC in COP therapy were identified through network pharmacology and bioinformatics analysis. Adult male Sprague-Dawley rats received oral mucosa lipopolysaccharide (LPS) injection to induce COP. Pain behaviors were evaluated by orofacial mechanical nociceptive assessment after intraganglionar injection. In vitro inflammatory cytokines in LPS-pretreated human periodontal ligament stem cells (hPDLSCs) and rat primary cultural trigeminal ganglion (TG) neurons were quantified by real-time quantitative polymerase chain reaction (RT-qPCR). Schrödinger software was used to verify the molecular docking of quercetin and critical targets. Whole-cell recording electrophysiology was used to evaluate the effect of quercetin on voltage-gated sodium (Na v ) channel in rat TG neurons. RESULTS: The assembled compound-target network consisted of 4 compounds and 46 targets. As 1 of the active components of MC correlated with most related targets, quercetin alleviated mechanical allodynia in LPS-induced rat model of COP (mechanical allodynia threshold median [interquartile range (IQR) 0.5 hours after drug administration: vehicle 1.3 [0.6-2.0] g vs quercetin 7.0 [6.0-8.5] g, P = .002). Gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that immune response and membrane functions play essential roles in MC-COP therapy. Five of the related targets were identified as core targets by protein-protein interaction analysis. Quercetin exerted an analgesic effect, possibly through blocking Na v channel in TG sensory neurons (peak current density median [IQR]: LPS -850.2 [-983.6 to -660.7] mV vs LPS + quercetin -589.6 [-711.0 to -147.8] mV, P = .006) while downregulating the expression level of proinflammatory cytokines-FOS (normalized messenger RNA [mRNA] level mean ± standard error of mean [SEM]: LPS [2. 22 ± 0.33] vs LPS + quercetin [1. 33 ± 0.14], P = .034) and TNF-α (normalized mRNA level mean ± SEM: LPS [8. 93 ± 0.78] vs LPS + quercetin [3. 77 ± 0.49], P < .0001). CONCLUSIONS: Identifying Na v as the molecular target of quercetin clarifies the analgesic mechanism of MC, and provides ideas for the development of novel selective and efficient chronic pain relievers.

Activation of 2D titanate nanosheet photocatalysts by nitrogen doping and solution plasma processing.

Novel two-dimensional (2D) oxides are of great interest for photocatalysis because of their superlative physical features, namely, large surface areas, short charge diffusion pathways, high crystallinity and easy surface modification. However, most 2D oxides suffer from weak visible light absorption and severe photogenerated carrier recombination. Nitrogen doping can successfully narrow the bandgap of 2D oxides but can hardly improve the charge separation. In this work, we pre-dope nitrogen into 2D titanate nanosheets (HTiO), followed by surface processing with solution plasma. By dual modification of nitrogen doping and solution plasma processing (SPP), the modified 2D titanate nanosheets (N-HTiO-SPP) display broad absorption extending to the visible light region and the healing of oxygen vacancies brought about by nitrogen doping. Compared with HTiO and nitrogen doped titanate (N-HTiO), a higher removal rate and mineralization rate towards the photocatalytic degradation of acetaldehyde were achieved over N-HTiO-SPP under solar light. This work provides a powerful way to activate 2D wide bandgap semiconductors for enhanced photocatalytic activity.

Resolvin D1 attenuates depressive-like behavior in LPS-challenged mice by promoting microglial autophagy.

It has been proven that neuroinflammation triggered by microglial activation is the pathogenesis of depression associated with sepsis. An endogenous lipid mediator known as resolvin D1 (RvD1) is known to have anti-inflammatory effects in a sepsis model. However, it remains unknown if the effects of RvD1 on inflammatory responses are regulated by microglial autophagy. The current study investigated the role of RvD1-induced microglial autophagy in neuroinflammation. The findings showed that RvD1 reverses LPS-induced autophagy inhibition in microglia. RvD1 treatment significantly inhibits inflammatory responses by preventing NF-κB nuclear translocation and microglial M1 phenotypic transition. RvD1 exhibits an attenuation of neurotoxicity in both in vivo and in vitro models of sepsis. Following RvD1 injection, depressive-like behaviors in SAE mice were significantly improved. Notably, the aforesaid effects of RvD1 were eliminated by 3-MA, demonstrating that microglial autophagy was modulated. In conclusion, our findings shed new light on the involvement of microglial autophagy in SAE and emphasize the potential benefits of RvD1 as a promising therapeutic agent in the treatment of depression.

OVOL2 inhibits macrophage M2 polarization by regulating IL-10 transcription, and thus inhibits the tumor metastasis by modulating the tumor microenvironment.

Invasion and metastasis of breast cancer cells is an important cause of death in breast cancer patients. In the tumor microenvironment, M2 polarization of macrophages can promote the invasion and metastasis of tumor cells. OVOL2 is an evolutionarily conserved transcription regulator, but its effect in macrophages has not been described previously. The aim of this study was to investigate the effects of OVOL2 on macrophage polarity and the role of these effects in the tumor metastasis. We found that overexpression of OVOL2 in macrophages significantly inhibited M2 polarization and thus inhibits breast cancer metastasis. We propose a novel mechanism in which OVOL2 inhibits M2 polarization of macrophages and thus reduces their ability to induce invasion and metastasis of breast cancer. By shedding new light on the regulation of metastasis in cancers, our study provides a new strategy for the targeted therapy of cancer.

Sevoflurane Aggravates the Progress of Alzheimer's Disease Through NLRP3/Caspase-1/Gasdermin D Pathway.

Background: Alzheimer's disease (AD) is the most common form of dementia worldwide. Previous studies have reported that sevoflurane, a frequently used anesthetic, can induce cognitive impairment in preclinical and clinical settings. However, the mechanism underlying the development of this neurotoxicity is currently unclear. Methods: Seven-month-old APP/PS1 mice were placed in an anesthesia induction box containing 3% sevoflurane in 100% O2 for 6 h, while BV2 cells were cultured with 4% sevoflurane for 6 h. Pyroptosis and tau protein expression in excised hippocampus tissues and cells were measured using Western blotting and immunofluorescence assay. Caspase-1 and NLRP3 were knocked out in BV2 microglia using CRISPR/Cas9 technology to determine whether they mediate the effects induced by sevoflurane. Results: Sevoflurane directly activated caspase-1 to induce pyroptosis in the mouse model of AD via NLRP3 and AIM2 activation. In addition, sevoflurane mediated cleavage of gasdermin D (GSDMD) but not gasdermin E (GSDME), promoted the biosynthesis of downstream interleukin-1ß and interleukin-18, and increased ß-amyloid (Aß) deposition and tau phosphorylation. The nontoxic caspase-1 small-molecule inhibitor VX-765 significantly inhibited this activation process in microglia, while NLRP3 deletion suppressed sevoflurane-induced caspase-1 cleavage and subsequently pyroptosis, as well as tau pathology. Furthermore, silencing caspase-1 alleviated the sevoflurane-induced release of IL-1ß and IL-18 and inhibited tau-related enzymes in microglia. Conclusion: This study is the first to report that clinical doses of sevoflurane aggravate the progression of AD via the NLRP3/caspase-1/GSDMD axis. Collectively, our findings elucidate the crucial mechanisms of NLRP3/caspase-1 in pyroptosis and tau pathogenesis induced by sevoflurane and suggest that VX-765 could represent a novel therapeutic intervention for treating AD.

Construction of the 1D Covalent Organic Framework/2D g-C3N4 Heterojunction with High Apparent Quantum Efficiency at 500 nm.

The reasonable construction of heterojunction photocatalysts with clear nanostructures and a good interface contact especially the one-dimensional/two-dimensional (1D/2D) composite heterojunction with unique morphology is considered one of the most effective strategies for designing highly efficient photocatalysts. Herein, a series of the 1D ß-keto-enamine-based covalent organic framework (COF)/2D g-C3N4 composite materials COF-CN (1:x; where 1:x represents the mass ratio of COF and g-C3N4, x = 2.5, 5, 10, 15, 20) is prepared through the in situ reaction of 2,4,6-triformylphloroglucinol (Tp) and benzidine (BD) in stripped g-C3N4 suspension. A series of characterizations, such as X-ray diffraction (XRD), Fourier transform infrared (FT-IR), scanning electron microscopy (SEM), and transmission electron microscopy (TEM), have verified their 1D/2D heterojunction structure. With the introduction of 1D COF nanobelts, the absorption of the composite is largely extended to 560 nm. Photocatalytic experiments reveal that the composite COF/CN shows evidently superior photocatalytic performance than individual COF and g-C3N4. The optimized COF-CN (1:10) exhibits a H2 production rate of 12.8 mmol g-1·h-1 under visible-light (λ ≥ 420 nm) irradiation, which is about 62 and 284 times higher than those of COF and g-C3N4, respectively. The apparent quantum efficiency (AQE) of COF-CN (1:10) is about 15.09% under 500 nm light irradiation, which is one of the highest among previous COF- or g-C3N4-based materials. This work provides important strategies for designing and constructing high-efficiency heterojunction photocatalysts with multidimensional features.

Propofol directly induces caspase-1-dependent macrophage pyroptosis through the NLRP3-ASC inflammasome.

Propofol infusion syndrome (PRIS) is an uncommon life-threatening complication observed most often in patients receiving high-dose propofol. High-dose propofol treatment with a prolonged duration can damage the immune system. However, the associated molecular mechanisms remain unclear. An increasing number of clinical and experimental observations have demonstrated that tissue-resident macrophages play a critical role in immune regulation during anaesthesia and procedural sedation. Since the inflammatory response is essential for mediating propofol-induced cell death and proinflammatory reactions, we hypothesised that propofol overdose induces macrophage pyroptosis through inflammasomes. Using primary cultured bone marrow-derived macrophages, murine macrophage cell lines (RAW264.7, RAW-asc and J774) and a mouse model, we investigated the role of NLRP3 inflammasome activation and secondary pyroptosis in propofol-induced cell death. We found that high-dose propofol strongly cleaved caspase-1 but not caspase-11 and biosynthesis of downstream interleukin (IL)-1ß and IL-18. Inhibition of caspase-1 activity blocks IL-1ß production. Moreover, NLRP3 deletion moderately suppressed cleaved caspase-1 as well as the proportion of pyroptosis, while levels of AIM2 were increased, triggering a compensatory pathway to pyroptosis in NLRP3-/- macrophages. Here, we show that propofol-induced mitochondrial reactive oxygen species (ROS) can trigger NLRP3 inflammasome activation. Furthermore, apoptosis-associated speck-like protein (ASC) was found to mediate NLRP3 and AIM2 signalling and contribute to propofol-induced macrophage pyroptosis. In addition, our work shows that propofol-induced apoptotic initiator caspase (caspase-9) subsequently cleaved effector caspases (caspase-3 and 7), indicating that both apoptotic and pyroptotic cellular death pathways are activated after propofol exposure. Our studies suggest, for the first time, that propofol-induced pyroptosis might be restricted to macrophage through an NLRP3/ASC/caspase-1 pathway, which provides potential targets for limiting adverse reactions during propofol application. These findings demonstrate that propofol overdose can trigger cell death through caspase-1 activation and offer new insights into the use of anaesthetic drugs.

pH-Sensitive Black Phosphorous-Incorporated Hydrogel as Novel Implant for Cancer Treatment.

In this study, black phosphorus nanosheets (BPNSs) were incorporated into a hydrogel formed from dibenzaldehyde-functionalized polymer (DF-PEG) and polyaspartylhydrazide (PAHy) polymer to create an injectable and pH-sensitive DF-PEG-PAHy/BPNSs hydrogel, which can be used as a smart depot for synergistic chemo-photothermal cancer therapy. The DF-PEG-PAHy/BPNSs hydrogel exhibited excellent gelation characteristics, pH sensitivity, and near-infrared responsiveness. The nanocomposite hydrogel provided controlled drug release and near-infrared irradiation speeded up release of drug from the hydrogel because of the photothermal effect of the BPNSs. Cytotoxicity tests confirmed that the hydrogel has good biocompatibility and exerts its photothermal effect in vitro. Antitumor tests in mice demonstrated the capacity of DF-PEG-PAHy/BPNSs hydrogel for synergistic chemo-photothermal therapy in vivo. The hydrogel showed reduced adverse effects because of stable drug release in the tumor area and an efficient photothermal effect. Together, these data demonstrated the potential of DF-PEG-PAHy/BPNSs hydrogel containing a chemotherapy drug to serve as a novel smart delivery system for combined chemo-photothermal cancer therapy.

Synthesis and spectroscopic properties of silver-fluorescein co-doped phosphotungstate hollow spheres.

Herein, silver-fluorescein co-doped phosphotungstate hollow spheres have been successfully synthesized using a chemical precipitation method by introducing silver ions and fluorescein at room temperature. This hybrid nanomaterial possesses a uniform structure, exhibiting a strong emission spectrum with a peak centered at 517 nm. It is anticipated that silver and fluorescein as functionalized dopants for the phosphotungstate hollow sphere structure would provide this material with multiple properties. The control experiments indicate that fluorescein plays a key role in the formation of the hollow sphere structure. Hence, the co-doping strategy is proposed to be a general method to endow polyoxometalate nanomaterials with new structures, new functions and potential applications in bioimaging, fluorescent chemical sensors, and antibacterial or optoelectronic devices.

Lup-20(29)-en-3ß,28-di-yl-nitrooxy acetate affects MCF-7 proliferation through the crosstalk between apoptosis and autophagy in mitochondria.

Betulin (BT), a pentacyclic lupine-type triterpenoid natural product, possesses antitumor activity in various types of cancers. However, its clinical development was discouraged due to its low biological activities and poor solubility. We prepared lup-20(29)-en-3ß,28-di-yl-nitrooxy acetate (NBT), a derivative of BT, that was chemically modified at position 3 of ring A and C-28 by introducing a NO-releasing moiety. This study mainly explored the mechanism of NBT in treating breast cancer through the crosstalk between apoptosis and autophagy in mitochondria. NBT possessed a potent antiproliferative activity in MCF-7 cells both in vitro and in vivo. Mechanically, NBT affected cell death through the mitochondrial apoptosis pathway and autophagy. NBT induced cell cycle arrest in the G0/G1 phase by decreasing the expression of cyclin D1. It also induced mitochondrial apoptosis by increasing the expression of Bax, caspase-9, and poly(ADP-ribose) polymerase and mitochondrial membrane potential loss and leaks of cytochrome c (Cyt C) from mitochondria in MCF-7 cells and decreasing the expression of mitochondrial Bcl-2. We further demonstrated whether chloroquine (CQ), which inhibits the degradation of autophagosome induced by NBT, affects the proliferation of MCF-7 cells compared with NBT. The experiments inferred that the combination of NBT and CQ significantly promoted MCF-7 cell mitochondria to divide and Cyt C to be released from mitochondria to the cytoplasm, resulting in an increased apoptosis rate. The in vivo experiments showed that NBT inhibited the growth of MCF-7 tumor via the apoptosis pathway, and its effect was similar to 5-fluorouracil.

Ex vivo 2D and 3D HSV-2 infection model using human normal vaginal epithelial cells.

Herpes simplex virus type 2 (HSV-2) infects human genital mucosa and establishes life-long latent infection. It is unmet need to establish a human cell-based microphysiological system for virus biology and anti-viral drug discovery. One of barriers is lacking of culture system of normal epithelial cells in vitro over decades. In this study, we established human normal vaginal epithelial cell (HNVEC) culture using co-culture system. HNVEC cells were then propagated rapidly and stably in a defined culture condition. HNVEC cells exhibited a normal diploid karyotype and formed the well-defined and polarized spheres in matrigel three-dimension (3D) culture, while malignant cells (HeLa) formed disorganized and nonpolar solid spheres. HNVEC cells had a normal cellular response to DNA damage and had no transforming property using soft agar assays. HNVEC expressed epithelial marker cytokeratin 14 (CK14) and p63, but not cytokeratin 18 (CK18). Next, we reconstructed HNVEC-derived 3D vaginal epithelium using air-liquid interface (ALI) culture. This 3D vaginal epithelium has the basal and apical layers with expression of epithelial markers as its originated human vaginal tissue. Finally, we established an HSV-2 infection model based on the reconstructed 3D vaginal epithelium. After inoculation of HSV-2 (G strain) at apical layer of the reconstructed 3D vaginal epithelium, we observed obvious pathological effects gradually spreading from the apical layer to basal layer with expression of a viral protein. Thus, we established an ex vivo 2D and 3D HSV-2 infection model that can be used for HSV-2 virology and anti-viral drug discovery.

[Effect of Borneol on the Permeability of Blood Tumor Barrier Model and its Mechanism Study].

OBJECTIVE: To observe the effect of natural borneol on the permeability of blood tumor barrier (BTB) model and the expression and activation of mitogen-activated protein kinase (MAPKs) signal transduction pathway related protein kinase in vitro. METHODS: C6 rat glioma cells and human umbilical vein endothelial cells (HUVECs) were co-cultured to establish BTB model. Then 4 groups were set up, the blank control group, low, middle, and high dose borneol groups (25, 50, 100 µg/mL), 3 samples collected at 7 time points (0, 10, 30, 60, 120, 180, 240 min, respectively). Blank culture medium was exchanged in the blank control group while medication. Different doses of natural borneol were administered to the 3 borneol groups. Cells were collected at different time points. BTB permeability was determined using horseradish peroxidase (HRP). Expression levels of extracellular signal regulated protein kinase (ERK), phosphorylation extracellular signal regulated protein kinase (P-ERK), P38MAPK, phosphor-P38MAPK, c-Jun N-terminal kinase (JNK), and phosphorylation c-Jun N-terminal kinase (P-JNK) were detected using Western blot. RESULTS: Compared with the same group at min 0, the permeation rate obviously increased (P < 0.01) in the 3 borneol groups at the rest time points. P-ERK expression was elevated first, reached the peak at 30 min, and gradually recovered to the initial level (P > 0.05). Compared with the blank control group, HRP permeation rate increased from 10 min to 240 min (P < 0.01), and expression of P-ERK protein increased at 30 min and 60 min (P < 0.05) in the low dose borneol group; expression of P-JNK protein decreased in the 3 borneol groups at 180 min and 240 min (P < 0.05). Compared with the low dose borneol group, expression of P-ERK protein increased from 10 min to 180 min (P < 0.05), HRP permeation rate increased from 30 min to 180 min (P < 0.05), expression of P-JNK protein decreased at 180 and 240 min (P < 0.05) in the middle dose borneol group. Compared with the middle dose borneol group, HRP permeation rate increased from 10 min to 180 min (P < 0.05), expression of P-ERK protein increased from 10 min to 180 min (P < 0.05), expression of P-JNK protein increased at 180 min and decreased at 240 min (both P < 0.05) in the high dose borneol group. CONCLUSION: Natural borneol arrived at the effect of regulating reversible BTB patency possibly through activating phosphorylation of ERK in MAPKs signal transduction pathway, and further reversibly down-regulating expression of associated proteins.

Borneol increases blood-tumour barrier permeability by regulating the expression levels of tight junction-associated proteins.

CONTEXT: Selectively opening the blood-tumour barrier (BTB) is critical to deliver antitumour drugs from blood to tumour tissues. The BTB problem is attributed to the tight junctions (TJs), which consist of several transmembrane proteins. OBJECTIVE: To investigate whether borneol could open the BTB by affecting TJ-associated proteins ZO-1, occludin, claudin-5 and F-actin in the rat model of C6 glioma. MATERIALS AND METHODS: The plasma and brain tissue of C6 glioma rats were collected at different points after rats were administered with 35 or 140 mg/kg borneol and 0.5% CMC-Na, respectively. The permeability of BTB was assessed by cisplatin extravasation. The mRNA and protein expression levels of TJ-associated proteins were determined by QPCR, ELISA and immunohistochemistry. RESULTS: The cisplatin bioavailability in the brain tissue of C6 glioma rats administered either 35 or 140 mg/kg borneol and 0.5% CMC-Na were 415.07, 227.04 and 192.07 (mg/mL/h), respectively. The mRNA and protein expression levels of ZO-1 and F-actin began to decrease from the time point of 2 min; the lowest levels in the borneol high-dose (46.7% decrease for ZO-1 and 63.3% for F-actin compared with control) and low-dose groups (54.3% for ZO-1; 77.9% for F-actin) appeared at the time points of 30 and 45 min, respectively. Thereafter, the levels were gradually restored to the level of borneol at 0 h. Occludin and claudin-5 expression levels were not significantly modified. CONCLUSION: Borneol could selectively open the BTB and consequently increase BTB permeability, and this mechanism is associated with the down-regulation of ZO-1 and F-actin.

Reference ranges for serum IGF-1 and IGFBP-3 levels in Chinese children during childhood and adolescence.

Serum levels of insulin-like growth factor-1(IGF-1) and insulin-like growth factor binding protein-3(IGFBP-3) reflect endogenous growth hormone secretion, and serum IGF-1 and IGFBP-3 values should be ethnic-specific, thus we established the reference ranges for serum IGF-1 and IGFBP-3 in Chinese children aged 6-18 yr according to age, sex, puberty stage and BMI. The study was included 837 children (age 6-18 yr, 416 boys and 421 girls) from different schools in Daqing, Beijing and Shanghai. Serum IGF-1 and IGFBP-3 were determined by a chemiluminescent assay system (IMMULITE 1000). The results show that IGF1 reached peak levels at around 13 yr in boys and 11 yr in girls while IGFBP-3 peaked at 14 yr in boys, and 11 yr in girls. Both IGF-1 and IGFBP-3 were at platform or decreased slightly after these ages. At each corresponding age, IGF-1 levels tended to be higher in boys compared to girls, while girls had higher IGFBP-3 levels than boys. A model for calculation of standard deviation scores of IGF-1 and IGFBP3 according to age, sex and pubertal stage was established. These normative data should facilitate child care, growth monitoring, clinical diagnosis and to follow up on GH treatment.