Mechanisms of biased agonism by Gαi/o-biased stapled peptide agonists of the relaxin-3 receptor.

The neuropeptide relaxin-3 is composed of an A chain and a B chain held together by disulfide bonds, and it modulates functions such as anxiety and food intake by binding to and activating its cognate receptor RXFP3, mainly through the B chain. Biased ligands of RXFP3 would help to determine the molecular mechanisms underlying the activation of G proteins and ß-arrestins downstream of RXFP3 that lead to such diverse functions. We showed that the i, i+4 stapled relaxin-3 B chains, 14s18 and d(1-7)14s18, were Gαi/o-biased agonists of RXFP3. These peptides did not induce recruitment of ß-arrestin1/2 to RXFP3 by GPCR kinases (GRKs), in contrast to relaxin-3, which enabled the GRK2/3-mediated recruitment of ß-arrestin1/2 to RXFP3. Relaxin-3 and the previously reported peptide 4 (an i, i+4 stapled relaxin-3 B chain) did not exhibit biased signaling. The staple linker of peptide 4 and parts of both the A chain and B chain of relaxin-3 interacted with extracellular loop 3 (ECL3) of RXFP3, moving it away from the binding pocket, suggesting that unbiased ligands promote a more open conformation of RXFP3. These findings highlight roles for the A chain and the N-terminal residues of the B chain of relaxin-3 in inducing conformational changes in RXFP3, which will help in designing selective biased ligands with improved therapeutic efficacy.

Lipidomics profiling reveals distinct patterns of plasma sphingolipid alterations in Alzheimer's disease and vascular dementia.

BACKGROUND: Alzheimer's disease (AD) and vascular dementia (VaD) are two of the commonest causes of dementia in the elderly. Of the myriad biomolecules implicated in dementia pathogenesis, sphingolipids have attracted relatively scant research attention despite their known involvement in multiple pathophysiological processes. The potential utility of peripheral sphingolipids as biomarkers in dementia cohorts with high concomitance of cerebrovascular diseases is also unclear. METHODS: Using a lipidomics platform, we performed a case-control study of plasma sphingolipids in a prospectively assessed cohort of 526 participants (non-cognitively impaired, NCI = 93, cognitively impaired = 217, AD = 166, VaD = 50) using a lipidomics platform. RESULTS: Distinct patterns of sphingolipid alterations were found in AD and VaD, namely an upregulation of d18:1 species in AD compared to downregulation of d16:1 species in VaD. In particular, GM3 d18:1/16:0 and GM3 d18:1/24:1 showed the strongest positive associations with AD. Furthermore, evaluation of sphingolipids panels showed specific combinations with higher sensitivity and specificity for classification of AD (Cer d16:1/24:0. Cer d18:1/16:0, GM3 d16:1/22:0, GM3 d18:1/16:0, SM d16:1/22:0, HexCer d18:1/18:0) and VAD (Cer d16:1/24:0, Cer d18:1/16:0, Hex2Cer d16:1/16:0, HexCer d18:1/18:0, SM d16:1/16:0, SM d16:1/20:0, SM d18:2/22:0) compared to NCI. CONCLUSIONS: AD and VaD are associated with distinct changes of plasma sphingolipids, warranting further studies into underlying pathophysiological mechanisms and assessments of their potential utility as dementia biomarkers and therapeutic targets.

Differences in the ultrastructure of neurons in the spinal ganglion and dorsal rootlet between rats treated with cisplatin only versus co-administration with a sphingosine 1-phosphate receptor 2 agonist in attenuating neuropathy and allodynia.

BACKGROUND AND AIMS: Cisplatin is a chemotherapeutic agent for many types of cancer. The neurotoxicity of cisplatin includes neuropathy and allodynia. We aimed to study structural changes by using CYM54-78, attenuating cisplatin-induced neuropathy and blocking the pathogenesis in neurons, and promoting axonal regeneration. METHODS: TEM (transmission electron microscopy) was used to distinguish ultrastructural changes in dorsal root ganglion (DRG) and dorsal rootlets (DR) between rats treated with cisplatin alone and rats co-treated with cisplatin and sphingosine -1-phosphate receptor2 (S1P2) agonist, CYM-5478. RESULTS: In DRG of rats treated with cisplatin alone, TEM micrographs showed necrosis and apoptotic cells. Neuronal cytoplasm showed numerous vacuole (stage C) and swelling (stage B➔C) mitochondrial degeneration. Neurons in DRG from cisplatin+CYM-5478 group showed a higher percentage of healthy mitochondria (from 5.3% to 75.6%) than those treated with cisplatin alone. DR of cisplatin only group showed abnormal axoplasm, axolemma, and focal detached myelin sheaths, especially in Aδ (fast pain) and Aß (touch) fibers, and revealed collateral branches that sprouted from Aß fibers, which is characteristic of allodynia. Moreover, vasoconstriction was observed in DRG and DR. Rats in cisplatin+CYM-5478 group showed not only fewer abnormal structures than those in cisplatin only group, but also showed Bands of Büngner and onion bulb-like structures, which are characteristic of nerve regeneration. INTERPRETATION: Together with our previous study, showed that CYM-5478 attenuated neuropathy and allodynia in a rat model of cisplatin-induced neuropathy, these results suggest S1P2 agonists as a potential approach the for treatment of cancer due to the reduction of side effects of cisplatin.

Decreased DHA-containing phospholipids in the neocortex of dementia with Lewy bodies are associated with soluble Aß42 , phosphorylated α-synuclein, and synaptopathology.

Docosahexaenoic acid (DHA) is an essential omega-3 polyunsaturated fatty acid implicated in cognitive functions by promoting synaptic protein expression. While alterations of specific DHA-containing phospholipids have been described in the neocortex of patients with Alzheimer's disease (AD), the status of these lipids in dementia with Lewy bodies (DLB), known to manifest aggregated α-synuclein-containing Lewy bodies together with variable amyloid pathology, is unclear. In this study, post-mortem samples from the parietal cortex of 25 DLB patients and 17 age-matched controls were processed for phospholipidomics analyses using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) platform. After controlling for false discovery rate, six out of the 46 identified putative DHA-phospholipid species were significantly decreased in DLB, with only one showing increase. Altered putative DHA-phospholipid species were subsequently validated with further LC-MS/MS measurements. Of the DHA-containing phospholipid (DCP) species showing decreases, five negatively correlated with soluble beta-amyloid (Aß42) levels, whilst three also correlated with phosphorylated α-synuclein (all p < 0.05). Furthermore, five of these phospholipid species correlated with deficits of presynaptic Rab3A, postsynaptic neurogranin, or both (all p < 0.05). Finally, we found altered immunoreactivities of brain lysolipid DHA transporter, MFSD2A, and the fatty acid binding protein FABP5 in DLB parietal cortex. In summary, we report alterations of specific DCP species in DLB, as well as their associations with markers of neuropathological burden and synaptopathology. These results support the potential role of DHA perturbations in DLB as well as therapeutic targets.

What Do Randomized Controlled Trials Inform Us About Potential Disease-Modifying Strategies for Parkinson's Disease?

Research advances have shed new insight into cellular pathways contributing to PD pathogenesis and offer increasingly compelling therapeutic targets. In this review, we made a broad survey of the published literature that report possible disease-modifying effects on PD. While there are many studies that demonstrate benefits for various therapies for PD in animal and human studies, we confined our search to human "randomised controlled trials" and with the key words "neuroprotection" or "disease-modifying". It is hoped that through studying the results of these trials, we might clarify possible mechanisms that underlie idiopathic PD. This contrasts with studying the effect of pathophysiology of familial PD, which could be carried out by gene knockouts and animal models. Randomised controlled trials indicate promising effects of MAO-B inhibitors, dopamine agonists, NMDA receptor antagonists, metabotropic glutamate receptor antagonists, therapies related to improving glucose utilization and energy production, therapies related to reduction of excitotoxicity and oxidative stress, statin use, therapies related to iron chelation, therapies related to the use of phytochemicals, and therapies related to physical exercise and brain reward pathway on slowing PD progression. Cumulatively, these approaches fall into two categories: direct enhancement of dopaminergic signalling, and reduction of neurodegeneration. Overlaps between the two categories result in challenges in distinguishing between symptomatic versus disease-modifying effects with current clinical trial designs. Nevertheless, a broad-based approach allows us to consider all possible therapeutic avenues which may be neuroprotective. While the traditional standard of care focuses on symptomatic management with dopaminergic drugs, more recent approaches suggest ways to preserve dopaminergic neurons by attenuating excitotoxicity and oxidative stress.

Mass Spectrometry Analysis of the Human Brain Sphingolipidome.

In recent decades, mass spectrometry-based lipidomics has provided a fertile environment for scientific investigations of biochemical and mechanistic processes in biological systems. Notably, this approach has been used to characterize physiological and pathological processes relevant to the central nervous system by identifying changes in the sphingolipid content in the brain, cerebral spinal fluid, and blood plasma. However, despite a preponderance of studies identifying correlations between specific lipids and disease progression, this powerful resource has not yet substantively translated into clinically useful diagnostic assays. Part of this gap may be explained by insufficient depth of the lipidomic profiles in many studies, by lab-to-lab inconsistencies in methodology, and a lack of absolute quantification. These issues limit the identification of specific molecular species and the harmonization of results across independent studies. In this chapter, we contextualize these issues with recent reports identifying correlations between brain lipids and neurological diseases, and we describe the workflow our group has optimized for analysis of the blood plasma sphingolipidome, adapted to the characterization of the human brain tissue.

Anti-Inflammatory Effects of Phytochemical Components of Clinacanthus nutans.

Recent studies on the ethnomedicinal use of Clinacanthus nutans suggest promising anti-inflammatory, anti-tumorigenic, and antiviral properties for this plant. Extraction of the leaves with polar and nonpolar solvents has yielded many C-glycosyl flavones, including schaftoside, isoorientin, orientin, isovitexin, and vitexin. Aside from studies with different extracts, there is increasing interest to understand the properties of these components, especially regarding their ability to exert anti-inflammatory effects on cells and tissues. A major focus for this review is to obtain information on the effects of C. nutans extracts and its phytochemical components on inflammatory signaling pathways in the peripheral and central nervous system. Particular emphasis is placed on their role to target the Toll-like receptor 4 (TLR4)-NF-kB pathway and pro-inflammatory cytokines, the antioxidant defense pathway involving nuclear factor erythroid-2-related factor 2 (NRF2) and heme oxygenase 1 (HO-1); and the phospholipase A2 (PLA2) pathway linking to cyclooxygenase-2 (COX-2) and production of eicosanoids. The ability to provide a better understanding of the molecular targets and mechanism of action of C. nutans extracts and their phytochemical components should encourage future studies to develop new therapeutic strategies for better use of this herb to combat inflammatory diseases.

ACE2, Circumventricular Organs and the Hypothalamus, and COVID-19.

The SARS-CoV-2 virus gains entry to cells by binding to angiotensin-converting enzyme 2 (ACE2). Since circumventricular organs and parts of the hypothalamus lack a blood-brain barrier, and immunohistochemical studies demonstrate that ACE2 is highly expressed in circumventricular organs which are intimately connected to the hypothalamus, and the hypothalamus itself, these might be easy entry points for SARS-CoV-2 into the brain via the circulation. High ACE2 protein expression is found in the subfornical organ, area postrema, and the paraventricular nucleus of the hypothalamus (PVH). The subfornical organ and PVH are parts of a circuit to regulate osmolarity in the blood, through the secretion of anti-diuretic hormone into the posterior pituitary. The PVH is also the stress response centre in the brain. It controls not only pre-ganglionic sympathetic neurons, but is also a source of corticotropin-releasing hormone, that induces the secretion of adrenocorticotropic hormone from the anterior pituitary. It is proposed that the function of ACE2 in the circumventricular organs and the PVH could be diminished by binding with SARS-CoV-2, thus leading to a reduction in the ACE2/Ang (1-7)/Mas receptor (MasR) signalling axis, that modulates ACE/Ang II/AT1R signalling. This could result in increased presympathetic activity/neuroendocrine secretion from the PVH, and effects on the hypothalamic-pituitary-adrenal axis activity. Besides the bloodstream, the hypothalamus might also be affected by SARS-CoV-2 via transneuronal spread along the olfactory/limbic pathways. Exploring potential therapeutic pathways to prevent or attenuate neurological symptoms of COVID-19, including drugs which modulate ACE signalling, remains an important area of unmet medical need.

Colonization of nasal cavities by Staphylococcus epidermidis mitigates SARS-CoV-2 nucleocapsid phosphoprotein-induced interleukin (IL)-6 in the lung.

Infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can trigger excessive interleukin (IL)-6 signalling, leading to a myriad of biological effects including a cytokine storm that contributes to multiple organ failure in severe coronavirus disease 2019 (COVID-19). Using a mouse model, we demonstrated that nasal inoculation of nucleocapsid phosphoprotein (NPP) of SARS-CoV-2 increased IL-6 content in bronchoalveolar lavage fluid (BALF). Nasal administration of liquid coco-caprylate/caprate (LCC) onto Staphylococcus epidermidis (S. epidermidis)-colonized mice significantly attenuated NPP-induced IL-6. Furthermore, S. epidermidis-mediated LCC fermentation to generate electricity and butyric acid that promoted bacterial colonization and activated free fatty acid receptor 2 (Ffar2) respectively. Inhibition of Ffar2 impeded the effect of S. epidermidis plus LCC on the reduction of NPP-induced IL-6. Collectively, these results suggest that nasal S. epidermidis is part of the first line of defence in ameliorating a cytokine storm induced by airway infection of SARS-CoV-2.

Electricity-producing Staphylococcus epidermidis counteracts Cutibacterium acnes.

Staphylococcus epidermidis (S. epidermidis) ATCC 12228 was incubated with 2% polyethylene glycol (PEG)-8 Laurate to yield electricity which was measured by a voltage difference between electrodes. Production of electron was validated by a Ferrozine assay. The anti-Cutibacterium acnes (C. acnes) activity of electrogenic S. epidermidis was assessed in vitro and in vivo. The voltage change (~ 4.4 mV) reached a peak 60 min after pipetting S. epidermidis plus 2% PEG-8 Laurate onto anodes. The electricity produced by S. epidermidis caused significant growth attenuation and cell lysis of C. acnes. Intradermal injection of C. acnes and S. epidermidis plus PEG-8 Laurate into the mouse ear considerably suppressed the growth of C. acnes. This suppressive effect was noticeably reversed when cyclophilin A of S. epidermidis was inhibited, indicating the essential role of cyclophilin A in electricity production of S. epidermidis against C. acnes. In summary, we demonstrate for the first time that skin S. epidermidis, in the presence of PEG-8 Laurate, can mediate cyclophilin A to elicit an electrical current that has anti-C. acnes effects. Electricity generated by S. epidermidis may confer immediate innate immunity in acne lesions to rein in the overgrowth of C. acnes at the onset of acne vulgaris.

Quantitative circular flow immunoassays with trained object recognition to detect antibodies to SARS-CoV-2 membrane glycoprotein.

Amidst infectious disease outbreaks, a practical tool that can quantitatively monitor individuals' antibodies to pathogens is vital for disease control. The currently used serological lateral flow immunoassays (LFIAs) can only detect the presence of antibodies for a single antigen. Here, we fabricated a multiplexed circular flow immunoassay (CFIA) test strip with YOLO v4-based object recognition that can quickly quantify and differentiate antibodies that bind membrane glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or hemagglutinin of influenza A (H1N1) virus in the sera of immunized mice in one assay using one sample. Spot intensities were found to be indicative of antibody titers to membrane glycoprotein of SARS-CoV-2 and were, thus, quantified relative to spots from immunoglobulin G (IgG) reaction in a CFIA to account for image heterogeneity. Quantitative intensities can be displayed in real time alongside an image of CFIA that was captured by a built-in camera. We demonstrate for the first time that CFIA is a specific, multi-target, and quantitative tool that holds potential for digital and simultaneous monitoring of antibodies recognizing various pathogens including SARS-CoV-2.

Hippocampal transcriptome profiling reveals common disease pathways in chronic hypoperfusion and aging.

Vascular dementia (VaD) is a progressive cognitive impairment of vascular etiology. VaD is characterized by cerebral hypoperfusion, increased blood-brain barrier permeability and white matter lesions. An increased burden of VaD is expected in rapidly aging populations. The hippocampus is particularly susceptible to hypoperfusion, and the resulting memory impairment may play a crucial role in VaD. Here we have investigated the hippocampal gene expression profile of young and old mice subjected to cerebral hypoperfusion by bilateral common carotid artery stenosis (BCAS). Our data in sham-operated young and aged mice reveal an age-associated decline in cerebral blood flow and differential gene expression. In fact, BCAS and aging caused broadly similar effects. However, BCAS-induced changes in hippocampal gene expression differed between young and aged mice. Specifically, transcriptomic analysis indicated that in comparison to young sham mice, many pathways altered by BCAS in young mice resembled those already present in sham aged mice. Over 30 days, BCAS in aged mice had minimal effect on either cerebral blood flow or hippocampal gene expression. Immunoblot analyses confirmed these findings. Finally, relative to young sham mice the cell type-specific profile of genes in both young BCAS and old sham animals further revealed common cell-specific genes. Our data provide a genetic-based molecular framework for hypoperfusion-induced hippocampal damage and reveal common cellular signaling pathways likely to be important in the pathophysiology of VaD.

The S1P2 receptor regulates blood-brain barrier integrity and leukocyte extravasation with implications for neurodegenerative disease.

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid which modulates vascular integrity through its receptors, S1P1-S1P5. Notably, S1P2 has been shown to mediate the disruption of cerebrovascular integrity in vitro and in vivo. However, the mechanism underlying this process has not been fully elucidated. We evaluated the role of S1P2 in blood-brain barrier (BBB) disruption induced by lipopolysaccharide (LPS)-mediated systemic inflammation and found that BBB disruption and neutrophil infiltration were significantly attenuated in S1pr2-/- mice relative to S1pr2+/- littermates. This is concomitant with attenuation of LPS-induced transcriptional activation of IL-6 and downregulation of occludin. Furthermore, S1pr2-/- mice had significantly reduced expression of genes essential for neutrophil infiltration: Sele, Cxcl1, and Cxcl2. Conversely, pharmacological agonism of S1P2 induced transcriptional activation of E-selectin in vitro and in vivo. Although S1P2 does not appear to be required for activation of microglia, stimulation of microglial cells with the S1P2 potentiated the response of endothelial cells to LPS. These results demonstrate that S1P2 promotes LPS-induced neutrophil extravasation by inducing expression of endothelial adhesion molecule gene, Sele, and potentiating microglial inflammation of endothelial cells. It is likely that S1P2 is a mediator of cerebrovascular inflammation and represents a potential therapeutic target for neurodegenerative disease such as vascular cognitive impairment.

Dietary Fat and Protein Intake in Relation to Plasma Sphingolipids as Determined by a Large-Scale Lipidomic Analysis.

Sphingolipid concentrations have been associated with risk of type 2 diabetes and cardiovascular diseases. Because sphingolipids can be synthesized de novo from saturated fatty acids (SFA), dietary fatty acids may affect plasma sphingolipid concentrations. We aimed to evaluate dietary fat and protein intakes in relation to circulating sphingolipid levels. We used cross-sectional data from 2860 ethnic Chinese Singaporeans collected from 2004-2007. Nutrient intakes were estimated on the basis of a validated 159-item food frequency questionnaire. We quantified 79 molecularly distinct sphingolipids in a large-scale lipidomic evaluation from plasma samples. Higher saturated fat intake was associated with higher concentrations of 16:1;O2 sphingolipids including ceramides, monohexosylcermides, dihexosylceramides, sphingomyelins, and sphingosine 1-phosphates. Higher polyunsaturated fat intake was associated with lower plasma long-chain ceramides and long-chain monohexosylcermide concentrations. Protein intake was inversely associated with concentrations of most subclasses of sphingolipids, with the exception of sphingolipids containing a 16:1;O2 sphingoid base. Lower intake of saturated fat and higher intake of polyunsaturated fat and protein may decrease plasma concentrations of several sphingolipid classes. These findings may represent a novel biological mechanism for the impact of nutrient intakes on cardio-metabolic health.

The noncanonical chronicles: Emerging roles of sphingolipid structural variants.

Sphingolipids (SPs) are structurally diverse and represent one of the most quantitatively abundant classes of lipids in mammalian cells. In addition to their structural roles, many SP species are known to be bioactive mediators of essential cellular processes. Historically, studies have focused on SP species that contain the canonical 18­carbon, mono-unsaturated sphingoid backbone. However, increasingly sensitive analytical technologies, driven by advances in mass spectrometry, have facilitated the identification of previously under-appreciated, molecularly distinct SP species. Many of these less abundant species contain noncanonical backbones. Interestingly, a growing number of studies have identified clinical associations between these noncanonical SPs and disease, suggesting that there is functional significance to the alteration of SP backbone structure. For example, associations have been found between SP chain length and cardiovascular disease, pain, diabetes, and dementia. This review will provide an overview of the processes that are known to regulate noncanonical SP accumulation, describe the clinical correlations reported for these molecules, and review the experimental evidence for the potential functional implications of their dysregulation. It is likely that further scrutiny of noncanonical SPs may provide new insight into pathophysiological processes, serve as useful biomarkers for disease, and lead to the design of novel therapeutic strategies.

Effect of Ergothioneine on 7-Ketocholesterol-Induced Endothelial Injury.

Ergothioneine (ET) is a naturally occurring antioxidant that is synthesized by non-yeast fungi and certain bacteria. ET is not synthesized by animals, including humans, but is avidly taken up from the diet, especially from mushrooms. In the current study, we elucidated the effect of ET on the hCMEC/D3 human brain endothelial cell line. Endothelial cells are exposed to high levels of the cholesterol oxidation product, 7-ketocholesterol (7KC), in patients with cardiovascular disease and diabetes, and this process is thought to mediate pathological inflammation. 7KC induces a dose-dependent loss of cell viability and an increase in apoptosis and necrosis in the endothelial cells. A relocalization of the tight junction proteins, zonula occludens-1 (ZO-1) and claudin-5, towards the nucleus of the cells was also observed. These effects were significantly attenuated by ET. In addition, 7KC induces marked increases in the mRNA expression of pro-inflammatory cytokines, IL-1ß IL-6, IL-8, TNF-α and cyclooxygenase-2 (COX2), as well as COX2 enzymatic activity, and these were significantly reduced by ET. Moreover, the cytoprotective and anti-inflammatory effects of ET were significantly reduced by co-incubation with an inhibitor of the ET transporter, OCTN1 (VHCL). This shows that ET needs to enter the endothelial cells to have a protective effect and is unlikely to act via extracellular neutralizing of 7KC. The protective effect on inflammation in brain endothelial cells suggests that ET might be useful as a nutraceutical for the prevention or management of neurovascular diseases, such as stroke and vascular dementia. Moreover, the ability of ET to cross the blood-brain barrier could point to its usefulness in combatting 7KC that is produced in the CNS during neuroinflammation, e.g. after excitotoxicity, in chronic neurodegenerative diseases, and possibly COVID-19-related neurologic complications.

Anti-inflammatory and Cytoprotective Effect of Clinacanthus nutans Leaf But Not Stem Extracts on 7-Ketocholesterol Induced Brain Endothelial Cell Injury.

Clinacanthus nutans (Lindau) (C. nutans) has diverse uses in traditional herbal medicine for treating skin rashes, insect and snake bites, lesions caused by herpes simplex virus, diabetes mellitus and gout in Singapore, Malaysia, Indonesia, Thailand and China. We previously showed that C. nutans has the ability to modulate the induction of cytosolic phospholipase A2 (cPLA2) expression in SH-SY5Y cells through the inhibition of histone deacetylases (HDACs). In the current study, we elucidated the effect of C. nutans on the hCMEC/D3 human brain endothelial cell line. Endothelial cells are exposed to high levels of the cholesterol oxidation product, 7-ketocholesterol (7KC), in patients with cardiovascular disease and diabetes, and this process is thought to mediate pathological inflammation. 7KC induced a dose-dependent loss of hCMEC/D3 cell viability, and such damage was significantly inhibited by C. nutans leaf extracts but not stem extracts. 7KC also induced a marked increase in mRNA expression of pro-inflammatory cytokines, IL-1ß IL-6, IL-8, TNF-α and cyclooxygenase-2 (COX-2) in brain endothelial cells, and these increases were significantly inhibited by C. nutans leaf but not stem extracts. HPLC analyses showed that leaf extracts have a markedly different chemical profile compared to stem extracts, which might explain their different effects in counteracting 7KC-induced inflammation. Further study is necessary to identify the putative phytochemicals in C. nutans leaves that have anti-inflammatory properties.

Clinacanthus nutans Mitigates Neuronal Death and Reduces Ischemic Brain Injury: Role of NF-κB-driven IL-1ß Transcription.

Neuroinflammation has been shown to exacerbate ischemic brain injury, and is considered as a prime target for the development of stroke therapies. Clinacanthus nutans Lindau (C. nutans) is widely used in traditional medicine for treating insect bites, viral infection and cancer, due largely to its anti-oxidative and anti-inflammatory properties. Recently, we reported that an ethanol extract from the leaf of C. nutans could protect the brain against ischemia-triggered neuronal death and infarction. In order to further understand the molecular mechanism(s) for its beneficial effects, two experimental paradigms, namely, in vitro primary cortical neurons subjected to oxygen-glucose deprivation (OGD) and in vivo rat middle cerebral artery (MCA) occlusion, were used to dissect the anti-inflammatory effects of C. nutans extract. Using promoter assays, immunofluorescence staining, and loss-of-function (siRNA) approaches, we demonstrated that transient OGD led to marked induction of IL-1ß, IL-6 and TNFα, while pretreatment with C. nutans suppressed production of inflammatory cytokines in primary neurons. C. nutans inhibited IL-1ß transcription via preventing NF-κB/p65 nuclear translocation, and siRNA knockdown of either p65 or IL-1ß mitigated OGD-mediated neuronal death. Correspondingly, post-ischemic treatment of C. nutans attenuated IκBα degradation and decreased IL-1ß, IL-6 and TNFα production in the ischemic brain. Furthermore, IL-1ß siRNA post-ischemic treatment reduced cerebral infarct, thus mimicking the beneficial effects of C. nutans. In summary, our findings demonstrated the ability for C. nutans to suppress NF-κB nuclear translocation and inhibit IL-1ß transcription in ischemic models. Results further suggest the possibility for using C. nutans to prevent and treat stroke patients.

Preclinical and Clinical Evidence for the Involvement of Sphingosine 1-Phosphate Signaling in the Pathophysiology of Vascular Cognitive Impairment.

Sphingosine 1-phosphates (S1Ps) are bioactive lipids that mediate a diverse range of effects through the activation of cognate receptors, S1P1-S1P5. Scrutiny of S1P-regulated pathways over the past three decades has identified important and occasionally counteracting functions in the brain and cerebrovascular system. For example, while S1P1 and S1P3 mediate proinflammatory effects on glial cells and directly promote endothelial cell barrier integrity, S1P2 is anti-inflammatory but disrupts barrier integrity. Cumulatively, there is significant preclinical evidence implicating critical roles for this pathway in regulating processes that drive cerebrovascular disease and vascular dementia, both being part of the continuum of vascular cognitive impairment (VCI). This is supported by clinical studies that have identified correlations between alterations of S1P and cognitive deficits. We review studies which proposed and evaluated potential mechanisms by which such alterations contribute to pathological S1P signaling that leads to VCI-associated chronic neuroinflammation and neurodegeneration. Notably, S1P receptors have divergent but overlapping expression patterns and demonstrate complex interactions. Therefore, the net effect produced by S1P represents the cumulative contributions of S1P receptors acting additively, synergistically, or antagonistically on the neural, vascular, and immune cells of the brain. Ultimately, an optimized therapeutic strategy that targets S1P signaling will have to consider these complex interactions.