Neuroprotection by Alstonia boonei De Wild., Anacardium occidentale L., Azadirachta indica A.Juss. and Mangifera indica L.

ETHNOPHARMACOLOGY RELEVANCE: Alstonia boonei De Wild. (stem bark), Anacardium occidentale L. (stem bark), Azadirachta indica A.Juss (leaves), Enantia chlorantha Oliv. (stem bark), Khaya senegalensis A.Juss (stem bark) Mangifera indica L. (stem bark), and Nauclea latifolia Sm. (stem bark) are used for treating malaria in southwest Nigeria. Surveys revealed that these plants are also employed for treating symptoms of malaria and cerebral malaria in the region. AIM OF THE STUDY: In this study, the effects of freeze-dried extracts of these plants were investigated on synthetic hemozoin (HZ)-induced neuroinflammation, neuronal damage, and increased permeability of brain microvascular endothelial cells. MATERIALS AND METHODS: Effects of freeze-dried plant extracts were investigated on neuroinflammation by measuring levels of pro-inflammatory mediators in culture supernatants, while in-cell western assays were used to measure protein levels of iNOS and NLRP3. Effects on HZ-induced neurotoxicity and ROS generation was measured using MTT and DCFDA assays, respectively. HZ-induced permeability of hCMEC/D3 endothelial cells was determined using the in vitro vascular permeability assay kit. RESULTS: The extracts produced significant (p < 0.05) reduction in TNFα, IL-6, IL-1ß, MCP-1, RANTES and iNOS/NO production in HZ-stimulated BV-2 microglia. Pre-treatment with 50 µg/mL of A. boonei, A. indica, A. occidentale, E. chlorantha and M. indica also resulted in the inhibition of NF-κB activation. Pre-treatment with A. indica produced, A. occidentale, M. indica and A. boonei reduced HZ-induced increased NLRP3 protein expression. HZ-induced increased caspase-1 activity was also reduced by A. boonei, A. occidentale, A. indica, E. chlorantha, and M. indica. Freeze-dried extracts of A. boonei, A. occidentale, A. indica and M. indica produced neuroprotective effect in HT-22 neuronal cells incubated with HZ by preventing HZ-induced neurotoxicity, ROS generation, DNA fragmentation and caspase 3/7 activity. Inhibition of HZ-induced increase in permeability of human hCMEC/D3 brain endothelial cells was also observed with A. boonei, A. occidentale, A. indica and M. indica, while reducing the release of TNFα and MMP-9. CONCLUSIONS: These results suggest that A. boonei, A. occidentale, A. indica and M. indica are neuroprotective through inhibition of neuroinflammation, neuronal damage and increased permeability of blood brain barrier. The outcome of the study provides pharmacological evidence for the potential benefits of plants as herbal treatments for cerebral malaria symptoms.

Neuroprotection by Skimmianine in Lipopolysaccharide-Activated BV-2 Microglia.

Skimmianine is a furoquinoline alkaloid which is found in the Zanthoxylum genus and also in other plants of the Rutaceae family. This study evaluated the effects of skimmianine on the production of pro-inflammatory mediators in LPS-activated BV-2 microglia. Cultured BV-2 cells were treated with skimmianine (10, 20 and 30 µM), followed by stimulation with LPS (100 ng/mL). Levels of TNFα and IL-6 in cell supernatants were measured using ELISA, while NO and PGE2 levels were evaluated with Griess assay and EIA, respectively. Western blotting was used to determine the protein expression of iNOS, COX-2, phospho-p65 and phospho-IκBα. Results showed that Skimmianine reduced LPS-induced elevated the secretion of TNFα, IL-6, NO, and PGE2, as well as the increased protein expression of iNOS and COX-2. Experiments to elucidate the mechanisms of the anti-neuroinflammatory activity of skimmianine revealed the significant inhibition of LPS-induced increased NF-κB-mediated luciferase activity. Pre-treatment with skimmianine also reduced LPS-induced the increased phosphorylation of NF-κB/p65 and IκBα proteins. Furthermore, skimmianine interfered with the binding capacity of NF-κB to consensus sites. Skimmianine pre-treatment protected HT-22 cells from toxicity induced by microglia-conditioned media, as well as increasing MAP-2 expression. The results of this study suggest that skimmianine inhibits neuroinflammation in LPS-activated microglia by targeting the NF-κB activation pathway. Skimmianine also produced neuroprotection against neurotoxicity induced by microglia-conditioned media.

Iminosugar Amino Acid idoBR1 Reduces Inflammatory Responses in Microglia.

(1) Background. Inflammation is reported to be a key factor in neurodegeneration. The microglia are immune cells present in the central nervous system; their activation results in the release of inflammatory cytokines and is thought to be related to aging and neurodegenerative disorders, such as Alzheimer's disease. (2) Methods. A mouse BV-2 microglia cell line was activated using LPS and the anti-inflammatory cucumber-derived iminosugar amino acid idoBR1, (2R,3R,4R,5S)-3,4,5-trihydroxypiperidine-2-carboxylic acid, was used alongside dexamethasone as the control to determine whether it could reduce the inflammatory responses. (3) Results. A dose-dependent reduction in the LPS-induced production of the proinflammatory factors TNFα, IL-6, and nitric oxide and the transcription factor NF-κB was found. (4) Conclusions. Further investigations of the anti-inflammatory effects of idoBR1 in other models of neurodegenerative diseases are warranted.

SARS-CoV-2 Spike Glycoprotein S1 Induces Neuroinflammation in BV-2 Microglia.

In addition to respiratory complications produced by SARS-CoV-2, accumulating evidence suggests that some neurological symptoms are associated with the disease caused by this coronavirus. In this study, we investigated the effects of the SARS-CoV-2 spike protein S1 stimulation on neuroinflammation in BV-2 microglia. Analyses of culture supernatants revealed an increase in the production of TNF-α, IL-6, IL-1ß and iNOS/NO. S1 also increased protein levels of phospho-p65 and phospho-IκBα, as well as enhanced DNA binding and transcriptional activity of NF-κB. These effects of the protein were blocked in the presence of BAY11-7082 (1 µM). Exposure of S1 to BV-2 microglia also increased the protein levels of NLRP3 inflammasome and enhanced caspase-1 activity. Increased protein levels of p38 MAPK was observed in BV-2 microglia stimulated with the spike protein S1 (100 ng/ml), an action that was reduced in the presence of SKF 86,002 (1 µM). Results of immunofluorescence microscopy showed an increase in TLR4 protein expression in S1-stimulated BV-2 microglia. Furthermore, pharmacological inhibition with TAK 242 (1 µM) and transfection with TLR4 small interfering RNA resulted in significant reduction in TNF-α and IL-6 production in S1-stimulated BV-2 microglia. These results have provided the first evidence demonstrating S1-induced neuroinflammation in BV-2 microglia. We propose that induction of neuroinflammation by this protein in the microglia is mediated through activation of NF-κB and p38 MAPK, possibly as a result of TLR4 activation. These results contribute to our understanding of some of the mechanisms involved in CNS pathologies of SARS-CoV-2.

Garcinia kola and garcinoic acid suppress SARS-CoV-2 spike glycoprotein S1-induced hyper-inflammation in human PBMCs through inhibition of NF-κB activation.

Symptoms and complications associated with severe SARS-CoV-2 infection such as acute respiratory distress syndrome (ARDS) and organ damage have been linked to SARS-CoV-2 spike protein S1-induced increased production of pro-inflammatory cytokines by immune cells. In this study, the effects of an extract of Garcinia kola seeds and garcinoic acid were investigated in SARS-CoV-2 spike protein S1-stimulated human PBMCs. Results of ELISA experiments revealed that Garcinia kola extract (6.25, 12.5, and 25 µg/ml) and garcinoic acid (1.25, 2.5, and 5 µM) significantly reduced SARS-CoV-2 spike protein S1-induced secretion of TNFα, IL-6, IL-1ß, and IL-8 in PBMCs. In-cell western assays showed that pre-treatment with Garcinia kola extract and garcinoic acid reduced expressions of both phospho-p65 and phospho-IκBα proteins, as well as NF-κB DNA binding capacity and NF-κB-driven luciferase expression following stimulation of PBMCs with spike protein S1. Furthermore, pre-treatment of PBMCs with Garcinia kola extract prior to stimulation with SARS-CoV-2 spike protein S1 resulted in reduced damage to adjacent A549 lung epithelial cells. These results suggest that the seed of Garcinia kola and garcinoic acid are natural products which may possess pharmacological/therapeutic benefits in reducing cytokine storm in severe SARS-CoV-2 and other coronavirus infections.

Induction of Exaggerated Cytokine Production in Human Peripheral Blood Mononuclear Cells by a Recombinant SARS-CoV-2 Spike Glycoprotein S1 and Its Inhibition by Dexamethasone.

An understanding of the pathological inflammatory mechanisms involved in SARS-CoV-2 virus infection is necessary in order to discover new molecular pharmacological targets for SARS-CoV-2 cytokine storm. In this study, the effects of a recombinant SARS-CoV-2 spike glycoprotein S1 was investigated in human peripheral blood mononuclear cells (PBMCs). Stimulation of PBMCs with spike glycoprotein S1 (100 ng/mL) resulted in significant elevation in the production of TNFα, IL-6, IL-1ß and IL-8. However, pre-treatment with dexamethasone (100 nM) caused significant reduction in the release of these cytokines. Further experiments revealed that S1 stimulation of PBMCs increased phosphorylation of NF-κB p65 and IκBα, and IκBα degradation. DNA binding of NF-κB p65 was also significantly increased following stimulation with spike glycoprotein S1. Treatment of PBMCs with dexamethasone (100 nM) or BAY11-7082 (1 µM) resulted in inhibition of spike glycoprotein S1-induced NF-κB activation. Activation of p38 MAPK by S1 was blocked in the presence of dexamethasone and SKF 86002. CRID3, but not dexamethasone pre-treatment, produced significant inhibition of S1-induced activation of NLRP3/caspase-1. Further experiments revealed that S1-induced increase in the production of TNFα, IL-6, IL-1ß and IL-8 was reduced in the presence of BAY11-7082 and SKF 86002, while CRID3 pre-treatment resulted in the reduction of IL-1ß production. These results suggest that SARS-CoV-2 spike glycoprotein S1 stimulated PBMCs to release pro-inflammatory cytokines through mechanisms involving activation of NF-κB, p38 MAPK and NLRP3 inflammasome. It is proposed that the clinical benefits of dexamethasone in COVID-19 are possibly due to its anti-inflammatory activity in reducing SARS-CoV-2 cytokine storm.