SARS/Corona Virus and NanoStilbene

Pterostilbene is a potent inhibitor of NLRP3


The SARS (severe acute respiratory syndrome) outbreak was caused by a coronavirus (CoV) named the SARS-CoV.

SARS pathology is propagated both by direct cytotoxic effects of the virus and aberrant activation of the innate immune response [1].

Here, we identify several mechanisms by which a SARS-CoV open reading frame (ORF) activates intracellular stress pathways and targets the innate immune response. We show that ORF8b forms insoluble intracellular aggregates dependent on a valine at residue 77.

Aggregated ORF8b induces endoplasmic reticulum (ER) stress, lysosomal damage, and subsequent activation of the master regulator of the autophagy and lysosome machinery, Transcription factor EB (TFEB). ORF8b causes cell death in epithelial cells, which is partially rescued by reducing its ability to aggregate. In macrophages, ORF8b robustly activates the NLRP3 inflammasome by providing a potent signal 2 required for activation.

Mechanistically, ORF8b interacts directly with the Leucine Rich Repeat domain of NLRP3 and localizes with NLRP3 and ASC in cytosolic dot-like structures. ORF8b triggers cell death consistent with pyroptotic cell death in macrophages.

While in those cells lacking NLRP3, accumulating ORF8b cytosolic aggregates cause ER stress, mitochondrial dysfunction, and caspase-independent cell death.

The NLRP3 inflammasome is a caspase-1-containing multi-protein complex that controls the release of IL-1β and plays important roles in the development of inflammatory disease.

Here, we report that resveratrol (RESV), a polyphenolic compound naturally produced by plants, inhibits NLRP3 inflammasome-derived IL-1β secretion and pyroptosis in macrophages [2]. RESV inhibits the activation step of the NLRP3 inflammasome by suppressing mitochondrial damage. RESV also induces autophagy by activating p38, and macrophages treated with an autophagy inhibitor are resistant to the suppressive effects of RESV.

In addition, RESV administration mitigates glomerular proliferation, glomerular sclerosis, and glomerular inflammation in a mouse model of progressive IgA nephropathy. These findings were associated with decreased renal mononuclear leukocyte infiltration, reduced renal superoxide anion levels, and inhibited renal NLRP3 inflammasome activation. Our data indicate that RESV suppresses NLRP3 inflammasome activation by preserving mitochondrial integrity and by augmenting autophagy.

Pterostilbene (PTER) is a methyl ether of RESV, known to possess anti-inflammatory and anticancer activity in various model systems [3]. Found in blueberries and grapes, it has a polyphenolic structure that is very similar to that of resveratrol.

However, there are also some important differences. RESV has three hydroxy groups versus only one hydroxy group on PTER. In place of the other two hydroxy groups found on RESV, it has two methoxy groups.

This structural difference renders PTER more oil-soluble and therefore much more bioavailable after oral administration than RESV because they are more readily absorbed into the body [4].

When consumed orally, these stilbenoid compounds are absorbed into the body through the intestine. As they move into the blood stream, they pass through the liver. They are altered by the liver, where glucuronide or sulfates are attached to them, marking them for elimination from the body as part of the liver’s function in eliminating toxins.

These tags are attached to the hydroxy groups (HO/OH) on the stilbenoid compounds. This does not happen to PTER’s methoxy molecules (H3CO/OCH3), so there is only one site that can be marked. With three hydroxy groups, RESV can more easily be tagged and therefore eliminated rapidly from the body. This gives PTER over 7 times more time to reach areas where it is needed via the circulatory system and be absorbed into cells.

Studies shows p38 mitogen-activated protein kinase cascade is a key signal transduction pathway for eliciting the anti-inflammatory action of PTER [5].

PTER has been shown to attenuates early brain injury (EBI) following subarachnoid hemorrhage (SAH) via inhibition of the NLRP3 Inflammasome and Nox2-Related oxidative stress [6].

After PTER treatment, the neurological score and brain water content of the mice were assessed. Oxidative stress and neuronal injury were also evaluated. Nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome activity was assessed using western blot analysis.

The results indicated that PTER treatment reduces the SAH grade, neurological score, and brain water content following SAH. PTER treatment also reduced NLRP3 inflammasome activation. PTER alleviated the oxidative stress following SAH as evidenced by the dihydroethidium staining, superoxide dismutase activity, malondialdehyde content, 3-nitrotyrosie and 8-hydroxy-2-deoxyguanosine levels, and gp91phox and 4-hydroxynonenal expression levels. Additionally, PTER treatment reduced neuronal apoptosis. This study suggests that PTER attenuates EBI following SAH possibly via the inhibition of NLRP3 inflammasome and Nox2-related oxidative stress.

In fructose fed diabetic rats PTER decreases cardiac oxidative stress and inflammation via activation of AMPK/Nrf2/HO-1 pathway. The fructose fed rats demonstrated cardiac hypertrophy, hypertension, enhanced myocardial oxidative stress, inflammation and increased NF-κB expression. Administration of PTER significantly decreased cardiac hypertrophy, hypertension, oxidative stress, inflammation, NF-κB expression and NLRP3 inflammasome.

Collectively, the PTER seemed to reduce cardiac oxidative stress and inflammation in the diabetic rats through stimulation of AMPK/Nrf2/HO-1 signaling [7].

PTER has been shown to inhibit amyloid-β-induced (Aβ) neuroinflammation in microglia cell lines by inactivating the NLRP3/caspase-1 inflammasome pathway.

The results indicated that pterostilbene attenuated Aβ induced cytotoxicity of BV-2 cells. Aβ induced NO production and iNOS mRNA and protein expression, while PTER inhibited the induction.

The expression and secretion levels of IL-6, IL-1β, and TNF-α were enhanced by Aβ treatment, whereas PTER decreased them.

Aβ activated NLRP3/caspase-1 inflammasome was inactivated by PTER. In addition, the inhibitor of caspase-1 Z-YVAD-FMK attenuated the Aβ induced neuroinflammation in BV-2 cells. In conclusion, PTER attenuated the neuroinflammatory response induced by Aβ in microglia through inhibiting the NLRP3/caspase-1 inflammasome pathway, indicating that PTER might be an effective therapy for Alzheimer’s [8].

In contact dermatitis (CD) PTER prevents cell apoptosis and inhibits IL-1β-related NLRP3 inflammasome activation.

Hexavalent chromium (Cr(VI)) is widely used in many industries but can induce contact dermatitis especially in cement industries. Possible mechanisms involved were investigated to see whether chromium-induced CD could be effectively inhibited by treating with PTER.

In vivo, epidermal Cr(VI) administration causes cutaneous inflammation in mice ear skin, and the pro-inflammatory cytokines, TNF-α and IL-1β, were found in the epidermis, presenting the level of increase after Cr(VI) treatment.

In vitro experiments showed that apoptosis and endoplasmic reticulum (ER) stress were induced after treatment with different concentrations of Cr(VI) in HaCaT cells (human keratinocyte). Cr(VI) also induced TNF-α and IL-1β mRNA expressions, through the activation of the p38 mitogen-activated protein kinase (MAPK)/MAPK-activated protein kinase 2 (MK2) pathway.

Notably, the severity of the skin reactions in the epicutaneous elicitation test significantly diminished when the mouse was treated with PTER. Likewise, PTER intervention also ameliorated the inflammation and apoptosis of HaCaT cells in vitro. Furthermore, the findings demonstrated that the NLRP3 inflammasome could be involved in the Cr(VI)-mediated inflammation and apoptosis of CD. Thus, interrupting this mechanism with proper nontoxic agents, such as PTER, could be a new option to improve occupational chromium toxicity and hypersensitivity [9].

Nod-like receptor family, pyrin domain-containing 3 (NLRP3) regulates the secretion of proinflammatory cytokines interleukin 1 beta (IL-1β) and IL-18. We previously showed that influenza virus M2 or encephalomyocarditis virus (EMCV) 2B proteins stimulate IL-1β secretion following activation of the NLRP3 inflammasome. However, the mechanism by which severe acute respiratory syndrome coronavirus (SARS-CoV) activates the NLRP3 inflammasome remains unknown.

Here, we provide direct evidence that SARS-CoV 3a protein activates the NLRP3 inflammasome in lipopolysaccharide-primed macrophages. SARS-CoV 3a was sufficient to cause the NLRP3 inflammasome activation. The ion channel activity of the 3a protein was essential for 3a-mediated IL-1β secretion. While cells uninfected or infected with a lentivirus expressing a 3a protein defective in ion channel activity expressed NLRP3 uniformly throughout the cytoplasm, NLRP3 was redistributed to the perinuclear space in cells infected with a lentivirus expressing the 3a protein. K+ efflux and mitochondrial reactive oxygen species were important for SARS-CoV 3a-induced NLRP3 inflammasome activation. These results highlight the importance of viroporins, transmembrane pore-forming viral proteins, in virus-induced NLRP3 inflammasome activation [1].

PTER inactivated the NLRP3/caspase-1 inflammasome and Nox2-related oxidative stress in early brain injury. Administration of PTER significantly decreased NLRP3 inflammasome in diabetic rats through stimulation of AMPK/Nrf2/HO-1 signaling. In amyloid-β-induced (Aβ) neuroinflammation PTER has been shown to inhibit Aβ by inactivating the NLRP3/caspase-1 inflammasome pathway. Therefore, Aβ activated NLRP3/caspase-1 inflammasome was inactivated by PTER. PTER prevents cell apoptosis and inhibits IL-1β-related NLRP3 inflammasome activation in contact dermatitis. PTER appears to be a potent inhibitor of NLRP3 and related cytokines.

NanoStilbene is a highly concentrated oral formulation of nano particle PTER that has been shown to have a superior pharmacokinetic profile when compared to powder PTER administration [10].

NanoStilbene is prepared by low-energy emulsification which allows for better solubility, stability, and the release performance of PTER nanoparticles. The PTER placed in a nanoemulsion droplet is free from air, light, and hard environment; therefore, as a delivery system, nanoemulsion’s improve the bioavailability of PTER, and also protects it from oxidation and hydrolysis, while it possesses an ability of sustained release at the same time.

Therapeutic uses of nanotechnology typically involve the delivery of small-molecule drugs, peptides, proteins, and nucleic acids. Nanoparticles have advanced pharmacological effects compared with the therapeutic entities they contain. Active intracellular delivery and improved pharmacokinetics and pharmacodynamics of drug nanoparticles depend on various factors, including their size and surface properties.

Nanoparticle therapeutics is an emerging treatment modality for cancer and other inflammatory disorders. The National Cancer Institute has recognized nanotechnology as an emerging field with the potential to revolutionize modern medicine for the detection, treatment, and prevention of cancer.

NanoStilbene™ is manufactured and sold under US Patent No.: 9,682,047.

On 09-25-18 TSOI filed a patent application titled “Pterostilbene and Formulations Thereof for Treatment of Pathological Immune Activation”covering such infectious diseases as influenza; bird flu; severe acute respiratory syndrome (SARS); Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (HLH); bacterial sepsis; gram-negative sepsis; Dengue virus; malaria; Ebola virus; variola virus; and a systemic Gram-negative bacterial infection.