Novel Anti-Viral Properties of the Herbal Extract of Davallia mariesii against Influenza A Virus.

Gu-Sui-Bu, the dried rhizome of Davallia mariesii, is a traditional Chinese herbal remedy with a significant history of treating osteoporosis and inflammatory conditions. However, its potential as an anti-influenza agent and its underlying mechanisms of action remain unexplored. To obtain a more potent extract from D. mariesii and gain insights into its mechanism of action against influenza A virus (IAV), we utilized a partitioning process involving organic solvents and water, resulting in the isolation of butanolic subfractions of the D. mariesii extract (DMBE). DMBE exhibited a broad anti-viral spectrum, effectively inhibiting IAV, with an EC50 of 24.32 ± 6.19 µg/mL and a selectivity index of 6.05. We subsequently conducted a series of in vitro assays to evaluate the antiviral effects of DMBE and to uncover its mechanisms of action. DMBE was found to inhibit IAV during the early stages of infection by hindering the attachment of the virus onto and its penetration into host cells. Importantly, DMBE was observed to hinder IAV-mediated cell-cell fusion. It also inhibited neuraminidase activity, plaque size, and the expression levels of phospho-AKT. In summary, this study provides evidence for the effectiveness of D. mariesii as a complementary and alternative herbal remedy against IAV. Specifically, our data highlight DMBE's capabilities in inhibiting viral entry and the release of virions.

Synthesis of bioactive evodiamine and rutaecarpine analogues under ball milling conditions.

Mechanochemical reactions achieved by processes such as milling and grinding are promising alternatives to traditional solution-based chemistry. This approach not only eliminates the need for large amounts of solvents, thereby reducing waste generation, but also finds applications in chemical and materials synthesis. The focus of this study is on the synthesis of quinazolinone derivatives by ball milling, in particular evodiamine and rutaecarpine analogues. These compounds are of interest due to their diverse bioactivities, including potential anticancer properties. The study examines the reactions carried out under ball milling conditions, emphasizing their efficiency in terms of shorter reaction times and reduced environmental impact compared to conventional methods. The ball milling reaction of evodiamine and rutaecarpine analogues resulted in yields of 63-78% and 22-61%, respectively. In addition, these compounds were tested for their cytotoxic activity, and evodiamine exhibited an IC50 of 0.75 ± 0.04 µg mL-1 against the Ca9-22 cell line. At its core, this research represents a new means to synthesise these compounds, providing a more environmentally friendly and sustainable alternative to traditional approaches.

Design and synthesis of sirtinol analogs as human neutrophil elastase inhibitors.

Human neutrophil elastase (HNE) overexpression has a crucial role in most acute inflammation and alpha1-antitrypsin deficiency syndromes observed in humans, triggering neutrophil invasion and activation of macrophage inflammatory and proteolytic effects, leading to tissue damage. Manipulating HNE level homeostasis could potentially help treat neutrophilic inflammation. Previous studies have shown that sirtinol (1) has a specific influence on HNE and potently attenuates acute lung injury and hepatic injury mediated by lipopolysaccharide or trauma hemorrhage. Therefore, 1 was chosen as the model structure to obtain more potent anti-HNE agents. In the present study, we synthesized a series of sirtinol analogues and determined their inhibitory effects on HNE. Structure-activity relationship (SAR) studies showed that swapping the imine and methyl groups of the sirtinol scaffold with diazene and carboxyl groups, respectively, enhances the HNE inhibiting potency. Compound 29 exhibited the highest potency in the SAR study and showed dual inhibitory effects on HNE and proteinase 3 with IC50 values of 4.91 and 20.69 µM, respectively. Furthermore, 29 was confirmed to have dual impacts on inhibiting O2•- generation and elastase release in cell-based assays with IC50 values of 0.90 and 1.86 µM, respectively. These findings suggest that 29 is a promising candidate for developing HNE inhibitors in the treatment of neutrophilic inflammatory diseases.

Ursolic acid inhibits the synaptic release of glutamate and prevents glutamate excitotoxicity in rats.

The present study evaluated the effect of ursolic acid, a natural pentacyclic triterpenoid, on glutamate release in rat cortical nerve terminals (synaptosomes) and its neuroprotection in a kainic acid-induced excitotoxicity rat model. In cortical synaptosomes, ursolic acid produced a concentration-dependent inhibition of evoked glutamate release with a half-maximum inhibition of release value of 9.5 µM, and calcium-free medium and the P/Q -type Ca2+ channel blocker, ω-agatoxin IVA, but not ω-conotoxin GVIA, an N-type Ca2+ channel blocker, prevented the ursoloic acid effect. The molecular docking study indicated that ursolic acid interacted with P/Q-type Ca2+ channels. Ursolic acid also significantly decreased the depolarization-induced activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and the subsequent phosphorylation of synapsin I, and the ursolic acid effect on evoked glutamate release was inhibited by the CaMKII inhibitor KN 62 in synaptosomes. In addition, in rats that were intraperitoneally injected with ursolic acid 30 min before kainic acid intraperitoneal injection, cortical neuronal degeneration was attenuated. This effect of ursolic acid in the improvement of kainic acid-induced neuronal damage was associated with the reduction of kainic acid-induced glutamate increase in the cortex of rats; this was characterized by the reduction of glutamate and glutaminase levels and elevation of glutamate dehydrogenase, glutamate transporter 1, glutamate-aspartate transporter, and glutamine synthetase protein levels. These results suggest that ursolic acid inhibits glutamate release from cortical synaptosomes by decreasing P/Q-type Ca2+ channel activity and subsequently suppressing CaMKII and exerts a preventive effect against glutamate neurotoxicity by controlling glutamate levels.

Cynarin, a caffeoylquinic acid derivative in artichoke, inhibits exocytotic glutamate release from rat cortical nerve terminals (synaptosomes).

The purpose of this study was to evaluate the effect of cynarin, a caffeoylquinic acid derivative in artichoke, on glutamate release elicited by 4-aminopyridine (4-AP) in rat cortical nerve terminals (synaptosomes). We observed that cynarin decreased 4-aminopyridine-elicited glutamate release, which was prevented by the removal of external free Ca2+ with ethylene glycol bis (ß-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA) or the blockade of P/Q-type calcium channels with ω-agatoxin IVA. Molecular docking also revealed that cynarin formed a hydrogen bond with the P/Q-type Ca2+ channel, indicating a mechanism of action involving Ca2+ influx inhibition. Additionally, the inhibitory effect of cynarin on glutamate release is associated with a change in the available synaptic vesicles, as cynarin decreased 4-AP-elicited FM1-43 release or hypertonic sucrose-evoked glutamate release from synaptosomes. Furthermore, the suppression of protein kinase A (PKA) prevented the effect of cynarin on 4-AP-elicited glutamate release. 4-AP-elicited PKA and synapsin I or synaptosomal-associated protein of 25 kDa (SNAP-25) phosphorylation at PKA-specific residues were also attenuated by cynarin. Our data indicate that cynarin, through the suppression of P/Q-type Ca2+ channels, inhibits PKA activation and attenuates synapsin I and SNAP-25 phosphorylation at PKA-specific residues, thus decreasing synaptic vesicle availability and contributing to glutamate release inhibition in cerebral cortex terminals.

Melastoma malabathricum L. Suppresses Neutrophil Extracellular Trap Formation Induced by Synthetic Analog of Viral Double-Stranded RNA Associated with SARS-CoV-2 Infection.

Platelet hyper-reactivity and neutrophil extracellular trap (NET) formation contribute to the development of thromboembolic diseases for patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This study investigated the pathophysiological effects of SARS-CoV-2 surface protein components and the viral double-stranded RNA (dsRNA) on platelet aggregation and NET formation. Traditional Chinese medicine (TCM) with anti-viral effects was also delineated. The treatment of human washed platelets with SARS-CoV-2 spike protein S1 or the ectodomain S1 + S2 regions neither caused platelet aggregation nor enhanced agonists-stimulated platelet aggregation. Moreover, NET formation can be induced by polyinosinic-polycytidylic acid (poly(I:C)), a synthetic analog of viral dsRNA, but not by the pseudovirus composed of SARS-CoV-2 spike, envelope, and membrane proteins. To search for TCM with anti-NET activity, the plant Melastoma malabathricum L. which has anticoagulant activity was partially purified by fractionation. One of the fractions inhibited poly(I:C)-induced NET formation in a dose-dependent manner. This study implicates that SARS-CoV-2 structural proteins alone are not sufficient to promote NET and platelet activation. Instead, dsRNA formed during viral replication stimulates NET formation. This study also sheds new insight into using the active components of Melastoma malabathricum L. with anti-NET activity in the battle of thromboembolic diseases associated with SARS-CoV-2 infection.

Protective role of casuarinin from Melastoma malabathricum against a mouse model of 5-fluorouracil-induced intestinal mucositis: Impact on inflammation and gut microbiota dysbiosis.

BACKGROUND: 5-FU-induced intestinal mucositis (FUIIM) is a common gastrointestinal side effect of chemotherapy, leading to gastric pain in clinical cancer patients. In a previous study, we demonstrated that neutrophil elastase (NE) inhibitors could alleviate FUIIM and manipulate the homeostasis of the gut microbiota. The root of Melastoma malabathricum, also called Ye-Mu-Dan, has been used as a traditional Chinese medicine for gastrointestinal disease. Water extract of the roots of M. malabathricum exhibits an inhibitory effect on NE, with an IC50 value of 9.13 µg/ml. PURPOSE: In this study, we aimed to isolate an anti-NE compound from the root of M. malabathricum and to determine the protective effect of the bioactive component on a mouse model of FUIIM with respect to tissue damage, inflammation, intestinal barrier dysfunction, and gut microbiota dysbiosis. METHODS: A water extract of the roots of M. malabathricum was prepared and its major bioactive compound, was identified using bioactivity-guided fractionation. The effects of samples on the inhibition of NE activity were evaluated using enzymatic assays. To evaluate the effects of the bioactive compound in an FUIIM animal model, male C57BL/6 mice treated with or without casuarinin (50 and 100 mg/kg/day, p.o.), and then received of 5-fluorouracil (50 mg/kg/day) intraperitoneally for 5 days to induce FUIIM. Histopathological staining was used to monitor the tissue damage, proliferation of intestinal crypts, and expression of tight junction proteins. The inflammation score was estimated by determining the levels of oxidative stress, neutrophil-related proteases, and proinflammatory cytokines in tissue and serum. The ecology of the gut microbiota was evaluated using 16S rRNA gene sequencing. RESULTS: Casuarinin had the most potent and selective effect against NE, with an IC50 value of 2.79 ± 0.07 µM. Casuarinin (100 mg/kg/day, p.o.) significantly improved 5-FU-induced body weight loss together with food intake reduction, and it also significantly reversed villus atrophy, restored the proliferative activity of the intestinal crypts, and suppressed inflammation and intestinal barrier dysfunction in the mouse model of FUIIM. Casuarinin also reversed 5-FU-induced gut microbiota dysbiosis, particularly the abundance of Actinobacteria, Candidatus Arthromitus, and Lactobacillus murinus, and the Firmicutes-to-Bacteroidetes ratio. CONCLUSION: This study firstly showed that casuarinin isolated from the root part of M. malabathricum could be used as a NE inhibitor, whereas it could improve FUIIM by modulating inflammation, intestinal barrier dysfunction, and gut microbiota dysbiosis. In summary, exploring anti-NE natural product may provide a way to find candidate for improvement of FUIIM.

3,4-Dicaffeoylquinic Acid from the Medicinal Plant Ilex kaushue Disrupts the Interaction Between the Five-Fold Axis of Enterovirus A-71 and the Heparan Sulfate Receptor.

While infections by enterovirus A71 (EV-A71) are generally self-limiting, they can occasionally lead to serious neurological complications and death. No licensed therapies against EV-A71 currently exist. Using anti-virus-induced cytopathic effect assays, 3,4-dicaffeoylquinic acid (3,4-DCQA) from Ilex kaushue extracts was found to exert significant anti-EV-A71 activity, with a broad inhibitory spectrum against different EV-A71 genotypes. Time-of-drug-addition assays revealed that 3,4-DCQA affects the initial phase (entry step) of EV-A71 infection by directly targeting viral particles and disrupting viral attachment to host cells. Using resistant virus selection experiments, we found that 3,4-DCQA targets the glutamic acid residue at position 98 (E98) and the proline residue at position 246 (P246) in the 5-fold axis located within the VP1 structural protein. Recombinant viruses harboring the two mutations were resistant to 3,4-DCQA-elicited inhibition of virus attachment and penetration into human rhabdomyosarcoma (RD) cells. Finally, we showed that 3,4-DCQA specifically inhibited the attachment of EV-A71 to the host receptor heparan sulfate (HS), but not to the scavenger receptor class B member 2 (SCARB2) and P-selectin glycoprotein ligand-1 (PSGL1). Molecular docking analysis confirmed that 3,4-DCQA targets the 5-fold axis to form a stable structure with the E98 and P246 residues through noncovalent and van der Waals interactions. The targeting of E98 and P246 by 3,4-DCQA was found to be specific; accordingly, HS binding of viruses carrying the K242A or K244A mutations in the 5-fold axis was successfully inhibited by 3,4-DCQA.The clinical utility of 3,4-DCQA in the prevention or treatment of EV-A71 infections warrants further scrutiny. IMPORTANCE The canyon region and the 5-fold axis of the EV-A71 viral particle located within the VP1 protein mediate the interaction of the virus with host surface receptors. The three most extensively investigated cellular receptors for EV-A71 include SCARB2, PSGL1, and cell surface heparan sulfate. In the current study, a RD cell-based anti-cytopathic effect assay was used to investigate the potential broad spectrum inhibitory activity of 3,4-DCQA against different EV-A71 strains. Mechanistically, we demonstrate that 3,4-DCQA disrupts the interaction between the 5-fold axis of EV-A71 and its heparan sulfate receptor; however, no effect was seen on the SCARB2 or PSGL1 receptors. Taken together, our findings show that this natural product may pave the way to novel anti-EV-A71 therapeutic strategies.

An Anthranilate Derivative Inhibits Glutamate Release and Glutamate Excitotoxicity in Rats.

The neurotransmitter glutamate plays an essential role in excitatory neurotransmission; however, excessive amounts of glutamate lead to excitotoxicity, which is the most common pathogenic feature of numerous brain disorders. This study aimed to investigate the role of butyl 2-[2-(2-fluorophenyl)acetamido]benzoate (HFP034), a synthesized anthranilate derivative, in the central glutamatergic system. We used rat cerebro-cortical synaptosomes to examine the effect of HFP034 on glutamate release. In addition, we used a rat model of kainic acid (KA)-induced glutamate excitotoxicity to evaluate the neuroprotective potential of HFP034. We showed that HFP034 inhibits 4-aminopyridine (4-AP)-induced glutamate release from synaptosomes, and this inhibition was absent in the absence of extracellular calcium. HFP034-mediated inhibition of glutamate release was associated with decreased 4-AP-evoked Ca2+ level elevation and had no effect on synaptosomal membrane potential. The inhibitory effect of HFP034 on evoked glutamate release was suppressed by blocking P/Q-type Ca2+ channels and protein kinase C (PKC). Furthermore, HFP034 inhibited the phosphorylation of PKC and its substrate, myristoylated alanine-rich C kinase substrate (MARCKS) in synaptosomes. We also observed that HFP034 pretreatment reduced neuronal death, glutamate concentration, glial activation, and the levels of endoplasmic reticulum stress-related proteins, calpains, glucose-regulated protein 78 (GRP 78), C/EBP homologous protein (CHOP), and caspase-12 in the hippocampus of KA-injected rats. We conclude that HFP034 is a neuroprotective agent that prevents glutamate excitotoxicity, and we suggest that this effect involves inhibition of presynaptic glutamate release through the suppression of P/Q-type Ca2+ channels and PKC/MARCKS pathways.

Chlorogenic Acid Decreases Glutamate Release from Rat Cortical Nerve Terminals by P/Q-Type Ca2+ Channel Suppression: A Possible Neuroprotective Mechanism.

The glutamatergic neurotransmitter system has received substantial attention in research on the pathophysiology and treatment of neurological disorders. The study investigated the effect of the polyphenolic compound chlorogenic acid (CGA) on glutamate release in rat cerebrocortical nerve terminals (synaptosomes). CGA inhibited 4-aminopyridine (4-AP)-induced glutamate release from synaptosomes. This inhibition was prevented in the absence of extracellular Ca2+ and was associated with the inhibition of 4-AP-induced elevation of Ca2+ but was not attributed to changes in synaptosomal membrane potential. In line with evidence observed through molecular docking, CGA did not inhibit glutamate release in the presence of P/Q-type Ca2+ channel inhibitors; therefore, CGA-induced inhibition of glutamate release may be mediated by P/Q-type Ca2+ channels. CGA-induced inhibition of glutamate release was also diminished by the calmodulin and Ca2+/calmodilin-dependent kinase II (CaMKII) inhibitors, and CGA reduced the phosphorylation of CaMKII and its substrate, synapsin I. Furthermore, pretreatment with intraperitoneal CGA injection attenuated the glutamate increment and neuronal damage in the rat cortex that were induced by kainic acid administration. These results indicate that CGA inhibits glutamate release from cortical synaptosomes by suppressing P/Q-type Ca2+ channels and CaMKII/synapsin I pathways, thereby preventing excitotoxic damage to cortical neurons.

Natural Product Isoliquiritigenin Activates GABAB Receptors to Decrease Voltage-Gate Ca2+ Channels and Glutamate Release in Rat Cerebrocortical Nerve Terminals.

Reduction in glutamate release is a key mechanism for neuroprotection and we investigated the effect of isoliquiritigenin (ISL), an active ingredient of Glycyrrhiza with neuroprotective activities, on glutamate release in rat cerebrocortical nerve terminals (synaptosomes). ISL produced a concentration-dependent inhibition of glutamate release and reduced the intraterminal [Ca2+] increase. The inhibition of glutamate release by ISL was prevented after removing extracellular Ca2+ or blocking P/Q-type Ca2+ channels. This inhibition was mediated through the γ-aminobutyric acid type B (GABAB) receptors because ISL was unable to inhibit glutamate release in the presence of baclofen (an GABAB agonist) or CGP3548 (an GABAB antagonist) and docking data revealed that ISL interacted with GABAB receptors. Furthermore, the ISL inhibition of glutamate release was abolished through the inhibition of Gi/o-mediated responses or Gßγ subunits, but not by 8-bromoadenosine 3',5'-cyclic monophosphate or adenylate cyclase inhibition. The ISL inhibition of glutamate release was also abolished through the inhibition of protein kinase C (PKC), and ISL decreased the phosphorylation of PKC. Thus, we inferred that ISL, through GABAB receptor activation and Gßγ-coupled inhibition of P/Q-type Ca2+ channels, suppressed the PKC phosphorylation to cause a decrease in evoked glutamate release at rat cerebrocortical nerve terminals.

Identification of Potential Drug Targets of Broad-Spectrum Inhibitors with a Michael Acceptor Moiety Using Shotgun Proteomics.

The Michael addition reaction is a spontaneous and quick chemical reaction that is widely applied in various fields. This reaction is performed by conjugating an addition of nucleophiles with α, ß-unsaturated carbonyl compounds, resulting in the bond formation of C-N, C-S, C-O, and so on. In the development of molecular materials, the Michael addition is not only used to synthesize chemical compounds but is also involved in the mechanism of drug action. Several covalent drugs that bond via Michael addition are regarded as anticarcinogens and anti-inflammatory drugs. Although drug development is mainly focused on pharmaceutical drug discovery, target-based discovery can provide a different perspective for drug usage. However, considerable time and labor are required to define a molecular target through molecular biological experiments. In this review, we systematically examine the chemical structures of current FDA-approved antiviral drugs for potential Michael addition moieties with α, ß-unsaturated carbonyl groups, which may exert an unidentified broad-spectrum inhibitory mechanism to target viral or host factors. We thus propose that profiling the targets of antiviral agents, such as Michael addition products, can be achieved by employing a high-throughput LC-MS approach to comprehensively analyze the interaction between drugs and targets, and the subsequent drug responses in the cellular environment to facilitate drug repurposing and/or identify potential adverse effects, with a particular emphasis on the pros and cons of this shotgun proteomic approach.

Eupafolin Suppresses P/Q-Type Ca2+ Channels to Inhibit Ca2+/ Calmodulin-Dependent Protein Kinase II and Glutamate Release at Rat Cerebrocortical Nerve Terminals.

Eupafolin, a constituent of the aerial parts of Phyla nodiflora, has neuroprotective property. Because reducing the synaptic release of glutamate is crucial to achieving pharmacotherapeutic effects of neuroprotectants, we investigated the effect of eupafolin on glutamate release in rat cerebrocortical synaptosomes and explored the possible mechanism. We discovered that eupafolin depressed 4-aminopyridine (4-AP)-induced glutamate release, and this phenomenon was prevented in the absence of extracellular calcium. Eupafolin inhibition of glutamate release from synaptic vesicles was confirmed through measurement of the release of the fluorescent dye FM 1-43. Eupafolin decreased 4-AP-induced [Ca2+]i elevation and had no effect on synaptosomal membrane potential. The inhibition of P/Q-type Ca2+ channels reduced the decrease in glutamate release that was caused by eupafolin, and docking data revealed that eupafolin interacted with P/Q-type Ca2+ channels. Additionally, the inhibition of calcium/calmodulindependent protein kinase II (CaMKII) prevented the effect of eupafolin on evoked glutamate release. Eupafolin also reduced the 4-AP-induced activation of CaMK II and the subsequent phosphorylation of synapsin I, which is the main presynaptic target of CaMKII. Therefore, eupafolin suppresses P/Q-type Ca2+ channels and thereby inhibits CaMKII/synapsin I pathways and the release of glutamate from rat cerebrocortical synaptosomes.

Rosmarinic Acid, a Bioactive Phenolic Compound, Inhibits Glutamate Release from Rat Cerebrocortical Synaptosomes through GABAA Receptor Activation.

Rosmarinic acid, a major component of rosemary, is a polyphenolic compound with potential neuroprotective effects. Asreducing the synaptic release of glutamate is crucial to achieving neuroprotectant's pharmacotherapeutic effects, the effect of rosmarinic acid on glutamate release was investigated in rat cerebrocortical nerve terminals (synaptosomes). Rosmarinic acid depressed the 4-aminopyridine (4-AP)-induced glutamate release in a concentration-dependent manner. The removal of extracellular calcium and the blockade of vesicular transporters prevented the inhibition of glutamate release by rosmarinic acid. Rosmarinic acid reduced 4-AP-induced intrasynaptosomal Ca2+ elevation. The inhibition of N-, P/Q-type Ca2+ channels and the calcium/calmodulin-dependent kinase II (CaMKII) prevented rosmarinic acid from having effects on glutamate release. Rosmarinic acid also reduced the 4-AP-induced activation of CaMKII and the subsequent phosphorylation of synapsin I, the main presynaptic target of CaMKII. In addition, immunocytochemistry confirmed the presence of GABAA receptors. GABAA receptor agonist and antagonist blocked the inhibitory effect of rosmarinic acid on 4-AP-evoked glutamate release. Docking data also revealed that rosmarinic acid formed a hydrogen bond with the amino acid residues of GABAA receptor. These results suggested that rosmarinic acid activates GABAA receptors in cerebrocortical synaptosomes to decrease Ca2+ influx and CaMKII/synapsin I pathway to inhibit the evoked glutamate release.

Randialic acid B and tomentosolic acid block formyl peptide receptor 1 in human neutrophils and attenuate psoriasis-like inflammation in vivo.

Psoriasis is a long-lasting inflammatory skin disease lacking proper cure. Dysregulated activation of neutrophils is a major pathogenic factor in psoriasis. Formyl peptide receptor 1 (FPR1) triggers neutrophil activation in response to bacteria- or mitochondria-derived N-formyl peptides, but its significance in neutrophilic psoriasis remains unknown. In this study, we discovered two derivatives of ursolic acid, 3ß-hydroxyurs-12,18-dien-28-oic acid (randialic acid B, RAB) and 3ß-hydroxyurs-12,19-dien-28-oic acid (tomentosolic acid, TA), as FPR1 inhibitors in human neutrophils with ability to suppress psoriatic symptoms in mice. Both RAB and TA, triterpenoids of traditional medicinal plant Ilex kaushue, selectively inhibited reactive oxygen species production, elastase release, and CD11b expression in human neutrophils activated by FPR1, but not non-FPR1 agonists. Importantly, RAB and TA inhibited the binding of N-formyl peptide to FPR1 in human neutrophils, neutrophil-like THP-1 cells, and hFPR1-transfected HEK293 cells, indicating FPR1 antagonism. Moreover, in assays induced by various concentrations of FPR1 agonist, both RAB and TA acted competitively for its binding to the FPR1 receptor. The FPR1-downstream signaling such as Ca2+ mobilisation and activation of Akt and MAPKs was also competitively inhibited. In addition, imiquimod-induced psoriasis-like symptoms, including epidermal hyperplasia, desquamation with scaling, neutrophil skin infiltration, and transepidermal water loss were significantly reduced by both RAB and TA. The results illustrate a possible role of human neutrophils FPR1 receptor in psoriasis-like inflammation. Accordingly, triterpenoids RAB and TA represent novel FPR1 antagonists and exhibit therapeutic potential for treating neutrophilic inflammatory skin diseases.

Neutrophil elastase inhibitor (MPH-966) improves intestinal mucosal damage and gut microbiota in a mouse model of 5-fluorouracil-induced intestinal mucositis.

BACKGROUND: 5-Fluorouracil (5-FU)-based chemotherapy is first-line chemotherapy for colorectal cancer. However, 5-FU-induced intestinal mucositis (FUIIM) is a common adverse effect that severely impairs drug tolerance and results in poor patient health. METHODS: Male C57BL/6 mice were given 5-FU (50 mg/kg/day, i.p.) and treated with MPH-966 (5 and 7.5 mg/kg/day, p.o.) for five days. The body weight loss and the amount of food intake, and histopathological findings were recorded and analyzed. In addition, the neutrophil infiltration, levels of neutrophil serine proteases and pro-inflammatory cytokines, and tight junction proteins expression in intestinal tissues were determined. The ecology of gut microbiota was performed through next-generation sequencing technologies. RESULTS: Neutrophil elastase (NE) overexpression is a key feature in FUIIM. This study showed that treatment with the specific NE inhibitor MPH-966 (7.5 mg/kg/day, p.o.) significantly reversed 5-FU-induced loss in body weight and food intake; reversed villous atrophy; significantly suppressed myeloperoxidase, NE, and proteinase 3 activity; and reduced pro-inflammatory cytokine expression in an FUIIM mouse model. In addition, MPH-966 prevented 5-FU-induced intestinal barrier dysfunction, as indicated by the modulated expression of the tight junction proteins zonula occludin-1 and occludin. MPH-966 also reversed 5-FU-induced changes in gut microbiota diversity and abundances, specifically the Firmicutes-to-Bacteroidetes ratio; Muribaculaceae, Ruminococcaceae, and Eggerthellaceae abundances at the family level; and Candidatus Arthromitus abundance at the genus level. CONCLUSION: These data indicate that NE inhibitor is a key treatment candidate to alleviate FUIIM by regulating abnormal inflammatory responses, intestinal barrier dysfunction, and gut microbiota imbalance.

Anti-inflammatory, hepatoprotective and antioxidant activity of ellagitannin isolated from Melaleuca styphelioides.

Ellagitannins have a marked antioxidant effect and can prevent liver injury induced by free radicals. An undescribed ellagitannin named styphelioidin was isolated from Melaleuca styphelioides Sm. The structure of styphelioidin was elucidated by using various spectroscopic methods. The hepatoprotective activity of styphelioidin (25, 50, and 100 µM) was tested using the CCl4-challenged HepG2 cell model by measuring alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in HepG2 cells treated with styphelioidin for 1 h followed by 40 mM CCl4. Glutathione (GSH), superoxide dismutase activity (SOD) and lipid peroxidation (MDA) were evaluated to determine the mechanisms of the hepatoprotective activity. Styphelioidin significantly reduced the levels of ALT, AST, and MDA at all tested concentrations. Moreover, it conferred a marked increase in the GSH levels and the SOD activity compared to the CCl4-treated groups. Styphelioidin also exerted DPPH· radical-scavenging effects with an IC50 value of 3.67 µM. Results indicated the hepatoprotective therapeutic potential of styphelioidin comparable to silymarin. Moreover, anti-inflammatory activity was assessed and styphelioidin inhibited fMLF/CB-induced elastase release in human neutrophils with IC50 2.51 µM. Cell-free experiments with human neutrophil elastase indicated a direct enzymatic inhibitory effect of styphelioidin on the enzyme activity (IC50 2.58 µM). The potential of styphelioidin to interact with human neutrophil elastase binding sites was further confirmed by molecular docking of styphelioidin into human neutrophil elastase crystal structure using AutoDock 4.2. Styphelioidin represents a potent hepatoprotective and antioxidant agent with effects on ALT, AST, MDA, GSH, and SOD comparable to silymarin. The beneficial anti-elastase properties hold the potential for drug development against elastase-related inflammatory diseases. This study highlights a promising natural hepatoprotective and anti-inflammatory candidate derived from M. styphelioides.

Rosmarinic acid exhibits broad anti-enterovirus A71 activity by inhibiting the interaction between the five-fold axis of capsid VP1 and cognate sulfated receptors.

Enterovirus A71 (EV-A71), a positive-stranded RNA virus of the Picornaviridae family, may cause neurological complications or fatality in children. We examined specific factors responsible for this virulence using a chemical genetics approach. Known compounds from an anti-EV-A71 herbal medicine, Salvia miltiorrhiza (Danshen), were screened for anti-EV-A71. We identified a natural product, rosmarinic acid (RA), as a potential inhibitor of EV-A71 by cell-based antiviral assay and in vivo mouse model. Results also show that RA may affect the early stage of viral infection and may target viral particles directly, thereby interfering with virus-P-selectin glycoprotein ligand-1 (PSGL1) and virus-heparan sulfate interactions without abolishing the interaction between the virus and scavenger receptor B2 (SCARB2). Sequencing of the plaque-purified RA-resistant viruses revealed a N104K mutation in the five-fold axis of the structural protein VP1, which contains positively charged amino acids reportedly associated with virus-PSGL1 and virus-heparan sulfate interactions via electrostatic attraction. The plasmid-derived recombinant virus harbouring this mutation was confirmed to be refractory to RA inhibition. Receptor pull-down showed that this non-positively charged VP1-N104 is critical for virus binding to heparan sulfate. As the VP1-N104 residue is conserved among different EV-A71 strains, RA may be useful for inhibiting EV-A71 infection, even for emergent virus variants. Our study provides insight into the molecular mechanism of virus-host interactions and identifies a promising new class of inhibitors based on its antiviral activity and broad spectrum effects against a range of EV-A71.

Polysaccharide-containing fraction from Artemisia argyi inhibits tumor cell-induced platelet aggregation by blocking interaction of podoplanin with C-type lectin-like receptor 2.

Tumor cell-induced platelet aggregation (TCIPA) is a mechanism that involves the protection of tumor cells in the circulation and the promotion of tumor cell invasion and metastases. The C-type lectin-like receptor 2 (CLEC-2) that binds podoplanin (PDPN) is on the platelet surface and facilitates the TCIPA. Selective blockage of the PDPN-mediated platelet-tumor cell interaction is thereby a plausible strategy for inhibiting metastases. In a search for antagonists of PDPN- and tumor cell-induced platelet aggregation, traditional Chinese medicines were screened and it was found that the water extract of Artemisia argyi leaves selectively inhibited the PDPN-induced platelet aggregation. Bioactivity-guided fractionation analysis was performed for defining a polysaccharide-containing fraction (AAWAP) characterized by inhibition of PDPN activity and tumor cell-induced platelet aggregation. The pharmacological effects of AAWAP on PDPN-activated CLEC-2 signaling were determined by using Western blot and alpha screening analyses. AAWAP was non-toxic to the cells and platelets and it suppressed PDPN- and tumor cell-induced platelet aggregation by irreversibly blocking the interaction between PDPN and CLEC-2 in a dose-dependent manner. These findings indicate that AAWAP is an antagonist of the PDPN-CLEC-2 interaction. This action by AAWAP may result in the prevention of tumor cell metastases, and if so, could become an effective pharmacological agent in treating cancer patients.