Luteolin-3′-O-Phosphate Inhibits Lipopolysaccharide-Induced Inflammatory Responses by Regulating NF-κB/MAPK Cascade Signaling in RAW 264.7 Cells

Luteolin (LT), present in most plants, has potent anti-inflammatory properties both in vitro and in vivo. Furthermore, some of its derivatives, such as luteolin-7-O-glucoside, also exhibit anti-inflammatory activity. However, the molecular mechanisms underlying luteolin-3′-O-phosphate (LTP)-mediated immune regulation are not fully understood. In this paper, we compared the anti-inflammatory properties of LT and LTP and analyzed their molecular mechanisms of action; we obtained LTP via the biorenovation of LT. We investigated the anti-inflammatory activities of LT and LTP in macrophage RAW 264.7 cells. We confirmed from previously reported literature that LT inhibits the production of nitric oxide and prostaglandin E₂, as well as the expression of inducible NO synthetase and cyclooxygenase-2. In addition, expressions of inflammatory genes and mediators, such as tumor necrosis factor-α, interleukin-6, and interleukin-1β, were suppressed. LTP showed anti-inflammatory activity similar to LT, but better anti-inflammatory activity in all the experiments, while also inhibiting mitogen-activated protein kinase and nuclear factor-kappa B more effectively than LT. At a concentration of 10 μM, LTP showed differences of 2.1 to 44.5% in the activity compared to LT; it also showed higher anti-inflammatory activity. Our findings suggest that LTP has stronger anti-inflammatory activity than LT.     

Anti-Inflammatory Effects of 6-Methylcoumarin in LPS-Stimulated RAW 264.7 Macrophages via Regulation of MAPK and NF-κB Signaling Pathways

Persistent inflammatory reactions promote mucosal damage and cause dysfunction, such as pain, swelling, seizures, and fever. Therefore, in this study, in order to explore the anti-inflammatory effect of 6-methylcoumarin (6-MC) and suggest its availability, macrophages were stimulated with lipopolysaccharide (LPS) to conduct an in vitro experiment. The effects of 6-MC on the production and levels of pro-inflammatory cytokines (interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α) and inflammatory mediators (nitric oxide (NO), prostaglandin E₂ (PGE₂)) in LPS-stimulated RAW 264.7 cells were examined. The results showed that 6-MC reduced the levels of NO and PGE₂ without being cytotoxic. In addition, it was demonstrated that the increase in the expression of pro-inflammatory cytokines caused by LPS stimulation, was decreased in a concentration-dependent manner with 6-MC treatment. Moreover, Western blot results showed that the protein levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), which increased with LPS treatment, were decreased by 6-MC treatment. Mechanistic studies revealed that 6-MC reduced the phosphorylation of the mitogen-activated protein kinase (MAPK) family and IκBα in the MAPK and nuclear factor-kappa B (NF-κB) pathways, respectively. These results suggest that 6-MC is a potential therapeutic agent for inflammatory diseases that inhibits inflammation via the MAPK and NF-κB pathways.     

Sophorolipid Suppresses LPS-Induced Inflammation in RAW264.7 Cells through the NF-κB Signaling Pathway

Objectives: Biosurfactants with anti-inflammatory activity may alleviate skin irritation caused by synthetic surfactants in cleaning products. Sophorolipid (SL) is a promising alternative to synthetic surfactants. However, there are few reports on the anti-inflammatory activity of SL and the underlying mechanism. The purpose of this work is to verify that lipopolysaccharide (LPS)-induced inflammation could be inhibited through targeting the pathway of nuclear factor-κB (NF-κB) in RAW264.7 cells. Methods: The influence of SL on cytokine release was investigated by LPS-induced RAW264.7 cells using ELISA. The quantification of the protein expression of corresponding molecular markers was realized by Western blot analysis. Flow cytometry was employed to determine the levels of Ca²⁺ and reactive oxygen species (ROS). The relative expression of inducible nitric oxide synthase (INOS) and cyclooxygenase-2 (COX-2) was determined by RT-PCR. An immunofluorescence assay and confocal microscope were used to observe the NF-κB/p65 translocation from the cytoplasm into the nucleus. The likely targets of SL were predicted by molecular docking analysis. Results: SL showed anti-inflammatory activity and reduced the release of inflammatory cytokines including interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and nitric oxide (NO). The experimental results show that SL suppressed the Ca²⁺ and ROS levels influx in the LPS-induced RAW264.7 cells and alleviated the LPS-induced expression of iNOS and COX-2, the LPS-induced translocation of NF-κB (p65) from the cytoplasm into the nucleus, and the expression of phosphorylated proteins such as p65 and IκBα. Furthermore, molecular docking analysis showed that SL may inhibit inflammatory signaling by competing with LPS to bind TLR4/MD-2 through hydrophobic interactions and by inhibiting IKKβ activation through the hydrogen bonding and hydrophobic interactions. Conclusion: This study demonstrated that SL exerted anti-inflammatory activity via the pathway of NF-κB in RAW264.7 cells.     

Scutellarein Inhibits LPS-Induced Inflammation through NF-κB/MAPKs Signaling Pathway in RAW264.7 Cells

Inflammation is a severe topic in the immune system and play a role as pro-inflammatory mediators. In response to such inflammatory substances, immune cells release cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). Lipopolysaccharide (LPS) is known as an endotoxin in the outer membrane of Gram-negative bacteria, and it catalyzes inflammation by stimulating the secretion of inflammatory-mediated cytokines such as cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) by stimulated immune cells. Among the pathways involved in inflammation, nuclear factor kappa (NF-кB) and mitogen-activated protein kinases (MAPKs) are important. NF-kB is a diploid composed of p65 and IkBα and stimulates the pro- gene. MAPKs is a family consisting of the extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38, JNK and p38 play a role as proinflammatory mediators. Thus, we aim to determine the scutellarein (SCU) effect on LPS stimulated RAW264.7 cells. Furthermore, since scutellarein has been shown to inhibit the SARS coronavirus helicase and has been used in Chinese medicine to treat inflammatory disorders like COVID-19, it would be required to examine scutellarein’s anti-inflammatory mechanism. We identified inflammation-inducing substances using western blot with RAW264.7 cells and SCU. And we discovered that was reduced by treatment with SCU in p-p65 and p-IκBα. Also, we found that p-JNK and p-ERK were also decreased but there was no effect in p-p38. In addition, we have confirmed that the iNOS was also decreased after treatment but there is no change in the expression of COX-2. Therefore, this study shows that SCU can be used as a compound to treat inflammation.     

Effects of 2′,4′-Dihydroxy-6′-methoxy-3′,5′-dimethylchalcone from Syzygium nervosum Seeds on Antiproliferative,DNA Damage,Cell Cycle Arrest,and Apoptosis in Human Cervical Cancer Cell Lines

2′,4′-Dihydroxy-6′-methoxy-3′,5′-dimethylchalcone (DMC), a natural product derived from Syzygium nervosum A. Cunn. ex DC., was investigated for its inhibitory activities against various cancer cell lines. In this work, we investigated the effects of DMC and available anticervical cancer drugs (5-fluorouracil, cisplatin, and doxorubicin) on three human cervical cancer cell lines (C-33A, HeLa, and SiHa). DMC displayed antiproliferative cervical cancer activity in C-33A, HeLa, and SiHa cells, with IC₅₀ values of 15.76 ± 1.49, 10.05 ± 0.22, and 18.31 ± 3.10 µM, respectively. DMC presented higher antiproliferative cancer activity in HeLa cells; therefore, we further investigated DMC-induced apoptosis in this cell line, including DNA damage, cell cycle arrest, and apoptosis assays. As a potential anticancer agent, DMC treatment increased DNA damage in cancer cells, observed through fluorescence inverted microscopy and a comet assay. The cell cycle assay showed an increased number of cells in the G₀/G₁ phase following DMC treatment. Furthermore, DMC treatment-induced apoptosis cell death was approximately three- to four-fold higher compared to the untreated group. Here, DMC represented a compound-induced apoptosis for cell death in the HeLa cervical cancer cell line. Our findings suggest that DMC, a phytochemical agent, is a potential candidate for antiproliferative cervical cancer drug development.     

Identification of Novel 4′-O-Demethyl-epipodophyllotoxin Derivatives as Antitumor Agents Targeting Topoisomerase II

C4 variation of 4′-O-demethyl-epipodophyllotoxin (DMEP) is an effective approach to optimize the antitumor spectra of this compound class. Accordingly, two series of novel DMEP derivatives were synthesized, and as expected, the antitumor spectra of these derivatives varied with different C4 substituents. Notably, most compounds showed significant inhibition against the etoposide (2)-resistant KBvin cells. Four of the compounds (11, 18, 27 and 28) induced protein-linked DNA break (PLDB) levels higher than those of GL-331 (6) and 2, and are assumed to be topoisomerase II (topo II) poisons more potent than 6 and 2. Compound 28, a potent topo II poison highly effective against KBvin cells, was further evaluated with a panel of tumor cells and was most active against HepG2. This compound also exhibited apparent in vivo antitumor efficacy in hepatoma 22 (H22) mouse model. The results indicated that C4 derivation of DMEP is a feasible approach to identify potent topo II inhibitors with optimized antitumor profiles.     

Wistin Exerts an Anti-Inflammatory Effect via Nuclear Factor-κB and p38 Signaling Pathways in Lipopolysaccharide-Stimulated RAW264.7 Cells

Inflammation is an immune response to cellular damage caused by various stimuli (internal or external) and is essential to human health. However, excessive inflammatory responses may be detrimental to the host. Considering that the existing drugs for the treatment of inflammatory diseases have various side effects, such as allergic reactions, stomach ulcers, and cardiovascular problems, there is a need for research on new anti-inflammatory agents with low toxicity and fewer side effects. As 4′,6-dimethoxyisoflavone-7-O-β-d-glucopyranoside (wistin) is a phytochemical that belongs to an isoflavonoid family, we investigated whether wistin could potentially serve as a novel anti-inflammatory agent. In this study, we found that wistin significantly reduced the production of nitric oxide and intracellular reactive oxygen species in lipopolysaccharide-stimulated RAW 264.7 cells. Moreover, wistin reduced the mRNA levels of pro-inflammatory enzymes (inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX-2)) and cytokines (interleukin (IL)-1β and IL-6) and significantly reduced the protein expression of pro-inflammatory enzymes (iNOS and COX-2). Furthermore, wistin reduced the activation of the nuclear factor-κB and p38 signaling pathways. Together, these results suggest that wistin is a prospective candidate for the development of anti-inflammatory drugs.     

β-Myrcene Mitigates Colon Inflammation by Inhibiting MAP Kinase and NF-κB Signaling Pathways

Inflammatory bowel diseases (IBDs) are chronic inflammatory disorders that include Crohn’s disease (CD) and ulcerative colitis (UC). The incidence of IBD is rising globally. However, the etiology of IBD is complex and governed by multiple factors. The current clinical treatment for IBD mainly includes steroids, biological agents and need-based surgery, based on the severity of the disease. Current drug therapy is often associated with adverse effects, which limits its use. Therefore, it necessitates the search for new drug candidates. In this pursuit, phytochemicals take the lead in the search for drug candidates to benefit from IBD treatment. β-myrcene is a natural phytochemical compound present in various plant species which possesses potent anti-inflammatory activity. Here we investigated the role of β-myrcene on colon inflammation to explore its molecular targets. We used 2% DSS colitis and TNF-α challenged HT-29 adenocarcinoma cells as in vivo and in vitro models. Our result indicated that the administration of β-myrcene in dextran sodium sulfate (DSS)-treated mice restored colon length, decreased disease activity index (DAI), myeloperoxidase (MPO) enzyme activity and suppressed proinflammatory mediators. β-myrcene administration suppressed mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB) pathways to limit inflammation. β-myrcene also suppressed mRNA expression of proinflammatory chemokines in tumor necrosis factor-α (TNF-α) challenged HT-29 adenocarcinoma cells. In conclusion, β-myrcene administration suppresses colon inflammation by inhibiting MAP kinases and NF-κB pathways.     

Synthesis,Neuroprotection, and Antioxidant Activity of 1,1′-Biphenylnitrones as α-Phenyl-N-tert-butylnitrone Analogues in In Vitro Ischemia Models

Herein, we report the neuroprotective and antioxidant activity of 1,1′-biphenyl nitrones (BPNs) 1–5 as α-phenyl-N-tert-butylnitrone analogues prepared from commercially available [1,1′-biphenyl]-4-carbaldehyde and [1,1′-biphenyl]-4,4′-dicarbaldehyde. The neuroprotection of BPNs 1-5 has been measured against oligomycin A/rotenone and in an oxygen–glucose deprivation in vitro ischemia model in human neuroblastoma SH-SY5Y cells. Our results indicate that BPNs 1–5 have better neuroprotective and antioxidant properties than α-phenyl-N-tert-butylnitrone (PBN), and they are quite similar to N-acetyl-L-cysteine (NAC), which is a well-known antioxidant agent. Among the nitrones studied, homo-bis-nitrone BPHBN5, bearing two N-tert-Bu radicals at the nitrone motif, has the best neuroprotective capacity (EC₅₀ = 13.16 ± 1.65 and 25.5 ± 3.93 μM, against the reduction in metabolic activity induced by respiratory chain blockers and oxygen–glucose deprivation in an in vitro ischemia model, respectively) as well as anti-necrotic, anti-apoptotic, and antioxidant activities (EC₅₀ = 11.2 ± 3.94 μM), which were measured by its capacity to reduce superoxide production in human neuroblastoma SH-SY5Y cell cultures, followed by mononitrone BPMN3, with one N-Bn radical, and BPMN2, with only one N-tert-Bu substituent. The antioxidant activity of BPNs 1-5 has also been analyzed for their capacity to scavenge hydroxyl free radicals (82% at 100 μM), lipoxygenase inhibition, and the inhibition of lipid peroxidation (68% at 100 μM). Results showed that although the number of nitrone groups improves the neuroprotection profile of these BPNs, the final effect is also dependent on the substitutent that is being incorporated. Thus, BPNs bearing N-tert-Bu and N-Bn groups show better neuroprotective and antioxidant properties than those substituted with Me. All these results led us to propose homo-bis-nitrone BPHBN5 as the most balanced and interesting nitrone based on its neuroprotective capacity in different neuronal models of oxidative stress and in vitro ischemia as well as its antioxidant activity.     

Polyphenols Extracted from Shanxi-Aged Vinegar Inhibit Inflammation in LPS-Induced RAW264.7 Macrophages and ICR Mice via the Suppression of MAPK/NF-κB Pathway Activation

Shanxi-aged vinegar, a traditional Chinese grain-fermented food that is rich in polyphenols, has been shown to have therapeutic effects on a variety of diseases. However, there has been no comprehensive evaluation of the anti-inflammatory activity of polyphenols extracted from Shanxi-aged vinegar (SAVEP) to date. The anti-inflammatory activities of SAVEP, both in RAW 264.7 macrophages and mice, were extensively investigated for the potential application of SAVEP as a novel anti-inflammatory agent. In order to confirm the notion that polyphenols could improve inflammatory symptoms, SAVEP was firstly detected by gas chromatography mass spectrometry (GC-MS). In total, 19 polyphenols were detected, including 12 phenolic acids. The study further investigated the protective effect of SAVEP on lipopolysaccharide-induced inflammation in RAW264.7 macrophages and ICR mice. The results showed that compared with those of the model group, SAVEP could remarkably recover the inflammation of macrophage RAW264.7 and ICR mice. SAVEP can normalise the expression of related proteins via the suppression of MAPK/NF-κB pathway activation, inhibiting the expression of iNOS and COX-2 proteins, and consequently the production of inflammatory factors, thus alleviating inflammatory stress. These results suggest that SAVEP may have a potential function against inflammation.     

Rosmanol and Carnosol Synergistically Alleviate Rheumatoid Arthritis through Inhibiting TLR4/NF-κB/MAPK Pathway

Callicarpalongissima has been used as a Yao folk medicine to treat arthritis for years in China, although its active anti-arthritic moieties have not been clarified so far. In this study, two natural phenolic diterpenoids with anti-rheumatoid arthritis (RA) effects, rosmanol and carnosol, isolated from the medicinal plant were reported on for the first time. In type II collagen-induced arthritis DBA/1 mice, both rosmanol (40 mg/kg/d) and carnosol (40 mg/kg/d) alone alleviated the RA symptoms, such as swelling, redness, and synovitis; decreased the arthritis index score; and downregulated the serum pro-inflammatory cytokine levels of interleukin 6 (IL-6), monocyte chemotactic protein 1 (MCP-1), and tumor necrosis factor α (TNF-α). Additionally, they blocked the activation of the Toll-like receptor 4 (TLR4)/nuclear factor κB (NF-κB)/c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) pathways. Of particular interest was that when they were used in combination (20 mg/kg/d each), the anti-RA effect and inhibitory activity on the TLR4/NF-κB/MAPK pathway were significantly enhanced. The results demonstrated that rosmanol and carnosol synergistically alleviated RA by inhibiting inflammation through regulating the TLR4/NF-κB/MAPK pathway, meaning they have the potential to be developed into novel, safe natural combinations for the treatment of RA.     

Polyphenols from the Peels of Punica granatum L. and Their Bioactivity of Suppressing Lipopolysaccharide-Stimulated Inflammatory Cytokines and Mediators in RAW 264.7 Cells via Activating p38 MAPK and NF-κB Signaling Pathways

Punica granatum L. (Punicaceae) is a popular fruit all over the world. Owning to its enriched polyphenols, P. granatum has been widely used in treating inflammation-related diseases, such as cardiovascular diseases and cancer. Twenty polyphenols, containing nine unreported ones, named punicagranins A–I (1–9), along with eleven known isolates (10–20), were obtained from the peels. Their detailed structures were elucidated based on UV, IR, NMR, MS, optical rotation, ECD analyses and chemical evidence. The potential anti-inflammatory activities of all polyphenols were examined on a lipopolysaccharide (LPS)-induced inflammatory macrophages model, which indicated that enhancing nitric oxide (NO) production in response to inflammation stimulated in RAW 264.7 cells was controlled by compounds 1, 3, 5–8, 10, 11, 14 and 16–20 in a concentration-dependent manner. The investigation of structure–activity relationships for tannins 6–8 and 12–20 suggested that HHDP, flavogallonyl and/or gallagyl were key groups for NO production inhibitory activity. Western blotting indicated that compounds 6–8 could down-regulate the phosphorylation levels of proteins p38 MAPK, IKKα/β, IκBα and NF-κB p65 as well as inhibit the levels of inflammation-related cytokines and mediators, such as IL-6, TNF-α, iNOS and COX-2, at the concentration of 30 μM. In conclusion, polyphenols are proposed to be the potential anti-inflammatory active ingredients in P. granatum peels, and their molecular mechanism is likely related to the regulation of the p38 MAPK and NF-κB signaling pathways.     

Tabersonine Inhibits the Lipopolysaccharide-Induced Neuroinflammatory Response in BV2 Microglia Cells via the NF-κB Signaling Pathway

The occurrence and development of neurodegenerative diseases is related to a variety of physiological and pathological changes. Neuroinflammation is one of the major factors that induces and aggravates neurodegenerative diseases. The most important manifestation of neuroinflammation is the activation of microglia. Therefore, inhibiting the abnormal activation of microglia is an important way to alleviate the occurrence of neuroinflammatory diseases. In this research, the inhibitory effect of tabersonine (Tab) on neuroinflammation was evaluated by establishing the BV2 neuroinflammation model induced by lipopolysaccharide (LPS). It was found that Tab significantly inhibited the production and expression of nitric oxide (NO), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and reactive oxygen species (ROS) in BV-2 cells stimulated by LPS. In addition, Tab can also inhibit the activation of nuclear factor-κB (NF-κB) induced by LPS, thus regulating inflammatory mediators such as inducible nitric oxide synthase (iNOS). These results indicated that Tab regulated the release of inflammatory mediators such as NO, IL-1β, TNF-α, and IL-6 by inhibiting NF-κB signaling pathway, and exerting its anti-neuroinflammatory effect. This is the first report regarding the inhibition on LPS-induced neuroinflammation in BV2 microglia cells of Tab, which indicated the drug development potential of Tab for the treatment of neurodegenerative diseases.     

Anti-Inflammatory Activity of 4-(4-(Heptyloxy)phenyl)-2,4-dihydro-3H-1,2,4-triazol-3-one via Repression of MAPK/NF-κB Signaling Pathways in β-Amyloid-Induced Alzheimer’s Disease Models

Alzheimer’s disease (AD) is a major neurodegenerative disease, but so far, it can only be treated symptomatically rather than changing the process of the disease. Recently, triazoles and their derivatives have been shown to have potential for the treatment of AD. In this study, the neuroprotective effects of 4-(4-(heptyloxy)phenyl)-2,4-dihydro-3H-1,2,4-triazol-3-one (W112) against β-amyloid (Aβ)-induced AD pathology and its possible mechanism were explored both in vitro and in vivo. The results showed that W112 exhibits a neuroprotective role against Aβ-induced cytotoxicity in PC12 cells and improves the learning and memory abilities of Aβ-induced AD-like rats. In addition, the assays of the protein expression revealed that W112 reversed tau hyperphosphorylation and reduced the production of proinflammatory cytokines, tumor necrosis factor-α and interleukin-6, both in vitro and in vivo studies. Further study indicated that the regulation of mitogen-activated protein kinase/nuclear factor-κB pathways played a key role in mediating the neuroprotective effects of W112 against AD-like pathology. W112 may become a potential drug for AD intervention.     

The Synthesis,Characterization and Anti-Tumor Activity of a Cu-MOF Based on Flavone-6,2′-dicarboxylic Acid

A novel two-dimensional copper(II) framework (LDU-1), formulated as {[Cu₂(L)₂·2NMP}n (H₂L = flavone-6,2′-dicarboxylic acid, NMP = N-Methyl pyrrolidone), has been constructed under solvothermal conditions and characterized by single-crystal X-ray diffraction, infrared spectroscopy (IR), thermogravimetric analysis and powder X-ray diffraction (PXRD). In the crystal structure, the Cu(II) shows hex-coordinated with the classical Cu paddle-wheel coordination geometry, and the flavonoid ligand coordinates with the Cu(II) ion in a bidentate bridging mode. Of particular interest of LDU-1 is the presence of anti-tumor activity against three human cancer cell lines including lung adenocarcinoma(A549), Michigan cancer foundation-7 (MCF-7), erythroleukemia (K562) and murine melanoma B16F10, indicating synergistic enhancement effects between metal ions and organic linkers. A cell cycle assay indicates that LDU-1 induces cells to arrest at S phase obviously at a lower concentration.     

(−)-Naringenin 4′,7-dimethyl Ether Isolated from Nardostachys jatamansi Relieves Pain through Inhibition of Multiple Channels

(−)-Naringenin 4′,7-dimethyl ether ((−)-NRG-DM) was isolated for the first time by our lab from Nardostachys jatamansi DC, a traditional medicinal plant frequently used to attenuate pain in Asia. As a natural derivative of analgesic, the current study was designed to test the potential analgesic activity of (−)-NRG-DM and its implicated mechanism. The analgesic activity of (−)-NRG-DM was assessed in a formalin-induced mouse inflammatory pain model and mustard oil-induced mouse colorectal pain model, in which the mice were intraperitoneally administrated with vehicle or (−)-NRG-DM (30 or 50 mg/kg) (n = 10 for each group). Our data showed that (−)-NRG-DM can dose dependently (30~50 mg/kg) relieve the pain behaviors. Notably, (−)-NRG-DM did not affect motor coordination in mice evaluated by the rotarod test, in which the animals were intraperitoneally injected with vehicle or (−)-NRG-DM (100, 200, or 400 mg/kg) (n = 10 for each group). In acutely isolated mouse dorsal root ganglion neurons, (−)-NRG-DM (1~30 μM) potently dampened the stimulated firing, reduced the action potential threshold and amplitude. In addition, the neuronal delayed rectifier potassium currents (I_K) and voltage-gated sodium currents (I_Na) were significantly suppressed. Consistently, (−)-NRG-DM dramatically inhibited heterologously expressed Kv2.1 and Nav1.8 channels which represent the major components of the endogenous I_K and I_Na. A pharmacokinetic study revealed the plasma concentration of (−)-NRG-DM is around 7 µM, which was higher than the effective concentrations for the I_K and I_Na. Taken together, our study showed that (−)-NRG-DM is a potential analgesic candidate with inhibition of multiple neuronal channels (mediating I_K and I_Na).     

Anti-Hair Loss Effect of Adenosine Is Exerted by cAMP Mediated Wnt/β-Catenin Pathway Stimulation via Modulation of Gsk3β Activity in Cultured Human Dermal Papilla Cells

In the present study, we investigated the molecular mechanisms of adenosine for its hair growth promoting effect. Adenosine stimulated the Wnt/β-catenin pathway by modulating the activity of Gsk3β in cultured human dermal papilla cells. It also activated adenosine receptor signaling, increasing intracellular cAMP level, and subsequently stimulating the cAMP mediated cellular energy metabolism. The phosphorylation of CREB, mTOR, and GSK3β was increased. Furthermore, the expression of β-catenin target genes such as Axin2, Lef1, and growth factors (bFGF, FGF7, IGF-1) was also enhanced. The inhibitor study data conducted in Wnt reporter cells and in cultured human dermal papilla cells demonstrated that adenosine stimulates Wnt/β-catenin signaling through the activation of the adenosine receptor and Gsk3β plays a critical role in transmitting the signals from the adenosine receptor to β-catenin, possibly via the Gαs/cAMP/PKA/mTOR signaling cascade.     

Pharmacophore Mapping Combined with dbCICA Reveal New Structural Features for the Development of Novel Ligands Targeting α4β2 and α7 Nicotinic Acetylcholine Receptors

The neuronal nicotinic acetylcholine receptors (nAChRs) belong to the ligand-gated ion channel (GLIC) group, presenting a crucial role in several biological processes and neuronal disorders. The α4β2 and α7 nAChRs are the most abundant in the central nervous system (CNS), being involved in challenging diseases such as epilepsy, Alzheimer’s disease, schizophrenia, and anxiety disorder, as well as alcohol and nicotine dependencies. In addition, in silico-based strategies may contribute to revealing new insights into drug design and virtual screening to find new drug candidates to treat CNS disorders. In this context, the pharmacophore maps were constructed and validated for the orthosteric sites of α4β2 and α7 nAChRs, through a docking-based Comparative Intermolecular Contacts Analysis (dbCICA). In this sense, bioactive ligands were retrieved from the literature for each receptor. A molecular docking protocol was developed for all ligands in both receptors by using GOLD software, considering GoldScore, ChemScore, ASP, and ChemPLP scoring functions. Output GOLD results were post-processed through dbCICA to identify critical contacts involved in protein-ligand interactions. Moreover, Crossminer software was used to construct a pharmacophoric map based on the most well-behaved ligands and negative contacts from the dbCICA model for each receptor. Both pharmacophore maps were validated by using a ROC curve. The results revealed important features for the ligands, such as the presence of hydrophobic regions, a planar ring, and hydrogen bond donor and acceptor atoms for α4β2. Parallelly, a non-planar ring region was identified for α7. These results can enable fragment-based drug design (FBDD) strategies, such as fragment growing, linking, and merging, allowing an increase in the activity of known fragments. Thus, our results can contribute to a further understanding of structural subunits presenting the potential for key ligand-receptor interactions, favoring the search in molecular databases and the design of novel ligands.