Neurodegenerative diseases (ND) can be characterized by degradation and subsequent loss of neurons. ND has been identified as the leading cause of disability-adjusted life years (DALYs) worldwide and is associated with various risk factors such as ageing, certain genetic polymorphisms, inflammation, immune and metabolic conditions that may induce elevated reactive oxygen species (ROS) release and subsequent oxidative stress. Presently, no specific cure or prevention is available for ND patients; the symptoms can be only alleviated via drug treatment or surgery. The existing pharmacological treatments are only available for partial treatment of the symptoms. A natural product known as oil palm phenolics (OPP), which is high in antioxidant, could become a potential supplementary antioxidant for neurodegenerative health. OPP is a water-soluble extract from palm fruit that demonstrated medicinal properties including anti-tumor, anti-diabetic and neuroprotective effects. In this review, OPP was proposed for its neuroprotective effects via several mechanisms including antioxidant and anti-inflammatory properties. Besides, OPP has been found to modulate the genes involved in neurotrophic activity. The evidence and proposed mechanism of OPP on the neuroprotective health may provide a comprehensive natural medicine approach to alleviate the symptoms of neurodegenerative diseases. Neurodegenerative diseases (ND) is a heterogeneous group of disorders that can be characterized by degradation and subsequent loss of neurons . The degDuring ageing, irreversible changes may occur such as the loss of irreplaceable cells in brain, heart and skeletal muscles. Specifically, neurons in the brain may shrink and disappear, whereas alterations may take place in neuronal synapses and networks. Due to the neuron loss particularly in hypothalamus region of the brain, certain physiological changes such as altered metabolism and circadian rhythm, which might be closely related to the mental and emotional aberrations in the elder group, may occur . Most neApart from that, neurodegenerative diseases have been associated with aggregation of cytoskeletal protein, which then developed to inclusions in neurons and/or glial cells . The accElaies guineensis) and is the product filtrate recovered from aqueous waste stream during palm milling process. Globally, there are approximately 85 million tonnes of vegetation liquor are discarded annually into the aqueous waste stream. The development of a novel recovery procedure to isolate phenolic compounds from the aqueous waste stream contributes to the OPP production. With the growing evidence that phenolics originated from plants may contribute to health benefits, the assessment of OPP for its potential bioactivities has been actively explored. The main active ingredients of OPP have been identified, which include caffeoylshikimic acid (CFA), p-hydroxybenzoic acid (PHBA), protocatechuic acid (PCA) and hydroxytyrosol [®), a drug used for prophylaxis and treatment of several influenza viruses such as swine-origin influenza virus H1N1, types A and B seasonal influenza virus and avian influenza virus H5N1 [Illicium verum) are limited [Pseudomonas aeruginosa PAO1 [A natural product known as oil palm phenolics (OPP), which is high in antioxidants, could become a potential supplementary antioxidant for neurodegenerative health. OPP is a water-soluble extract from palm fruit  compared to control and high-cholesterol groups. It was also found that OPP-treated group preserved more healthy neurons in H&E staining while demonstrating lower density of plaque deposition in the hippocampal CA-1 and dentate gyrus (DG) areas when compared to high-cholesterol fed diet rats [It was shown that the OPP-fed rats had a significant improvement in EL (iet rats . This fiiet rats . The OPPiet rats . β-amyloiet rats . OPP-fediet rats . This suiet rats . Ketone iet rats . Howeveriet rats . In the iet rats . ApoE geiet rats .Another in vitro study had supplemented 800 µg/mL OPP for 12 h to U87 MG glioma cell line . In the Another in vitro study has been conducted to determine the effects of OPP on liposaccharide (LPS) activated BV-2 cell line , which iAn in vitro study using IL-1β-activated normal human astrocytes has been performed by Weinberg et al. 2018 . In the Free radicals can be described as molecules with unpaired electron in their outer orbit. In a normal controlled physiological process, free radicals have a very important role in many biochemical activities such as signal transduction, gene transcription and regulation of soluble guanylate cyclase activity ,62. For -) groups that may be responsible for the potent antioxidant activity of OPP. Moreover, other phenolic acid contents in OPP such as protocatechuic acid and p-hydroxybenzoic acid may also contribute to the antioxidant property, which might act synergistically.Plant phenolics have been well-known for their antioxidant properties due to the high redox potential that allows them to act as reducing agents, hydrogen donors, singlet oxygen quenchers and metal chelators. In neurons, antioxidants have the potential to prevent and treat neuronal disorders concerning the oxidative stress ,72. OPP Oxidative stress (OS) is commonly considered as the initial point for the onset of several aging and chronic diseases, which undoubtedly plays a major role in the development of degenerative disorders . OS in n2+) and biliverdin. With the action of biliverdin reductase, biliverdin can be converted into the antioxidant bilirubin, that is able to scavenge free radical molecules [Sundaresan (2013)  has consolecules ,81. Therolecules ,83.Nrf2 is an essential transcription factor that coordinates the induction of genes encoding numerous cytoprotective enzymes such as NAD(P)H: quinone oxidoreductase-1 (NQO1), superoxide dismutase (SOD), glutathione S-transferase (GST), glutathione peroxidase (GPx), heme oxygenase-1 (HO-1), glutamate cysteine ligase (GCL), catalase, and thioredoxin ,85. It hThe main cause of oxidative stress in neurodegenerative diseases is the inflammation caused by activated microglial cells that constitute up to 20% of the brain cell population . Highly Polyphenols have been reported to downregulate the pro-inflammatory cytokines such as iNOS and COX-2 via the suppression of Nuclear factor kappa-light-chain-enhancer of activated B cells (NF- κB) ,98,99. NTherefore, as OPP are able to inhibit the NF-κB pathway, the pro-inflammatory response during neuroinflammation could also be halted. Meanwhile, the non-canonical NF-κB pathway might not be responsible for the anti-inflammatory actions of OPP since all the mediators involved are not downregulated by OPP. In brief, the non-canonical NF-κB pathway requires NF-κB inducing kinase (NIK) synthesis and is considered a slower kinetics compared to canonical pathway. When stimulated by specific stimuli such as LPS, lymphotoxin (LT) α1β2 and receptor activator of NF-κB (RANK), NIK is activated and recruits I κKα to the p100 complex to phosphorylate p100 leading to p100 ubiquitination. P52, the processing product of p100, generates the activated p52/RelB NF-κB complex, which can be translocated to the nucleus and induces the downstream gene expressions. Based on our readings, none of the pro-inflammatory molecules involved in non-canonical pathway have been downregulated by OPP .Since OPP have been reported to significantly increase Nrf2 , this maAnti-inflammatory effects of OPP have been also observed in the microarray assay performed by Leow et al. . The resFor example, protein level of Spp1 was increased in multiple sclerosis during disease relapses . MoreoveOther than that, the anti-inflammatory of OPP can be seen via its ability to inhibit Aβ42 aggregation, which is the primary component of amyloid plaque in the brains of Alzheimer’s patients. Alzheimer’s disease is associated with the aggregation of amyloid-β peptide (Aβ42/Aβ40) that develops into soluble neurotoxic oligomers, activating microglia and astrocytes to an acute inflammatory state that releases cytokines such as TNF-α, IL-1b, and IFN-γ. The Aβ peptide-induced neuroinflammatory state results in synaptic loss and neuronal death in the brain regions essential for cognitive function, causing the loss of memories, personalities and cognitive function resulting in dementia. The inhibitory effects by OPP may prevent the formation of neurotoxic oligomers and hold the potential to reduce neuroinflammation.BDNF is a part of neurotrophin family, which also consists of neural growth factor (NGF), neurotrophin 3 (NT3) and neurotrophin 4 (NT4). It has a wide array of functions within the brain and is highly present in several brain structures. Additionally, it also can be found in peripheral organs including the thymus, spleen, gut and heart. The major storage site for peripheral BDNF is platelet (90%) and only a small amount of free BDNF is present in plasma. In the brain, its functions include activity-dependent plasticity, neuronal survival, formation of new synapses, dendritic branching as well as the regulation of excitatory and inhibitory neurotransmitter profiles ,114. DurIn the microarray gene expression analysis harvested from the brain of OPP-treated mice, there is an up-regulation of genes involved in neurotrophic activities such as calcium ion binding, calmodulin binding, potassium ion transport and transmembrane receptor protein tyrosine phosphatase activity. Besides, OPP also up-regulated genes involved in nervous system development, neurotransmitter transport, striated muscle contraction, synaptic transmission and synaptogenesis. Remarkably, majority of these genes are regulated by BDNF, which plays an important role in learning and memory. Previous studies have demonstrated the role of BDNF in learning and memory whereby increased BDNF mRNA were detected in hippocampus brain region, which is responsible for cognitive functions ,118. ParGingko biloba leaves, which are a well-known dietary supplement with good efficiency to counteract depression, short-term memory loss as well as lack of attention. Those up-regulated genes are namely as tyrosine phosphatases (Ptprn and Ptprt) and ionotropic glutamate receptor (Gria3). Tyrosine phosphatase is important for the regulation of synapse dynamics and is closely associated with the breakdown of intracellular neurofibrillary tangles, an important feature of AD [Interestingly, it was found that OPP shares identical up-regulated neurotrophic genes with extracts of re of AD ,121. MeaAnother gene up-regulated by OPP is Arc , which is a master regulator of synaptic function that has a critical role in late-phase of long-term potentiation and memory consolidation . The expApart from that, OPP may also improve the condition of neurodegenerative diseases via postsynaptic regulation. This is due to the ability of OPP to upregulate Dlgap2 and Homer1 gene expression, which are associated with postsynaptic density. Dlgap is a member of a family of Discs large associated proteins that function as scaffold proteins in the postsynaptic density (PSD). PSD is a highly specialized matrix that can be found in the synaptic terminal of postsynaptic neurons and participates in the neuronal signal transmission across the synaptic junction . MeanwhiThis review has summarized the direct effects and potential mechanisms of OPP on neuroprotective actions. Both in vitro and in vivo studies have shown promising results of this valuable nutraceutical derived from the oil palm fruit on neurodegenerative diseases, especially for Alzheimer’s disease. To our concern, apart from taking good supplementation to prevent and treat neurodegenerative diseases, people should remain mentally and socially active throughout their lives. For instance, people may opt to try new activities and taking part in group gatherings to obtain a healthy mental and social life.For future direction, clinical trials should be performed to evaluate the applicability of these reported pre-clinical results and the proposed mechanisms of OPP in the human neuronal health. More studies using OPP on other important neurodegenerative diseases such as Parkinson’s disease and Huntington’s disease should be explored. In this review, the antioxidant and anti-inflammatory properties have been proposed as the responsible pathways for the neuroprotective actions of OPP. Besides, the modulation of genes involved in brain-derived neurotrophic factor is a potential mechanism for the therapeutic action of OPP on neuronal health. Further in-depth studies are warranted to explore a conclusive mechanism. The understanding of the proposed mechanism that leads to neuronal health will deliver more insight into the development of potent neuroprotective agents that specifically target the pathways. In conclusion, the direct effects of OPP related to neuronal health have been evidently revealed by literature with promising results in preventing and treating the neurodegenerative diseases.


The effects of combining 0.1–5 wt % graphene nanoplatelet (GNP) and 3–30 wt % phosphorous flame retardant, 9,10- dihydro-9-oxa-10-phosphaphenanthrene-10-oxide (DOPO) as fillers in epoxy polymer on the mechanical, flame retardancy, and electrical properties of the epoxy nanocomposites was investigated. GNP was homogeneously dispersed into the epoxy matrix using a solvent-free three-roll milling process, while DOPO was incorporated into the epoxy resin by mechanical stirring at elevated temperature. The incorporation of DOPO reduced the crosslinking density of the epoxy resin. When using polyetheramine as a hardener, the structural rigidity effect of DOPO overshadowed the crosslinking effect and governed the flexural moduli of epoxy/DOPO resins. The flexural moduli of the nanocomposites were improved by adding GNP up to 5 wt % and DOPO up to 30 wt %, whereas the flexural strengths deteriorated when the GNP and DOPO loading were higher than 1 wt % and 10 wt %, respectively. Limited by the adverse effects on mechanical property, the loading combinations of GNP and DOPO within the range of 0–1 wt % and 0–10 wt %, respectively, in epoxy resin were further studied. Flame retardancy index (FRI), which depended on three parameters obtained from cone calorimetry, was considered to evaluate the flame retardancy of the epoxy composites. DOPO showed better performance than GNP as the flame retardant additive, while combining DOPO and GNP could further improve FRI to some extent. With the combination of 0.5 wt % GNP and 10 wt % DOPO, improvement in both mechanical properties and flame retardant efficiency of the nanocomposite was observed. Such a combination did not affect the electrical conductivity of the nanocomposites since the percolation threshold was at 1.6 wt % GNP. Our results enhance the understanding of the structure–property relationship of additive-filled epoxy resin composites and serve as a property constraining guidance for the composite manufacturing. Epoxy resin (denoted as EP) is a thermoset polymer that has been extensively used for composite manufacturing due to its chemical and thermal resistance, excellent adhesion, and ease of processing . DespiteVarious flame retardants and reinforcing nanomaterials have been investigated as epoxy resin fillers to overcome the flammability of epoxy thermosets. 9,10- dihydro-9-oxa-10-phosphaphenanthrene-10-oxide (DOPO) is one of the phosphorous flame retardants (PFRs), which were proposed as an alternative to halogenated flame retardants due to concerns about the latter’s toxic effects and environmental impacts ,6. OwingGraphene nanoplatelets (GNP) have been investigated as a filler in epoxy resin to enhance mechanical properties, thermal conductivity, electrical conductivity, and flame retardancy. Improved mechanical properties when adding GNP to epoxy resin have been reported, such as an increase in fracture toughness, flexural modulus, and hardness of the nanocomposites ,16,17,182 [The combination of GNP with other flame retardants has gained attention to further enhance the properties of the nanocomposites in terms of mechanical performance as well as flame retarding efficiency. Recent studies reported that the combination of DOPO and GNP could improve flame retardancy of epoxy thermosets ,25. For 2 . Althoug2 ,13. Howe2 ,27. Wang2 . In addi2 . To our Based on structural classification, the above mentioned studies ,26,27 em−1. The curing agent was polyetheramine Jeffamine D-230 , which has molecular weight (MW) of 230 g·mol−1 and amine hydrogen equivalent weight (AHEW) of 60 g·eq−1. The phosphorous-containing flame retardant 9,10- dihydro-9-oxa-10-phosphaphenanthrene-10-oxide  was purchased from Tokyo Chemical Industry . Graphene nanoplatelet xGNP M25  was purchased from XG Sciences, St. Louis, MO, USA.The epoxy resin used in this study was diglycidyl ether of bisphenol A , which has an epoxy equivalent of 183–189 g·eqThe desired amount of DOPO was added into the epoxy resin. The mixture was then heated with continuous stirring until the temperature reached 160 °C and kept constant with continuous stirring for 5 h for complete reaction between DOPO and epoxy resin . The amoThe dispersion method and manufacturing process of the composites was adapted from a previous study . The desThe ratio of resin/hardener was 100:32 for the composites without DOPO. For the EP/DOPO mixtures, since DOPO reacted with the epoxy groups, the amount of hardener added was adjusted according to the available epoxy groups obtained from the theoretical value as shown in the Attenuated total reflection-Fourier transform infrared spectrometer  was used to characterize the functional groups of DOPO, epoxy resin, cured epoxy resins and cured EP/DOPO. The dispersion state of GNPs in the epoxy matrix was determined using an optical microscope .A discharge test was performed using an electrostatic Wimshurst machine as a high voltage source and the samples were placed adjacent to the spark gap formed by two metal spheres. The surface resistance was measured using a Keithley multimeter  with two-point electrodes. The samples had dimensions of 10 cm × 10 cm and 4 mm thickness.−1. Five specimens from each sample were tested. The dimensions of the specimen were 1 cm × 8 cm × 4 cm (width × length × thickness). Scanning electron microscope  was employed to analyze the fracture surface of the samples after a three-point bending test.The flexural modulus was evaluated by the three-point bending test according to ISO 178:2001  using th−1. The sample dimensions were 10 mm × 50 mm and 4 mm thickness. The crosslinking density −3 was evaluated using the kinetic theory of rubber elasticity as follows:E’ is storage elastic modulus (Pa) of cured resin at the peak temperature of tan δ + 40 °C to ensure the rubbery stage of the sample. R is the gas constant (8.3145 m3·Pa·mol−1·K−1) and T is the absolute temperature (K) at which E’ is determined, in this case at the peak temperature of tan δ + 40 °C.In order to determine the crosslinking density, dynamic mechanical thermal analysis (DMTA) was performed using an advanced rheometric expansion system  to obtain the storage elastic modulus and tan δ of the cured resins, which were important parameters for an estimation of the crosslinking density. The samples tested were neat epoxy and EP/10DOPO. Samples were analyzed with applying constant static force at 1 Hz and a strain of 0.05%. The scanning temperature was from −150 to 180 °C at a heating rate of 3 °C·ming) was analyzed using a differential scanning calorimeter  with a heating rate of 20 °C·min−1. The scanned temperature ranged from 20 to 200 °C. Thermal stability was measured by a thermogravimetric analysis using Thermobalance Netzsch TG209 F1  under a nitrogen environment at the heating rate of 20 °C·min−1 from 28 to 1008 °C. Several parameters were obtained to describe the thermal stability of cured epoxy resins. The onset temperature or initial decomposition temperature (Td) is the temperature at which the sample starts to decompose. The mid-point temperature (T−50%) is the temperature at which 50% of the weight loss occurred. Tmax stands for the decomposition temperature at the maximum mass loss rate. The amount of final residue presented by % char is the percentage of final residual weight at 1008 °C with relative to the initial sample weight.The glass transition temperature (T−2. The experiments were terminated after the flame stopped for 100 s. The sample size was 10 cm × 10 cm × 4 mm (length × width × thickness).The FTT Cone calorimeter  was employed to analyze the flammability of the composites according to ISO 5660-1 with the heat flux of 50 kW·m−1. The disappearance of this peak confirmed the bonding of DGEBA and DOPO [3 carbon [−1 and the existence of hydroxyl groups as a broad peak between 3200 and 3600 cm−1, which was broader and more intense as compared to the O–H peak of the neat epoxy resin, which appeared between 3400 and 3600 cm−1. Moreover, the shift of the P–C stretching vibration from 682 cm−1 in DOPO to 686 cm−1 in EP/DOPO could also be a sign of the bonding between the phosphorus atom of DOPO and a carbon atom of epoxide ring.The successful incorporation of DOPO into the epoxy resin, whose reaction is presented in and DOPO . The rea3 carbon . The for3 carbon . The occThe discharge measurement  revealedc, and critical exponent, t, obtained from fitting the experimental data with the power law c of 0.006 or 1.6 wt % GNP. σc and σf are the conductivity of the nanocomposite and the filler, respectively, and ϕ is the filler concentration.Percolation theory can be applied to predict the critical amount of the filler that can form the connected network and affect the behaviors of the nanocomposite. The electrical percolation threshold, ϕp is the thickness of the platelet [The percolation threshold values published earlier ranged from 0.0052 to 0.021 volume fraction, while the corresponding t values were in the range of 1.36–5.37 as summarized in platelet . The sugplatelet , at whicProperties of the epoxy resins were influenced by the chemical network structure, which could be explained by the crosslinking state of the cured epoxy resins . In ordeAs shown in The flexural fracture surface of the epoxy composites was determined using SEM as shown in Flexural modulus and flexural strength were investigated to describe the mechanical performance of the EP/GNP nanocomposites as displayed in The flexural moduli and strengths of the nanocomposites measured in this study were compared with the values from other studies as shown in With an increasing amount of DOPO, our study showed a continuous improvement in the flexural modulus of the EP/DOPO thermosets, which does not completely agree with other studies, as shown in Such a discrepancy in the DOPO effect on the flexural modulus of the cured DOPO-modified epoxy resin could result from different types of hardeners used in various studies since the structure of the hardeners could also play an important role on the mechanical properties of the epoxy thermosets ,45,46. Hd around 369 °C and the maximum mass loss rate around 387 °C. However, GNP significantly increased the char residues of the epoxy. The addition of GNP up to 1 wt % loading can increase the char content up to 50% by forming the structure containing smaller pores as compared to the char of neat epoxy, which led to a compact and continuous char structure [d of neat epoxy resin at 369 °C. After 480 °C, DOPO completely decomposed, as there was almost no residue left. Regarding EP/DOPO samples, when the DOPO loading increased, Td and Tmax of the EP/DOPO decreased. Our results clearly showed significant improvement in char yields as the amount of DOPO increased. This could be due to the fact that the decomposition products of DOPO could catalyze the formation of char, which can insulate the underneath layer from burning [d and Tmax of the epoxy resin as compared to EP/DOPO, but it could further improve the char residues.TGA curves and their derivatives , which represent the thermal stability of the samples are demonstrated in tructure . DOPO un burning ,24,47. Ag) of epoxy nanocomposites determined by DSC and the important parameters obtained from thermal stability investigation by TGA. GNP slightly reduced Tg of the cured epoxy resin as compared to the neat epoxy resin. The reduction in Tg could be the result of GNP acting as a heat flow barrier in the polymer matrix [g because polymer chain mobility was more enhanced with respect to the neat epoxy resin [r matrix . Moreovexy resin ,50.g due to the reaction of epoxide groups and DOPO, which depleted the available crosslinking sites between epoxy resin and hardener and resulted in less crosslinking network of the cured DOPO-incorporated epoxy resin. The addition of GNP to EP/DOPO did not make a notable difference in Tg and Td of the resulting nanocomposites as compared to EP/DOPO samples; however, the significant improvement in char yield was observed in samples containing GNP.An increase in the DOPO content resulted in a decrease in T2 formation (ave-CO2 yield) were obtained from cone calorimetry. −2 at 140 s. GNP did not significantly influence pHRR, but another HRR peak around 200–300 s was more pronounced as compared to that of the neat epoxy. The development of the second peak in the HRR profile of EP/GNP could be an effect of the breakage of the char layer formed by GNP. When the char layer broke, the combustible gases were released, which enhanced the combustion. As a result, THR of the EP/GNP was higher than that of neat epoxy. Liu et al. reported a similar effect of GNP on the development of small HRR peaks with the addition of 5 wt % of GNP; however, they showed substantial reduction of pHRR by 57% as compared to the epoxy resin without a filler [The important parameters for flame retardancy including time to ignition (TTI), peak heat release rate (pHRR), average heat release rate (ave-HRR), average effective heat of combustion (ave-EHC), total heat release (THR), total smoke production (TSP), average CO formation (ave-CO yield), and average COa filler , while oThe incorporation of DOPO could decrease the pHRR and THR in relation to the epoxy resin because DOPO could act in both the condensed phase by forming the protective char layer and gas phase by scavenging the reactive combustible species . By addid of DOPO as compared to the neat epoxy resin as shown in TGA results. TSP, ave-CO yield, and ave-CO2 yield are indicators of the completeness of the combustion. The products of complete combustion are water and CO2, while incomplete combustion produces smoke and CO [2 production, which indicated more incomplete combustion as compared to the neat epoxy resin. The incorporation of DOPO reduced pHRR, ave-HRR, ave-EHC, and THR of the composites as compared to the cured neat epoxy resin. According to our finding, GNP up to 1 wt % did not significantly promote the flame retarding efficiency of the epoxy resin in terms of pHRR and THR. However, the combination of DOPO and GNP could further reduce pHRR, ave-HRR, and THR of the nanocomposites as compared to the EP/DOPO or EP/GNP alone.omposite , was calomposite . On the e and CO . AdditioThe flame retarding mechanism of combining DOPO and GNP has been proposed as GNP is acting in the condensed phase, while DOPO is acting in both the condensed phase and gas phase ,47. Ave-2 production, which reflected the chemical reaction process [d of DOPO as compared to the neat epoxy resin, meaning that EP/3DOPO decomposed earlier than neat epoxy resin, as evidenced in the TGA results. Combining 0.5 wt % GNP with 3 wt % DOPO could delay TTI of the epoxy composite, but THR was increased, which was a major contribution to the poor FRI. In both cases, avg-EHC did not decrease, indicating that no gas phase reaction occurred. Since DOPO was responsible for the effect on the gas phase reaction, we concluded that the loading of 3 wt % DOPO in our epoxy system was not enough for DOPO to affect the gas phase reaction. The combination of 10 wt % DOPO and 0.5 wt % GNP could additionally promote the FRI to 1.9 as compared to the epoxy resin loaded with 10 wt % DOPO, which showed FRI at 1.7.The change in TTI, pHRR, and THR with respect to the neat epoxy resin was reflected in FRI values of the composites. Three performance categories were proposed including poor, good, and excellent, corresponding to FRI < 1, 1 < FRI < 10, and FRI > 10, respectively . The res process , of EP/0Since FRI is a relative value indicating the flame retardancy efficiency of the composites with respect to the neat epoxy resin, this allows us to compare the influence of additives on the flame retardancy efficiency of different epoxy resin systems. Our results demonstrated that, in terms of the FRI values, DOPO showed better flame retardancy performance as compared to GNP. Our finding was consistent with the previous review work, which extracted the information regarding flame retardancy efficiency from a number of literature . They reStudies have reported enhanced flame retardancy property by adding graphene-related materials. For example, when 5 wt % of multilayer graphene was added to polypropylene and 5 wt % of graphene nanosheets were added to epoxy resin, they could achieve FRI of 2.9  and 4.4 As shown in our study, the flame retardancy efficiency of epoxy resins could be enhanced by the incorporation of DOPO since DOPO could act in both the condensed phase and gas phase to retard the flame. By combining DOPO and GNP, the flame retardancy efficiency could be further improved to some extent as compared to the EP/DOPO composite due to the effect of GNP on delaying the ignition time and improving char formation. One might consider increasing the GNP loading to achieve better flame retardancy performance; however, a balance must be considered due to the adverse effect on the mechanical properties of the composites resulting from agglomeration of GNP at high loading.Epoxy-based composites with homogeneously dispersed GNP were produced using a three-roll milling process. DOPO was also successfully incorporated into the epoxy resin. The electrical percolation threshold was detected at 0.006 volume fraction of GNP or 1.6 wt % GNP. Both DOPO and GNP could enhance the flexural modulus and flexural strength of the epoxy composites. Unlike aromatic hardeners, the linear structure of polyetheramine, which was the hardener used in this study, allowed the rigidity of grafted DOPO to dominate the flexural modulus of the composites; therefore, the flexural modulus increased continuously as the DOPO loading increased, although the crosslinking density decreased when DOPO was incorporated. However, higher DOPO and GNP contents (more than 10 wt % DOPO and 1 wt % GNP) could cause difficulty in composite manufacturing due to an increase in viscosity, which resulted in poorer dispersion of GNP and poorer flexural strength of the composites. Since incorporation of DOPO depleted the crosslinking density of the epoxy thermoset, the glass transition temperature decreased as DOPO loading increased. Thermogravimetric analysis showed that both GNP and DOPO could enhance the char residues. Incorporating 10 wt % DOPO could reduce the peak heat release rate (pHRR) and the total heat release (THR) of epoxy composites by 20% and 18%, respectively, compared to the neat epoxy resin. Increasing GNP loading up to 1 wt % did not significantly affect the pHRR of epoxy composites, but increased the THR by 8% and delayed TTI. These three parameters, pHRR, THR, and TTI could be converted to FRI. The results revealed that EP/DOPO had better FRI than EP/GNP. Using DOPO and GNP together could further improve FRI to some extent and achieve good performance regarding the FRI criterion.The effects of GNP and DOPO loading on the composites’ properties are illustrated in


Metastasis is one of the main contributors to the poor prognosis of hepatocellular carcinoma (HCC). However, the underlying mechanism of HCC metastasis remains largely unknown. Here, we showed that TXNDC12, a thioredoxin-like protein, was upregulated in highly metastatic HCC cell lines as well as in portal vein tumor thrombus and lung metastasis tissues of HCC patients. We found that the enforced expression of TXNDC12 promoted metastasis both in vitro and in vivo. Subsequent mechanistic investigations revealed that TXNDC12 promoted metastasis through upregulation of the ZEB1-mediated epithelial–mesenchymal transition (EMT) process. We subsequently showed that TXNDC12 overexpression stimulated the nuclear translocation and activation of β-catenin, a positive transcriptional regulator of ZEB1. Accordingly, we found that TXNDC12 interacted with β-catenin and that the thioredoxin-like domain of TXNDC12 was essential for the interaction between TXNDC12 and β-catenin as well as for TXNDC12-mediated β-catenin activation. Moreover, high levels of TXNDC12 in clinical HCC tissues correlated with elevated nuclear β-catenin levels and predicted worse overall and disease-free survival. In summary, our study demonstrated that TXNDC12 could activate β-catenin via protein–protein interaction and promote ZEB1-mediated EMT and HCC metastasis. Liver cancer, of which 70–90% are hepatocellular carcinoma (HCC), is the second leading cause of cancer death worldwide . AlthougEpithelial–mesenchymal transition (EMT) is a highly conserved cellular program during which epithelial cells lose their polarized organization and acquire migratory and invasive capabilities . For theThioredoxin domain-containing protein 12 (TXNDC12), which is also known as ERp16, ERp18, ERp19, or hTLP19, is a member of the protein disulfide isomerase (PDI) family that plays important roles in cancer development and progression –17. TXNDIn this study, we found that TXNDC12 was upregulated in HCC and correlated with HCC metastasis. Enforced expression of TXNDC12 stimulated the metastatic potential of HCC cells via induction of the EMT process. In addition, we revealed that TXNDC12 interacted with and activated β-catenin, thereby contributing to ZEB1 upregulation and EMT. Moreover, we showed a positive correlation between TXNDC12 and the nuclear expression of β-catenin, as well as the prognostic potential of this combination. Taken together, our data indicate that the overexpression of TXNDC12 promotes HCC metastasis through β-catenin-mediated induction of EMT and that TXNDC12 has the potential to be a prognostic factor and therapeutic target of HCC.To clarify the underlying role of TXNDC12 in HCC, the expression of TXNDC12 was evaluated in 106 paired human HCC samples , Vimentin , and ZEB1   Fig. . Taken tr = 0.6949, P < 0.001)  Fig. . In addi01) Fig. . These rTo verify this finding, the cytosolic and nuclear fractions of cell lysates were separated. Neither overexpression nor knockdown of TXNDC12 significantly affected the total β-catenin level Fig. . HoweverTo determine whether the activation of β-catenin is essential for the TXNDC12-mediated promotion of EMT, we knocked down the expression of β-catenin in TXNDC12-overexpressing cells and observed that knockdown of β-catenin eliminated the TXNDC12-mediated upregulation of ZEB1 and Vimentin, as well as the TXNDC12-mediated downregulation of E-cadherin Fig. . ConsistTo further confirm the role of TXNDC12 in the EMT process, TGFβ1 was used to induce EMT in HCC cells. As shown in Supplementary Fig. The formation of the E-cadherin/β-catenin complex has been demonstrated to negatively regulate the activation of β-catenin . To explTo further investigate whether the PDI enzymatic activity of TXNDC12 is required for β-catenin activation, we constructed a TXNDC12-CS mutant in which both cysteines in the active site were mutated. As shown in Fig. n = 60) and the low TXNDC12 expression group (n = 46). As shown in Supplementary Table P < 0.001), multiple tumors (P = 0.006), a lack of encapsulation (P < 0.001), high serum α-fetoprotein (AFP) levels , microvascular invasion (P = 0.003), and macrovascular invasion (P = 0.019). For both the West China Hospital (WCH) and TCGA datasets, the unadjusted survival curves showed that the high TXNDC12 expression group had worse OS (hazard ratio (HR), 3.80; 95% confidence interval (CI), 1.91–7.54) and DFS  than the low TXNDC12 expression group (P < 0.05). The 3-year OS rate of the low TXNDC12 expression group was higher than that of the high TXNDC12 expression group. Similarly, the 3-year DFS rate of the low TXNDC12 expression group was higher than that of the high TXNDC12 expression group , and streptomycin (10 mg/L) at 37 °C and 5% CO2.Hep3B, Huh7, PLC/PRF/5, and HCCLM3 cells were obtained from the Cell Bank Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China. THLE-2 and SNU449 cells were obtained from ATCC . All cells were cultivated in Dulbecco’s modified Eagle’s medium  containing 10% FBS (HyClone), penicillin . A total of 106 tumor tissues and paired adjacent normal tissues were collected from patients who underwent resection for HCC at WCH between 2010 and 2014. The detailed protocols for tissue collection and clinical data processing  were performed as described previously . The stu4 cells per well 24 h before transfection. Cells were transfected with Lef/Tcf luciferase reporter  or FOP-Flash reporter (Upstate) plus the pRL-CMV plasmid . After 48 h, luciferase activity was measured using a dual luciferase reporter assay system (Promega) following the manufacturer’s protocol.Cells were plated in 24-well plates at 2 × 106 cells were injected into nude mice   through the tail vein. After 4 weeks, mice were sacrificed, and the lungs were excised, imaged, and embedded in paraffin. The orthotopic mouse model of HCC was established as described previously [6 cells were injected into the subcapsular region of the middle lobe. After 6 weeks, mice were sacrificed, and the livers were excised, imaged, and embedded in paraffin. Tumor metastases were confirmed by hematoxylin and eosin staining. All animal experiments were approved by the Institutional Animal Care and Use Committee of Sichuan University.For the mouse lung metastasis model, 2 × 10eviously . Brieflywww.cbioportal.org). X-tile plots  were used to select the optimum cutoff for the expression of TXNDC12 based on the association of gene mRNA levels with the patients’ OS. Microarray gene expression data of HCC patient cohorts were downloaded from the GEO database . The R package “GEOquery” was used to extract the expression values of the genes of interest.Level 3 RNA-sequencing data and clinical information for 370 HCC patients were downloaded from the cBioPortal for Cancer Genomics  and R software (R 3.3.2). Cox proportional hazards models were used to evaluate the association of TXNDC12 and β-catenin with overall and disease-specific mortality. One-way ANOVA and the Pearson Supplementary Information


To demonstrate the effect of Ginsenoside Rg1 on the differentiation of human bone marrow-derived mesenchymal stem cells (hBM-MSCs). Subsequently, a rational mechanism for the detection of Rg1 which affects mesenchymal stem cell differentiation was explored. β-gal staining is used to detect cell senescence levels. Western blot and immunofluorescence were used to determine protein expression levels. RT-qPCR is used to detect mRNA expression levels.  Flow cytometry is used for cell identification. The differentiation ability of hBM-MSCs was studied by differentiation culture. SA-β-catenin signaling pathway and significantly adjusted the phosphorylation of GSK-3β. GSK-3β inhibitor (Licl) significantly increased Rg1-induced phosphorylation of GSK-3β, which in turn reduced Rg1-induced differentiation of hBM-MSCs.  Rg1 regulates the differentiation of hBM-MSCs. Differentiation culture analysis showed that Rg1 promoted cells to osteogenesis and chondrogenesis. Western blot results showed that Rg1 regulated the overactivation of the β and regulate the mesenchymal stem cell differentiation ability. Ginsenoside Rg1 can reduce the excessive activation of the Wnt pathway in senescent cells by inhibiting the phosphorylation of GSK-3 Adult stem cell senescence is the latest theory of the occurrence and development of human aging and senile diseases –4. Stem Panax ginseng is a traditional Chinese medicine to replenish “qi”. According to Shen Nong's Herbal Classic , P. ginsβ-catenin signaling pathway is an important stem-cell related pathway [β-catenin signaling pathway is closely related to the level of stem cell senescence. In our previous studies [β-catenin signaling pathway have a wide and important relationship with changes in stem cell function. Recent studies [β-catenin signaling pathway has a wide range of biological effects on hematopoietic stem cell senescence, and hematopoietic stem cell senescence can be produced by overactivation of the β-catenin signaling pathway. In contrast, inhibition of the β-catenin signaling pathway can attenuate the senescence of hematopoietic stem cells. So, is aging of human bone marrow mesenchymal stem cells (hBM-MSCs) associated with the β-catenin signaling pathway?The Wnt/ pathway . The Wnt pathway –21. It h pathway –25 that  studies –28, we f studies , 29, 30 β-catenin signaling pathway were studied. These findings may support the use of Rg1 in promoting stem cell viability and differentiation, as well as in tissue engineering and clinical therapy.In the current study, the effect of Rg1 on the differentiation of hBM-MSCs was determined, and the roles of the Wnt/n > 20/group) was collected from volunteers who received bone marrow puncture at the First Affiliated Hospital of Chongqing Medical University, China. The study was approved by the ethics committee of Chongqing medical university.Human bone marrow-derived mesenchymal stem cells were isolated from the bone marrow. The bone marrow from healthy donors aged 18 to 80  were used for the experiment. The growth and morphology of the cells were observed with an inverted microscope .The bone marrow cells were mixed with the red blood cell cracking liquid and the sedimentation (volume 5 : 1) at 4°C for 5 min. Mononuclear cells were separated from the residue by using an isolated lymphocyte separation medium. The isolated hBM-MSCs were resuspended in DMEM/F12 supplemented with 10% FBS, 1% penicillin, and 1% streptomycin. Cells were cultured at a density of 5 × 106 in each group) of each group were suspended in PBS containing 2% BSA, fluorescein isothiocyanate- (FITC-) labeled or phycoerythrin- (PE-) labeled specific antibodies FITC-CD105, FITC-CD45, FITC-CD34, FITC-CD19, FITC-CD14, FITC HLA-DR, FITC-CD90, PE-CD73, PE-CD11b which were incubated in accordance with the specification at 4°C for 30 min in dark. The results were used Cell Quest software for data processing.Flow cytometry was used to detect the expression of hBM-MSC surface antigen markers. The cells . The optimal concentration and time of the drug were determined by a CCK-8 method and cell proliferation analysis.HBM-MSCs were inoculated in 24-well plates at a concentration of 1.5 × 105/mL and cultured in Edu solution for 24 h. 4% paraformaldehyde was fixed 30 min at room temperature. Washed cells were permeabilized 0.5% Triton X-100 for 20 min. Then, the cells were incubated with 1X Apollo® reaction cocktail for 30 min and stained using 1X Hoechst33342 for 30 min and imaged under a fluorescent microscope (Olympus Corporation). The proliferation rate was defined as the ratio of Edu-positive cells (labeled red fluorescence) to hoechst33342-positive cells (labeled blue fluorescence).β-gal staining was performed according to the manufacturer's instructions. Cells were prepared as previously described, fix in 4% paraformaldehyde for 5 minutes, washed twice with PBS, and plastic wrap sealed in a dyeing box, stained at 37°C overnight. The cytoplasm stained blue is senescent cells. Collection of images was done by an inverted microscope and optical density analysis by Image-Pro Plus 6.0 software.The SA-When the cell adherence rate reached 50%-70%, the conventional medium was replaced with ODM, and the culture was replaced with ODM every 3 d for 21 d. Rg1 was added to ODM media in the Rg1 treatment group for 21 days. After 21 d, it was soaked in 4% paraformaldehyde for 30 min and washed with PBS for 3 times. Then, 1% alizarin red S (pH = 4.2) was used for 5 minutes.When cell adherent reached 100%, use ADM instead of conventional culture medium, replace ADM culture every 3 d, and cultivate for 21 d. Rg1 was added to ADM media in the Rg1 treatment group for 21 days. After 21 d, the cells were washed with 4% paraformaldehyde for 30 min and washed with PBS for 3 times. Then, dye with oil red O for 60 minutes.When cell adherent reached 70%-80%, use CDM instead of conventional culture medium, replace CDM culture every 3 d, and cultivate for 21 d. Rg1 was added to CDM media in the Rg1 treatment group for 21 days. After 21 d, the cells were washed with 4% paraformaldehyde for 30 min and washed with PBS for 3 times. Then, dye with Alcian blue for 30 minutes.Differentiation kits and staining solution were purchased from Cyagen Biosciences Inc. . Images were observed and collected using an inverted microscope (Olympus Corporation).β-catenin (1 : 100) overnight at 4°C. In the next day, the cells were warmed at room temperature for 30 mins and washed three times. Then, the cy3-labeled secondary antibody (1 : 300) was incubated at 37°C for 1 h. DAPI is used for nuclear staining. All slides were observed directly under a fluorescence microscope . Antibodies were purchased from Cell Signaling Technology Inc. .Cells prepared as previously described were fixed by 4% paraformaldehyde for 20 mins. After washing with 0.5% Triton X-100 for 20 min, blocking with 10% goat serum was done for 30 mins at room temperature. Then, the cells were incubated with antibodies against β-catenin, GSK-3β, β-actin , LEF, TCF-4, and C-myc . Incubate at room temperature for 90 minutes with a secondary antibody (1 : 10000 TBS-tween). Use an enhanced chemiluminescence detection system , with β-actin  as internal control. The integrated optical density was quantified by Image Lab 5.2.1 .Total protein was extracted by radioimmunoprecipitation, the lysate buffer was mixed with a 1% protease inhibitor cocktail (Beyotime Institute of Biotechnology), and the concentration was determined by the bicinchoninic acid method. Samples containing 40 g proteins were separated by 12% SDS PAGE and transferred to polyvinylidene fluoride membrane. At room temperature, it was sealed with skim milk powder (5% dissolved in TBS tween 20) for 2 h, incubated overnight in 4°C (1 : 1000 diluted in primary antibody dilution buffer) with specific primary antibodies for Total RNA was isolated with trizol reagent (Invitrogen. USA) according to the manufacturer's agreement. The first strand of cDNA was constructed by TaqMan RT reagent . SYBR green Supermix (Bio-Rad) was used to perform real-time quantitative PCR on the circulator real-time detection system (Bio-Rad). The mRNA expression level and GAPDH were normalized, and the comparative cycle threshold method was used for analysis. The mean ± standard deviation of three independent experiments was measured. The PCR primers used are provided in the supporting information Tables  and 2.P < 0.05 was considered statistically significant.All data were expressed as mean ± standard deviation. All statistical analyses were performed using one-way anova, and Fisher's least significant difference test was performed using SPSS v20.0 . Bone marrow biopsy is a way to understand the bone marrow. However, the biopsy is more detailed and comprehensive than the puncture on the bone marrow status. For certain diseases, such as myelofibrosis, bone marrow punctures often fail due to “dry pumping”, and bone marrow biopsy is often successful. We clinically collected patient biopsy specimens for Wright's staining. The content of each tissue was examined using morphometric software. The results  showed tThe cell morphology, the potential of multipotential differentiation, and the surface markers would be the key to identify the stem cells. Accordingly, experiments were under ways to ensure the validity of cells. The result of cell culture shows that the cells are generally elongated and spindle-shaped and its growth is characteristics of screw growth . AdipogeP < 0.05) as the Rg1 concentration increased. However, there were no significant differences between the obviously increased groups (P > .05). At 48 h, cells treated with 40 μM Rg1 had a significantly higher viability than that of the other group, while those treated with higher concentrations were not changed substantially.In order to screen the optimal dose and exposure time of Rg1 for hBM-MSCs, we conducted CCK-8 experiment. In general, the mean OD value of the Rg1 group was higher than that of the control group (>65 years old group) . The celμM at 48 h.Subsequently, hBM-MSC diffusion was studied by Edu in different groups, and the results showed that the proliferation of hBM-MSCs increased significantly when treated with Rg1 in 40 7 per group. After 72 h, 60%-70% of hBM-MSCs were observed to adhere to the culture dish, and some spindle cells were observed  significantly reduced β-catenin, C-myc, LEF, and TCF-4 levels after treatment of senescent mesenchymal stem cells . The levels of β-catenin increased significantly and GSK-3β decreased significantly after pretreatment with Licl, indicating that Licl effectively activates the Wnt/β-catenin pathway in hBM-MSCs.To determine whether the effect of Rg1 on the differentiation of aging hBM-MSCs depends on Wnt/β-catenin, TCF, LEF, and C-myc increased, while the expression of GSK-3β decreased. After Rg1 treatment, β-catenin protein, TCF, LEF, and C-myc decreased, while GSK-3β expression increased  treated group and the control group Figures . The resMyelofibrosis (MF) is referred to as myelin. It is a kind of myeloproliferative disease caused by collagen hyperplasia in bone marrow hematopoietic tissue, and its fibrous tissue seriously affects hematopoietic function. The age of onset is between 50 and 70 years old. Some scholars believe that myelofibrosis is caused by abnormal stimulation of hematopoietic stem cells, leading to fibrous tissue hyperplasia, even new bone formation, and bone marrow hematopoietic tissue involvement eventually leading to hematopoietic failure.During the bone marrow puncture process, it was found that the older the patient, the greater the probability of “dry pumping”. Statistics on bone marrow biopsy results show that as the age increases, the proportion of hematopoietic tissue and trabecular bone in human bone marrow decreases, while the proportion of adipose tissue increases.In this study, hBM-MSCs were successfully isolated from the human bone marrow. The cultured hBM-MSCs displayed fibroblastic morphology. Previous experiments  have shoMesenchymal stem cells (MSC) are a kind of pluripotent stem cells belonging to the mesoderm . The difβ-catenin signaling pathway regulated the differentiation of hBM-MSCs. The Wnt/β-catenin signaling pathway is a canonical pathway to regulate stem cell functions by regulating the localization of β-catenin, thus modulating the expression of downstream proteins and genes [β is an important part of the complex degrading β-catenin protein in the Wnt/β-catenin signal pathway [β-catenin forms polyprotein complexes with Axin, APC (adenomatous polyposis coli), GSK3β, and CK1α. Then, β-catenin is phosphorylated by the kinase in this complex and degraded by E3 ubiquitin ligase. TCF-4 and LEF are important binding sites of β-catenin protein after entering the nucleus. C-myc and Cyclin D1 genes are important target genes of the Wnt/β-catenin signal pathway. Overactivation of the Wnt/β-catenin signal pathway tends β-catenin protein to cluster together to enter into the nucleus. Overactivation of the Wnt/β-catenin signaling pathway increases the production of β-catenin by degrading the complex of APC and GSK-3β. As the level of β-catenin in the cytoplasm increased, it will transfer to the nucleus and form a complex with family members such as transcription factor TCF/LEF [Our study found that Rg1-inhibited Wnt/nd genes , 20, 55. TCF/LEF –58. By rβ, Licl can activate the Wnt/β-catenin signaling pathway and aggravate the senescence of mesenchymal stem cells. When ginsenoside Rg1 was added to the Licl group, it was seen that the ability of Rg1+Licl to form bone nodules and cartilage nodules was enhanced compared to the Licl group. It is shown that Rg1 can regulate the decrease of Licl-induced differentiation ability. Western blot showed that expression of β-catenin, TCF, C-myc, and LEF in the Rg1+Licl group decreased, and the expression of GSK-3β increased. These results indicate that ginsenoside Rg1 can interfere with the activation of the Wnt/β-catenin signaling pathway and delay the senescence of hBM-MSCs.In this study, the mechanism of Rg1 delaying the senescence of mesenchymal stem cells was further investigated. As an inhibitor of GSK-3β-catenin signaling pathway remains unclear. It has been reported [α (TNF-α). Zhou et al. [κB transcription factor, which leads to induction of Wnt ligands such as Wnt1 and Wnt7a and activation of β-catenin. The finding showed that oxidative stress is an upstream regulator of Wnt/β-catenin, a developmental signal usually silenced in normal adult kidney. Bi et al. [β-Catenin and GSK-3β play a regulatory role in these processes [β and β-catenin. However, this speculation needs to be further studied.However, the exact mechanism by which Rg1 regulates the Wnt/reported  that Rg1reported , 61 haveu et al.  found thi et al.  found throcesses . Thus, wβ-catenin signaling pathway may be involved in this process. The findings of this study provide important experimental evidence for the application of Rg1 in the adjuvant therapy of MSCs.In conclusion, Rg1 can regulate the differentiation of hBM-MSCs, and the Wnt/


Nuclear factor-κB (NF-κB) plays a central role in psoriasis and canonical Wnt/β-catenin pathway blunts the immune-mediated inflammatory cascade in psoriasis. Adenosine A2A receptor activation blocks NF-κB and boosts the Wnt/β-catenin signaling. PDRN (Polydeoxyribonucleotide) is a biologic agonist of the A2A receptor and its effects were studied in an experimental model of psoriasis. Psoriasis-like lesions were induced by a daily application of imiquimod (IMQ) on the shaved back skin of mice for 7 days. Animals were randomly assigned to the following groups: Sham psoriasis challenged with Vaseline; IMQ animals challenged with imiquimod; and IMQ animals treated with PDRN (8 mg/kg/ip). An additional arm of IMQ animals was treated with PDRN plus istradefylline  as an A2A antagonist. PDRN restored a normal skin architecture, whereas istradefylline abrogated PDRN positive effects, thus pointing out the mechanistic role of the A2A receptor. PDRN decreased pro-inflammatory cytokines, prompted Wnt signaling, reduced IL-2 and increased IL-10. PDRN also reverted the LPS repressed Wnt-1/β-catenin in human keratinocytes and these effects were abolished by ZM241385, an A2A receptor antagonist. Finally, PDRN reduced CD3+ cells in superficial psoriatic dermis. PDRN anti-psoriasis potential may be linked to a “dual mode” of action: NF-κB inhibition and Wnt/β-catenin stimulation. Psoriasis is an inflammatory skin disease with an important impact on patient quality of life. Plaques psoriasis is the most common type of the disease and presents red, well delineated, and silver plaques mainly localized in the umbilical and lumbosacral area as well as in the elbows, knees, and scalp. From a histological point of view, psoriasis plaques show a high proliferation rate and are characterized by immature keratinocytes and by an incomplete development of cornification, causing a marked thickness in epidermis with the occurrence of elongated rete ridges. The dermis is also characterized by the infiltration of an exaggerated number of macrophages, dendritic cells and T-cells. Furthermore, psoriasis is frequently associated with an uncontrolled inflammation of the musculoskeletal system; the main interested sites being the joints (arthritis), the tendons and ligaments of the bone (enthesitis), and the bone of the peripheral and assail skeleton. Indeed, it has been shown that in experimental models of psoriasis an impaired activity of T-cells represents a key fingerprint of the disease and an imbalance between immune system, inflammatory response and skin turnover is responsible for the appearance of psoriatic lesions . InflammMethotrexate, one of the main prescribed drugs for the treatment of psoriasis, acts, at least in part, by augmenting the endogenous levels of adenosine, thus unmasking a role for this mediator in the inflammatory reaction that occurs under this pathological condition. Indeed, adenosine is an endogenous regulator of inflammatory processes, mediating the resolution of inflammation . Four tyThese receptors differ in affinity for adenosine and have a different level of expression in the several cell types. It has been proposed that targeting adenosine receptors, specifically the A2A subtype, might be considered a good strategy for the treatment of skin diseases characterized by inflammation ,5.Indeed, A2A receptor stimulation produces anti-inflammatory effects by inhibiting Nuclear factor κB (NF-κB) translocation to the nucleus and consequently suppressing TNF-α expression ,7. This Besides the NF-κB triggered inflammatory cascade, the Wnt/β-catenin signaling has also been suggested to positively modulate the inflammatory reaction of psoriasis . InteresPDRN, polydeoxyribonucleotide, is a biologic compound that engages the A2A receptor and exerts a significant anti-inflammatory effect . MoreoveTherefore, the aim of this study was to investigate the efficacy of PDRN in an experimental model of psoriasis-like dermatitis.Shaved back skin of mice challenged with Vaseline showed a normal appearance during all the experimental procedure, with no phenotypical or histological alterations A. IMQ apA previous experiment has already demonstrated that the expression of the phosphorylated transcriptional factor NF-κB and consequently of the pro-inflammatory cytokine TNF-α increases following IMQ administration because of an inflammatory process triggering . In thisSkin challenged with IMQ alone showed an increased mRNA expression of other pro-inflammatory cytokines, such as IL-6 and IL-12 compared to Sham mice. PDRN administration caused a marked decrease of both IL-6 and IL-12 , likely Skin of IMQ mice showed a reduced expression of both Wnt-1 and β-catenin , thus dePDRN treatment also boosted the activation of Wnt-1 and β-catenin in psoriatic animals. Moreover, PDRN reduced IL-2 expression and increased IL-10 mRNA expression, as a consequence of Wnt/β-catenin pathway activation .An inflammatory in vitro model was reproduced to confirm the anti-inflammatory effect of PDRN by treating human keratinocytes with LPS for 24 h. The stimulation with LPS reduced the expression of both Wnt-1 and β-catenin compared to control cells. PDRN treatment significantly increased both molecules involved in Wnt/β-catenin signaling , thus coMoreover, HaCaT cells were stimulated with TNF-α for 6 h and then treated with PDRN until 24 h to evaluate whether PDRN might modulate the inflammatory response induced by TNF-α. TNF-α incubation produced a marked increase of IL-6 expression, whereas as expected, PDRN significantly reduced the mRNA expression following 24 h of treatment .Immunohistochemical evaluation of representative skin samples revealed that skin collected from Sham mice showed a small amount of CD3+ cells A, whereaHuman keratinocytes were used to demonstrate PDRN effects on Wnt/β-catenin signaling. PDRN treatment significantly increased both Wnt1 and β-catenin mRNA expression compared to untreated keratinocytes.CGS21680, a specific A2A receptor agonist, also augmented mRNA expression of Wnt-1 and β-catenin in human keratinocytes, thus confirming the A2A receptor involvement in Wnt/β-catenin pathway. The use of the A2A antagonist ZM241385 abrogated PDRN effects on Wnt-1 and β-catenin, thus confirming PDRN mode of action .Inflammation plays an important role in psoriasis and represents one of the most relevant features of the disease, worsening the prognosis . Hyper-aAmong the several pro-inflammatory cytokines involved in psoriasis, TNF-α, IL-6, and IL-12 are usually increased in psoriatic patients, creating a negative loop that exacerbates the inflammatory condition . PreclinIn the intracellular cascade of events, nuclear factor κB plays a central role. This is a nuclear transcription factor that modulates the expression of the genes involved in the inflammatory processes. NF-κB is usually in its inactive state in the cytosol and associated with I-κB. When I-κBα is phosphorylated, NF-κB separates from I-κBα and translocates into the nucleus, thus activating the transcription of the genes encoding pro-inflammatory cytokines.Under the present experimental design, IMQ application activated the transcriptional factor NF-κB and consequently promoted the expression of pro-inflammatory cytokines such as TNF-α, IL-6, and IL-12. In this context, IMQ caused characteristic histological skin alterations: more specifically epidermal hyperplasia, hyperparakeratosis and inflammatory infiltrate accumulation were observed.The adenosine A2A receptor activation determines an important and useful anti-inflammatory effect, for this reason PDRN, thanks to its activity on adenosine receptors, may be considered as a new approach in pharmacotherapy. DNA nucleases located in plasma or in tissues are involved in the degradation of PDRN, thus forming oligo and mononucleotides. This is one of the main processes that characterize PDRN mechanism of action: the production of nucleosides and nucleotides following PDRN degradation mediates the binding to the A2A receptor . MoreoveRecently, a role of the canonical Wnt/β-catenin pathway has been described in psoriasis: more specifically it has been pointed out that Wnt signaling may be involved in the modulation of immune/inflammatory cascade. Interestingly, Wnt/β-catenin pathway is involved in a plethora of events, in particular during embryogenesis, stimulating developmental processes . The actCanonical Wnt/β-catenin pathway activation modulates immune-inflammatory mediators, such as IL-2 and IL-10 expression. In this experimental model, IMQ administration diminished Wnt1 and consequently β-catenin mRNA expression. Moreover, IMQ caused a significant increase and a marked decrease of IL-2 and IL-10 mRNA expression, respectively, likely as a consequence of Wnt1 and β-catenin pathway down-regulation.Therefore “reawakening” the Wnt/β-catenin signaling may be an innovative strategy for the treatment of psoriasis. However, it remains to identify an effective tool that accomplishes this task. Targeting the adenosine A2A receptor offers an interesting potential. Indeed, canonical Wnt/β-catenin signaling may be activated by A2A receptor stimulation because cyclic AMP (adenosine monophosphate) accumulation increases Wnt/β-catenin expression . In agreIn addition, TNF-α and LPS were used as stimuli to induce a psoriasis-like in vitro model. PDRN treatment significantly reduced the mRNA expression of IL-6 following stimulation of keratinocytes with TNF-α, confirming the anti-inflammatory effect of PDRN, as previously described ,12. SchöWnt/β-catenin pathway was studied following LPS incubation: LPS reduced the expression of Wnt-1 and β-catenin in stimulated keratinocytes whereas PDRN significantly increased the mRNA expression of both, thus confirming the data obtained in psoriatic skin and in normal keratinocytes.As a read-out of this triggering, PDRN blunted IL-2 and increased IL-10 mRNA expression in treated psoriatic mice compared to IMQ animals.Psoriasis is a complex immune-mediated disease and, in this specific immunological setting, T cells activation plays a fundamental role . IL-2 maThe present experimental data suggest that PDRN anti-psoriasis potential may be linked to a “dual mode” of action: a NF-κB inhibitory activity that is combined with a stimulation of canonical Wnt/β-catenin pathway. Thus, the A2A receptor orchestrates a crosstalk between the two intracellular signaling cascades that results in positive modulation of both the inflammatory and immune reactions. PDRN is a biologic A2A agonist already in the market, even if, at the present, with different therapeutic claims: therefore, this study has a strong translational impact. However, the anti-psoriatic effect deserves additional studies and needs to be confirmed in a clinical setting. In addition, it is worthy of interest to stress another high translational potential linked to the PDRN pharmacology . Indeed,Indeed, PDRN may share with apremilast, a benchmark drug for the treatment of psoriasis, an overlapping mode of action, at least on the intracellular signaling events that lead to the downregulation of the inflammatory cascade that plays a fundamental role in the pathogenesis of psoriasis. Activation of the A2A receptor induces an increase in the intracellular cyclic AMP as apremilast does through phosphodiesterase inhibition. The augmentation in cyclic AMP synergizes with other molecular signaling that cooperate to reduce the translocation of NF-κB to the nucleus and to turn off genes codifying for the master cytokines involved in the pathogenesis of psoriasis. However, stimulation by PDRN of this specific subtype of adenosine receptor has, as we have showed in the present paper, further effects on another signaling cascade that concur to the blunting of the exaggerated inflammatory reaction: the Wnt/β-catenin signaling. The modulation of this intracellular molecular pathway may cause a more robust and sustained suppression of the keratinocyte inflammatory phenotype, in turn limiting keratinocyte proliferation and plaque formation. However, at the moment, we do not know whether PDRN exerts a greater therapeutic activity when compared to apremilast, and future experimental work should be carried out to investigate this point. Finally, it should be pointed out that PDRN has a good safety profile . ClinicaBalb/c mice  were used in this study. During the experiment, mice were housed in plastic cages under controlled environmental conditions  in the Animal Facility of the Department of Clinical and Experimental Medicine. Animals were provided with standard food and water ad libitum and all the experiments were performed in compliance with the standards for care and use of animals as stated in the Directive 2010/63/EU, and the ARRIVE guidelines [n = 30) for 7 consecutive days. Animals were randomly assigned to the following groups: sham psoriasis, which were challenged with Vaseline cream; IMQ animals, only challenged with imiquimod cream and IMQ animals challenged with IMQ and treated with PDRN (8 mg/kg/ip). In histological experiments an additional arm of IMQ animals was treated with PDRN plus istradefylline , an A2A antagonist. Both PDRN and istradefylline were daily administered starting on day 3, following psoriasis-like lesions induction. Mice from each group were killed 7 days following the first imiquimod or Vaseline administration.Psoriasis-like lesions were induced by a topical daily application of imiquimod cream  on the shaved back skin of mice  and the lysates were centrifuged at 15.000 rpm for 15 min at 4 °C. Total protein content was measured using the Bio-Rad protein-assay kit  and the supernatant was mixed with Laemmli sample buffer , and stored at −20 °C. Thirty micrograms of proteins were run by electrophoresis on a 10% sodium dodecyl sulphate (SDS) polyacrylamide gel. Following separation, proteins were transferred onto a PVDF membrane using a transfer buffer  at 100 V for 1 h and then blocked with 5% non-fat dry milk in TBS 0.1% for 1 h at room temperature. Membranes were incubated with primary antibodies for TNF-α , pNF-κB p65 and β-actin  in TBS-0.1% Tween overnight at 4 °C. Primary antibodies were removed, membranes were washed 3 times with TBS-0.1% Tween and incubated with a secondary peroxidase-conjugated goat anti-rabbit antibody  for 1 h at room temperature. Membranes were analyzed by the enhanced chemiluminescence system (LumiGlo reserve) according to the manufacture’s protocol (KPL) and the protein detection was quantified by scanning densitometry using a bio-image analysis system . The results were expressed as relative integrated intensity compared to normal skin tissue measured within the same batch.Total RNA was extracted from skin of mice at the end of the experiment (7th day) and from human keratinocytes using Trizol LS reagent . RNA was quantified with a spectrophotometer  and 2 μg was reverse transcribed in a volume of 20 μL using the Superscript VILO kit . One microliter of cDNA was added to the EvaGreen qPCR Master Mix , in a volume of 20 μL per well. Samples were run in duplicate and GADPH was used as housekeeping gene; the reaction was performed using the 2-step thermal protocol recommended by the manufacturer. Ten micromoles was the primer concentration selected to perform the analysis. Target genes were Wnt-1, β-catenin, IL-6, IL-10, IL-12, and IL-2.Primers used for target and reference genes are listed below:GADPHFw:5′GTCAAGGCTGAGAATGGGAA3′Rv:5′ATACTCAGCACCAGCATCAC3′;Wnt-1Fw:5′ATAGCCTCCTCCACGAACCT3′Rv:5′ GGAATTGCCACTTGCACTCT3′;β-cateninFw:5′CGGCACCTTCCTATTTCTTCT3′Rv:5′TCTGGAAATTAACTTCAGGCAAC3′;IL-6Fw:5′AGTTGCCTTCTTGGGACTGA3′Rv:5′ TCCACGATTTCCCAGAGAAC3′;IL-10Fw:5′CCAAGCCTTATCGGAAATGA3′Rv:5′ TTTTCACAGGGGAGAAATCG3′;IL-12Fw:5′CATCGATGAGCTGATGCAGT3′Rv:5′CAGATAGCCCATCACCCTGT3′;IL-2Fw:5′CCCACTTCAAGCTCCACTTC3′Rv:5′ATCCTGGGGAGTTTCAGGTT′;Primers used for human target genes are listed below:GAPDHFw:5′ GAGTCAACGGATTTGGTCGT3′Rv:5′ TTGATTTTGGAGGGATCTCG3′;IL6Fw:5′ TACCCCCAGGAGAAGATTCC3′Rv:5′ TTTTCTGCCAGTGCCTCTTT3′;Wnt-1Fw:5′ CGGCGTTTATCTTCGCTATC3′Rv:5′ GCCTCGTTGTTGTGAAGGTT3′;β-cateninFw:5′ATCTGCCTCCAGAGCAGGTA3′Rv:5′CCTCAGGATTGCCTTTACCA3′;t method, and expressed as n-fold increase in gene expression using the CTRL group as calibrator.Results were calculated using the 2-ΔΔCParaffin-embedded tissues were sectioned (5 μm) and the immunohistochemical analysis was performed with a rabbit primary antibody anti-CD3  using the Ventana BenchMark automated slide stainer in combination with Ventana detection kits according to manufacturer specifications. Reaction was observed with diaminobenzidine tetra-hydrochloride . Slides were counterstained with Mayer’s Haemalum, dehydrated, and mounted with coverslips. All slides were coded and evaluated by a pathologist at 40× magnification with a Nikon eclipse Ci-e microscope.2. Starved HaCaT cells were stimulated with LPS (10 μg/mL) for 24 h or TNF-α (10 ng/mL) for 6h to reproduce an inflammatory condition in psoriatic keratinocytes. In addition, keratinocytes were treated with PDRN (1 μM), or CGS21680 (1 μM), or with PDRN in association with an A2A antagonist, ZM241385 (1 μM), for 4 h. ZM241385 was dissolved in DMSO to obtain a concentration of 1000 μM and then diluted in DMEM for a final concentration of 1 μM.Human HaCaT cells were obtained from ATCC  and cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM) with 10% inactivated FBS, 100-U/mL penicillin and 100-μg/mL streptomycin. Cells were incubated at 37 °C under 5% COP < 0.05 was considered as the possibility of error and was considered as statistically significant. Graphs were drawn using GraphPad Prism .All data are expressed as means ± standard deviation (S.D.). Different treatments were compared and analyzed by one-way ANOVA with Tukey post-test for intergroup comparisons.


APC and the β-catenin encoding gene (CTNNB1), detected in a large majority of CRC patients. Wnt/β-catenin signaling is involved in the basic types of vascularization (sprouting and nonsprouting angiogenesis), vasculogenic mimicry as well as the formation of mosaic vessels. The number of known Wnt/β-catenin signaling components and other pathways interacting with Wnt signaling, regulating angiogenesis, and enabling CRC progression continuously increases. This review summarizes the current knowledge about the role of the Wnt/Fzd/β-catenin signaling pathway in the process of CRC angiogenesis, aiming to improve the understanding of the mechanisms of metastasis as well as improvements in the management of this cancer.Angiogenesis belongs to the most clinical characteristics of colorectal cancer (CRC) and is strongly linked to the activation of Wnt/β-catenin signaling. The most prominent factors stimulating constitutive activation of this pathway, and in consequence angiogenesis, are genetic alterations (mainly mutations) concerning Aberrant activation of the Wnt/Fzd/β-catenin signaling pathway is one of the major molecular mechanisms of colorectal cancer (CRC) development and progression. On the other hand, one of the most common clinical CRC characteristics include high levels of angiogenesis, which is a key event in cancer cell dissemination and distant metastasis. The canonical Wnt/β-catenin downstream signaling regulates the most important pro-angiogenic molecules including vascular endothelial growth factor (VEGF) family members, matrix metalloproteinases (MMPs), and chemokines. Furthermore, mutations of the β-catenin gene associated with nuclear localization of the protein have been mainly detected in microsatellite unstable CRC. Elevated nuclear β-catenin increases the expression of many genes involved in tumor angiogenesis. Factors regulating angiogenesis with the participation of Wnt/β-catenin signaling include different groups of biologically active molecules including Wnt pathway components , and non-Wnt pathway factors . Several lines of evidence argue for the use of angiogenesis inhibition in the treatment of CRC. In the context of this paper, components of the Wnt pathway are among the most promising targets for CRC therapy. This review summarizes the current knowledge about the role of the Wnt/Fzd/β-catenin signaling pathway in the process of CRC angiogenesis, aiming to improve the understanding of the mechanisms of metastasis as well as improvements in the management of this cancer. Drosophila as well as the discovery of the first mammalian Wnt gene (1982), the role of Wnt signaling was mostly implied in cell growth regulation during embryonic development and maintenance of adult tissue structure , and endoglin (CD105), all involved in tumor progression and prognosis. Moreover, endoglin expression in tumor cells was positively correlated with E-cadherin, β-catenin, and Syndecan-1 . It was demonstrated that exosomes derived from hypoxic CRC cells promote angiogenesis. These exosomes, enriched with Wnt4, promoted the proliferation and migration of ECs through Wnt4-induced β-catenin signaling. It was proved that Wnt4 increased nuclear translocation of β-catenin in ECs. Furthermore, an increase in tumor size and angiogenesis via CRC cell-derived exosomes was also confirmed in an animal in vivo model . CSCs of human CRC are unique cell types able to maintain tumor mass, modify the tumoral microenvironment by expressing angiogenic factors and enhanced neovascularization, and survive outside of the primary tumor at metastatic sites ,110,111.The mutual relations between CRCSCs and the canonical Wnt/β-catenin signaling pathway are also described in the case of CRC. This signaling pathway is a master regulator of a balance between stemness and differentiation in several adult stem cell niches including colon CSCs population in intestinal crypts of Lieberkühn. The colon-crypt base is characterized by high activity of Wnt signaling, especially in the bottom third of the crypts (where CSCs reside) due to signals from the stromal microenvironment cells ,32. In HAPC/CTNNB1 mutations) in a paracrine manner. Fzd8, a putative Wnt2 receptor, was identified on CAFs. It was demonstrated that Wnt2 activates autocrine canonical Wnt signaling in primary fibroblasts, which was connected to the pro-migrative and pro-invasive phenotype. These studies indicate the major role of Wnt2 in the promotion of growth, invasion, and CRC metastasis in vivo [Cancer-associated fibroblasts, as a major component of tumor stroma, play an underestimated role in the development and progression of various solid tumors (including CRC) ,118. Act in vivo . Further in vivo . The res in vivo . In physiology, ECs are responsible for the formation of a semi-permeable barrier, a process enabled by the structure of intercellular connections as well as the presence of VE-cadherin (cadherin 5/CD144) and β-catenin, linking the VE-cadherin junction complex to the cytoskeleton ,123. TheIn tumor (including CRC) blood vessels, structural and functional changes can be observed, connected to alterations in leukocyte trafficking. It was demonstrated that VE-cadherin expression and downstream activation of the Akt/GSK3β/β-catenin signaling caused an increase in the expression of the chemokine (C-C motif) ligand 2 (CCL2) and CXCL10, which facilitate CD8+ T cell transmigration into tumor parenchyma. Restoration of proper EC junctions not only inhibits vascular leak, but also regulates immune cell infiltration into tumors . The endThe blood vessels produced within the tumor are lined by TECs, characterized by abnormal proliferation and apoptosis . TECs exIn the case of CRC, in contrast to normal ECs, TECs originate not only from EPCs but also from the differentiation of CSCs. It was shown that CRC cells (HCT116 line) can transform into TECs under hypoxia conditions via a VEGFR-2-dependent mechanism. These cells expressed EC markers and formed tube-like structures in vitro . CharactWhen it comes to the expression of Wnt signaling components in CRC tissues, nuclear localization of β-catenin is described in the invasive front, in close proximity of the tumor microenvironment cells (known as the β-catenin paradox) ,132,133.APC and CTNNB, while crucial for constitutive Wnt pathway activation, are not sufficient for nuclear β-catenin accumulation and full action of this signaling pathway [It was also noted that the cytoplasmic levels of β-catenin increased in response to hypoxia . Dilek e pathway ,16. Another protein of the Wnt pathway, increased expression of which in tumor cells is important for the initiation of inhibition of CRC angiogenesis process, is Wnt2 ,120,121.When it comes to the role of serum concentration of Wnt signaling components, as markers of CRC angiogenesis, it was proven that serum VE-cadherin was about fourfold higher in CRC patients compared with the controls, but it was not correlated with the VEGF level and any clinicopathological data . Hence, the authors suggest that these proteins can be considered as independent markers of CRC angiogenesis . The number of known Wnt/β-catenin signaling components and other pathways interacting with Wnt signaling, regulating angiogenesis, and enabling CRC progression continuously increases ,136,137.In turn, anti-angiogenic signaling pathways interacting with Wnt signaling are TGF-β1 ,98, HIF-MicroRNAs  and long noncoding RNAs (lncRNAs) are two major families of non-protein-coding transcripts . This grMiRNAs are the most commonly studied form of non-coding RNAs, responsible for modulating up to 60% of protein-coding gene expression . An incrNotably, increased expression of β-catenin in CRC tissues of mice (C57BL/6Apc(min/+) and human CRC cells positively correlated with significantly upregulated miR-574-5p. This miRNA changed the expression of β-catenin and p27 (Kip1 protein) as well as intensified the migration and invasion of cancer cells. Furthermore, in CRC tissues, miR-574-5p was negatively correlated with the expression of RNA binding protein Quaking (Qki)  . Another study, among 26 deregulated miRNAs in an APC-inducible cell line, identified members of the miR-17-92 cluster that were inhibited by APC. In this process, the stabilized form of β-catenin (as a result of APC mutation) bound to and activated the miR-17-92 promoter. The main mechanism by which APC exerted its tumor suppressor activity was the reduction of miR-19a, the most important member of the miR-17-92 cluster. Therefore, the expression of miR-19a correlated with the level of β-catenin in the CRC samples, and was associated with an aggressive stage of cancer . MiR-92aUpregulation of miR-452 in ~70% CRC tissue samples vs. normal tissues was also reported, correlating with the clinical data. This MiR-452 promotes nuclear relocalization of β-catenin and the expression of target genes . In turn, in vitro and xenograft mice models showed that MiR-452 can activate Wnt/β-catenin signaling and promotes an aggressive CRC phenotype through direct regulation of the 3’ untranslated region (3’UTR) of GSK3β. The miR-452 promoter is affected by the same transcription factors (TCF/LEF family of transcription factors). The authors conclude that a miR-452-GSK3β-TCF4/LEF1 positive feedback loop has an important role in CRC initiation and progression (including angiogenesis) . Other MiRs promoting CRC proliferation, migration, invasion, and suppression of apoptosis in vitro, and in vivo include miR-27a-3p. This molecule acts through downregulation of nuclear receptor retinoid x receptor alpha (RXRα). On the tissue level, an increased expression of this MiR was demonstrated, correlating negatively with RXRα, and positively with various clinical  and histological data (tumor differentiation). The authors also noted that RXRα negatively regulates the expression of β-catenin by its ubiquitination in CRC . This coMiR-224  or epigeOn the other hand, miR-490-3p inhibits β-catenin and suppresses cell proliferation as well as lowers cell invasiveness by repressing EMT. Its direct target was identified as the protooncogene frequently rearranged in advanced T-cell lymphoma 1 (FRAT1) protein, which is linked with nuclear accumulation of β-catenin. Furthermore, hypermethylation of the miR-490-3p promoter downregulated the expression of this miR in CRC cells. The authors conclude that alterations in the miR-490-3p/FRAT1/β-catenin pathway can play an important role in CRC progression (including angiogenesis) . Antagonistic action in transactivation of Wnt signaling is also exhibited by ectopic miR-29b expression. This miR acts through downregulation of β-catenin coactivators  in colon cancer cells (SW480). It binds the 3’UTR of BCL9L, lowering its expression and reducing nuclear translocation of β-catenin. As a consequence, MiR-29b inhibits anchorage-independent cell growth, promotes EMT reversal, and reduces the ability of CRC cell-conditioned medium to induce in vitro tube formation in ECs . Other commonly investigated molecules taking part in different stages of CRC progression (including angiogenesis) also include long non-coding RNAs ,156,157.In turn, inhibition of tumorigenesis and progression (including angiogenesis and metastasis) in CRC is caused by lncRNA-CTD903  and lncRInhibition of the Wnt signaling is also mediated by upregulation of lncRNA growth arrest specific 5 (lncRNA GAS5). This type of lncRNA plays a pivotal role in the prevention of angiogenesis, inhibiting invasion and CRC metastasis . Other tOne circRNA, namely circular decaprenyl-diphosphate synthase subunit 1 (PDSS1) was upregulated in CRC tissue compared to the control samples. All experiments showed that circPDSS1 is linked with local and distant metastasis as well as poor prognosis in CRC patients. Moreover, it was reported to stimulate angiogenesis in CRC via Wnt/ β-catenin signaling. Knockdown experiments resulted in attenuated migratory ability and angiogenesis in CRC cells. The authors noted a downregulation of Wnt/β-catenin signaling proteins including β-catenin, GSK3β, C-Myc, MMP-9, and cyclin D1 protein levels in CRC transfected with sh-cicrPDSS1 . The main types of non-coding RNAs in CRC angiogenesis regulated by Wnt/β-catenin signaling-mediated mechanisms are summarized in RAS expression) [Treatment of CRC patients, especially those affected by metastatic CRC (mCRC), still poses a major challenge and requires significant treatment personalization. Different forms of anti-angiogenic therapy have been attempted, taking into account the mechanisms of CRC angiogenesis, in which a major role is played by the VEGF pathway. There have been approaches based on the application of anti-angiogenic small-molecule TKIs , with or without chemotherapy. Furthermore, monoclonal antibodies have also been used, both anti-VEGF pathway and EGFR targeting (cetuximab and panitumumab). The effectiveness of typical anti-VEGF-R TKIs  turned out to greatly vary in mCRC treatment. The first, most effective multikinase inhibitor of angiogenic , stromal and oncogenic receptor TK, was regorafenib ,161,162.ression) . Furtherression) . However, anti-angiogenic CRC therapies  are not fully effective, being a matter of discussion in many excellent reviews ,161,165.There are currently a few anti-angiogenic agents approved by the U.S. FDA for mCRC treatment: anti-VEGF/VEGF-R agents , anti-EGFR agents , or immune-check-point inhibitors . However, bevacizumab is the only anti-angiogenic compound for the first-line treatment of mCRC (from 2004) ,165. TheApart from anti-angiogenic therapy based on the VEGF pathway, Wnt/β-catenin signaling is among the pathways offering potential sites for targeting ,167,168.The most commonly mentioned existing anti-angiogenic drugs targeting the Wnt/β-catenin pathway in CRC include non-steroidal anti-inflammatory drugs (NSAIDs) , which can “bypass” many carcinogenic effects, also regulating the increased expression of PTEN and GSK3β, inhibition of Akt (and β-catenin), and MMPs as well as iNOS activation, all of which induce cancer cell apoptosis ,167. Oth2D3 in human CRC occurs through the DKK-1 gene induction [2D3 on C-Myc. Methylation of this factor increased the anti-tumor effects of vitamin D in CRC [Apart from NSAIDs, vitamin A and D derivatives also showed efficacy in the disruption of a number of signaling pathways  including Wnt. This fact, together with the introduction of new generations of their derivatives, creates a perspective for potential new interesting clinical trials ,173,174.nduction  and DKK-nduction . The usenduction . Other mD in CRC . In turn, when it comes to vitamin A and its derivatives, it is worth noting that the pathways directing β-catenin for proteasome degradation (in addition to p53/Siah-1/APC and Wnt/GSK3β/APC) include the RXR-mediated pathway ,179. It A growing number of novel agents targeting the Wnt pathway are subjected to clinical trials including specific small molecules ,168. ThiIt was proven that aberrant activation of Wnt/β-catenin signaling mediates resistance of CRC cells to irradiation and 5-FU-based chemotherapy. Higher levels of active β-catenin and increased TCF/LEF reporter activity were observed in SW1463 cells that evolved radiation resistance. It was also demonstrated that inhibition of β-catenin , sensitized CRC cells to chemoradiotherapy . Other sBugula neritina) for CRC treatment [Recently, previous observations on some Wnt/β-catenin signaling inhibitors and downstream targets involving PKCα came back to light, indirectly related to the progression of CRC and angiogenesis. It was proven that PKCα is rarely mutated in CRC samples, hence its function might be activated with no side effects for the intestinal epithelium. Additionally, PKCα activation results in increased cell death and is drug-inducible. According to the authors of the study, there are ongoing phase II clinical trials on the application of natural PKCα activators (found in the Bryozoan species reatment . The usereatment . Thymus vulgaris L [Tetrastigma hemsleyani flavone (RTHF), it was proven that this compound causes downregulation of β-catenin activation and downstream protein expression . It also decreased the size of tumors in vivo in mice through the inhibition of pro-proliferative properties of the Wnt pathway [Curcuma longa, is curcumin. This phytochemical also shows an anti-inflammatory, anti-oxidant, and anti-cancer activity [Psoralea corylifolia, also inhibits growth and colony formation of CRC tumor cells as well as the induction of apoptosis through the inhibition of the AKT/GSK3β/β-catenin pathway have been noted [Coptis sp. and Berberis sp. [275, Arg316, and Arg371 residues. As a result, a promotion of this receptor’s interaction with nuclear β-catenin occurs, leading to c-Cbl mediated degradation of β-catenin, hence the inhibition of cell proliferation. Moreover, human CRC xenograft in nude mice also demonstrated the inhibition of tumor growth in an RXRα-dependent manner [Moreover, a growing number of publications have documented the action of anti-Wnt/β-catenin signaling plant based compounds  However, anti-angiogenic actions linked to Wnt signaling are only attributed to some of them, for example, Raddeanin ,104,105 lgaris L ,185. Onelgaris L . In turn pathway . Anotheractivity . In studactivity . Recentlactivity . The isoen noted . Promisien noted ,193, an eris sp. . Specialeris sp. . It was The basic drawbacks of anti-angiogenic and anti-Wnt signaling targeted therapies have been presented in several reviews ,165. TheDirect CRC angiogenesis inhibition mechanisms based on Wnt/β-catenin signaling are only described in a small number of existing or potential therapeutics. Nevertheless, previously mentioned results of studies  demonstrAPC and the β-catenin encoding gene (CTNNB1), detected in a large majority of CRC patients. These mutations lead to an intensification of CRC cell proliferation, migration, and invasion in vitro as well as tumor growth, angiogenesis, and distant metastases in vivo. In addition to the mutations mentioned, there are more and more genetic and epigenetic biomarkers used to determine CRC diagnosis, prognosis, and response to therapy, as summarized in excellent reviews [Angiogenesis belongs to the most clinical characteristics of CRC and is strongly linked to the activation of Wnt/β-catenin signaling. The most prominent factors stimulating constitutive activation of this pathway, and in consequence angiogenesis, are genetic alterations (mainly mutations) concerning  reviews . There a reviews ).Wnt/β-catenin signaling is involved in the basic types of vascularization (sprouting and nonsprouting angiogenesis) and vasculogenic mimicry as well as the formation of mosaic vessels. In vascular cells, expression of Wnt ligands, Wnt receptors, and Wnt inhibitors has been reported. The main type of angiogenesis with the participation of Wnt signaling is currently assumed to occur through the hypoxia-adaptation mechanism mediated by VEGF-signaling and upregulation of the HIF-1 complex. β-catenin itself induces the expression of VEGF in colon cancer cells in the early steps of CRC neoangiogenesis. Furthermore, tissue VEGF expression positively correlates with the cytoplasmic expression of β-catenin in tumor cells and tumor progression in vivo. In turn, the influence of HIF-1α (increasing) and HIF-2α (decreasing) on β-catenin levels/transcriptional activity in CRC cells remains much more varied. Moreover, non-endothelial interactions between both VEGF receptor types  and Wnt/β-catenin signaling have also been reported. It was confirmed that VEGFR-1 positively regulates Wnt signaling in a GSK3β-independent manner. In contrary to the previous paradigm, the presence of both VEGF receptor types was also demonstrated on tumor CRC cells, suggesting the possibility of autocrine VEGF action. Factors regulating angiogenesis with the participation of Wnt/β-catenin signaling include different groups of biologically active molecules, namely selected molecules belonging to Wnt family proteins  as well as various factors outside the Wnt family . A direct influence of several pro-angiogenic factors  on Wnt signaling has been demonstrated (binding β-catenin) in the angiogenesis process. Other factors promoting angiogenesis  regulate Wnt signaling through β-catenin stabilization and increase Wnt gene expression as well as the intensification of EMT-related transcription factor expression (including β-catenin). This regulation results in EC migration and the formation of capillary-like tubules of human microvascular ECs. The opposite effects are evoked by the anti-angiogenic factors through the inhibition of production and transcriptional activity of β-catenin . Recently, an increasingly important role in Wnt signaling involving CRC angiogenesis is attributed to non-coding RNAs. A number of these molecules activate , while other inhibit Wnt signaling . The active cellular components of CRC-related angiogenesis consist of tumor cells, CRC stem cells, and cancer-associated fibroblasts (CAFs) as well as cells directly linked to blood vessels . Moreover, complex intercellular interactions have been reported in tumors during angiogenesis. CRC cells produce β-catenin (mRNA and protein), which intensifies VEGF expression and increases vessel density. The norrin protein produced by cancer cells binds to Fzd4, regulating EC proliferation and motility. In turn, norrin/Fzd4 interactions are modulated via regulation of Fzd4 expression by Wnt2. Furthermore, exosomes enriched in Wnt4 produced by CRC cancer cells promote angiogenesis by increasing ECs proliferation and migration via Wnt signaling. Both tumor CRC cells and CAFs (main source) produce the Wnt2 protein, which plays a major role in the initiation and maintenance of the CRC angiogenesis process. Wnt2 expression in CAFs correlates with a number of clinicopathological data (including venous invasion) of CRC patients. Wnt2 intensifies EC migration and invasion, enhanced vessel density, and tumor volume. Wnt2 expression positively correlates with the expression of vascular markers and an increase in pro-angiogenic function of many proteins . When it comes to CRC stem cells, high Wnt activity is mostly present in the bottom third of the crypts (where CSCs reside). These cells have the ability of transdifferentiation into human TECs as well as the generation of functional blood vessels. The list of Wnt/β-catenin signaling components and pathways interacting with Wnt signaling, regulating angiogenesis, and conditioning CRC progression, continuously increases. CTNNB1 mutations, there are ongoing studies on the action of a number of inhibitors of β-catenin itself as well as knockdown strategies. However, no results of such research have yet been introduced into CRC oncological practice, due to the relatively low effectiveness as well as significant intestinal toxicity. Small molecules blocking Wnt signaling in CRC also include tankyrase inhibitors (G007-LK and G244-LM). There are several clinical trials  on the use of novel Wnt targeting agents in CRC . Positive anti-angiogenic effects, disrupting Wnt/β-catenin signaling have been demonstrated for a number of NSAIDs  and vitamin A and D derivatives. Furthermore, many natural plant-derived compounds used in traditional Chinese medicine inhibits Wnt/β-catenin signaling and, directly or indirectly, CRC angiogenesis . As for now, the available results mostly concern in vitro and mouse in vivo models. As β-catenin is considered as a primary cause of dysregulated Wnt signaling in CRC as a consequence of APC/As the reviewed literature shows, the role of aberrant Wnt/β-catenin signaling in CRC-related angiogenesis is undisputed. These activities mostly occur due to canonical APC/β-catenin pathway activation in tumor colorectal cells, CRC stem cells, cancer-associated fibroblasts and tumor ECs, intensification of β-catenin expression, and translocation to the nucleus as well as positive correlations with other typical pro-angiogenic factors . Furthermore, the role of a number of active polypeptides, proteins, and non-coding RNAs is indicated in this process. However, when it comes to anti-angiogenic CRC treatments based on targeting the Wnt/β-catenin signaling, studied inhibitors of this pathway are still mostly in preclinical stages, with only a few compounds reaching phase 1 or 2 clinical trials. Individualized targeted CRC therapeutic strategies should take into account the newest findings of molecular biology, explaining the role of direct tumor cell interactions, and all pro- and anti-angiogenic factors acting on this type of signaling as well as other related pathways. An especially large number of publications in the last five years focusing on the role of Wnt/β-catenin signaling in cancer progression (including CRC) has certainly resulted in a better understanding of the mechanisms of metastasis as well as improvements in the management of this cancer.