Bioactivated Glucoraphanin Modulates Genes Involved in Necroptosis on Motor-Neuron-like Nsc-34: A Transcriptomic Study.

Research on bioactive compounds has grown recently due to their health benefits and limited adverse effects, particularly in reducing the risk of chronic diseases, including neurodegenerative conditions. According to these observations, this study investigates the activity of sulforaphane (RS-GRA) on an in vitro model of differentiated NSC-34 cells. We performed a transcriptomic analysis at various time points (24 h, 48 h, and 72 h) and RS-GRA concentrations (1 µM, 5 µM, and 10 µM) to identify molecular pathways influenced by this compound and the effects of dosage and prolonged exposure. We found 39 differentially expressed genes consistently up- or downregulated across all conditions. Notably, Nfe2l2, Slc1a5, Slc7a11, Slc6a9, Slc6a5, Sod1, and Sod2 genes were consistently upregulated, while Ripk1, Glul, Ripk3, and Mlkl genes were downregulated. Pathway perturbation analysis showed that the overall dysregulation of these genes results in a significant increase in redox pathway activity (adjusted p-value 1.11 × 10-3) and a significant inhibition of the necroptosis pathway (adjusted p-value 4.64 × 10-3). These findings suggest RS-GRA's potential as an adjuvant in neurodegenerative disease treatment, as both increased redox activity and necroptosis inhibition may be beneficial in this context. Furthermore, our data suggest two possible administration strategies, namely an acute approach with higher dosages and a chronic approach with lower dosages.

Cannabinol (CBN) Influences the Ion Channels and Synaptic-Related Genes in NSC-34 Cell Line: A Transcriptomic Study.

Neurological disorders such as Alzheimer's, Parkinson's, amyotrophic lateral sclerosis, and schizophrenia are associated with altered neuronal excitability, resulting from dysfunctions in the molecular architecture and physiological regulation of ion channels and synaptic transmission. Ion channels and synapses are regarded as suitable therapeutic targets in modern pharmacology. Cannabinoids have received great attention as an original therapeutic approach for their effects on human health due to their ability to modulate the neurotransmitter release through interaction with the endocannabinoid system. In our study, we explored the effect of cannabinol (CBN) through next-generation sequencing analysis of NSC-34 cell physiology. Our findings revealed that CBN strongly influences the ontologies related to ion channels and synapse activity at all doses tested. Specifically, the genes coding for calcium and potassium voltage-gated channel subunits, and the glutamatergic and GABAergic receptors (Cacna1b, Cacna1h, Cacng8, Kcnc3, Kcnd1, Kcnd2, Kcnj4, Grik5, Grik1, Slc17a7, Gabra5), were up-regulated. Conversely, the genes involved into serotoninergic and cholinergic pathways (Htr3a, Htr3b, Htr1b, Chrna3, Chrnb2, Chrnb4), were down-regulated. These findings highlight the influence of CBN in the expression of genes involved into ion influx and synaptic transmission.

α-Cyclodextrin/Moringin Induces an Antioxidant Transcriptional Response Activating Nrf2 in Differentiated NSC-34 Motor Neurons.

Oxidative stress is a common feature of neurodegenerative diseases. Different natural compounds mediate neuroprotective effects by activating the Nrf2 antioxidant response. Some isothiocyanates are Nrf2 activators, including Moringin (MOR). In this study, the transcriptional profile of differentiated NSC-34 motor neurons was evaluated after treatment for 48 h and 96 h with concentrations of 0.5, 5, and 10 µM of a new MOR formulation obtained with α-cyclodextrin (α-CD). All the concentrations increased gene expression and cytoplasmic protein levels of Nrf2 at 96 h. However, the highest dose also increased nuclear Nrf2 levels at 96 h. Then, Nrf2 interactors were selected using STRING, and common biological process (BP) terms between the groups were evaluated. α-CD/MOR was able to modulate BP related to responses to oxidative stress, proteostasis, and autophagy. Specifically, the treatment with 10 µM of α-CD/MOR for 96 h induced genes involved in glutathione synthesis and proteasome subunits and reduced the expression of genes related to endoplasmic reticulum stress. Moreover, this group showed the lowest levels of the apoptotic markers Bax, cleaved caspase 9, and cleaved caspase 3. These results indicate the beneficial effects of prolonged α-CD/MOR supplementation that are mediated, at least in part, by Nrf2 activation. Then, α-CD/MOR could be a valuable treatment against neurodegenerative diseases, in particular motor neuron degeneration.

Cannabinol Regulates the Expression of Cell Cycle-Associated Genes in Motor Neuron-like NSC-34: A Transcriptomic Analysis.

Cannabinoids are reported to have neuroprotective properties and play a role in neurogenesis and neuroplasticity in in vitro and in vivo models. Cannabinol (CBN) is a minor cannabinoid produced by the degradation of Δ9-tetrahydrocannabinol in Cannabis sativa L. and exhibits anti-oxidant, analgesic, anti-bacterial, and anti-inflammatory effects. In this study, we explored the biological effects of 20 µM CBN (6.20 µg/mL) on differentiated NSC-34 cells by MTT assay and next-generation sequencing analysis on the transcriptome. KEGG and Gene Ontology enrichment analyses have been performed to evaluate potential CBN-associated processes. Our results highlighted the absence of any cytotoxic effect of CBN. The comparative transcriptomic analysis pointed out the downregulation of Cdkn2a, Cdkn2c and Cdkn2d genes, which are known to suppress the cell cycle. Ccne2, Cdk2, Cdk7, Anapc11, Anapc10, Cdc23, Cdc16, Anapc4, Cdc27, Stag1, Smc3, Smc1a, Nipbl, Pds5a, Pds5b, and Wapl genes, renowned for their role as cell cycle progression activators, were instead upregulated. Our work suggests that CBN regulates the expression of many genes related to the cell cycle, which are required for axonal maturation, migration, and synaptic plasticity, while not affecting the expression of genes involved in cell death or tumorigenesis.

Cannabinerol Prevents Endoplasmic Reticulum and Mitochondria Dysfunctions in an In Vitro Model of Alzheimer's Disease: A Network-Based Transcriptomic Analysis.

Neurodegenerative disorders are affecting millions of people worldwide, impacting the healthcare system of our society. Among them, Alzheimer's disease (AD) is the most common form of dementia, characterized by severe cognitive impairments. Neuropathological hallmarks of AD are ß-amyloid (Aß) plaques and neurofibrillary tangles, as well as endoplasmic reticulum and mitochondria dysfunctions, which finally lead to apoptosis and neuronal loss. Since, to date, there is no definitive cure, new therapeutic and prevention strategies are of crucial importance. In this scenario, cannabinoids are deeply investigated as promising neuroprotective compounds for AD. In this study, we evaluated the potential neuroprotective role of cannabinerol (CBNR) in an in vitro cellular model of AD via next-generation sequencing. We observed that CBNR pretreatment counteracts the Aß-induced loss of cell viability of differentiated SH-SY5Y cells. Moreover, a network-based transcriptomic analysis revealed that CBNR restores normal mitochondrial and endoplasmic reticulum functions in the AD model. Specifically, the most important genes regulated by CBNR are related mainly to oxidative phosphorylation (COX6B1, OXA1L, MT-CO2, MT-CO3), protein folding (HSPA5) and degradation (CUL3, FBXW7, UBE2D1), and glucose (G6PC3) and lipid (HSD17B7, ERG28, SCD) metabolism. Therefore, these results suggest that CBNR could be a new neuroprotective agent helpful in the prevention of AD dysfunctions.

Cannabinerol (CBNR) Influences Synaptic Genes Associated with Cytoskeleton and Ion Channels in NSC-34 Cell Line: A Transcriptomic Study.

Cannabinoids are receiving great attention as a novel approach in the treatment of cognitive and motor disabilities, which characterize neurological disorders. To date, over 100 phytocannabinoids have been extracted from Cannabis sativa, and some of them have shown neuroprotective properties and the capacity to influence synaptic transmission. In this study, we investigated the effects of a less-known phytocannabinoid, cannabinerol (CBNR), on neuronal physiology. Using the NSC-34 motor-neuron-like cell line and next-generation sequencing analysis, we discovered that CBNR influences synaptic genes associated with synapse organization and specialization, including genes related to the cytoskeleton and ion channels. Specifically, the calcium, sodium, and potassium channel subunits (Cacna1b, Cacna1c, Cacnb1, Grin1, Scn8a, Kcnc1, Kcnj9) were upregulated, along with genes related to NMDAR (Agap3, Syngap1) and calcium (Cabp1, Camkv) signaling. Moreover, cytoskeletal and cytoskeleton-associated genes (Actn2, Ina, Trio, Marcks, Bsn, Rtn4, Dgkz, Htt) were also regulated by CBNR. These findings highlight the important role played by CBNR in the regulation of synaptogenesis and synaptic transmission, suggesting the need for further studies to evaluate the neuroprotective role of CBNR in the treatment of synaptic dysfunctions that characterize motor disabilities in many neurological disorders.

Transcriptomic Profiling after In Vitro Δ8-THC Exposure Shows Cytoskeletal Remodeling in Trauma-Injured NSC-34 Cell Line.

Neuronal cell death is a physiological process that, when uncontrollable, leads to neurodegenerative disorders like spinal cord injury (SCI). SCI represents one of the major causes of trauma and disabilities worldwide for which no effective pharmacological intervention exists. Herein, we observed the beneficial effects of Δ8-Tetrahydrocannabinol (Δ8-THC) during neuronal cell death recovery. We cultured NSC-34 motoneuron cell line performing three different experiments. A traumatic scratch injury was caused in two experiments. One of the scratched was pretreated with Δ8-THC to observe the role of the cannabinoid following the trauma. An experimental control group was neither scratched nor pretreated. All the experiments underwent RNA-seq analysis. The effects of traumatic injury were observed in scratch against control comparison. Comparison of scratch models with or without pretreatment highlighted how Δ8-THC counteracts the traumatic event. Our results shown that Δ8-THC triggers the cytoskeletal remodeling probably due to the activation of the Janus Kinase Signal Transducer and Activator of Transcription (JAK/STAT) signaling pathway and the signaling cascade operated by the Mitogen-Activated Protein (MAP) Kinase signaling pathway. In light of this evidence, Δ8-THC could be a valid pharmacological approach in the treatment of abnormal neuronal cell death occurring in motoneuron cells.

An In Silico Analysis Reveals Sustained Upregulation of Neuroprotective Genes in the Post-Stroke Human Brain.

Ischemic stroke is a cerebrovascular disease caused by an interruption of blood flow to the brain, thus determining a lack of oxygen and nutrient supply. The ischemic event leads to the activation of several molecular signaling pathways involved in inflammation and the production of reactive oxygen species, causing irreversible neuronal damage. Several studies have focused on the acute phase of ischemic stroke. It is not clear if this traumatic event can influence some of the molecular processes in the affected area even years after the clinical event. In our study, we performed an in silico analysis using freely available raw data with the purpose of evaluating the transcriptomic state of post-mortem brain tissue. The samples were taken from non-fatal ischemic stroke patients, meaning that they suffered an ischemic stroke and lived for a period of about 2 years after the event. These samples were compared with healthy controls. The aim was to evaluate possible recovery processes useful to mitigating neuronal damage and the detrimental consequences of stroke. Our results highlighted differentially expressed genes codifying for proteins along with long non-coding genes with anti-inflammatory and anti-oxidant functions. This suggests that even after an amount of time from the ischemic insult, different neuroprotective mechanisms are activated to ameliorate brain conditions and repair post-stroke neuronal injury.

Δ8-THC Induces Up-Regulation of Glutamatergic Pathway Genes in Differentiated SH-SY5Y: A Transcriptomic Study.

Cannabinoids, natural or synthetic, have antidepressant, anxiolytic, anticonvulsant, and anti-psychotic properties. Cannabidiol (CBD) and delta-9-tetrahydrocannabinol (Δ9-THC) are the most studied cannabinoids, but recently, attention has turned towards minor cannabinoids. Delta-8-tetrahydrocannabinol (Δ8-THC), an isomer of Δ9-THC, is a compound for which, to date, there is no evidence of its role in the modulation of synaptic pathways. The aim of our work was to evaluate the effects of Δ8-THC on differentiated SH-SY5Y human neuroblastoma cells. Using next generation sequencing (NGS), we investigated whether Δ8-THC could modify the transcriptomic profile of genes involved in synapse functions. Our results showed that Δ8-THC upregulates the expression of genes involved in the glutamatergic pathway and inhibits gene expression at cholinergic synapses. Conversely, Δ8-THC did not modify the transcriptomic profile of genes involved in the GABAergic and dopaminergic pathways.

Δ8-THC Protects against Amyloid Beta Toxicity Modulating ER Stress In Vitro: A Transcriptomic Analysis.

Alzheimer's disease (AD) represents the most common form of dementia, characterized by amyloid ß (Aß) plaques and neurofibrillary tangles (NFTs). It is characterized by neuroinflammation, the accumulation of misfolded protein, ER stress and neuronal apoptosis. It is of main importance to find new therapeutic strategies because AD prevalence is increasing worldwide. Cannabinoids are arising as promising neuroprotective phytocompounds. In this study, we evaluated the neuroprotective potential of Δ8-THC pretreatment in an in vitro model of AD through transcriptomic analysis. We found that Δ8-THC pretreatment restored the loss of cell viability in retinoic acid-differentiated neuroblastoma SH-SY5Y cells treated with Aß1-42. Moreover, the transcriptomic analysis provided evidence that the enriched biological processes of gene ontology were related to ER functions and proteostasis. In particular, Aß1-42 upregulated genes involved in ER stress and unfolded protein response, leading to apoptosis as demonstrated by the increase in Bax and the decrease in Bcl-2 both at gene and protein expression levels. Moreover, genes involved in protein folding and degradation were also deregulated. On the contrary, Δ8-THC pretreatment reduced ER stress and, as a consequence, neuronal apoptosis. Then, the results demonstrated that Δ8-THC might represent a new neuroprotective agent in AD.

Cannabichromene Induces Neuronal Differentiation in NSC-34 Cells: Insights from Transcriptomic Analysis.

Phytocannabinoids, with their variety of beneficial effects, represent a valid group of substances that could be employed as neurogenesis-enhancers or neuronal differentiation inducers. We focused our attention on the neuronal-related potential of cannabichromene (CBC) when administered to undifferentiated NSC-34 for 24 h. Transcriptomic analysis showed an upregulation of several neuronal markers, such as Neurod1 and Tubb3, as well as indicators of neuronal differentiation process progression, such as Pax6. An in-depth investigation of the processes involved in neuronal differentiation indicates positive cytoskeleton remodeling by upregulation of Cfl2 and Tubg1, and active differentiation-targeted transcriptional program, suggested by Phox2b and Hes1. After 48 h of treatment, the markers previously examined in the transcriptomic analysis are still overexpressed, like Ache and Hes1, indicating that the differentiation process is still in progress. The lack of GFAP protein suggests that no astroglial differentiation is taking place, and it is reasonable to indicate the neuronal one as the ongoing one. These results indicate CBC as a potential neuronal differentiation inducer for NSC-34 cells.

Cannabigerol Activates Cytoskeletal Remodeling via Wnt/PCP in NSC-34: An In Vitro Transcriptional Study.

Cannabigerol (CBG) is a non-psychoactive phytocannabinoid present in the Cannabis sativa L. plant. In our study, CBG at the concentration of 10 µM was used to treat NSC-34 motor neuron-like cells. The aim of the study was to evaluate the effects of CBG on NSC-34 cells, using next-generation sequencing (NGS) technology. Analysis showed the activation of the WNT/planar cell polarity (PCP) pathway and Ephrin-Eph signaling. The results revealed that CBG increases the expression of genes associated with the onset process of cytoskeletal remodeling and axon guidance.

A Comprehensive Exploration of the Transcriptomic Landscape in Multiple Sclerosis: A Systematic Review.

Multiple Sclerosis (MS) is, to date, an incurable disease of the nervous system characterized by demyelination. Several genetic mutations are associated with the disease but they are not able to explain all the diagnosticated cases. Thus, it is suggested that altered gene expression may play a role in human pathologies. In this review, we explored the role of the transcriptomic profile in MS to investigate the main altered biological processes and pathways involved in the disease. Herein, we focused our attention on RNA-seq methods that in recent years are producing a huge amount of data rapidly replacing microarrays, both with bulk and single-cells. The studies evidenced that different MS stages have specific molecular signatures and non-coding RNAs may play a key role in the disease. Sex-dependence was observed before and after treatments used to alleviate symptomatology activating different biological processes in a drug-dependent manner. New pathways, such as neddylation, were found deregulated in MS and inflammation was linked to neuron degeneration areas through spatial transcriptomics. It is evident that the use of RNA-seq in the study of complex pathologies, such as MS, is a valid strategy to shed light on new involved mechanisms.

Cannabidiol Promotes Neuronal Differentiation Using Akt and Erk Pathways Triggered by Cb1 Signaling.

Recently, the scientific community has started to focus on the neurogenic potential of cannabinoids. The phytocompound cannabidiol (CBD) shows different mechanism of signaling on cannabinoid receptor 1 (CB1), depending on its concentration. In this study, we investigated if CBD may induce in vitro neuronal differentiation after treatment at 5 µM and 10 µM. For this purpose, we decided to use the spinal cord × neuroblastoma hybrid cell line (NSC-34) because of its proliferative and undifferentiated state. The messenger RNAs (mRNAs) expression profiles were tested using high-throughput sequencing technology and Western blot assay was used to determine the number of main proteins in different pathways. Interestingly, the treatment shows different genes associated with neurodifferentiation statistically significant, such as Rbfox3, Tubb3, Pax6 and Eno2. The CB1 signaling pathway is responsible for neuronal differentiation at 10 µM, as suggested by the presence of p-ERK and p-AKT, but not at 5 µM. A new correlation between CBD, neurodifferentiation and retinoic acid receptor-related orphan receptors (RORs) has been observed.

Does Childhood Obesity Trigger Neuroinflammation?

Childhood obesity is constantly increasing around the world, and it has become a major public health issue. Considerable evidence indicates that overweight and obesity are important risk factors for the development of comorbidities such as cognitive decline, neuroinflammation and neurodegenerative diseases. It is known that during obesity, adipose tissue undergoes immune, metabolic and functional changes which could induce a neuroinflammatory response of the central nervous system (CNS). In this context, to inspect if obesity can start to trigger the neuroinflammation from a pediatric age, we surgically collected and analyzed adipose tissue from the periumbilical area of three obese children (AT-OB) and two normal-weight children (AT-Ctrl). We considered the transcriptomic profile of our samples to detect alterations in different biological processes that might be also involved in the inflammatory and neuroinflammatory response. Our results show alterations of lipid and fatty acids metabolism in AT-OB compared to the AT-Ctrl. We also observed an onset of inflammatory response in AT-OB. Interestingly, among the genes involved in neuroinflammation, GRN and SMO were upregulated, while IFNGR1 and SNCA were downregulated. Our study highlights that obesity may trigger inflammation and neuroinflammation from a pediatric age.

Cannabinerol and NSC-34 Transcriptomic Analysis: Is the Dose Who Makes Neuronal Differentiation?

Cannabis sativa L. proved to be a source of several phytocompounds able to help patients facing different diseases. Moreover, these phytocompounds can help ameliorate general conditions and control certain unpleasant effects of diseases. Some cannabinoids, however, provided more benefits applicable to settings other than palliative care. Using the NSC-34 cell line, we evaluated the barely known phytocompound named cannabinerol (CBNR) at different doses, in order to understand its unique characteristics and the ones shared with other cannabinoids. The transcriptomic analysis suggests a possible ongoing neuronal differentiation, principally due to the activation of cannabinoid receptor 1 (CB1), to which the phosphorylation of serine-threonine protein kinase (Akt) followed, especially between 20 and 7.5 µM. The increase of Neurod1 and Map2 genes at 7.5 µM, accompanied by a decrease of Vim, as well as the increase of Syp at all the other doses, point toward the initiation of differentiation signals. Our preliminary results indicate CBNR as a promising candidate to be added to the list of cannabinoids with neuronal differentiation-enhancer properties. However, further studies are needed to confirm this initial insight.

The Role of Hypoxia in Improving the Therapeutic Potential of Mesenchymal Stromal Cells. A Comparative Study From Healthy Lung and Congenital Pulmonary Airway Malformations in Infants.

Mesenchymal stromal cells (MSCs) play an important role in the field of regenerative medicine thanks to their immunomodulatory properties and their ability to secrete paracrine factors. The use of MSCs has also been tested in children with congenital lung diseases inducing fibrosis and a decrease in lung function. Congenital malformations of the pulmonary airways (CPAM) are the most frequently encountered lung lesion that results from defects in early development of airways. Despite the beneficial properties of MSCs, interventions aimed at improving the outcome of cell therapy are needed. Hypoxia may be an approach aimed to ameliorate the therapeutic potential of MSCs. In this regard, we evaluated the transcriptomic profile of MSCs collected from pediatric patients with CPAM, analyzing similarities and differences between healthy tissue (MSCs-lung) and cystic tissue (MSCs-CPAM) both in normoxia and in cells preconditioned with hypoxia (0.2%) for 24 h. Study results showed that hypoxia induces cell cycle activation, increasing in such a way the cell proliferation ability, and enhancing cell anaerobic metabolism in both MSCs-lung and MSCs-CPAM-lung. Additionally, hypoxia downregulated several pro-apoptotic genes preserving MSCs from apoptosis and, at the same time, improving their viability in both comparisons. Finally, data obtained indicates that hypoxia leads to a greater expression of genes involved in the regulation of the cytoskeleton in MSCs-lung than MSCs-CPAM.

Artificial Intelligence Predictor for Alzheimer's Disease Trained on Blood Transcriptome: The Role of Oxidative Stress.

Alzheimer's disease (AD) is an incurable neurodegenerative disease diagnosed by clinicians through healthcare records and neuroimaging techniques. These methods lack sensitivity and specificity, so new antemortem non-invasive strategies to diagnose AD are needed. Herein, we designed a machine learning predictor based on transcriptomic data obtained from the blood of AD patients and individuals without dementia (non-AD) through an 8 × 60 K microarray. The dataset was used to train different models with different hyperparameters. The support vector machines method allowed us to reach a Receiver Operating Characteristic score of 93% and an accuracy of 89%. High score levels were also achieved by the neural network and logistic regression methods. Furthermore, the Gene Ontology enrichment analysis of the features selected to train the model along with the genes differentially expressed between the non-AD and AD transcriptomic profiles shows the "mitochondrial translation" biological process to be the most interesting. In addition, inspection of the KEGG pathways suggests that the accumulation of ß-amyloid triggers electron transport chain impairment, enhancement of reactive oxygen species and endoplasmic reticulum stress. Taken together, all these elements suggest that the oxidative stress induced by ß-amyloid is a key feature trained by the model for the prediction of AD with high accuracy.

Sphingolipid Metabolism as a New Predictive Target Correlated with Aging and AD: A Transcriptomic Analysis.

Background and objectives: Alzheimer's disease (AD) is the most common form of dementia characterized by memory loss and executive dysfunction. To date, no markers can effectively predict the onset of AD and an early diagnosis is increasingly necessary. Age represents an important risk factor for the disease but it is not known whether it is the trigger event. Materials and Methods: We downloaded transcriptomic data related to post-mortem brain of thirty samples gathered as young without AD (Young), old without AD (Old), and old suffering from AD (OAD) groups. Results: Our results showed that steroid biosynthesis was enriched and associated with aging, while sphingolipid metabolism was related to both aging and AD. Specifically, sphingolipid metabolism is involved in the deregulation of CERS2, UGT8, and PLPP2. These genes are downregulated in Young and Old groups as compared with upregulated between Old and OAD groups. Moreover, the analysis of the interaction networks revealed that GABAergic synapse and Hippo signaling pathways were altered in AD condition along with mitochondrial metabolism and RNA processing. Conclusions: Observing the particular trend of genes related to sphingolipid metabolism that are downregulated during normal aging and start to be upregulated with the onset of AD, we suppose that sphingolipids could be early markers for the disease.

Potential Anti-Inflammatory Effects of a New Lyophilized Formulation of the Conditioned Medium Derived from Periodontal Ligament Stem Cells.

The mesenchymal stem cells' (MSCs) secretome includes the bioactive molecules released in the conditioned medium (CM), such as soluble proteins, free nucleic acids, lipids and extracellular vesicles. The secretome is known to mediate some of the beneficial properties related to MSCs, such as anti-inflammatory, anti-apoptotic and regenerative capacities. In this work, we aim to evaluate the anti-inflammatory potential of a new lyophilized formulation of CM derived from human periodontal ligament stem cells (hPDLSCs). With this aim, we treat hPDLSCs with lipopolysaccharide (LPS) and test the anti-inflammatory potential of lyophilized CM (LYO) through the evaluation of wound closure, transcriptomic and immunofluorescence analysis. LPS treatment increased the expression of TLR4 and of genes involved in its signaling and in p38 and NF-κB activation, also increasing the expression of cytokines and chemokines. Interestingly, LYO downregulated the expression of genes involved in Toll-like receptor 4 (TLR-4), nuclear factor kappa light chain enhancer of activated B cells (NF-κB) and p38 signaling. As a consequence, the genes encoding for cytokines and chemokines were also downregulated. Immunofluorescence acquisitions confirmed the downregulation of TLR-4 and NF-κB with the LYO treatment. Moreover, the LYO treatment also increased hPDLSCs' migration. LYO was demonstrated to contain transforming growth factor (TGF)-ß3 and vascular endothelial growth factor (VEGF). These results suggest that LYO represents an efficacious formulation with anti-inflammatory potential and highlights lyophilization as a valid method to produce stable formulations of MSCs' secretome.