Novel insights about albumin in cardiovascular diseases: Focus on heart failure.

The Human Plasma Proteome has always been the most investigated compartment in proteomics-based biomarker discovery, and is considered the largest and deepest version of the human proteome, reflecting the state of the body in health and disease. Even if efforts have been always dedicated to the refinement of proteomic approaches to investigate more deeply the plasma proteome, it should not be forgotten that also highly abundant plasma proteins, like human serum albumin (HSA), often neglected in these studies, might provide fundamental physiological functions in plasma, and should be better considered. This review summarizes the important roles of HSA in the context of cardiovascular diseases (CVD), and in particular in heart failure. Notwithstanding much attention has been historically directed toward the association of HSA levels and CVD risk, the advances in the field of mass spectrometry research allow also a better characterization of the effects of oxidative modifications that could alter not only the structure but also the function of HSA.

Quantitative proteomics study of carnosine effect in an animal model of Western diet-induced nonalcoholic fatty liver disease.

The nonalcoholic fatty liver disease (NAFLD), which is closely related to westernized dietary (WD) patterns, displays a rising epidemiological and economic burden. Since there is no pharmacological therapy approved for this disease, mechanistic studies are warranted. In this work, we investigated the action of carnosine (CAR), a natural dipeptide with several protection roles against oxidative stress in the liver of NAFLD rats. NAFLD was induced by WD-rich sugars and fat, verifying the histological evidence of steatosis. As intraperitoneal administration of CAR reversed liver steatosis, the protein profiles of NAFLD liver and CAR NAFLD liver were evaluated by label-free proteomics approach. A total of 2531 proteins were identified and the 230 and 276 were significantly up- and downregulated, respectively, by CAR treatment of NAFLD rats and involved in fundamental pathways such as oxidative stress and lipid metabolism. Perilipin 2 and apolipoprotein E, components of the plasma membrane of vesicle, resulted in highly downregulated in the CAR-treated NAFLD liver. The advanced bioanalytical approach demonstrated the efficacy of CAR in overcoming the main symptoms of NAFLD, ameliorating the steatosis in the liver.

Discovery of a septin-4 covalent binder with antimetastatic activity in a mouse model of melanoma.

Cancer spreading through metastatic processes is one of the major causes of tumour-related mortality. Metastasis is a complex phenomenon which involves multiple pathways ranging from cell metabolic alterations to changes in the biophysical phenotype of cells and tissues. In the search for new effective anti-metastatic agents, we modulated the chemical structure of the lead compound AA6, in order to find the structural determinants of activity, and to identify the cellular target responsible of the downstream anti-metastatic effects observed. New compounds synthesized were able to inhibit in vitro B16-F10 melanoma cell invasiveness, and one selected compound, CM365, showed in vivo anti-metastatic effects in a lung metastasis mouse model of melanoma. Septin-4 was identified as the most likely molecular target responsible for these effects. This study showed that CM365 is a promising molecule for metastasis prevention, remarkably effective alone or co-administered with drugs normally used in cancer therapy, such as paclitaxel.

Screening of Mpro Protease (SARS-CoV-2) Covalent Inhibitors from an Anthocyanin-Rich Blueberry Extract Using an HRMS-Based Analytical Platform.

BACKGROUND: The viral main protease (Mpro) of SARS-CoV-2 has been recently proposed as a key target to inhibit virus replication in the host. Therefore, molecules that can bind the catalytic site of Mpro could be considered as potential drug candidates in the treatment of SARS-CoV-2 infections. Here we proposed the application of a state-of-the-art analytical platform which combines metabolomics and protein structure analysis to fish-out potential active compounds deriving from a natural matrix, i.e., a blueberry extract. METHODS: The experiments focus on finding MS covalent inhibitors of Mpro that contain in their structure a catechol/pyrogallol moiety capable of binding to the nucleophilic amino acids of the enzyme's catalytic site. RESULTS: Among the potential candidates identified, the delphinidin-3-glucoside showed the most promising results. Its antiviral activity has been confirmed in vitro on Vero E6 cells infected with SARS-CoV-2, showing a dose-dependent inhibitory effect almost comparable to the known Mpro inhibitor baicalin. The interaction of delphinidin-3-glucoside with the Mpro pocket observed was also evaluated by computational studies. CONCLUSIONS: The HRMS analytical platform described proved to be effective in identifying compounds that covalently bind Mpro and are active in the inhibition of SARS-CoV-2 replication, such as delphinidin-3-glucoside.

Carnosine supplementation and retinal oxidative parameters in a high-calorie diet rat model.

BACKGROUND: To assess oxidative effects induced by a high-calorie diet on the retina of Wistar rats and test the antioxidative effects of carnosine supplementation. METHODS: Wistar rats were randomly divided into the following groups: standard diet (SD), high-calorie diet (HcD), standard diet + carnosine (SD + Car), and high-calorie diet + carnosine (HcD + Car). The body weight, adiposity index, plasma glucose, total lipids, high-density lipoprotein (HDL), low-density lipoprotein (LDL), uric acid, creatinine, and triglycerides of the animals were evaluated. The retinas were analyzed for markers of oxidative stress. Hydrogen peroxide production was assessed by 2',7'-dichlorodihydrofluorescein diacetate (DCF) oxidation. The total glutathione (tGSH), total antioxidant capacity (TAC), protein carbonyl, and sulfhydryl groups of the antioxidant system were analyzed. RESULTS: TAC levels increased in the retinas of the SD + Car group compared to the SD group (p < 0.05) and in the HcD + Car group compared to the HcD group (p < 0.05). The levels of GSH and the GSSH:GSSG ratio were increased in the HcD + Car group compared to the SD + Car group (p < 0.05). An increase in the retinal carbonyl content was observed in the HcD group compared to the SD group (p < 0.05) and in the HcD + Car group compared to the SD + Car group (p < 0.05). A high-calorie diet (HcD) was also associated with a decrease in retinal sulfhydryl-type levels compared to the SD group (p < 0.05). CONCLUSION: The results suggest that feeding a high-calorie diet to rats can promote an increase in carbonyl content and a reduction in sulfhydryl groups in their retinas. The administration of carnosine was not effective in attenuating these oxidative markers. TRIAL REGISTRATION: Animal Ethics Committee of Botucatu Medical School - Certificate number 1292/2019.

Bergamot (Citrus bergamia) leaf extract improves metabolic, antioxidant and anti-inflammatory activity in skeletal muscles in a metabolic syndrome experimental model.

Metabolic Syndrome (MetS), inflammation and oxidative stress contribute to impairment of skeletal muscle function. Bergamot (Citrus bergamia) leaf extract (BLE) has shown protective effects against comorbidities associated with MetS through its anti-inflammatory and antioxidant effects. The aim of this work was to elucidate the antioxidant and anti-inflammatory activity of BLE in skeletal muscles in an experimental model of MetS. Once metabolic syndrome was diagnosed, animals were divided into groups receiving different treatments for 10 weeks, including control diet (n = 10), control + BLE (n = 10), High Sugar-fat diet (HSF) (n = 10), HSF + BLE (n = 10). Evaluation included nutritional, metabolic and hormonal analyses, along with measurements of inflammatory status and oxidative stress in soleus and extensor digitorum longus (EDL) muscles. BLE showed positive metabolic effects, with a reduction of plasma triglycerides and insulin resistance and an increase in high-density lipoprotein cholesterol, and protective activity against oxidative stress and inflammation in Soleus and EDL muscles in animals with MetS.

Lipidome Investigation of Carnosine Effect on Nude Mice Skin to Prevent UV-A Damage.

The lipid profile of skin is fundamental in the maintenance of the protective barrier against the external environment. Signaling and constitutive lipids of this large organ are involved in inflammation, metabolism, aging, and wound healing, such as phospholipids, triglycerides, FFA, and sphingomyelin. Skin exposure to ultraviolet (UV) radiation results in a photoaging process that is an accelerated form of aging. UV-A radiation deeply penetrates the dermis and promotes damage to DNA, lipids, and proteins by increasing the generation of reactive oxygen species (ROS). Carnosine, an endogenous ß-alanyl-L-histidine dipeptide, demonstrated antioxidant properties that prevent photoaging and modification of skin protein profiling, making carnosine a compelling ingredient to consider for use in dermatology. The aim of this research was to investigate the modification of skin lipidome after UV-A treatment in presence or not of topic administration of carnosine. Quantitative analyses based on high-resolution mass spectrometry of nude mice skin-extracted lipids resulted in several modifications of barrier composition after UV-A radiation, with or without carnosine treatment. In total, 328 out of 683 molecules showed significant alteration-262 after UV-A radiation and 126 after UV-A and carnosine treatment versus controls. Importantly, the increased oxidized TGs after UV-A radiation, responsible of dermis photoaging, were completely reverted by carnosine application to prevent the UV-A damage. Network analyses also showed that the production of ROS and the calcium and TNF signaling were modulated by UV-A and carnosine. In conclusion, lipidome analyses attested the carnosine activity to prevent the UV-A damage, reducing the lipid oxidation, the inflammation, and the dysregulation of lipid skin barrier.

New Insights into Bitopic Orthosteric/Allosteric Ligands of Cannabinoid Receptor Type 2.

Very recently, we have developed a new generation of ligands targeting the cannabinoid receptor type 2 (CB2R), namely JR compounds, which combine the pharmacophoric portion of the CB2R positive allosteric modulator (PAM), EC21a, with that of the CB2R selective orthosteric agonist LV62, both synthesized in our laboratories. The functional examination enabled us to identify JR14a, JR22a, and JR64a as the most promising compounds of the series. In the current study, we focused on the assessment of the bitopic (dualsteric) nature of these three compounds. Experiments in cAMP assays highlighted that only JR22a behaves as a CB2R bitopic (dualsteric) ligand. In parallel, computational studies helped us to clarify the binding mode of these three compounds at CB2R, confirming the bitopic (dualsteric) nature of JR22a. Finally, the potential of JR22a to prevent neuroinflammation was investigated on a human microglial cell inflammatory model.

Targeting Nrf2 and NF-κB Signaling Pathways in Cancer Prevention: The Role of Apple Phytochemicals.

Plant secondary metabolites, known as phytochemicals, have recently gained much attention in light of the "circular economy", to reutilize waste products deriving from agriculture and food industry. Phytochemicals are known for their onco-preventive and chemoprotective effects, among several other beneficial properties. Apple phytochemicals have been extensively studied for their effectiveness in a wide range of diseases, cancer included. This review aims to provide a thorough overview of the main studies reported in the literature concerning apple phytochemicals, mostly polyphenols, in cancer prevention. Although there are many different mechanisms targeted by phytochemicals, the Nrf2 and NF-κB signaling pathways are the ones this review will be focused on, highlighting also the existing crosstalk between these two systems.

Targeting Nrf2 and NF-κB Signaling Pathways in Inflammatory Pain: The Role of Polyphenols from Thinned Apples.

Diet can modulate the different stages of inflammation due to the presence of bioactive compounds such as polyphenols. Apples are a great source of phenolic compounds that show anti-inflammatory and antioxidant properties, and these might be used as a dietary supplement and/or functional element in the treatment of chronic inflammatory illnesses. The aim of our study was to evaluate the anti-inflammatory and antioxidant actions of thinned apple polyphenol (TAP) extracts in a model of paw edema. The experimental model was induced in rats via subplantar injections of 1% λ-Carrageenan (CAR) in the right hind leg, and TAP extract was administered via oral gavage 30 min before and 1 h after the CAR injection at doses of 5 mg/kg and 10 mg/kg, respectively. The inflammatory response is usually quantified by the increase in the size of the paw (edema), which is maximal about 5 h after the injection of CAR. CAR-induced inflammation generates the release of pro-inflammatory mediators and reactive oxygen species (ROS). Furthermore, the inflammatory state induces the pain that involves the peripheral nociceptors, but above all it acts centrally at the level of the spinal cord. Our results showed that the TAP extracts reduced paw histological changes, neutrophil infiltration, mast cell degranulation, and oxidative stress. Additionally, the oral administration of TAP extracts decreased thermal and mechanical hyperalgesia, along with a reduction in spinal microglia and the markers of nociception. In conclusion, we demonstrate that TAP extract is able to modulate inflammatory, oxidative, and painful processes, and is also useful in the treatment of the symptoms associated with paw edema.

Chemical, Nutritional and Biological Evaluation of a Sustainable and Scalable Complex of Phytochemicals from Bergamot By-Products.

The present paper reports a sustainable raw material obtained from the by-products derived from the industrial production of bergamot (Citrus × Bergamia Risso & Poiteau) essential oils. The procedure to obtain the raw material is designed to maintain as much of the bioactive components as possible and to avoid expensive chemical purification. It consists of spray-drying the fruit juice obtained by squeezing the fruits, which is mixed with the aqueous extract of the pulp, i.e., the solid residue remained after fruit pressing. The resulting powder bergamot juice (PBJ) contains multiple bioactive components, in particular, among others, soluble fibers, polyphenols and amino-acid betaines, such as stachydrine and betonicine. LC-MS analysis identified 86 compounds, with hesperetin, naringenin, apigenin and eridictyol glucosides being the main components. In the second part of the paper, dose-dependent anti-inflammatory activity of PBJ and of stachydrine was found, but neither of the compounds were effective in activating Nrf2. PBJ was then found to be effective in an in vivo model of a metabolic syndrome induced by a high-sugar, high-fat (HSF) diet and evidenced by a significant increase of the values related to a set of parameters: blood glucose, triglycerides, insulin resistance, systolic blood pressure, visceral adipose tissue and adiposity index. PBJ, when given to control rats, did not significantly change these values; in contrast, they were found to be greatly affected in rats receiving an HSF diet. The in vivo effect of PBJ can be ascribed not only to bergamot polyphenols with well-known anti-inflammatory, antioxidant and lipid-regulating effects, but also to the dietary fibers and to the non-phenolic constituents, such as stachydrine. Moreover, since PBJ was found to affect energy homeostasis and to regulate food intake, a mechanism on the regulation of energy homeostasis through leptin networking should also be considered and deserves further investigation.

In vivo detection of carnosine and its derivatives using chemical exchange saturation transfer.

PURPOSE: To detect carnosine, anserine and homocarnosine in vivo with chemical exchange saturation transfer (CEST) at 17.2 T. METHODS: CEST MR acquisitions were performed using a CEST-linescan sequence developed in-house and optimized for carnosine detection. In vivo CEST data were collected from three different regions of interest (the lower leg muscle, the olfactory bulb and the neocortex) of eight rats. RESULTS: The CEST effect for carnosine, anserine and homocarnosine was characterized in phantoms, demonstrating the possibility to separate individual contributions by employing high spectral resolution (0.005 ppm) and low CEST saturation power (0.15 µ$$ \mu $$ T). The CEST signature of these peptides was evidenced, in vivo, in the rat brain and skeletal muscle. The presence of carnosine and anserine in the muscle was corroborated by in vivo localized spectroscopy (MRS). However, the sensitivity of MRS was insufficient for carnosine and homocarnosine detection in the brain. The absolute amounts of carnosine and derivatives in the investigated tissues were determined by liquid chromatography-electrospray ionization-tandem mass spectrometry using isotopic dilution standard methods and were in agreement with the CEST results. CONCLUSION: The robustness of the CEST-linescan approach and the favorable conditions for CEST at ultra-high magnetic field allowed the in vivo CEST MR detection of carnosine and related peptides. This approach could be useful to investigate noninvasively the (patho)-physiological roles of these molecules.

Oxidative Stress Modulation by Carnosine in Scaffold Free Human Dermis Spheroids Model: A Proteomic Study.

Carnosine is an endogenous ß-alanyl-L-histidine dipeptide endowed with antioxidant and carbonyl scavenger properties, which is able to significantly prevent the visible signs of aging and photoaging. To investigate the mechanism of action of carnosine on human skin proteome, a 3D scaffold-free spheroid model of primary dermal fibroblasts from a 50-year-old donor was adopted in combination with quantitative proteomics for the first time. The label free proteomics approach based on high-resolution mass spectrometry, integrated with network analyses, provided a highly sensitive and selective method to describe the human dermis spheroid model during long-term culture and upon carnosine treatment. Overall, 2171 quantified proteins allowed the in-depth characterization of the 3D dermis phenotype during growth and differentiation, at 14 versus 7 days of culture. A total of 485 proteins were differentially regulated by carnosine at 7 days, an intermediate time of culture. Of the several modulated pathways, most are involved in mitochondrial functionality, such as oxidative phosphorylation, TCA cycle, extracellular matrix reorganization and apoptosis. In long-term culture, functional modules related to oxidative stress were upregulated, inducing the aging process of dermis spheroids, while carnosine treatment prevented this by the downregulation of the same functional modules. The application of quantitative proteomics, coupled to advanced and relevant in vitro scaffold free spheroids, represents a new concrete application for personalized therapies and a novel care approach.

Protein Profiling of a Cellular Model of NAFLD by Advanced Bioanalytical Approaches.

Advanced quantitative bioanalytical approaches in combination with network analyses allow us to answer complex biological questions, such as the description of changes in protein profiles under disease conditions or upon treatment with drugs. In the present work, three quantitative proteomic approaches-either based on labelling or not-in combination with network analyses were applied to a new in vitro cellular model of nonalcoholic fatty liver disease (NAFLD) for the first time. This disease is characterized by the accumulation of lipids, inflammation, fibrosis, and insulin resistance. Hepatic G2 cells were used as model, and NAFLD was induced by a complex of oleic acid and bovine albumin. The development of the disease was verified by lipid vesicle staining and by the increase in the expression of perilipin-2-a protein constitutively present in the vesicles during NAFLD. The nLC-MS/MS analyses of peptide samples obtained from three different proteomic approaches resulted in accurate and reproducible quantitative data of protein fold-change expressed in NAFLD versus control cells. The differentially regulated proteins were used to evaluate the involved and statistically enriched pathways. Network analyses highlighted several functional and disease modules affected by NAFLD, such as inflammation, oxidative stress defense, cell proliferation, and ferroptosis. Each quantitative approach allowed the identification of similar modulated pathways. The combination of the three approaches improved the power of statistical network analyses by increasing the number of involved proteins and their fold-change. In conclusion, the application of advanced bioanalytical approaches in combination with pathway analyses allows the in-depth and accurate description of the protein profile of an in vitro cellular model of NAFLD by using high-resolution quantitative mass spectrometry data. This model could be extremely useful in the discovery of new drugs to modulate the equilibrium NAFLD health state.

The Therapeutic Potential of Carnosine as an Antidote against Drug-Induced Cardiotoxicity and Neurotoxicity: Focus on Nrf2 Pathway.

Different drug classes such as antineoplastic drugs (anthracyclines, cyclophosphamide, 5-fluorouracil, taxanes, tyrosine kinase inhibitors), antiretroviral drugs, antipsychotic, and immunosuppressant drugs are known to induce cardiotoxic and neurotoxic effects. Recent studies have demonstrated that the impairment of the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway is a primary event in the pathophysiology of drug-induced cardiotoxicity and neurotoxicity. The Nrf2 pathway regulates the expression of different genes whose products are involved in antioxidant and inflammatory responses and the detoxification of toxic species. Cardiotoxic drugs, such as the anthracycline doxorubicin, or neurotoxic drugs, such as paclitaxel, suppress or impair the Nrf2 pathway, whereas the rescue of this pathway counteracts both the oxidative stress and inflammation that are related to drug-induced cardiotoxicity and neurotoxicity. Therefore Nrf2 represents a novel pharmacological target to develop new antidotes in the field of clinical toxicology. Interestingly, carnosine (ß-alanyl-l-histidine), an endogenous dipeptide that is characterized by strong antioxidant, anti-inflammatory, and neuroprotective properties is able to rescue/activate the Nrf2 pathway, as demonstrated by different preclinical studies and preliminary clinical evidence. Starting from these new data, in the present review, we examined the evidence on the therapeutic potential of carnosine as an endogenous antidote that is able to rescue the Nrf2 pathway and then counteract drug-induced cardiotoxicity and neurotoxicity.

Achillea moschata Wulfen: From Ethnobotany to Phytochemistry, Morphology, and Biological Activity.

A multidisciplinary investigation on Achillea moschata Wulfen (Asteraceae) is outlined herein. This work, part of the European Interreg Italy-Switzerland B-ICE project, originated from an ethnobotanical survey performed in Chiesa in Valmalenco (Sondrio, Lombardy, Northern Italy) in 2019-2021 which highlighted this species' relevance of use in folk medicine to treat gastrointestinal diseases. In addition, this contribution included analyses of the: (a) phytochemical profile of the aqueous and methanolic extracts of the dried flower heads using LC-MS/MS; (b) morpho-anatomy and histochemistry of the vegetative and reproductive organs through Light, Fluorescence, and Scanning Electron Microscopy; (c) biological activity of the aqueous extract concerning the antioxidant and anti-inflammatory potential through cell-based in vitro models. A total of 31 compounds (5 phenolic acids, 13 flavonols, and 13 flavones) were detected, 28 of which included in both extracts. Covering and secreting trichomes were observed: the biseriate 10-celled glandular trichomes prevailing on the inflorescences represented the main sites of synthesis of the polyphenols and flavonoids detected in the extracts, along with volatile terpenoids. Finally, significant antioxidant and anti-inflammatory activities of the aqueous extract were documented, even at very low concentrations; for the first time, the in vitro tests allowed us to formulate hypotheses about the mechanism of action. This work brings an element of novelty due to the faithful reproduction of the traditional aqueous preparation and the combination of phytochemical and micromorphological research approaches.

Prothrombin is a binding partner of the human receptor of advanced glycation end products.

The receptor for advanced glycation end products (RAGE) plays a key role in mammal physiology and in the etiology and progression of inflammatory and oxidative stress-based diseases. In adults, RAGE expression is normally high only in the lung where the protein concentrates in the basal membrane of alveolar Type I epithelial cells. In diseases, RAGE levels increase in the affected tissues and sustain chronic inflammation. RAGE exists as a membrane glycoprotein with an ectodomain, a transmembrane helix, and a short carboxyl-terminal tail, or as a soluble ectodomain that acts as a decoy receptor (sRAGE). VC1 domain is responsible for binding to the majority of RAGE ligands including advanced glycation end products (AGEs), S100 proteins, and HMGB1. To ascertain whether other ligands exist, we analyzed by MS the material pulled down by VC1 from human plasma. Twenty of 295 identified proteins were selected and associated to coagulation and complement processes and to extracellular matrix. Four of them contained a γ-carboxyl glutamic acid (Gla) domain, a calcium-binding module, and prothrombin (PT) was the most abundant. Using MicroScale thermophoresis, we quantified the interaction of PT with VC1 and sRAGE in the absence or presence of calcium that acted as a competitor. PT devoid of the Gla domain (PT des-Gla) did not bind to sRAGE, providing further evidence that the Gla domain is critical for the interaction. Finally, the presence of VC1 delayed plasma clotting in a dose-dependent manner. We propose that RAGE is involved in modulating blood coagulation presumably in conditions of lung injury.

Differentially expressed proteins obtained by label-free quantitative proteomic analysis reveal affected biological processes and functions in Western diet-induced steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is a pathological manifestation with a progressive incidence in response to the epidemic of hepatic steatosis caused primarily by excessive energy intake. The present study unravels affected biological processes and functions by the presence of NASH in rats using a label-free quantitative proteomic strategy. NASH was induced by a Western high-sugar and high-fat diet for 20 weeks. The liver tissue was collected for histology and for a mass spectrometry-based proteomic protocol. The NASH group showed severe lipidosis, hepatocyte ballooning, and the presence of collagen deposition. Among upregulated proteins in NASH perilipin-2 (Plin-2; F6QBA3; difference [diff]: 2.29), ferritin heavy (Fth1; Q66HI5; diff: 2.19) and light (Ftl1; P02793; diff: 1.75) chains, macrophage migration inhibitory factor 1 (Mif; P30904; diff: 1.69), and fibronectin (Fn1; F1LST1; diff: 0.35) were observed, whereas among downregulated proteins, plectin (Q6S399; diff: -3.34), some Cyp2 family proteins of the cytochrome P450 complex, glutathione S-transferases, flavin-containing monooxygenase 1 (Fmo1; P36365; diff: -2.08), acetyl-CoA acetyltransferase 2 (Acat2; Q5XI22; diff: -2.25), acyl-CoA oxidase 2 (Acox2; F1LNW3; diff: -1.59), and acyl-CoA oxidase 3 (Acox3; F1M9A7; diff: -2.41) were observed. Also, biological processes and functions such as LPS/IL-1 inhibition of RXR, fatty acid metabolism, Nrf2-mediated oxidative stress response, xenobiotic metabolism, and PXR/RXR and CAR/RXR activations were predicted to be affected. In conclusion, the liver of rats with NASH induced by Western diet shows a decreased capacity of metabolizing lipids, fatty acids, and xenobiotic compounds that predispose fibrosis development.

Effect of Extraction Solvent and Temperature on Polyphenol Profiles, Antioxidant and Anti-Inflammatory Effects of Red Grape Skin By-Product.

A fully-detailed LC-MS qualitative profiling of red grape skin, extracted with a mixture of ethanol and water (70:30 v:v) has permitted the identification of 65 compounds which can be classified into the following chemical classes: organic and phenolic acids (14 compounds), stilbenoids (1 compound), flavanols (21 compounds), flavonols (15 compounds) and anthocyanins (14 compounds). The extraction yield obtained with water at different temperatures (100 °C, 70 °C, room temperature) was then evaluated and the overall polyphenol content indicates that EtOH:H2O solvent is the most efficient and selective for polyphenol extraction. However, by analyzing the recovery yield of each single polyphenol, we found that water extraction under heating conditions is effective (extraction yield similar or even better in respect to the binary solvent) for some polyphenolic classes, such as hydrophilic procyanidins, phenolic acids, flavonol glucosides and stilbenoids. However, according to their lipophilic character, a poor yield was found for the most lipophilic components, such as flavonol aglycones, and in general for anthocyanins. The radical scavenging activity was in accordance with the polyphenol content, and hence, much higher for the extract obtained with the binary solvent in respect to water extraction. All the tested extracts were found to have an anti-inflammatory activity in the R3/1 cell line with NF-kb reporter challenged with 0.01 µg/mL of IL-1α, in a 1 to 250 µg/mL concentration range. An intriguing result was that the EtOH:H2O extract was found to be superimposable with that obtained using water at 100 °C despite the lower polyphenol content. Taken together, the results show the bioactive potentialities of grape skin extracts and the possibility to exploit this rich industrial waste. Water extraction carried out by heating is an easy, low-cost and environmentally friendly extraction method for some polyphenol classes and may have great potential for extracts with anti-inflammatory activities.

Integratomics of Human Dermal Fibroblasts Treated with Low Molecular Weight Hyaluronic Acid.

Hyaluronic acid (HA) is a glycosaminoglycan very common in commercial products from pharmaceuticals to cosmetics due to its widespread distribution in humans and its diversified physico-chemical proprieties. Despite its extended use and preliminary evidence showing even also opposite activities to the native form, the precise cellular effects of HA at low-molecular-weight (LWM-HA) are currently unclear. The 'omics sciences currently in development offer a new and combined perspective on the cellular and organismal environment. This work aims to integrate lipidomics analyses to our previous quantitative proteomics one for a multi-omics vision of intra- and extra-cellular impact of different concentrations (0.125, 0.25, and 0.50%) of LMW-HA (20-50 kDa) on normal human dermal fibroblasts by LC-high resolution mass spectrometry (LC-HRMS). Untargeted lipidomics allowed us to identify 903 unique lipids mostly represented by triacylglycerols, ceramides, and phosphatidylcholines. According to proteomics analyses, LMW-HA 0.50% was the most effective concentration also in the lipidome rearrangement especially stimulating the synthesis of ceramides involved in skin hydration and reparation, cell signaling, and energy balance. Finally, integrative analyses showed 25 nodes covering several intra- and extra-cellular functions. The more complete comprehension of intra- and extra-cellular effects of LMW-HA here pointed out will be useful to further exploit its features and improve current formulations even though further studies on lipids biosynthesis and degradation are necessary.