Usnic acid (UA) is a dibenzofuran derivative naturally present in lichens, organisms resulting from the symbiosis between a fungus and a cyanobacterium, or an alga. UA shows antimicrobial, antitumor, antioxidant, analgesic, anti-inflammatory as well as UV-protective activities. Its use as pharmacological agent is widely described in traditional medicine, and in the past few years, the product has been marketed as a food supplement for the induction of weight loss. However, the development of severe hepatotoxicity in a limited number of subjects prompted the FDA to issue a warning letter, which led to the withdrawal of the product from the market in November 2001. Data published in literature on UA toxicology, genotoxicity, mutagenesis, and teratogenicity have been reviewed, as well as the case reports of subjects who developed hepatotoxicity following oral administration of UA as a slimming agent. Finally, we reviewed the most recent studies on the topical use of UA, as well as studies aimed at improving UA pharmacologic activity and reducing toxicity. Indeed, advancements in this field of research could open the possibility to reintroduce the use of UA as therapeutical agent. Alectoria, Cladonia, Usnea, Lecanora, Ramalina, and Evernia  of UA are limited. A study carried out in rabbit plasma and bovine serum albumin has shown that 99.2% of UA binds to plasma proteins . Studiesμg/ml and it was reached after 12.2 ± 3.8 hours [A pharmacokinetics study in rabbits treated with either 5 mg/kg intravenously or 20 mg/kg orally have shown UA half-lives of 10.7 ± 4.6 hours and 18.9 ± 2.9 hours, respectively . The pea.8 hours .UA metabolism has been studied in plasma, hepatocytes, and subcellular fractions of human liver. UA was incubated in human liver S9 fractions, and samples were analysed by liquid chromatography/mass spectrometry (LC/MS). The hepatic clearance of UA was 13.86 ml/min/kg. This study showed that UA is metabolized by the 1A2 isoform of cytochrome P450 (CYP1A2) since furafylline (a specific inhibitor) increased the half-life of UA by tenfold. On the other hand, conjugation of UA with glycuronic acid depends on two isoforms of uridina diphosphate-glycuronosyl transferase (UGT), namely, 1A1 and 1A3. The combined activity of CYP1A2, UGT1A1, and UGT1A3 produce three different monohydroxylated metabolites and two regioisomeric glucuronide conjugates of the parent drug .μM of UA [Pretreatment with antioxidants (hydroxytoluenebutylate and vitamin E) reduced UA-induced necrosis by approximately 70%Mitochondrial glutathione (GSH) depletion with diethylmaleate increased hepatocyte susceptibility to UAUA treatment increased free radical productionSeveral studies aimed at identifying the molecular basis of UA hepatotoxicity have been carried out in human hepatoblastoma cell lines, as well as in primary hepatocyte cultures. Murine hepatocytes were cultured by Han et al. in a medium added with 5 μM of UA . After 1UA autoxidation and increased hydrogen peroxide production in mitochondria were responsible for the increased level of reactive oxygen species following the addition of UA to the medium. At the concentrations tested, UA was hepatotoxic, triggered oxidative stress, and disrupted the normal metabolic pathways .μM uncoupled the oxidative phosphorylation in mitochondria isolated from hepatocytes. In summary, high concentrations of UA induced loss of membrane integrity by impairing mitochondrial respiration and oxidative phosphorylation [Pramyothin et al. evaluated the hepatotoxic effects of UA in rats, isolated rat hepatocytes, and isolated rat liver mitochondria . Followirylation .μM does not induce CYP1A2, CYP2B6, or CYP3A4 in human hepatocytes [in vitro suggest that UA might potentiate hepatotoxicity induced by other drugs. However, the development of hepatotoxicity depends also on other cofactors, such as the induction of CYP1A, the coadministration of CYP1A2 inhibitors, the presence of specific UGT1A1 polymorphisms, the presence of hyperbilirubinemia, or the coadministration of CYP2C substrates with low therapeutic index [The aforementioned study by Foti et al. showed that UA at a concentration of 20 atocytes . UA is aic index .13C isomer distribution. Cells were exposed to UA concentrations of 0, 1, 5, or 10 μM for a time course of 2, 6, or 24 hours. At the end of the treatment, aliquots of supernatant were collected to determine the distribution of the 13C isomer isotope in CO2, lactate, glucose, and glutamate. The 1 μM concentration of UA did not produce significant changes in cell viability compared to controls. However, 5 and 10 μM concentrations significantly decreased cell viability with increasing exposure times. Overlapping results were obtained in ATP depletion experiments. The 1 and 5 μM concentrations increased oxidative phosphorylation, whereas the 10 μM concentration produced a significant inhibition of oxidative phosphorylation and gluconeogenesis. The increase in oxidative phosphorylation observed at the lower concentration could be an adaptive response to compensate the decreased mitochondrial function [Sonko et al. published a paper on UA toxicity in primary rat hepatocytes. In order to better understand the metabolic pathways involved in the development of cell toxicity, the authors assessed cell viability, ATP concentration and function .μM for 24 h. At the end of the treatment period, cytochrome P450 activity, cytotoxicity, oxidative stress, mitochondrial dysfunction, and changes in gene expression profiles were assessed. Exposure to UA increased the cytochrome P450 activity, cytotoxicity, oxidative stress, and mitochondrial dysfunction. UA significantly modified the expression of six of the 84 genes examined. In particular, it was observed an increased activity of C-C motif chemokine ligand 21 (CCL21), CCNC and UGT1A4, genes associated with inflammation, cell proliferation, and DNA damage and repairing processes, respectively. On the other hand, it was observed a decreased activity of colony stimulating factor 2 (CSF2), CYP7A1 and CYP2E1, genes associated with inflammation and oxidative stress, respectively. In summary, the biomarkers assessed confirmed UA toxicity in HepG2 cells, suggesting an oxidative mechanism of action [Sahu et al. evaluated the metabolism and toxicity of UA in human hepatoblastoma cell line G2 (HepG2). Cells were treated with control vehicle alone or with UA at concentrations ranging from 0 to 100 f action .2. After the treatment, cells were evaluated by analysis of traditional biochemical endpoints of toxicity in combination with toxicogenomics endpoints . Taken individually, low concentrations of UA and lipopolysaccharide did not affect cell viability. On the other hand, their association increased cytotoxicity, oxidative stress, and mitochondrial damage. Gene expression patterns analysis showed significant changes of several genes. Study results suggest some interaction between UA (at non-toxic concentrations) and lipopolysaccharide [A second study by Sahu et al. assessed the interaction between UA and lipopolysaccharide in hepatotoxicity induction . The ratccharide .μM), alpha-naphthoflavone (10 μM), or ketoconazole (25 μM) significantly increased the toxicity of UA at concentrations between 3 and 6 μM after 3 to 20 hours, as demonstrated by the release of lactate dehydrogenase (LDH) from the cells. Two hours after UA exposure and before LDH release from cells, CYP inhibitors potentiated the inhibition of cellular respiration and ATP depletion. These data demonstrate that UA is transformed in rat hepatocytes into less toxic metabolites, probably through the action of CYP1A and CYP3A [Shi et al. studied the toxicity of UA in primary cultures of rat hepatocytes. Inhibition of CYP activity with SKF-525A  or small interfering RNAs against autophagy-related 7 (Atg7) increased apoptosis and reduced cell viability, demonstrating that autophagy protects cells from UA-induced toxicity. The molecule activates the mitogen-activated protein kinase (MAPK) pathway, whereas suppression of JNKs with a specific inhibitor increases apoptosis by reducing autophagy. In turn, inhibition of autophagy reduced JNK activity. As a matter of facts, UA modifies cytoplasmic signals, and the induction of autophagy seems to be a defensive mechanism against molecule-induced toxicity .α (p-eIF2α), and X-box binding protein 1 (XBP1). In addition, the endoplasmic reticulum stress inhibitor 4-phenylbutyrate attenuated UA-induced apoptosis. UA significantly increased free calcium in the cytosol, the expression of calcium release-activated calcium channel protein 1 (ORAI1) and stromal interaction molecule 1 (SIM1), two key components of store-operated calcium entry (SOCE) that represents the main calcium transport system in nonexcitable cells. Furthermore, knockdown of ORAI1 prevented endoplasmic reticulum stress and ATP depletion in response to UA. Nevertheless, overexpression of ORAI1 increased endoplasmic reticulum stress and ATP depletion caused by UA. Overall, UA interferes with calcium homeostasis and induces stress in the endoplasmic reticulum. UA-induced cellular damage is produced, at least partially, by the activation of SOCE calcium channels [A second study by Chen et al. evaluated the effects of UA on the endoplasmic reticulum of Hep2 cells. The authors observed that UA induces stress in the endoplasmic reticulum, as demonstrated by the increased expression of markers such as C/EBP homologous protein (CHOP), activating transcription factor 4 (ATF-4), phosphorylation of eukaryotic initiation factor-2channels .5 cells/ml in 96-well multiwells. Cells were cultured for 24 hours before treatment with either 0.1% UA or vehicle alone (DMSO). The authors observed that UA-induced DNA damage and cell cycle arrest in S-phase. UA-induced oxidative stress, as demonstrated by the production of reactive oxygen species (ROS) and GSH depletion. Exposure for 6 hours to UA significantly increased Nrf2 level, promoted its translocation into the nucleus, increased the expression of proteins related to the antioxidant response, and induced the expression of Chen2-regulated targets such as glutathione reductase, glutathione S transferase, and NAD(P)H quinone oxidoreductase-1 (NQO1). Knockdown of Nrf2 with shRNA enhanced UA-induced damage and cytotoxicity. These data demonstrate that UA produces dysregulation of cell cycle, DNA damage, and oxidative stress, and UA-induced cytotoxicity activates the transcription factor Nrf2 [A third study by Chen et al. showed that UA activates nuclear factor erythroid 2-related factor 2 (Nrf2) and increases the activity of c-Jun N-terminal kinases (JNKs) and Akt/mammalian target of rapamycin (mTOR). HepG2 cells were plated at a concentration of 3 × 10tor Nrf2 .Piska et al. studied the biotransformation of UA enantiomers into reactive products using a GSH assay in human, mouse, and rat microsomes. The results of the study showed that each enantiomer produces two reactive glutathione-binding metabolites, which could at least partially explain the toxicity of UA .A recent study by Kwong et al. focused on the role played by porimin in UA-induced hepatotoxicity by several techniques  using both normal human L02 cells and ICR mice. Assays were performed to assess oxidative stress, GSH, malondialdehyde (MDA), and superoxide dismutase (SOD) levels. Identification of apoptotic cells was performed by double fluorescence. Expression of proteins such as glutathione S transferase, glutathione reductase, glutathione peroxidase 4, catalase, N-terminal kinase-Jun, caspases, gastamin-D, and porimin was assessed by Western blot. Exposure to UA induced a significant increase in the expression of glutathione reductase, glutathione S transferase, and glutathione peroxidase-4 in L02 cells, while the expression of catalase was decreased in a dose-dependent manner. The dextrorotatory isomer of UA did not activate caspase-3, caspase-1, or gasdermine-D, but significantly increased the expression of porimin. The dextrorotatory isomer of UA did not cause cytotoxicity in L02 cells silenced for porimin. The authors conclude that increased porimin expression and pore formation in the membrane could contribute to UA-induced hepatotoxicity .in vitro studies carried out on UA toxicity are summarized in In summary, the two main mechanisms of UA toxicity seem to be the uncoupling of oxidative phosphorylation and free radical generation. The former decreases ATP synthesis, and the latter induces cell membrane and mitochondrial injury, with a mechanism similar to that observed following the administration of carbon tetrachloride. The main outcomes of the Several studies have been carried out to assess UA toxicity in vivo. The study by Pramyothin et al. has been already described in Section 3 since it included tests in vitro, as well as in vivo .Joseph et al. examined the expression levels of 542 genes in the liver of B6C3F female mice using a mitochondria-specific microarray. Mice received UA at 0, 60, 180, and 600 ppm in their diet for two weeks. Microarray analysis showed a significant induction of genes associated with complexes I through IV of the electron transport chain only at the highest dose of 600 ppm. Several genes involved in fatty acid oxidation, Krebs cycle, apoptosis, and membrane transporters were also overexpressed. The authors argued that the upregulation of complexes I–IV may be a compensatory mechanism to maintain the proton gradient across the mitochondrial inner membrane, while induction of fatty acid oxidation and the Krebs cycle may be an adaptive response to uncoupling of mitochondria .Lu et al. studied the hepatotoxicity of the dextrorotatory enantiomer of UA on rats by mass spectrometry: Wistar rats were treated with three different doses of UA: 100, 200, and 240 mg/kg daily for 8 days. The main biochemical and histopathological parameters were assessed together with a metabolomic analysis performed by gas chromatography/mass spectrometry. The ratio of liver/body weight was significantly increased in UA-treated rats, as well as transaminase and total bilirubin levels. Signs of diffuse hepatocyte degeneration were observed in liver sections of rats treated with the higher dosages of UA. Exposure of rats to UA-induced changes in energy, amino acid, lipid, and nucleotide metabolism .Liu et al. studied the proteome expression in UA-induced hepatotoxicity in rats: expression profiles were analysed by two-dimensional gel electrophoresis followed by mass spectrometry. The results showed significant changes in the expression of ten proteins associated with oxidative stress and lipid metabolism, suggesting that endoplasmic reticulum and mitochondria might be the primary targets in UA-induced hepatotoxicity .μM), and stimulated glycolysis and glycogenolysis. UA increased the production of 14 CO2 from 1–14C octanoate and 1–14C oleate, but ketogenesis from octanoate was reduced (no differences were observed in ketogenesis from oleate). The authors conclude that, up to a concentration of 2.5 μM, UA is an uncoupler of oxidative phosphorylation. At higher concentrations, other effects occur, namely, inhibition of electron flow in mitochondria and inhibition of medium-chain fatty acid oxidation. From a metabolic point of view, it is possible to predict a more pronounced UA toxicity during fasting due to the decreased hepatic flux of glucose and ketone bodies [Moreira et al. studied the hepatotoxicity of UA in livers of male Wistar rats perfused in a system without recirculation . At low e bodies .in vivo studies confirm those observed in in vitro studies and show that UA, usually at doses equal or higher than 200 mg/kg/day, is an uncoupler of oxidative phosphorylation. The main outcomes of the in vivo studies carried out on UA toxicity are summarized in In summary, data from μg/ml. Swiss mice were treated with UA at doses of 25, 50, 100, and 200 mg/kg body weight. The same concentrations of UA were combined with methyl-methanesulfonate (MMS) to assess antigenotoxicity. In vitro studies showed that UA damages DNA at concentrations of 60 and 120 μg/ml in the Comet assay, whereas no genotoxic effect was observed in the micronucleus assay. In vivo, no genotoxic effect was observed, and combined administration of UA and MMS significantly decreased the frequency of micronuclei and DNA damage both in vitro and in vivo. Although the mechanisms underlying the protective effect demonstrated by UA are not completely understood, the antioxidant activity of the molecule could explain the protective effect against MMS-induced genotoxicity [Leandro et al. studied the genotoxicity of UA in vitro on V79 cells and in vivo on Swiss mice. In this study, V79 cells were exposed to UA concentrations of 15, 30, 60, and 120 toxicity .Koparal et al. evaluated the potential genotoxicity of UA on V79 (Chinese hamster lung fibroblasts), A549 (human lung carcinoma cells), and human lymphocytes cell lines by MTT and CBMN assays. The results of the study showed that both enantiomers of UA lacked genotoxicity but induced a significant cytotoxic and proapoptotic effect in tumour cell lines .μg/ml. Genotoxic damage was evaluated by chromosomal and micronucleus aberration assays. Furthermore, biochemical parameters, such as total antioxidant capacity and total oxidative status, were evaluated. The authors demonstrated that UA has no mutagenic effects on human lymphocytes. Overall, the results obtained indicate that low concentrations of UA (1 and 5 μg/ml) increase the total antioxidant capacity in cultured human cells and that the total oxidative status is maintained unchanged [The genotoxicity of UA was the subject of a study by Polat et al. carried out on human lymphocytes exposed to UA at concentrations ranging from 0 to 200 nchanged .Drosophila melanogaster. For this purpose, 72 ± 4 hr Drosophila larvae were treated with UA  and urethane (10 mM) as positive control or solvent . The results of the study indicate that, at the doses tested, UA can exert mutagenic effects on Drosophila melanogaster somatic cells [Machado et al. evaluated the mutagenic potential of the dextrorotatory enantiomer of UA by SMART (somatic mutation and recombination test) assay to detect epithelial tumour clones in ic cells .A study by Silva et al. focused on the potential teratogenic effects of UA. Female rats were randomized into three study groups and treated with (a) 25 mg/kg body weight of UA; (b) 15 mg/kg body weight of UA; and (c) control treated with vehicle only. Treatments were administered with drinking water between days 6 and 15 of pregnancy, and after 20 days, the foetuses were removed and analysed. Low weight gain, increased resorption, and reduced number and weight of viable foetuses were found. Morphologic changes ranging from eye exposure to limb atrophy were found at the highest dose. From a histological point of view, in the group treated with the highest dose of UA, fewer megakaryocytes and more hepatocytes were observed in the liver of the foetuses. The experimental model used suggests the possibility of teratogenic effects during organogenesis in the foetus .Prokopiev et al. studied the genotoxic effects of dextrorotatory and levorotatory enantiomers of UA on human mononuclear cells: the enantiomers of UA at concentrations between 0.04 and 0.30 mM induce a genotoxic effect. In this study, the genotoxicity of the levorotatory enantiomer was twice as high as that of the dextrorotatory enantiomer .In a second study, Prokopiev et al. evaluated the genotoxic effects of dextrorotatory and levorotatory enantiomers of UA in murine liver and kidney cells. DNA damage was observed 1 hour after UA administration at doses ranging from 100 to 50 mg/kg. Genotoxic damage is associated with oxidative stress in cells, with no significant difference between dextrorotatory and levorotatory enantiomer .High doses of the dextrorotatory isomer of UA produce uncoupling of oxidative phosphorylation and subsequent stress in mitochondria and endoplasmic reticulum. This mechanism appears to underlie the hepatotoxic effects observed when the product is administered orally at high doses. However, it is unclear whether UA induces toxicity to other mitochondria-rich organs such as the heart.Yokouchi et al. randomized 27 female rats into three study groups of 9 animals each treated with (a) 30 mg/kg body weight of UA; (b) 100 mg/kg body weight of UA; (c) vehicle alone (0.5% methylcellulose solution) . The treμg/ml. The authors conclude that the dextrorotatory isomer of UA is a naturally occurring compound with low toxicity in mice and it does not produce obvious toxic effects when administered at medium and low doses [Cheng et al. evaluated the acute toxicity of the dextrorotatory isomer of UA in mice and rat cardiac fibroblasts. The LD50 of the dextrorotatory isomer of UA in mice after oral administration was 388 mg/kg. The IC50 in rat cardiac fibroblasts was 322 ow doses .in vitro neurotoxicological effects of UA, the authors evaluated its effects on neuron-like cells named SH-SY5Y. At the highest concentrations tested, UA showed significant antioxidant activity evaluated as the total reactive antioxidant potential (TRAP) index. In addition, UA was also effective against hydroxyl radicals and decreased the nitric oxide production. The MTT reduction assay demonstrated increased in vitro lipoperoxidation and modified cell viability at the highest concentration of 20 μg/mL for 1 and 4 hours, as well as at 2 and 20 μg/mL for 24 hours. UA showed no protective effects against hydrogen peroxide-induced cellular damage. The production of reactive oxygen species assessed by DCFH assay showed that UA induces changes in their basal production at concentrations of 20 μg/ml for 1 h and from 2 to 20 μg/ml for 4 and 24 h. In summary, UA shows different oxidoreductive properties depending on the conditions and cellular environments tested. The study also suggests that the potentially neurotoxic effects of UA should be investigated by further approaches, i.e., by in vivo studies and clinical trials [The interactions of UA with genes, proteins, and other molecules that play a key role in cellular oxidoreductive balance and nitric oxide production were assessed by Rabelo et al. . The stul trials .in vitro and in vivo studies clearly demonstrate that UA is an uncoupler of oxidative phosphorylation. Since UA decreases the efficiency of energy use and increases thermogenesis, it has been included in the composition of several food supplements intended for the induction of weight loss in obese subjects. Two of these, namely, Lipokinetix  and UCP-1  have been marketed in the US until 2001 [The aforementioned til 2001 . Each catil 2001 . The rectil 2001 .Overall, Lipokinetix and UCP-1 were responsible for at least 21 cases of hepatotoxicity reported to the FDA. Among these, one subject died, one underwent a liver transplant, seven developed acute liver failure, ten experienced elevated transaminases, and four developed mild hepatotoxicity. On the basis of these findings, the FDA issued a warning letter, and the products were withdrawn from the market in November 2001. Several cases of UA-induced hepatotoxicity have been published in literature.Favreau et al. described five cases of Japanese nationals residing in the Los Angeles area treated for acute liver failure between July and December 2000 at the Cedars-Sinai Medical Center  . Two morIn twenty cases of acute liver failure published by the Oregon Health & Science University Hospital and the Portland Veterans Affairs Medical Center in Oregon, ten were regular users of dietary supplements and herbal products. In two of these cases (a 25-year-old woman who died and a 42-year-old man who recovered), Lipokinetix was identified as the sole cause of acute liver failure .A retrospective analysis has been carried out on 12 patients who developed hepatotoxicity related to the ingestion of herbal products for the induction of weight loss. Two out of the 12 patients reviewed took Lipokinetix according to the manufacturer's recommendations. One patient recovered, while the other one underwent liver transplantation .Durazo et al. described the case of a 28-year-old woman taking 500 mg of UA  daily as slimming agent. She stopped the treatment after two weeks because she began to feel sick. After two weeks, she resumed the treatment for 4 days. Due to the development of signs and symptoms of hepatotoxicity, she was transferred to the Medical Center of the University of California, Los Angeles (UCLA), where she underwent liver transplant. She recovered completely, and the examination of the injured liver demonstrated a shrunken organ  with scattered irregularly shaped nodules of residual parenchyma .Sanchez et al. described two subjects who developed severe hepatotoxicity while using UCP-1 at the recommended daily dose. The first one was a 38-year-old female fitness instructor who developed asthenia, abdominal pain, and jaundice after three months of treatment. The patient was taking oral contraceptives, but never had liver disease in the past, was not an alcohol abuser, and was not at risk of developing viral hepatitis. Laboratory tests showed elevated levels of bilirubin, alkaline phosphatase, aspartate aminotransferase, and alanine aminotransferase. Patient's condition progressively worsened, with the development of grade 2-3 hepatic encephalopathy. The patient underwent liver transplantation, and the explant pathology revealed a shrunken liver with diffuse parenchymal necrosis. The patient recovered rapidly and was discharged a week after the surgery intervention. Her husband was also taking UCP-1 with the same schedule of administration. He was asymptomatic; however, hematochemical examinations showed a marked increase in transaminases, which returned to basal four months after stopping the treatment .Krishna et al. described the case of a 28-year-old female bodybuilder treated at the Mount Sinai Medical Center  for progressive malaise and jaundice. The patient took two food supplements, namely, Somalyz and Lipolyz  containing 4 mg and 12 mg of UA per capsule, respectively. She was taking 1-2 capsules of Somalyz at bedtime and one capsule of Lipolyz three times per day with meals, corresponding to an overall daily UA intake of 44 mg for one month before symptoms onset. The patient underwent successful liver transplantation. Gross examination showed massive necrosis of the liver and parenchymal collapse .Topical application of lichen extracts containing UA has been used in folk medicine for wound healing, athlete's foot, and sore throat . UA is cRecently, Zhang et al. investigated the activity of UA applied topically in wound healing. The effects were measured by means of wound contraction experiments, histological analysis, and immunohistochemistry. Study results showed that wound healing rates were higher and re-epithelialization time shorter following topical application of UA compared to control. Treatment with UA decreased the number of inflammatory cells, while increasing fibroblast proliferation, granulation tissue, and vascular regeneration. UA-induced earlier complete re-epithelialization, production of well-organized bands of collagen, and epidermal keratinization. The authors conclude that the topical use of UA promotes wound healing thanks to the anti-inflammatory properties of the molecule . SeveralCoşkunmeriç et al. developed an innovative formulation of UA in nanogel for the treatment of oral ulcers. UA in nanogel accelerated wound healing in an animal model and showed an effective antimicrobial activity against Bacillus Cereus .Galanty et al. compared the photoprotective properties of the two UA enantiomers. Both the compounds showed good skin-penetrating properties; however, the left-handed enantiomer was slightly more toxic to keratinocytes compared to the right-handed one. On the other hand, (+)-UA showed good photoprotective properties and photostability, comparable to the UV filter octocrylene. Surprisingly, (+)-UA in combination with octocrylene showed enhanced photoprotective properties and improved photostability. The authors concluded that (+)-UA can be used in cosmetic products as a UV filter .μg/ml or 150 μg/ml . The spray is a medical device intended for the prevention of airborne viral infections. Rats were treated with 30 μl per nostril 3 times daily for one week. At the end of the treatment period, no signs of inflammation were observed in the nasal mucosa, as well as in the liver, kidney, spleen, lungs, brain, and heart. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were in the normal range, and UA serum concentration measured by LC/MS was very low (around 0–6 μg/ml).Recently, the ENEA Casaccia Research Center  carried out a study on 30 Wistar rats randomized to receive two nasal spray formulations containing UA at the concentration of 75 Overall, topical application of UA appears to be safe and well tolerated.Due to the numerous biological and therapeutical properties of UA, several studies investigated many strategies to improve its pharmacologic activity and reduce toxicity, including the use of diverse drug delivery systems. In particular, the encapsulation of UA into nano- and microcarriers, including liposomes and nanoemulsion, was considered. Indeed, besides enhancing drug stability, solubility, and intracellular uptake, these delivery systems can also grant a sustained and controlled release of the drug, thus modulating toxicity and favouring drug accumulation in the target tissues.Diverse in vitro studies reported the successful encapsulation of UA into liposomes, nanoemulsions, polymeric nanospheres, and polymeric or magnetic nanoparticles and demonstrated the positive impact of encapsulation on the enhancement of UA properties, especially focussing on antitumoral, antimicrobial, and antioxidant activities. In parallel, these studies also investigated the toxicity reduction of encapsulated UA –66.Mycobacterium tuberculosis, compared to free UA and suggested their potential in targeting M. tuberculosis-infected macrophages for the treatment of pulmonary tuberculosis [Staphylococcus aureus [S. epidermidis [Lira et al. were the first who studied the in vitro antimicrobial effects of UA encapsulated into liposomes. Authors demonstrated the enhanced antibacterial activity of UA-loaded liposomes on rculosis . Subsequrculosis . These rrculosis . In addis aureus  and S. edermidis .Several studies also evaluated the antitumoral activity of encapsuled UA . One of The effects of UA nanoencapsulation have been also assessed for the evaluation of toxicity both in in vivo and in vitro studies. In this context, results were controversial. Some authors reported no significative changes in encapsulated UA toxicity, compared to free UA, and some studies reported even an increase of the UA toxicity after encapsulation , 74. On Overall, the results obtained in these studies are promising and could lead to extend the use of UA also for systemic administration, opening the possibility of exploiting the UA properties for the therapy of various diseases, including infectious, tumoral, and inflammatory. However, although several studies have fully demonstrated the enhancement of UA therapeutical properties after encapsulation, further studies are needed to validate the in vivo effectiveness of encapsulation in reducing UA toxicity.UA is a promising therapeutic agent with antibacterial, antiviral, antifungal, antiprotozoal, and antitumor activities. Lichen extracts rich in UA have been used in traditional medicine for the treatment of malaria, wounds, snakebite, and cough. The molecule is currently used in industry to produce perfumes, cosmetics, and medical ointments, as well as toothpaste, mouthwash, deodorants, and sunscreen products.in vitro and in vivo confirmed that high concentrations of UA are hepatotoxic, due to the uncoupling of oxidative phosphorylation and free radical generation. The molecule is not genotoxic; however, data on mutagenesis and teratogenicity are quite controversial.Intake of food supplements containing high doses of UA  for slimming purposes resulted in 21 cases of hepatotoxicity documented by the FDA. Several studies On the other hand, topical use of UA seems to be safe and well tolerated. The product could be potentially used in a wide range of applications, ranging from bioactive wound dressing to nasal spray for the prevention of airborne infections. UA encapsulation in micro- and nanocarriers could potentially improve UA bioavailability and decrease its toxicity. However, further studies are needed to confirm these preliminary observations.


The wingless-related integration site (Wnt) signaling pathway plays an essential role in embryonic development and nervous system regulation. It is critically involved in multiple types of neuropathic pain (NP), such as HIV-related NP, cancer pain, diabetic neuralgia, multiple sclerosis-related NP, endometriosis pain, and other painful diseases. Wnt signaling is also implicated in the pain induced by sciatic nerve compression injury and selective spinal nerve ligation. Thus, the Wnt signaling pathway may be a potential therapeutic target for NP. The International Society for Pain Research defines neuropathic pain (NP) as caused by injury or disease of the somatosensory nervous system. The clinical features of NP include spontaneous pain, hyperalgesia, pain hypersensitivity, abnormal sensation, etc.  and in mammals. It regulates many physiological processes in the human body, including nervous system development. The Wnt protein family is a group of secreted glycoproteins that function as specific ligands for cells and tissues during brain development and maturation. These proteins participate in neuron formation, cell fate regulation, polarity and migration, axon development, synapse formation, nerve maturation, nerve maintenance, and regeneration , was identified in 1982 as a gene activated by the integration of virus DNA in mouse breast tumors , protein tyrosine kinases 7 (PTKs7), G-protein coupled receptor (GPCR), etc. Farin,  In addit2+ signaling pathways , casein kinase 1 (CK1), and GSK-3β . The antagonists for the Wnt/β-catenin signaling pathway include CK1, DSH, GSK-3β, and glucan-binding protein (GBP). When no Wnt ligand binds to cell-surface receptors, β-catenin is gradually phosphorylated by CK1 and GSK-3β. The phosphor-β-catenin is ubiquitinated and degraded by Skip-Cullin1-F-box beta-transducin repeat-containing protein (β-TRCP) Fig.  Azzolin. TankyraOnce the Wnt protein binds to the surface receptor FZD, CK1 is activated and leads to the phosphorylation of DSH, the activation and release of GBP, and the inhibition of GSK-3β activity. In this case, GSK-3β cannot phosphorylate β-catenin, making it unrecognizable to ubiquitin and stably accumulates in the cytoplasm.2+ signaling pathway. These pathways are also called the T-cell transcription factor-independent sexual signaling pathway since signal transduction is different from that of β-catenin and transcription factor LEF/TCF  signaling pathway, and the Wnt/CaThe Wnt/PCP signaling pathway is responsible for establishing cell polarity and regulating directional cell movement. The PCP signaling pathway was originally found in Drosophila, where it controls the planar polarity of the epithelial cells in the eyes, wings, and chest cavity , the cross-membrane co-receptor of the FZD protein, Prickle (the partner of Strabismus in the cytoplasm), and the small G-protein in the cytoplasm. RhoA and DSH can activate the Rac protein by binding their receptors, Rho-kinase (ROCK) and JNK, respectively.Six core genes, in addition to The Wnt/PCP ligands, including Wnt5a, Wnt7, and Wnt11, bind to the FZD receptor. DVL-mediated induction of small GTPase  activates JNK and the transcription of downstream effectors involved in cytoskeletal rearrangement and epithelial cell polarization  leads to the release of intracellular Ca2+ and the subsequent activation of three Ca2+ sensitive components: protein kinase C (PKC)   Hu, , calcine2+, which in turn activates Ca2+ sensitive components PKC and CaMKII  enters the nucleus, initiating the transcription of target genes  result in abnormal neuronal excitability through the release of neurotransmitters  and the activation of the CaThe CCI animal model has been commonly used to mimic the symptoms of NP induced by tumor compression, heavy metal ion poisoning, and hypoxia. Animals experience hyperalgesia and spontaneous pain after surgery. The dorsal root ganglion (DRG) and SC damage is the key driver of NP development. Both typical and atypical Wnt signaling pathways are involved in regulating chronic sciatic nerve compression in rats. In the model of tumor cell implantation (TCI)-induced pain, the Wnt protein is upregulated in the DRG and SC, and the Wnt/FZD8/β-catenin signaling pathway is activated in the SC. The level of activated Wnt3a elevated in the superficial layer of the SDH after surgery  is a nociceptive transmitter in normal animals. Among the main afferents, it is mainly expressed by C fiber neurons  and the increase in repeated discharge Song, . The RYKIt has recently been reported that 2 out of 20 A-fiber neurons expressed SP after CCI of the sciatic nerve, compared with 13 in the control group. The difference was not significant in the number of neurons investigated. The two A-fiber neurons expressing SP had higher conduction velocity  in astrocytes compared to the sham-operated group. Short hairpin RNA against RYK reduced inflammation and increased the threshold of mechanical pain in SNL-treated animals, indicating the implication of the Wnt1/RYK pathway in NP. The protein level of chemokine CCL2 in the SDH of a rat SNL model was significantly upregulated, while intrathecal injection of the RYK antibody reduced the production of CCL2 , WNT10A, platelet-derived growth factor alpha (PDGFA), and neuregulin 1 (NRG1) to sensitize sensory neurons, thereby promoting neuropathic pain associated with various cancers. The interaction between paracrine factors and their receptors can cause the activation of downstream transcription factors such as B-Cell lymphoma 3-encoded protein (BCL3), E2F transcription Factor 1 (E2F1) and SMAD family member 5 (Smad5), and RE1 silencing transcription factor (REST) can upregulate the expression of pain-related genes such as chromodomain helicase DNA binding protein 5 (CHD5) and pain including arrestin beta 2 (ARRB2). TNF, WNT10A, and Platelet-derived growth factor alpha (PDGFA) are widely expressed in various tumors, but their expression is not normally distributed in patients. These data indicate the presence of infiltrating dendritic cells in the tumor. The microenvironment promotes neuropathic pain by sensitizing nociceptive sensory neurons with paracrine factors. Blocking paracrine signaling may alleviate cancer pain  is a common complication of diabetes that affects more than half of diabetics throughout disease progression, leading to neuropathic pain and an increased risk of ulcers and amputations  can activate PI3K/AKT and B-catenin signaling pathways by downregulating Mir-503 under high glucose conditions  is a traditional Chinese medicine prescription for treating diabetic peripheral neuropathy (DPN). Relevant studies studied the effects of TLN on microRNA (MiRNA) expression in diabetic rats, and predicted the target genes.Further pathway and genetic analysis identified DVL1 from the Wnt signaling pathway and neurotrophin-3 (NTF3) from the neurotrophin signaling pathway. Previous studies have shown that Wnt signaling pathway may mediate oxidized low-density lipoprotein (ox-LDL)-induced endothelial cell injury through oxidative stress. That inhibition of oxidative stress and inhibition of Wnt signaling pathway can ameliorate endothelial cell injury , is a commonly used compound medicine. It consists of 12 kinds of Traditional Chinese medicine, including 10 types of Chinese medicinal materials and two types of animal medicinal materials, containing a variety of components with unique chemical structures and a variety of biological characteristics. Previous studies have verified the alleviating effect of the components on diabetic peripheral neuropathy (DPN). Among them, quercetin, cinnamaldehyde, and hirudin come from three main herbs  respectively, and their neuroprotective effects have been verified. As a result, all of these compounds have protective effects on rat dorsal root ganglion (DRG) neurons against in vitro damage induced by high glucose , and WNT inhibitory factor-1 (WIF-1) involved in WNT /β-catenin signaling pathway and Myelin protein zero (MPZ) in rat sciatic nerve was determined.The results showed that the expression of β-catenin mRNA and protein was significantly inhibited after sciatic nerve injury in rats. After 8, 12, 16 weeks, the mRNA and protein levels of β-catenin were increased considerably, and the expression of P-β-catenin decreased. These results are consistent with our previous in vitro studies, which found that JMT promotes myelin repair by stimulating Schwann cell proliferation in high glucose medium and enhances MPZ secretion by upregulation of β-catenin  cells Th1 and Th17 in the chronic phase. Th17 cells are the main immune participants involved in the pathogenesis of MS by mediating the production of pro-inflammatory cytokines   mice have been widely used to investigate MS-related neurological complications, including central nerve demyelination, neuroinflammation, movement disorders and sexual pain. Immunohistochemistry analysis showed that Wnt3a, β-catenin, Wnt5a, and its receptor Ror2 were significantly upregulated in the SDH of EAE mice. Intrathecal injection of Wnt5a antagonist Box5 and β-catenin inhibitor indomethacin effectively alleviated pain in EAE mice  to ensure that muscle membrane potential depolarization can be trigged. Receptor-associated protein at the synapse (Rapsyn) is a protein that promotes the aggregation of acetylcholine receptors (Chen et al., MG is an autoimmune disease involving impaired neuromuscular transmission and weakness of skeletal muscles Dominik, . The WntEMT is a pathological condition characterized by uncontrolled proliferation and distant metastasis of blood vessels and other circulatory system components to surrounding tissues Lang, . EstrogeThe Wnt/β-catenin signaling pathway is involved in ectopic endometrial adhesion, invasion, angiogenesis, and inflammation during the progression of EMT (Liu et al., As an important regulatory pathway of embryonic development and adult tissue homeostasis, Wnt signaling pathway is closely related to a variety of diseases and is an important target of drug therapy. A growing body of evidence supports the role of Wnt signaling in regulating NP and has been validated in many animal models. Studies have shown that WNT may be involved in the pathogenesis of NP by regulating central sensitization and inflammatory factor release. Application of WNT signaling pathway-related blockers can effectively alleviate NP.Further investigations on the Wnt signaling pathway may contribute to developing novel therapeutic options for NP patients, thereby improving their quality of life.


Radix Bupleuri, exert anti-inflammatory effects. However, the topical effects of SSA and SSC on chronic inflammatory skin diseases are unclear. In this study, we investigated the effects of SSA and SSC on TSLP suppression in an AD-like inflammatory environment. We observed that SSA and SSC suppressed tumor necrosis factor-α-induced TSLP expression by downregulating the expression of the transcription factor early growth response 1 (EGR1) via inhibition of the extracellular signal-regulated kinase 1/2, c-Jun N-terminal kinase 1/2, and p38 mitogen-activated protein kinase pathways. We also confirmed that topical application of SSA or SSC reduced AD-like skin lesions in BALB/c mice challenged with 2,4-dinitrochlorobenzene. Our findings suggest that suppression of EGR1-regulated TSLP expression in keratinocytes might be attributable to the anti-inflammatory effects of SSA and SSC in AD-like skin lesions.Atopic dermatitis (AD) is one of the most common chronic inflammatory skin diseases worldwide, characterized by intense pruritus and eczematous lesions. Aberrant expression of thymic stromal lymphopoietin (TSLP) in keratinocytes is associated with the pathogenesis of AD and is considered a therapeutic target for the treatment of this disease. Saikosaponin A (SSA) and saikosaponin C (SSC), identified from  It is characterized by intense pruritus and eczematous lesions, which considerably affect the quality of life of patients ,2. Howevtrophils . Analysetrophils . Furthertrophils , suggest lesions .Thymic stromal lymphopoietin (TSLP) is an IL-7-like cytokine expressed mainly by stromal and epithelial cells in the skin, lung, and intestine . TSLP acRadix Bupleuri, the dried root of Bupleurum species, has been widely used as an oriental folk medicine [Radix Bupleuri [Radix Bupleuri. However, there is little information regarding the anti-inflammatory activity of saikosaponins against chronic skin inflammatory diseases, such as AD. Of these, saikosaponin A  and saikosaponin C  exert anti-inflammatory effects [Natural products are a rich source of novel drug candidates with fewer side effects than those of many synthetic drugs ,17. Radimedicine . Its extmedicine ,19. SaikSSA, CAS 736-09-8  effects ,22. HoweIn this study, we investigated the biological activities of SSA and SSC in the regulation of TSLP expression in TNF-α-stimulated HaCaT keratinocytes. We also evaluated the potential of SSA and SSC for topical application in allergic skin inflammatory diseases using a 2,4-dinitrochlorobenzene (DNCB)-challenged BALB/c mouse model. We first determined the effects of SSA and SSC A on cytoKeratinocyte-derived TSLP is a master regulator of Th2 inflammatory responses in AD. Thus, upregulated TSLP expression is considered a hallmark of AD pathogenesis ,15. TNF-TSLP expression is altered upon exposure to SSA or SSC, we treated HaCaT keratinocytes with TNF-α and analyzed TSLP mRNA expression in these cells. Consistent with a previous report [TSLP mRNA levels was reduced by pretreatment with SSA (TSLP mRNA levels by 8.45-fold compared to those of the control (p < 0.001) decreased by pretreatment with 1 µM SSA or 10 µM SSC. To investigate whether s report , the TNFwith SSA A or SSC with SSA B in a do control C, which To determine whether SSA and SSC reduced TNF-α-induced increase in TSLP protein levels, we performed immunoblot analysis. Consistent with their mRNA levels, TNF-α-induced enhanced protein expression of TSLP was markedly decreased after pretreatment with SSA A or SSC TSLP promoter activity [TSLP promoter activity, we used a series of 5′-deletion constructs of the TSLP promoter region, including −1338/+18, −1214/+18, −1000/+18, and −389/+18, wherein the TSLP promoter drives luciferase reporter activity [p < 0.001) increase in luciferase reporter activity upon TNF-α stimulation, which was abrogated by SSA or SSC treatment , located in the region between −206 and −187, in response to TNF-α stimulation [TSLP promoter activity compared to that in the wild-type (WT) construct  suppressed TNF-α-induced EGR1 accumulation in a dose-dependent manner. Fluorescence immunocytochemical staining showed that TNF-α-induced nuclear localization of EGR1 was effectively reduced by SSA or SSC pretreatment  pathway in various cell types ,28,29. Ap < 0.001 in all cases) suppressed DNCB-induced epidermal and dermal thickness ) was used for promoter analysis. Anti-TSLP antibody was obtained from Novus Biologicals , and anti-EGR1, phospho-ERK1/2 (Thr202/Tyr204), phospho-p38 (Thr180/Tyr182), and phospho-JNK1/2 (Thr183/Tyr185) antibodies were obtained from Cell Signaling Technology . The anti-GAPDH antibody was purchased from Santa Cruz Biotechnology . Secondary antibodies conjugated to Alexa Fluor 488 and rhodamine red-X were obtained from Jackson ImmunoResearch Laboratories . The MAPK, p38 kinase, and JNK inhibitors , DNCB, TB, and an H&E staining kit were purchased from Sigma-Aldrich.SSA and SSC were purchased from Sigma-Aldrich . TNF-α was obtained from ProSpec-Tany TechnoGene Ltd. . The firefly luciferase assay system was purchased from Promega . The web-based program MatInspector  and cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum  and 0.5% penicillin-streptomycin (Sigma-Aldrich).3 cells/sample) were treated with either the vehicle or different concentrations of SSA or SSC . After 24 h, CCK-8 solution was added, and the cells were incubated for an additional hour. The CCK-8 solution contains water-soluble tetrazolium salt WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5--2H-tetrazolium, monosodium salt), which is colorless, but produces orange-colored WST-8 formazan dye when reduced by dehydrogenase in cells. The amount of WST-8 formazan dye was measured using the Emax Endpoint ELISA Microplate Reader  at 450 nm, which indicated the number of living cells.Cell viability assay was performed using the CCK-8  according to the manufacturer’s instructions. Briefly, HaCaT cells . First-strand cDNA was synthesized using the iScript cDNA synthesis kit . RT-PCR was performed using reverse transcriptase (Promega) and gene-specific PCR primers, as follows: The thermal cycling conditions were as follows: 94 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, and elongation at 72 °C for 1 min. The amplified PCR products were electrophoresed on a 2% agarose gel and visualized under a UV transilluminator.TSLP mRNA were normalized to those of GAPDH using the software provided by the manufacturer (Bio-Rad).qPCR was performed using the iCycler iQ system and IQ SYBR Green Supermix kit (Bio-Rad) and validated using commercial qPCR primers and SYBR Green-based fluorescent probes specific for TSLP (id: qHsaCIP0030468) and GAPDH (id: qHsaCEP0041396) purchased from Bio-Rad. The relative expression levels of 3VO4, 1 mM of NaF, 10 mg/mL of leupeptin, and 1 mM of PMSF. The protein extracts were electrophoresed on 8 or 12% sodium dodecyl sulfate (SDS) polyacrylamide gels and transferred onto nitrocellulose membranes. After incubation with appropriate primary and secondary antibodies, the enhanced chemiluminescence detection system  was used to develop the blots. The intensities of the immunoreactive bands were quantified using ImageJ software, version 1.52a. The relative band intensities were calculated as a ratio to GAPDH band intensity.HaCaT cells were lysed using the RIPA buffer containing 50 mM of Tris-HCl (pH 7.4), 1% NP-40, 0.25% Na-deoxycholate, 500 mM of NaCl, 1 mM of EDTA, 1 mM of NaHaCaT cells cultured on coverslips were treated with the drugs, fixed, permeabilized, and incubated with primary antibodies (for 24 h at 4 °C), followed by incubation with secondary antibodies (for 1 h at 25 °C) and 1 μg/mL Hoechst 33258 (for 1 h at 25 °C). The fluorescently stained cells were observed using the EVOS FL fluorescence microscope .TSLP promoter region-spanning nucleotides −1338/+18, −1214/+18, −1017/+18, and −369/+18 has been described elsewhere [−172F: 5′-GAGGGAAATTCCTGATGACT-3′;−187R: 5′-GCTTTGCCCTAAACACTTC-3′.Construction of a series of deletion constructs of the human lsewhere . Internalsewhere . The seqThe sequence of the deletion construct was verified by DNA sequencing .TSLP promoter-reporter constructs using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s instructions. After 24 h of transfection, the cells were pretreated with different concentrations of SSA or SSC for 30 min, followed by the addition of 10 ng/mL of TNF-α. After 8 h, firefly luciferase activity was measured using the Dual-Glo luciferase assay system (Promega) with the pRL-null plasmid encoding Renilla luciferase (Promega) according to the manufacturer’s instructions. Luciferase activity was measured using the dual luminometer . HaCaT cells were cultured in 12-well plates and transiently transfected with 0.2 μg ® shRNA; Sigma-Aldrich) targeting EGR1 (shEgr1), according to the manufacturer’s instructions. After two weeks, knockdown of EGR1 expression was verified by immunoblot analysis.HaCaT cells were transfected with lentiviral shRNA . A biotinylated deoxyoligonucleotide probe (5′-CAA AAA GGA GGA AGG TGA GGG AA-biotin-3′) corresponding to EBS was synthesized by Macrogen. Nuclear extracts were prepared from HaCaT cells using the Nuclear and Cytoplasmic Extraction kit (Thermo Fisher Scientific). Nuclear extracts (3 µg) were incubated with the biotin-EBS probe (50 fmol) and salmon sperm DNA (Sigma-Aldrich) in the absence or presence of unlabeled probes as a competitor (2500 fmol). DNA-protein complexes were electrophoresed on non-denaturing 6% polyacrylamide gels and visualized using the Amersham ECL western blotting detection kit .v/v). The mice were treated with DNCB once daily for 3 days on the SDS-sensitized area and then rested for 4 days. SSA and SSC were dissolved in 70% ethanol at a concentration of 0.5% and 0.3%, respectively. After a challenge with 0.5% DNCB, 120 µL of 0.5% SSA or 0.3% SSC was dropped (Group II and Group IV). After sacrificing all mice on day 22, paraffin-embedded tissue sections were prepared. All animal experiments were performed according to the protocols approved by the Konkuk University Institutional Animal Care and Use Committee .Male BALB/c mice (7-week-old) were purchased from Orient Bio Inc. . The mice were divided into four groups: Group I, naive; Group II, DNCB + vehicle ); Group III, DNCB + 0.5% SSA; Group Ⅳ, DNCB + 0.3% SSC. The mouse in vivo study was performed as described previously, with minor modifications . BrieflyFluorescence immunohistochemical staining was performed as described previously with minor modifications . Brieflyp value less than 0.05 indicated statistically significant differences for all analyses.Data are presented as mean ± standard deviation (SD). Statistical analyses were performed using one-way analysis of variance (ANOVA) followed by Dunnett’s or Sidak’s multiple comparisons test using GraphPad Prism version 9.0.1 . The results with a Radix Bupleuri suppressed TNF-α-induced TSLP expression at the transcriptional level by inhibiting MAPK signaling-mediated transcriptional activation of EGR1 in HaCaT keratinocytes. Topical application of SSA or SSC ameliorated AD-like skin lesions in a mouse model challenged with DNCB. In conclusion, SSA or SSC could be used as effective and promising candidates for topical therapeutic application in chronic skin inflammatory diseases such as AD. Our study demonstrated that SSA and SSC identified from


Human T-lymphotropic viruses 1 and 2 (HTLV-1 and HTLV-2) infection has been described in several Amazonian populations; however, there is still a lack of data on the prevalence of the virus in riparian populations living in rural areas of the state of Pará. The present study aimed to evaluate the prevalence of HTLV-1/2 infection in four riverine communities and one rural area in the state of Pará and to describe the possible risk factors for infection. A total of 907 individuals responded to an epidemiological survey and gave blood samples collected for anti-HTLV-1/2 antibodies by immunoenzymatic assay (EIA). The serum-reactive samples were subjected to confirmation by an in-line assay (Inno-Lia) and by proviral DNA screening using real-time PCR (qPCR). The total prevalence was 0.8% (7/907) for HTLV-1/2 (CI: 0.2−1.3%), with 0.66% HTLV-1 and 0.11% HTLV-2. The prevalence by sex was 0.7% in women (4/565) and 0.9% in men (3/342). Among seropositive patients, 83.3% (5/7) reported being sexually active, and 57.1% (4/7) reported not having the habit of using condoms during their sexual relations. Intrafamily infection was also observed. The results reinforce the need for public policies to prevent and block the spread of HTLV, especially in riparian communities that are subject to difficulties in accessing the Unified Health System (Sistema Único de Saúde/SUS) because infected individuals need clinical monitoring for surveillance and early diagnosis of symptoms associated with HTLV-1. Retroviridae family, was first described in 1980 [The human T-lymphotropic virus (HTLV), belonging to the  in 1980  and asso in 1980 ,5,6,7,8.It is estimated that Brazil has the highest number of absolute cases of HTLV infection, with approximately 800,000 to 2.5 million people infected ,10, and Among the main groups described in the literature and related to HTLV infection in the Amazon region of Pará are blood donors, people living with HIV, Japanese people and indigenous people ,18,19,20Although HTLV-1/2 infection has been described in some populations of the Brazilian Amazon region, especially in indigenous and urban communities ,18,19,20Thus, the objective of this study was to describe the prevalence of HTLV-1/2 epidemiological and behavioural aspects of risk for exposure to the virus in rural and riverine communities located in the state of Pará, Brazil.n = 357; estimated population of 985 inhabitants), Acará , Limoeiro do Ajurú  and the rural areas of the municipalities of Bonito  and Maracanã   . IndividA blood sample (5 mL) was collected from each participant for serological and molecular biology tests. Aliquots of plasma and leukocytes were separated by centrifugation (8000 rpm for 15 min) and stored at −20 °C until use.The present study was approved by the National Research Ethics Committee  (CAAE: 27290619.2.0000.0018), following resolution 466/12 of the Ministry of Health, responsible for regulating any research involving human beings.The detection of anti-HTLV-1/2 antibodies was performed using an ELISA kit  following the protocol suggested by the manufacturer. All samples considered reactive or indeterminate in this test were subjected to confirmatory tests .® HTLV I/II Score  was used as a method for confirmation and differentiation of viral types, following the manufacturer’s protocol.The INNO-LIAThe analysis of HTLV proviral DNA was performed using 200 µL of whole blood, which was subjected to DNA extraction with the aid of the QiaAmp DNA mini kit  according to the protocol defined by the manufacturer. Prior to the amplification of the targets, the samples were quantified using the Qubit 2.0 fluorometer .pol (186 bp) and HTLV-2 tax (75 bp) genes as molecular markers of the virus, adapted from [Molecular confirmation of HTLV infection was performed by a single-plex real-time PCR using the TaqMan system  on the Applied Biosystems StepOne Plus Real Time PCR platform with three target sequences: the albumin gene, as endogenous control, and non-homologous regions of the HTLV-1 ted from . In each reaction, 12.5 µL of TaqMan Universal PCR Master Mix (2X) , 6.0 µL of ultrapure water, 0.5 µL of each primer (10 pmol), 0.5 µL of each probe (5 mM) and 5.0 µL of DNA (50 ng) were used, resulting in a total volume of 25 µL. The temperature cycles used were 95 °C for 10 min, followed by 45 cycles of 95 °C for 15 s and 60 °C for 1 min.The following primers were used in the reactions: 5′-CCCTACAATCCAACCAGCTCAG-3′ (HTLV-1F), 5′-GTGGTGAAGCTGCCATCGGGTTTT-3′ (HTLV-1R), 5′-CGATTGTGTACAGGCCGATTG-3′ (HTLV-2F), 5′-CAGGAGGGCATGTCGATGTAG-3′ (HTLV-2R), 5′-GCTGTCATCTCTTGTGGGCTGT-3′ (Albumin F), and 5′-AAACTCATGGGAGCTGCTGGTT-3′ (Albumin R). The probe sequences were as follows: FAM-5′-CTTTACTGACAAACCCGACCTACCCATGGA-3′-MGB (HTLV-1), FAM-5′-TGTCCCGTCTCAGGTGGTCTATGTTCCA-3′-MGB (HTLV-2) and FAM-5′-CCTGTCATGCCCACACAAATCTC-3′-MGB (Albumin).p < 0.05).The data obtained based on the questionnaires answered by the participants were added to the Epi-Info 7.2 database. Statistical analyses were performed using BioEstat 5.3 . All varThe serological investigation showed a prevalence of anti-HTLV-1/2 antibodies of 0.8% (7/907) (CI: 0.2–1.3%) for the total of riparian and rural populations evaluated in the present study, with six individuals infected by HTLV-1 (0.7%) and one infected with HTLV-2 (0.1%). The analysis by community revealed seroprevalences of 0.3% (1/357) on Combú Island, 1.8% (2/111) in Limoeiro do Ajurú and 3.3% (4/118) in Maracanã. No reactive samples were found in the Acará and Bonito populations. Among the seropositive samples in the ELISA test, five were confirmed by INNO-LIA and by qPCR as HTLV-1. The only sample positive for HTLV-2 was confirmed only by INNO-LIA, and amplification was not detected in the qPCR .Regarding the risks of exposure to HTLV-1/2 infection among seropositive volunteers, it was observed that 83.3% (5/7) reported being sexually active, 100% (7/7) reported not having tattoos or piercings, not having received blood transfusion, and were breastfed during childhood. In addition, 57.1% (4/7) reported not having the habit of using condoms in their sexual relations, 83.3% (5/7) never received a diagnosis of sexually transmitted infections (STIs), and 85.7% (6/7) reported never having sex in exchange for money .The two individuals who had a positive diagnosis, residents of the municipality of Limoeiro do Ajurú, belonged to the same nuclear family, with individual #94 being the mother of individual #63, thus identifying a possible occurrence of intrafamily infection by mother-to-child transmission via the vertical route (delivery) or even through breastfeeding. The difficulty of access to the community and the nonadherence to participation in the study prevented the testing of other members of this family unit, such as the spouse of individual #94.The seroprevalence of HTLV-1/2 in the state of Pará has already been described heterogeneously in different groups and communities ,27,28. TA low prevalence of HTLV-1 and HTLV-2 among the quilombola population has been described, ranging from 0.11 to 2.6% and 0.34 to 1.06%, respectively ,33. In tThe main limitation of our study was the sample size obtained from the communities, a fact resulting from the health restrictions imposed by the COVID-19 pandemic, which made it difficult to access a more representative sample of the populations studied and, possibly, a more accurate estimation of HTLV-1/2 infection.Most participants in this study had a low level of education, which may be related to their level of knowledge about ways to contract STIs . During The difficulty of access to urban areas, where medical care can be found more easily, is also an unfavourable condition for access to health in general ,42. ThisAlthough the results are not statistically significant, the infection rates for HTLV-1/2 were higher for females, which is in agreement with what has been reported in previous studies ,44,45,46Most infected individuals reported being sexually active and not having the habit of using condoms during sexual intercourse, a fact that may explain the infection in these individuals, since sexual intercourse is considered the main means of HTLV infection ,49. In aThe Brazilian Protocol of Sexually Transmitted Infections 2020: Infection by the Human T-lymphotropic Virus describes breastfeeding as a route of viral transmission ,53. In tInstituto Brasileiro de Geografia e Pesquisa/IBGE) [According to the Brazilian Institute of Geography and Research (sa/IBGE) , the munsa/IBGE) ,38 and tsa/IBGE) , suggestAmong the study participants who were diagnosed with HTLV-1/2, it was possible to perform the second collection of only two individuals, who were confirmed as positive by means of real-time PCR. Because they are riparian populations, some communities where these individuals reside are located in areas of difficult access and communication; however, the team of the Virology Laboratory of the Federal University of UFPA is still seeking ways to contact positive individuals again for the performance of extended evaluation of intrafamily transmission.The frequency of HTLV-1/2 detected in the populations of the present study shows variations in prevalence, reinforcing the heterogeneity of the infection observed in different geographic areas. Riparian people are vulnerable populations due to socioeconomic factors such as a lack of education and access to health. In addition, the fact that HTLV is still neglected in Brazil has further contributed to the difficulty of detecting new cases. Public policies and strategies that can act in the control, prevention, and promotion of information and allow additional investigation of intrafamily viral infection are considered the best options to support these populations.