Effects of Two Different Rhodiola rosea Extracts on Primary Human Visceral Adipocytes

Rhodiola rosea (Rro) has been reported to have various pharmacological properties, including anti-fatigue, anti-stress and anti-inflammatory activity. It is also known to improve glucose and lipid metabolism, but the effects of Rhodiola rosea on adipocyte differentiation and metabolism are not still elucidated. In this study the anti-adipogenic and lipolytic activity of two extracts of Rhodiola rosea, containing 3% salidroside (RS) or 1% salidroside and 3% rosavines (RR) on primary human visceral adipocytes was investigated. Pre-adipocytes were analyzed after 10 and 20 days of treatment during differentiation and after 7 days of treatment when they reached mature shape. The RS extract significantly induced higher apoptosis and lipolysis in comparison to control cells and to RR extract. In contrast, RR extract significantly reduced triglyceride incorporation during maturation. Differentiation of pre-adipocytes in the presence of RS and RR extracts showed a significant decrease in expression of genes involved in adipocyte function such as SLC2A4 and the adipogenic factor FGF2 and significant increase in expression of genes involved in inhibition of adipogenesis, such as GATA3, WNT3A, WNT10B. Furthermore RR extract, in contrast to RS, significantly down-regulates PPARG, the master regulator of adipogenesis and FABP4. These data support the lipolytic and anti-adipogenetic activity of two different commercial extracts of Rhodiola rosea in primary human visceral pre-adipocytes during differentiation

Effect of plant extracts on H2O2-induced inflammatory gene expression in macrophages

Background: Arctium lappa (AL), Camellia sinensis (CS), Echinacea angustifolia, Eleutherococcus senticosus, Panax ginseng (PG), and Vaccinium myrtillus (VM) are plants traditionally used in many herbal formulations for the treatment of various conditions. Although they are well known and already studied for their anti-inflammatory properties, their effects on H2O2-stimulated macrophages are a novel area of study. Materials and methods: Cell viability was tested after treatment with increasing doses of H2O2 and/or plant extracts at different times of incubation to identify the optimal experimental conditions. The messenger (m)RNA expression of TNF\u3b1, COX2, IL1\u3b2, NF\u3baB1, NF\u3baB2, NOS2, NFE2L2, and PPAR\u3b3 was analyzed in macrophages under H2O2 stimulation. The same genes were also quantified after plant extract treatment on cells pre-stimulated with H2O2. Results: A noncytotoxic dose (200 \u3bcM) of H2O2 induced active mRNA expression of COX2, IL1\u3b2, NFE2L2, NF\u3baB1, NF\u3baB2, NOS2, and TNF\u3b1, while PPAR\u3b3 was depressed. The expression of all genes tested was significantly (P,0.001) regulated by plant extracts after pre-stimulation with H2O2. COX2 was downregulated by AL, PG, and VM. All extracts depressed IL1\u3b2 expression, but upregulated NFE2L2. NF\u3baB1, NF\u3baB2, and TNF\u3b1 were downregulated by AL, CS, PG, and VM. NOS2 was inhibited by CS, PG, and VM. PPAR\u3b3 was decreased only after treatment with E. angustifolia and E. senticosus. Conclusion: The results of the present study indicate that the stimulation of H2O2 on RAW267.4 cells induced the transcription of proinflammatory mediators, showing that this could be an applicable system by which to activate macrophages. Plant extracts from AL, CS, PG, and VM possess in vitro anti-inflammatory activity on H2O2-stimulated macrophages by modulating key inflammation mediators. Further in vitro and in vivo investigation into molecular mechanisms modulated by herbal extracts should be undertaken to shed light on the development of novel modulating therapeutic strategies

Identification of miRNAs of Strongyloides stercoralis L1 and iL3 larvae isolated from human stool

Strongyloidiasis is a neglected tropical disease caused by the soil-transmitted nematode by Strongyloides stercoralis, that affects approximately 600 million people worldwide. In immunosuppressed individuals disseminated strongyloidiasis can rapidly lead to fatal outcomes. There is no gold standard for diagnosing strongyloidiasis, and infections are frequently misdiagnosed. A better understanding of the molecular biology of this parasite can be useful for example for the discovery of potential new biomarkers. Interestingly, recent evidence showed the presence of small RNAs in Strongyloididae, but no data was provided for S. stercoralis. In this study, we present the first identification of miRNAs of both L1 and iL3 larval stages of S. stercoralis. For our purpose, the aims were: (i) to analyse the miRNome of L1 and iL3 S. stercoralis and to identify potential miRNAs of this nematode, (ii) to obtain the mRNAs profiles in these two larval stages and (iii) to predict potential miRNA target sites in mRNA sequences. Total RNA was isolated from L1 and iL3 collected from the stool of 5 infected individuals. For the miRNAs analysis, we used miRDeep2 software and a pipeline of bio-informatic tools to construct a catalog of a total of 385 sequences. Among these, 53% were common to S. ratti, 19% to S. papillosus, 1% to Caenorhabditis elegans and 44% were novel. Using a differential analysis between the larval stages, we observed 6 suggestive modulated miRNAs (STR-MIR-34A-3P, STR-MIR-8397-3P, STR-MIR-34B-3P and STR-MIR-34C-3P expressed more in iL3, and STR-MIR-7880H-5P and STR-MIR-7880M-5P expressed more in L1). Along with this analysis, we obtained also the mRNAs profiles in the same samples of larvae. Multiple testing found 81 statistically significant mRNAs of the total 1553 obtained (FDR < 0.05; 32 genes expressed more in L1 than iL3; 49 genes expressed more in L3 than iL1). Finally, we found 33 predicted mRNA targets of the modulated miRNAs, providing relevant data for a further validation to better understand the role of these small molecules in the larval stages and their valuein clinical diagnostics

<i>Strongyloides</i> questions-a research agenda for the future.

The Strongyloides genus of parasitic nematodes have a fascinating life cycle and biology, but are also important pathogens of people and a World Health Organization-defined neglected tropical disease. Here, a community of Strongyloides researchers have posed thirteen major questions about Strongyloides biology and infection that sets a Strongyloides research agenda for the future. This article is part of the Theo Murphy meeting issue 'Strongyloides: omics to worm-free populations'

Local synthesis and effect of sex steroids in human skin and the hair follicle and hormonal effects on the wound healing response

Humans, as others mammals, are able to synthesize steroid hormones from endocrine organs, such as gonads, adrenal gland and placenta. The expression and activity of steroidogenic enzymes in peripheral tissues provide the intracrine production of active androgens and estrogens by transformation of adrenal precursor dehydroepiandrosterone (DHEA). Only human and primates are unique in having large amounts of the inactive adrenal DHEA and especially DHEA-sulfate (DHEA-S). However, adrenal secretion decreases from the age of 30 years in both sexes, thus potentially providing an explanation for part of the mechanisms involved in the pathogenesis of age-related conditions, such as skin aging. In this study, it was determined whether human skin expresses the enzymes required for the local biosynthesis of active sex steroid hormones. Specifically, biopsies of skin and hair follicles (HFs) were collected from female healthy donors, and cultured dermal fibroblasts (DFs) and cultured epidermal keratinocytes (EKs) were established from female and male healthy donors. The mRNA expression of seven key steroidogenic proteins (P450arom, P450scc, P450c17, STS, OATP2B1, 5α-reductase 1 and 5α-reductase 2), and the estrogen receptors (ERα and ERß) and the androgen receptor (AR) were investigated using RT-PCR and qRT-PCR. All samples produced PCR products of the expected size for P450scc, P450c17, STS and 5α-reductase 1. The mRNA expression of 5α-reductase 2 was detected only in HF. EK did not produce the transcript of P450arom and DF did not express OATP2B1. Both ERα and ERß were expressed in EK as well as in DF. In particular, EK showed to strongly express ERß compared to ERα. In contrast, ERα has been detected with higher expression than ERß in DF. Differences in estrogen receptors expression have been demonstrated also in human HFs that have showed strongly expressed ERß in comparison with ERα. On the other hand, all the samples showed AR expression. In order to the possibility of steroids metabolism, a gene array analysis was performed on whole transcriptome of human HFs and cultured EKs after steroid treatment. Three different hormones were used for 24h of incubation: DHEA-S (10µM), testosterone (TST) (50nM) and 17ß-estradiol (E2) (1nM). The global mRNA expression profiling revealed changes in the expression of a large number of genes. In particular, interesting up-regulation of genes involved in inflammation, cell proliferation and differentiation and structural functions. The analysis on EKs was focused on four genes up-regulated with all three steroids investigated: CXCL1, ANGPTL4, TXNIP, and LMNB1. In addition, the in vitro scratch wound assay was used to determine the direct effects of E2, DHEA, DHEA-S and TST on the migration of mechanically wounded human DFs and EKs. All steroids stimulated cell migration, although both DHEA and TST were blocked by an aromatase inhibitor (Arimidex) and DHEA-S by the STS inhibitor. These results indicate an important role for local synthesis of E2 in skin from the circulating precursor DHEAS and expression of its action in the wound healing process. To verify the hypothesis of the local synthesis of E2, the activity of the aromatase, which is the enzyme that catalyzes the conversion of androgen to estrogen, was assessed in EKs and DFs using the tritiated water (3H2O) assay. Specifically, it was investigated whether the aromatase activity and mRNA expression were modulated in cultured skin cells in response to either dexamethasone or mechanical wounding. The analysis revealed that dexamethasone increased the activity and mRNA expression in particular in DFs, in contrast, the mechanically wounded showed in EKs an increase of the aromatase activity at 24h compared to the non-wounded confluent monolayer cells. All data suggest that human skin and individual cells as target and source of active androgens and estrogens. Further studies are required to understand the role of steroids in inflammation, proliferation and differentiation which have implications for hair growth, skin cancer, aging and wound-healing. In particular, a greater understanding of DHEA signalling may help develop wound-healing therapies, without the risks of estrogen therapy, benefiting the elderly and patients with impaired wound-healing.Gli umani, come altri mammiferi, sintetizzano ormoni steroidei da organi endocrini come le gonadi, la surrenale e la placenta. L’espressione e attività di enzimi steroidogenici a livello di tessuti periferici provvedono alla formazione intracrina di androgeni ed estrogeni attivi a partire dal precursore deidroepiandrosterone (DHEA). Solamente umani ed alcuni primati secernono alti livelli di DHEA e DHEA-S. Tuttavia con l’età si presenta in entrambi i sessi una riduzione della secrezione adrenalica che si riflette in un notevole declino della produzione periferica di ormoni sessuali. Questa diminuita formazione è potenzialmente correlata alla patogenesi di malattie che incorrono con la vecchiaia tra cui anche alcune condizioni della pelle. Nel presente studio è stata valutata l’espressione dell’mRNA di sette proteine chiave della steroidogenesi (P450arom, P450scc, P450c17, STS, OATP2B1, 5α-riduttasi 1 e 5α-riduttasi 2), oltre che dei recettori di estrogeni (ERα e ERß) ed androgeni (AR), in biopsie di pelle e follicoli piliferi derivati da donatrici donne sane e in fibroblasti dermici e cheratinociti epidermici primari di donatori femmine e un maschio sempre sani. L’analisi condotta con RT-PCR e qRT-PCR ha rilevato in particolare che solo i follicoli esprimono 5α-riduttasi 2, i cheratinociti non presentano espressione dell’aromatasi (P450arom) e i fibroblasti non risultano esprimere il trasportatore OATP2B1. Dall’altra parte, entrambi i recettori ERα e ERß e quello per androgeni sono espressi nelle cellule della pelle e nei follicoli piliferi. In particolare ERß risulta con più forte espressione in cheratinociti e follicoli piliferi rispetto a ERα e il contrario in fibroblasti. In relazione quindi alla possibilità di una locale formazione ed azione di ormoni sessuali nella pelle, è stato investigato l’effetto in vitro di tre steroidi sull’espressione genica di follicoli piliferi e cheratinociti ottenuti da pelle facciale di donne sane. L’intero trascrittoma è stato analizzato dopo un’incubazione di 24 ore con DHEA-S (10µM), testosterone (TST) (50nM) e 17ß-estradiolo (E2) (1nM). In particolare sono state individuate modulazioni dell’espressione di geni coinvolti in funzioni come infiammazione, proliferazione e differenziamento cellulare, e di struttura. L’analisi condotta su cheratinociti è stata focalizzata su quattro geni sovra-espressi da tutti e tre i diversi trattamenti steroidei: CXCL1, ANGPTL4, TXNPI e LMNB1. Dati gli effetti e funzioni implicati dagli ormoni in esame, è stato valutato in vitro l’effetto di E2, DHEA, DHEA-S e TST sulla migrazione di fibroblasti e cheratinociti mediante scratch wound assay. L’analisi ha rilevato l’accelerazione della migrazione di entrambi i tipi cellulari in presenza di tutti gli steroidi in esame. In particolare, la combinazione di un inibitore dell’aromatasi (Arimidex) e uno della steroide solfatasi (STX64) hanno determinato l’inibizione dell’effetto stimolatorio rispettivamente di DHEA e TST, e di DHEA-S. I risultati suggeriscono un importante ruolo della formazione locale di E2 nella pelle a partire dal precursore DHEA (-S) e della sua azione nel processo di wound healing. Per verificare tale ipotesi della sintesi locale di estradiolo, è stata investigata l’attività dell’enzima catalizzante la conversione di androgeno ad estrogeno, l’aromatasi. Le misurazioni dell’attività aromatasica sono state eseguite con il metodo dell’acqua triziata. Specificatamente, l’esame è stato condotto su fibroblasti e cheratinociti umani e individuando l’effetto su attività ed espressione dell’mRNA dell’enzima dopo trattamento con desametasone o eseguendo mechanical scratch wound. Nel primo caso, il desametasone ha indotto stimolazione sia di attività che di espressione del trascritto. Mentre nel secondo caso, è stato rilevato un incremento in cheratinociti dopo 24 ore dallo scratch. In generale questo studio suggerisce che la pelle umana, oltre che separatamente fibroblasti e cheratinociti, e follicolo pilifero, siano target e fonte degli ormoni sessuali. Ulteriori studi saranno necessari per comprendere il ruolo di estrogeni ed androgeni in meccanismi come infiammazione, proliferazione e differenziamento cellulare, che possono avere implicazioni nella crescita del pelo, cancro della pelle, invecchiamento e wound healing. In particolare, la comprensione del signalling di DHEA aiuterebbe lo sviluppo di terapie per il wound healing riducendo i rischi correlati alla somministrazione degli estrogeni

Effects of Two Different Rhodiola rosea Extracts on Primary Human Visceral Adipocytes

Rhodiola rosea (Rro) has been reported to have various pharmacological properties, including anti-fatigue, anti-stress and anti-inflammatory activity. It is also known to improve glucose and lipid metabolism, but the effects of Rhodiola rosea on adipocyte differentiation and metabolism are not still elucidated. In this study the anti-adipogenic and lipolytic activity of two extracts of Rhodiola rosea, containing 3% salidroside (RS) or 1% salidroside and 3% rosavines (RR) on primary human visceral adipocytes was investigated. Pre-adipocytes were analyzed after 10 and 20 days of treatment during differentiation and after 7 days of treatment when they reached mature shape. The RS extract significantly induced higher apoptosis and lipolysis in comparison to control cells and to RR extract. In contrast, RR extract significantly reduced triglyceride incorporation during maturation. Differentiation of pre-adipocytes in the presence of RS and RR extracts showed a significant decrease in expression of genes involved in adipocyte function such as SLC2A4 and the adipogenic factor FGF2 and significant increase in expression of genes involved in inhibition of adipogenesis, such as GATA3, WNT3A, WNT10B. Furthermore RR extract, in contrast to RS, significantly down-regulates PPARG, the master regulator of adipogenesis and FABP4. These data support the lipolytic and anti-adipogenetic activity of two different commercial extracts of Rhodiola rosea in primary human visceral pre-adipocytes during differentiation

Effect of Arctium lappa (Burdock) extract on canine dermal fibroblasts

Although the biological activities of Arctium lappa (burdock) have been already investigated in human and other species, no data evaluating the molecular mechanisms have not been reported in the dog. In this study we analyzed for the first time the effect of a root extract of burdock on molecular responses in canine dermal fibroblasts with H2O2 stimulation (H group), with burdock treatment (B group) and with H2O2 stimulation and burdock treatment out H2O2 stimulation (BH group), using RNAseq technology. Differentially expressed genes (P < 0.05) among treatmentsof H, B and BH groups in comparison to the untreated sample (negative control, C group) were identified with MeV software and were functional annotated and monitored for signaling pathways and candidate biomarkers using the Ingenuity Pathways Analysis (IPA). The expression profile of canine dermal fibroblasts treated with burdock extract with or without H2O2 stimulation, showed a significantan up-regulation of mitochondrial superoxide dismutase (SOD2), disheveled 3 (DVL3) and chondroitin sulfate N-acetylgalactosaminyltransferase 2 (CSGALNACT2). The data suggested that burdock has implications in cell adhesion and gene expression with anti-oxidative and regenerative effects including the stimulation modulation of the production of extracellular matrix elements through Wnt/ f catenin signaling and Chondroitin Sulphate Biosynthesis that are particularly important for the wound healing process

Effects of Rosmarinus officinalis extract on human primary omental pre- adipocytes and adipocytes

3The prevalence of obesity is increasing all over the world. Although it has been shown that natural substances influence fat metabolism, little is known about the effect on cellular and molecular mechanisms in human. In this in vitro study, the activity of Rosmarinus officinalis (RO) standardized extract in modulating human primary visceral preadipocytes differentiation, lipolysis, and apoptosis was investigated. Moreover, gene expression of key adipogenesis modulators and microRNAs-seq were evaluated. Preadipocytes treated with RO extract significantly reduced triglyceride incorporation during maturation in a dose-dependent manner without affecting cell viability. In addition, RO extract stimulated lipolytic activity in differentiating preadipocytes and mature adipocytes in treated cells compared to controls. Differentiating preadipocytes incubated in the presence of RO extract showed a decreased expression of cell cycle genes such as cyclin D1, cyclin-dependent kinase 4, cyclin-dependent kinase inhibitor 1A (p21, Cip1) and an increased expression of GATA binding protein 3, wingless-type MMTV integration site family, member 3A mRNA levels. Recent studies have demonstrated that some phytochemicals alter the expression of specific genes and microRNAs that play a fundamental role in the pathogenesis of obesity and related diseases. Interestingly, genes modulated in RO-treated cells were found to be validated miRNAs targets, such as let-7f-1, miR-17, and miR-143. The results indicated that RO extract modulates human adipocyte differentiation and significantly interferes with adipogenesis and lipid metabolism, supporting its interest as dietary supplement.PMID: 25710930reservedmixedStefanon B.; Pomari E.; Colitti M.Stefanon, Bruno; Pomari, Elena; Colitti, Monic

Digital PCR: a new technology for diagnosis of parasitic infections

Parasitic infections are responsible for a significant burden of disease worldwide with international travel and immigration. More accurate diagnostic tools are necessary in support to parasite control and elimination programmes in endemic regions as well as for rapid case detection in non-endemic areas. Digital PCR (dPCR) is a powerful technology with recent applications in parasitology